Carbohydrates and glycobiology: the "3rd alphabet of life" after DNA and proteins

Glycans – the third revolution in Molecular biology 1

https://reasonandscience.catsboard.com/t2071-carbohydrates-and-glycobiology-the-3rd-alphabet-of-life-after-dna-and-proteins



The development and maintenance of a complex organism composed of trillions of cells is an extremely complex task. At the molecular level every process requires a specific molecular structures to perform it, thus it is difficult to imagine how less than tenfold increase in the number of genes between simple bacteria and higher eukaryotes enabled this quantum leap in complexity. In this perspective article we present the hypothesis that the invention of glycans was the third revolution in evolution (the appearance of nucleic acids and proteins being the first two), which enabled the creation of novel molecular entities that do not require a direct genetic template. Contrary to proteins and nucleic acids, which are made from a direct DNA template, glycans are product of a complex biosynthetic pathway affected by hundreds of genetic and environmental factors. Therefore glycans enable adaptive response to environmental changes and, unlike other epiproteomic modifications, which act as off/on switches, glycosylation significantly contributes to protein structure and enables novel functions. The importance of glycosylation is evident from the fact that nearly all proteins invented after the appearance of multicellular life are composed of both polypeptide and glycan parts.

GLYCANS ARE ONE OF FOUR MAJOR GROUPS OF MACROMOLECULES

Carbohydrates are one of four major groups of biologically important macromolecules that can be found in all forms of life. They have many biochemical, structural, and functional features that could provide a number of evolutionary benefits or even stimulate or enhance some evolutionary events. During evolution, carbohydrates served as a source of food and energy, provided protection against UV radiation and oxygen free radicals and participated in molecular structure of complex organisms. With time, simple carbohydrates became more complex through the process of polymerization and evolved novel functions. According to the one origin of life theory, called glyco-world, carbohydrates are thought to be the original molecules of life, which provided molecular basis for the evolution of all living things. Ribose and deoxyribose are integral parts of RNA and DNA molecules and cellulose (glucose polymer) is the most abundant molecule on the planet. There is also evidence for catalytic properties of some carbohydrates which further support theory about the capacity of glycans to enable evolution of life.



Carbohydrates are essential for all forms of life, but the largest variety of their functions is now found in higher eukaryotes. The majority of eukaryotic proteins are modified by cotranslational and posttranslational attachment of complex oligosaccharides (glycans) to generate the most complex epiproteomic modification – protein glycosylation. Very large number of different glycans can be made by varying number, order and type of monosaccharide units. The most abundant monosaccharides that can be found in animal glycan are: fucose (Fuc), galactose (Gal), glucose (Glu), mannose (Man), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), sialic acid (Sia) and xylose (Xyl). There are two main ways for protein modification with glycans: O-glycosylation and N-glycosylation. In O-glycosylation, the glycan is bound to the oxygen (O) atom of serine or threonine amino acid in the protein. Another type of protein glycosylation is N-glycosylation, where glycan is bound to the nitrogen (N) atom of asparagine amino acid in the protein.


Surfaces of all eukaryotic cells are covered with a thick layer of complex glycans attached to proteins or lipids. Many cells in our organism can function without the nuclei, but there is no known living cell that can function without glycans on their surface. Anything approaching the cell, being it a protein, another cell, or a microorganism, has to interact with the cellular glycan coat. This appears to be a universal rule since even in sponges, which are the simplest multicellular organisms formed by more or less independent cells, the recognition between cells is based on glycans. One of the critical steps in the evolution of multicellularity was formation of extracellular matrix. Multicellular life evolved independently multiple times during evolution and there are two main theories how the initial multicellular group of cells was made. The first theory says that individual cell came together to create symbiotic colonies, and another theory is that cells stayed together after cell division. Appearance of extracellular matrix enabled this initial group of cells to start function as a coordinated unit. Extracellular matrix has huge importance for multicellular organisms . It has role in cell signaling, communication between cells, cell adhesion and in transmitting signal from the environment, and also provides structural support for cells, tissues and organs. Extracellular matrix plays essential role in numerous fundamental processes such as differentiation, proliferation, survival and migration of cells. The main components of ECM are glycoproteins and proteoglycans and the same molecules are responsible for functional properties of ECM. Extracellular matrix evolved in parallel with first multicellular organisms (Hynes, 2012), therefore, glycans of the early ECM probably participated in evolution of multicellular organisms by enabling communication between cells and thus provided signals for cooperation and differentiation.



Nearly all membrane and secreted proteins are modified by covalent addition of glycans with very high site occupancy. Absence of glycosylation is embryonically lethal.
glycan parts of (glyco) proteins are integral elements of the final molecular structure and together with amino acids in the polypeptide backbone they form a single molecular entity that performs biological functions. Contrary to other posttranslational modifications that generally function as on/off switches, glycosylation generates large complex structures with more profound functions. The role of glycans in biological process should not be ignored since large part of the picture is missing when proteins are being studied without its glycans.

Two large obstacles in the study of glycans are their non-linear complex chemical structure and the absence of a direct genetic template. Contrary to polypeptides, which are a direct translation of the corresponding gene, glycans are encoded in a complex dynamic network comprising hundreds of genes

GLYCANS PROVIDE HIGHER EUKARYOTES WITH UNIQUE ADVANTAGES

Glycosylation, as the most complex epiproteomic modification, gives higher organisms some unique advantages. For example, IgG is one of the most important weapons in our “arsenal,” which enables us to successfully fight with microorganisms, despite their high mutation and reproduction rates.

Notch protein is the main actor in Notch signaling pathway that plays role in proper development of multicellular organisms. Notch is a transmembrane receptor composed of extracellular, transmembrane and intracellular domains. Upon ligand binding intracellular domain is cleaved and recruited into the nucleus to regulate expression of target genes

GLYCANS ENABLE DYNAMIC EPIGENETIC ADAPTATION

It is generally assumed that the appearance of self-replicating nucleic acids (the first revolution in evolution) provided the basis for the development of early life. Nucleic acids then recruited amino acids to create proteins, which are still the main effectors of life at the cellular level (the second revolution). However, the integration of different cells into a complex multicellular organism required an additional layer of complexity. Here we propose that the invention of protein glycosylation (the third revolution) through its inherent ability to create novel structures without the need to alter genetic information enabled the development of multicellular life in its present complexity.

The biggest evolutionary advantage that glycans confer to higher eukaryotes is the ability to create new structures without introducing changes into the precious genetic heritage. In principle all posttranslational modifications enable this to some extent, but most of them function as simple on/off molecular switches, while glycans represent significant structural components contributing with up to 50% in mass and even much more to the molecular volume of many proteins . The fact that so large parts of the molecule are not hardwired in the genome provides a rapid and extensive epiproteomic adaptation mechanism.

One example of role of glycosylation in the process of adaptation is found to be important for function of mammalian sperm cell and for the reproduction process itself. Mammalian sperm cells are masked with sialylated sugars in order to prevent recognition as foreign cells in the female reproductive system. After successful adaptation of sperm cell to the new environment, the removal of sialic acid residues from sperm surface glycans is the necessary step in the process of sperm cell maturation and the establishment of interaction between sperm and egg cells . Another interesting example how glycosylation of proteins can ensure adaptation and survival comes from the kingdom of archaebacteria 

Epigenetic regulation of gene expression has been reported to be important for protein glycosylation  and this could explain the observed temporal stability of the glycome . Comparative studies of the glycome in different organisms are rare, but they indicate higher rates of divergence in glycans than in proteins or DNA . Interactions established through glycans are not restricted just to cell- cell interactions and communication that could have played significant role in the evolution of multicellular life forms. Glycans also play significant role in the interaction between different organisms, including host-pathogen interactions or interactions between symbionts. Effect of glycosylation on the composition of the human intestinal microbiota has been well examined. Intestinal symbiotic bacteria are very important to humans as they help in food digestion, produce some vitamins and provide protection against pathogenic bacteria. In return, symbiotic bacteria use host glycan molecules as receptors for colonization of intestine and, also, both host and dietary glycans serve as energy source for symbiotic bacteria. It is reported that individuals who don’t secrete blood group glycans into the intestinal mucosa have reduced number and diversity of probiotic bacteria in the intestine . Except for food, symbiotic bacteria also use sugars that are highly abundant in intestine for glycosylation of their surface in order to escape the human immune system. Furthermore, digestion of sugars by symbiotic bacteria enables activation of signaling system that control pathogenicity of some non-symbiotic bacteria . Based on these facts, it can be safely assumed that glycans play important role in evolution of symbiotic relationship between humans and intestinal bacteria.

In some biological systems, like for example AB0 blood groups, glycans act as simple molecular switches that introduce inter-individual variability of cellular surfaces. In other systems, like immunoglobulin glycosylation, they enable new physiological functions, which could not be performed without this complex posttranslational tool. Glycosylation is particularly complex in human brain, but currently available technologies do not allow detailed study of this highly intricate system. Since all eukaryotic cells are heavily glycosylated (at significant metabolic cost) and elaborate mechanisms that regulate glycosylation are being discovered, we propose that the invention of glycosylation was the third large revolution in evolution, which enabled the development of complex multicellular organisms.



Glycan 7

The terms glycan and polysaccharide are defined by IUPAC as synonyms meaning "compounds consisting of a large number of monosaccharides linked glycosidically". However, in practice the term glycan may also be used to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan, even if the carbohydrate is only an oligosaccharide. Glycans usually consist solely of O-glycosidic linkages of monosaccharides. For example, cellulose is a glycan (or, to be more specific, a glucan) composed of β-1,4-linked D-glucose, and chitin is a glycan composed of β-1,4-linked N-acetyl-D-glucosamine. Glycans can be homo- or heteropolymers of monosaccharide residues, and can be linear or branched.

Glycans can be found attached to proteins as in glycoproteins and proteoglycans. In general, they are found on the exterior surface of cells.

Glycans are carbohydrate chains that are considered to be one of the most essential bio-informative macromolecules as well as nucleic acids and proteins. 3

Distinct from nucleic acids, glycans are expressed on cell surfaces and in extracellular matrices as various forms of glycoconjugates. They are indispensable to cover vital cells and to protect against physical and biochemical attacks. Distinct from proteins, glycans are indirect products of so-called glycogenes, i.e., genes that encode glycosyltransfearses, glycosidases and sugar nucleotide transporters involved in glycan biosynthesis. Since individual steps of these processes are not complete,
a series of glycans are produced simultaneously as a consequence of collaboration of glycogenes. As another unique feature of glycans, they have a number of linkage and branching isomers.

Glycoprotein 8

Glycoproteins are proteins that contain oligosaccharide chains (glycans) covalently attached to polypeptide side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycosylation. Secreted extracellular proteins are often glycosylated. In proteins that have segments extending extracellularly, the extracellular segments are also glycosylated. Glycoproteins are often important integral membrane proteins, where they play a role in cell–cell interactions.

Jonathan Wells: Far from being all-powerful, DNA does not wholly determine biological form 

For years and years and years, scientists routinely ignored the glycosylation of proteins, because it often made their structural studies difficult (so they just cut them off) and also because carbohydrates stuctures of glycans are much more difficult to study experimentally than DNA or proteins. Over the past 20 years however, glycobiology has started to take off. We now know that the glycan structures on proteins can control everything from protein signaling, protein half life, and cellular trafficking to regulating DNA transcription. Carbohydrates are also part of the histone code, and a special type of carbohydrate heavily regulates your epigenetics.

The entire set of glycan structures --the glycome-- is believed to be orders of magnitude more complex than the genome. Couple that with the fact the the glycome is also post translationally modified as well with events like phosphorylation, sulfation, acetylation, and now you have the millions of distinct molecular species that are needed to define life. Chew on this:

While DNA and proteins have gotten all of the attention, the realm of carbohydrates will be the next explosive field of biological research, if it hasn't already started to become it. You can not have life without carbohydrates and protein glycosylation. Sugars are absolutely critical for life, with major, major importance far above and beyond for just use as energy and carbon building blocks.


Saccharides have chirality like amino acids. Naturally occurring saccharides are basically defined as "D-enantiomers", while L-fucose, L-rhamnose and some other L-sugars are actually biosynthesized from either D-mannose or D-glucose. Important notation is that only few component saccharides, i.e., D-glucose, D-mannose and D-galactose are utilized in nature among possible 16 aldohexoses. This observation implies that the first living organisms could make use of a relatively small number of simple saccharides that had been sufficiently available on the prebiotic earth


Glycan structures can be massive, and the amount of glycans on glycoproteins can often exceed the molecular weight of the core protein itself (which makes you wonder why in the first place scientists chose to ignore glycosylation in the firstplace). For example, think of all of the physiology that is controlled by ion channels in your brain. Nearly 30% of the molecular weight of ion channels comes from glycosylation. Furthermore, just changing one special sugar structure on a ion channel can radically change the gating physiology of the ion channel. There are even examples where the glycan structures' importance even supercededs the importance of the protein itself for overall glycoprotein function.


Sugars and the prebiotic soup 6

Evidence that supports the occurrence of sugars in the prebiotic soup:

Monosaccharides form readily in Miller's spark-discharge experiment.
Heating H2CO molecules in solution forms almost all the pentose and hexose monosaccharides.

Conclusion: The formation of sugars is not a real issue anymore

Unresolved Problems

Laboratory simulations of early Earth -- Different pentoses and hexoses form in approximately equal amounts; but for RNA to form, ribose should have been dominant.

Chirality-Why did only right-handed sugars emerge during chemical evolution?

Carbohydrates consist of numerous functions that are important to living organisms. They are also known as saccharides, or sugar if they exist in small quantities; these names are used interchangeably to describe the same thing. The simplest carbohydrates are the monosaccharides, also known as simple sugars. Disaccharides are double sugars, consisting of two monosaccharides joined by a covalent bond. Carbohydrates also include polysaccharides, which are polymers composed of many sugar building blocks. The name "carbohydrate" is derived from 'hydrates of carbon', and they arise from photosynthesis, where they exist as products.

Carbohydrates are the most abundant aldehyde compounds found in living organisms. They provide storage, transport starch and glycogen that provide energy to bodies, and contain structural components such as cellulose in plants and chitin in animals. Additionally, they contribute to the immune system, fertilization, pathogenesis, blood clotting, and development. 5

There are four general classes of carbohydrates: monosaccharides, disaccharides, oligosaccharides, and polysaccharides.

The most important carbohydrate is glucose. In general, monosaccharides have one carbonyl group (aldehyde, ketone, or acid), and the remaining carbons each bear one hydroxyl group. Monosaccharides can be linked together via ether and/or acetal bonds to form very large polymers called polysaccharides. A disaccharide consists of 2 linked monosaccharides and so on. Almost all saccharides in nature have at least one chiral carbon and they occur in nature as a single enantiomer. Glucose has 4 chiral carbons and has 15 other stereoisomers for a total of 16 possible stereoisomers of this gross structural formula.

The suffix –ose is often used in describing and naming carbohydrates. For example:

A carbohydrate with 6 carbons is called a hexose
A carbohydrate with 5 carbons is called a pentose
A carbohydrate with an aldehyde as its carbonyl unit is called an aldose
A carbohydrate with a ketone as its carbonyl unit is called a ketose

Glucogen Metabolism Glucose metabolism and various forms of it in the process is described by the process below. Glucose-containing compounds are digested and taken up by the body in the intestines, including starch, glycogen, disaccharides and as monosaccharide. Glucose is stored in mainly the liver and muscles as glycogen. It is distributed and utilized in tissues as free glucose.



There are three reasons why we study glycans. First, they play an important role in living organisms (functional importance). Second, compared with nucleic acids and proteins they are substantially more difficult to synthesize and characterize, which, once accomplished, should therefore have the potential to introduce a new paradigm in life science (an attractive and challenging target for scientists). Third, the origin of glycans is closely linked to the origin of life and its evolution (relationship with the origin of life and its evolution) although this cannot be verified by experiments. If the origin of glycans is as old as or older than that of nucleic acids and proteins, proteins which are associated with glycosyltransferases and sugar-nucleotide syntheses (synthesis systems of glycans), and recognition systems of glycans (lectins, cytokines and antibodies against glycans, etc.), which specifically recognize and identify glycans derived thereof, can be assumed to have evolved in conjunction with each other 4




The topic, “Comparative glycomics and life evolution” comprises “Glycans in various organisms”, “Evolution of glycosyltransferases” and “Evolution of lectins”, and elucidates a variety of biological activities from the viewpoint of “origin and evolution of glycans” or “comparative glycomics”.

Some sacharides are assumed to have evolved chemically prior to the beginning of life due to the fact that they are synthesized nonbiologically (formose reaction, aldol condensation, and Lobry de Bruyn transformation,





Carbohydrates

The same way aldehydes and ketones react with alcohols to form hemiacetals and hemiketals, respectively, carbohydrates react intermolecularly to form rings. When forming a ring 5 or 6 membered ring is most favorable and will only be formed. The Carbon 1 will be attacked by either the Carbon 5 or Carbon 6 hydroxyl group to form a 5 or 6 membered (respectively)carbohydrate ring.

The carbohydrates are a major source of metabolic energy, both for plants and for animals that depend on plants for food. Aside from the sugars and starches that meet this vital nutritional role, carbohydrates also serve as a structural material (cellulose), a component of the energy transport compound ATP, recognition sites on cell surfaces, and one of three essential components of DNA and RNA. Carbohydrates are called saccharides or, if they are relatively small, sugars.

Glycans, which are assumed to have been first synthesized in the form of simple homo-polysaccharides (amylose, cellulose, etc.), are understood to have evolved into more complex hetero-polysaccharides (Evolution of the “synthesis systems of glycans”, see “Glycogene”). This evolution is assumed to have triggered the advent of proteins (“lectins”, see “Lectin”) related to the “recognition system of glycans” that recognizes each structure, identifies molecules, introduces biological signaling and facilitates infections. The synthesis system and the recognition system of glycans depend on each other and are still considered to be undergoing coevolution.


You'll also read how metabolism is another primary regulator of differentiation and development:

http://www.nature.com/nchembio/journal/v6/n3/full/nchembio.324.html

http://www.ncbi.nlm.nih.gov/pubmed/23715547

The next revolution will come from metabolomics. Genomic sequencing is nice and all, but the more important question is how exactly do mutations physiologically manifest themselves? Genomics only offers some indirect insight into this. There can be mutations in genes that have no physiological relevance at all, but a genomics screen may flag them as important. Gene expression in tons of cases also doesn't correlate with protein quantities at all. Metabolomics OTOH tells you the exact picture of physiology in a snapshot of time to reveal which parts of the metabolome are perturbed.

We've been waiting for the revolution that genomics has promised for almost 20 years now, where are the spectacular results? We forgot that there's a huge gap between genetic expression, protein quantities, and how networks of proteins actually behave physiologically.

THe article is getting at the fact that the hbp is a major nexus for metabolism. The carbohydrates produced by the hbp can control everything from protein folding to epigentics and DNA transcriptional events. The hbp responds dynamically to stress and is believe to confer cardioprotection due to the upr that may occur after a stress event like ischaemia injury.

these pathways are affected by nutrient deprivation, for example see this review:

http://www.sciencedirect.com/science/article/pii/S0304416509002074

Imagine the HBP as a hidden biochemical super computer within the cell. It dynamically responds to nutritional states to titrate carbohydrate metabolic fluxes, which can modulate everything from protein/DNA expression, to protein folding, signaling, protein half lives and more.
It is known now that the carbohydrates produced by the HBP, specifically UDP-GlcNAc which is used for the O-GlcNAc modifcation (see review, O-GlcNAc if you aren't aware, is basically a master-master regulator of virtually all cellular functions), are part of the histone code and profoundly regulate epigenetics:

http://www.ncbi.nlm.nih.gov/pubmed/22522719

Nutritional states, through carbohydrates, get imprinted onto your genome epigentically, which can be passed down to offspring--so yes, these pathways are affected by glucose restricted diets. Gluconeogenesis probably can compensate a bit, but as for how much, probably no one knows yet.
Since these pathways are affected by nutritional environment, this could impact treatment strategies during cardiac stress. For example, one idea may be simply to feed people GlcNAc (which can be bought) after or during cardiac stress injuries since it bypasses many feedback control mechanisms of the HBP.
Also, to address your last question even more there is this article:

https://circ.ahajournals.org/content/116/8/884.full

Too much of a good thing may be bad though. You have of course the well known heart problems with diabetic patients (overactive HBP probably bad). But in response to acute injury, the HBP and glucose metabolism play a very important role in cardioprotection.
The O-GlcNAc modification (from the review) and protein glycosylation control so many different aspects of life and this is why glycobiology and carbohydrates have been called the 3rd alphabet of life after DNA and proteins.

2) http://crev.info/2006/09/nothing_in_evolution_makes_sense_except_in_the_light_of_speculation/
3) http://origin-life.gr.jp/2903/2903119/2903119.pdf
4) http://www.glycoforum.gr.jp/science/word/evolution/ES-00E.html
5) https://en.wikibooks.org/wiki/Structural_Biochemistry/Organic_Chemistry/Carbohydrates
6) http://www.uic.edu/classes/bios/bios100/mike/spring2003/lect04.htm
7) https://en.wikipedia.org/wiki/Glycan
8 )https://en.wikipedia.org/wiki/Glycoprotein
9) http://www.reddit.com/r/science/comments/2411zy/spark_of_life_metabolism_appears_in_lab_without/

http://blog.drwile.com/?p=12071

http://creation.com/lipid-rafts-evidence-of-biosyntax-and-biopragmatics

This situation is analogous to the sentences discussed above where the same words mean something different when the syntax is altered. The two conditions, ‘ErbB4 in lipid rafts’ and ‘ErbB4 not in lipid rafts’, differ only in the relative position of the relevant components of the system and not in the presence or absence of these components. The lipid rafts do not change the statistics of the information in the signalling pathway. Instead, they form a context in which the arrangements of the members take on defined meanings. Thus in these cultured cortical neurons, lipid rafts provide the molecular syntax in which NRG-induced activation of ErbB4 can bring about particular effects that do not occur in their absence

http://www.ncbi.nlm.nih.gov/pubmed/?term=Lauc+G%2C+Vojta+A%2C+Zoldo%C5%A1+V+%282014%29+Epigenetic+regulation+of+glycosylation+is+a+quantum+mechanics+of+biology.+BBA+General+Subjects%2C+1840%281%29%3B+65-70.

Most proteins are glycosylated, with glycans being integral structural and functional components of a glycoprotein. In contrast to polypeptides, which are fully encoded by the corresponding gene, glycans result from a dynamic interaction between the environment and a network of hundreds of genes

http://www.sciencemag.org/site/products/lst_20110107.xhtml

What's the most abundant posttranslational modification on eukaryotic proteins? It's not phosphorylation. Some 50 percent of eukaryotic proteins, and not just those on the cell surface, are dusted with sugars like some molecular pastry. Those glycan modifications mediate inter-molecular and intercellular binding events from fertility to immunity. Yet for years, researchers in the sugar and protein communities have operated independently of one another, cataloging sugars free of protein, or proteins free of sugar, and ne'er the twain shall meet. Today, though, the two communities are bridging their technical divide. "We are in a transition now," says James Paulson, principal investigator of the Consortium for Functional Glycomics. Glycoproteomics, he says, "is becoming mainstream."

Glycoproteomics might be "becoming mainstream," but it's not there yet, and it won't come easy. Proteomics—cataloging and quantifying proteins from a biological sample en masse—is more or less routine. Glycomics, proteomics' glycan analog, is considerably more challenging, but it too is doable.

Put them together, though, and the problem becomes exponentially greater, a reflection of the fact that glycoproteomics encompasses two completely different classes of molecules—molecules with very different chemistries, compositions, and structures.

"Many glycoproteins have 10, 20, 30, and in worst case, hundreds of glycosylation sites within a single glycoprotein," says Stuart Haslam of Imperial College London. "Some of those sites will be occupied, and some will not, and each can have a variety of glycans associated with it." One 2007 study from Haslam's research director, Anne Dell, documented over 100 different modifications on one site on a single protein in the mouse zona pellucida.

More recently, though, a small but growing set of publications demonstrate that it is actually possible to study glycans in the context of their protein scaffolds. Though researchers cannot yet do so in a high throughput proteomics mode, they're getting close

Glycoprotein sugars are not the stuff of baking and coffee. Sugars, says Paulson, are "the third alphabet" of molecular biology (the others being nucleic acids and protein). Theirs is an alphabet of mannose and fucose, of N-acetylglucosamine and sialic acid, and it is one that often is expressed not in letters but hieroglyphics of diamonds, circles, and squares. These "cartoons," as they are called, are both easier to read and write than the chemical entities they describe: oligosaccharides like "GalNAcα1-4GalNAcα1-4(Glcβ1-3)GalNAcα1-4GalNAcα1- 4GalNAcα1-3Baβ1-NAsn."

That alphabet can assume practically limitless arrangements. Synthesized sans template by enzymes called glycosyltransferases, glycan modifications run the gamut from simple monosaccharides to complex branching trees, with myriad compositions and chemical linkages. "There may be well over 10,000 carbohydrate structures in the human glycome," says Cummings.

One variable, for instance, is the glycan-peptide bond. Glycans couple to proteins primarily in two ways, though dozens of different linkages have actually been des cribed, says Cummings. In O-linked glycans, sugars are linked to the protein backbone through the hydroxyl oxygens of serine and threonine residues; N-linked carbohydrates couple via the nitrogen atoms in asparagine side chains.

With so many variables, glycosylation provides "a massive exponential enhancement to the information content in the genome," Cummings says. The molecular adage, one gene-one protein, still holds, but glycosylation turns that notion on its head. Glycodelin, for instance, is expressed both in males and females, in two very different glycoforms. In women, the glycoprotein is a contraceptive; in men, it promotes sperm-egg binding.

Plus, actually solving a glycan's complete structure—not just its composition, but also its order and inter-unit linkages—is a complex, time-consuming exercise, something many proteomics researchers are either unwilling or unable to do.

"You cannot understand the structure of a carbohydrate until you look at every intervening linkage between each monomer, because they all vary," he says. "It can vary by one residue and [that may] be the effective residue that gives the carbohydrate its function."

Glycans – the third revolution in evolution 1

The development and maintenance of a complex organism composed of trillions of cells is an extremely complex task. At the molecular level every process requires a specific molecular structures to perform it, thus it is difficult to imagine how less than tenfold increase in the number of genes between simple bacteria and higher eukaryotes enabled this quantum leap in complexity. In this perspective article we present the hypothesis that the invention of glycans was the third revolution in evolution (the appearance of nucleic acids and proteins being the first two), which enabled the creation of novel molecular entities that do not require a direct genetic template. Contrary to proteins and nucleic acids, which are made from a direct DNA template, glycans are product of a complex biosynthetic pathway affected by hundreds of genetic and environmental factors. Therefore glycans enable adaptive response to environmental changes and, unlike other epiproteomic modifications, which act as off/on switches, glycosylation significantly contributes to protein structure and enables novel functions. The importance of glycosylation is evident from the fact that nearly all proteins invented after the appearance of multicellular life are composed of both polypeptide and glycan parts.

GLYCANS ARE ONE OF FOUR MAJOR GROUPS OF MACROMOLECULES

Carbohydrates are one of four major groups of biologically important macromolecules that can be found in all forms of life. They have many biochemical, structural, and functional features that could provide a number of evolutionary benefits or even stimulate or enhance some evolutionary events. During evolution, carbohydrates served as a source of food and energy, provided protection against UV radiation and oxygen free radicals and participated in molecular structure of complex organisms. With time, simple carbohydrates became more complex through the process of polymerization and evolved novel functions. According to the one origin of life theory, called glyco-world, carbohydrates are thought to be the original molecules of life, which provided molecular basis for the evolution of all living things (Stern and Jedrzejas, 2008). Ribose and deoxyribose are integral parts of RNA and DNA molecules and cellulose (glucose polymer) is the most abundant molecule on the planet. There is also evidence for catalytic properties of some carbohydrates (Del Valle, 2004) which further support theory about the capacity of glycans to enable evolution of life.

Carbohydrates are essential for all forms of life, but the largest variety of their functions is now found in higher eukaryotes. The majority of eukaryotic proteins are modified by cotranslational and posttranslational attachment of complex oligosaccharides (glycans) to generate the most complex epiproteomic modification – protein glycosylation. Very large number of different glycans can be made by varying number, order and type of monosaccharide units. The most abundant monosaccharides that can be found in animal glycan are: fucose (Fuc), galactose (Gal), glucose (Glu), mannose (Man), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), sialic acid (Sia) and xylose (Xyl). There are two main ways for protein modification with glycans: O-glycosylation and N-glycosylation. In O-glycosylation, the glycan is bound to the oxygen (O) atom of serine or threonine amino acid in the protein. Another type of protein glycosylation is N-glycosylation, where glycan is bound to the nitrogen (N) atom of asparagine amino acid in the protein.

Surfaces of all eukaryotic cells are covered with a thick layer of complex glycans attached to proteins or lipids. Many cells in our organism can function without the nuclei, but there is no known living cell that can function without glycans on their surface. Anything approaching the cell, being it a protein, another cell, or a microorganism, has to interact with the cellular glycan coat (Gagneux and Varki, 1999; Varki and Lowe, 2009; Varki, 2011). This appears to be a universal rule since even in sponges, which are the simplest multicellular organisms formed by more or less independent cells, the recognition between cells is based on glycans (Misevic and Burger, 1993). One of the critical steps in the evolution of multicellularity was formation of extracellular matrix (ECM; Sachs, 2008; Hynes, 2012). Multicellular life evolved independently multiple times during evolution and there are two main theories how the initial multicellular group of cells was made. The first theory says that individual cell came together to create symbiotic colonies, and another theory is that cells stayed together after cell division (Sachs, 2008). Appearance of extracellular matrix enabled this initial group of cells to start function as a coordinated unit. Extracellular matrix has huge importance for multicellular organisms (Hynes, 2009). It has role in cell signaling, communication between cells, cell adhesion and in transmitting signal from the environment, and also provides structural support for cells, tissues and organs. Extracellular matrix plays essential role in numerous fundamental processes such as differentiation, proliferation, survival and migration of cells. The main components of ECM are glycoproteins and proteoglycans and the same molecules are responsible for functional properties of ECM (Hynes and Naba, 2012). Extracellular matrix evolved in parallel with first multicellular organisms (Hynes, 2012), therefore, glycans of the early ECM probably participated in evolution of multicellular organisms by enabling communication between cells and thus provided signals for cooperation and differentiation.

Nearly all membrane and secreted proteins are modified by covalent addition of glycans with very high site occupancy. Absence of glycosylation is embryonically lethal.
glycan parts of (glyco) proteins are integral elements of the final molecular structure and together with amino acids in the polypeptide backbone they form a single molecular entity that performs biological functions. Contrary to other posttranslational modifications that generally function as on/off switches, glycosylation generates large complex structures with more profound functions. The role of glycans in biological process should not be ignored since large part of the picture is missing when proteins are being studied without its glycans.

Two large obstacles in the study of glycans are their non-linear complex chemical structure and the absence of a direct genetic template. Contrary to polypeptides, which are a direct translation of the corresponding gene, glycans are encoded in a complex dynamic network comprising hundreds of genes

GLYCANS PROVIDE HIGHER EUKARYOTES WITH UNIQUE ADVANTAGES

Glycosylation, as the most complex epiproteomic modification, gives higher organisms some unique advantages. For example, IgG is one of the most important weapons in our “arsenal,” which enables us to successfully fight with microorganisms, despite their high mutation and reproduction rates.

Notch protein is the main actor in Notch signaling pathway that play role in proper development of multicellular organisms. Notch is a transmembrane receptor composed of extracellular, transmembrane and intracellular domains. Upon ligand binding intracellular domain is cleaved and recruited into the nucleus to regulate expression of target genes

GLYCANS ENABLE DYNAMIC EPIGENETIC ADAPTATION

It is generally assumed that the appearance of self-replicating nucleic acids (the first revolution in evolution) provided the basis for the development of early life. Nucleic acids then recruited amino acids to create proteins, which are still the main effectors of life at the cellular level (the second revolution). However, the integration of different cells into a complex multicellular organism required an additional layer of complexity. Here we propose that the invention of protein glycosylation (the third revolution) through its inherent ability to create novel structures without the need to alter genetic information enabled the development of multicellular life in its present complexity.

The biggest evolutionary advantage that glycans confer to higher eukaryotes is the ability to create new structures without introducing changes into the precious genetic heritage . In principle all posttranslational modifications enable this to some extent, but most of them function as simple on/off molecular switches, while glycans represent significant structural components contributing with up to 50% in mass and even much more to the molecular volume of many proteins . The fact that so large parts of the molecule are not hardwired in the genome provides a rapid and extensive epiproteomic adaptation mechanism.

One example of role of glycosylation in the process of adaptation is found to be important for function of mammalian sperm cell and for the reproduction process itself. Mammalian sperm cells are masked with sialylated sugars in order to prevent recognition as foreign cells in the female reproductive system. After successful adaptation of sperm cell to the new environment, the removal of sialic acid residues from sperm surface glycans is the necessary step in the process of sperm cell maturation and the establishment of interaction between sperm and egg cells . Another interesting example how glycosylation of proteins can ensure adaptation and survival comes from the kingdom of archaebacteria

Epigenetic regulation of gene expression has been reported to be important for protein glycosylation  and this could explain the observed temporal stability of the glycome . Comparative studies of the glycome in different organisms are rare, but they indicate higher rates of divergence in glycans than in proteins or DNA . Interactions established through glycans are not restricted just to cell- cell interactions and communication that could have played significant role in the evolution of multicellular life forms. Glycans also play significant role in the interaction between different organisms, including host-pathogen interactions or interactions between symbionts. Effect of glycosylation on the composition of the human intestinal microbiota has been well examined. Intestinal symbiotic bacteria are very important to humans as they help in food digestion, produce some vitamins and provide protection against pathogenic bacteria. In return, symbiotic bacteria use host glycan molecules as receptors for colonization of intestine and, also, both host and dietary glycans serve as energy source for symbiotic bacteria. It is reported that individuals who don’t secrete blood group glycans into the intestinal mucosa have reduced number and diversity of probiotic bacteria in the intestine . Except for food, symbiotic bacteria also use sugars that are highly abundant in intestine for glycosylation of their surface in order to escape the human immune system. Furthermore, digestion of sugars by symbiotic bacteria enables activation of signaling system that control pathogenicity of some non-symbiotic bacteria . Based on these facts, it can be safely assumed that glycans play important role in evolution of symbiotic relationship between humans and intestinal bacteria.

In some biological systems, like for example AB0 blood groups, glycans act as simple molecular switches that introduce inter-individual variability of cellular surfaces. In other systems, like immunoglobulin glycosylation, they enable new physiological functions, which could not be performed without this complex posttranslational tool. Glycosylation is particularly complex in human brain, but currently available technologies do not allow detailed study of this highly intricate system. Since all eukaryotic cells are heavily glycosylated (at significant metabolic cost) and elaborate mechanisms that regulate glycosylation are being discovered, we propose that the invention of glycosylation was the third large revolution in evolution, which enabled the development of complex multicellular organisms.

http://darwins-god.blogspot.com.br/search?q=glycans

Even by mid century contradictions to evolutionary expectations were becoming obvious in serological tests. As J.B.S.Haldane explained in 1949:

Now every species of mammal and bird so far investigated has shown quite a surprising biochemical diversity by serological tests. The antigens concerned seem to be proteins to which polysaccharides are attached. We do not know their functions in the organism, though some of them seem to be part of the structure of the cell membrane. I wish to suggest that they may play a part in disease resistance, a particular race of bacteria or virus being adapted to individuals of a certain range of biochemical constitutions, while those of other constitutions are relatively resistant.

Indeed these polysaccharides, or glycans, would become rather uncooperative with evolution. As one recent paper explained, glycans show “remarkably discontinuous distribution across evolutionary lineages,” for they “occur in a discontinuous and puzzling distribution across evolutionary lineages.” This dizzying array of glycans can be (i) specific to a particular lineage, (i) similar in very distant lineages, (iii) and conspicuously absent from very restricted taxa only. In other words, the evidence is not what evolution expected.

Here is how another paper described early glycan findings:

There is also no clear explanation for the extreme complexity and diversity of glycans that can be found on a given glycoconjugate or cell type. Based on the limited information available about the scope and distribution of this diversity among taxonomic groups, it is difficult to see clear trends or patterns consistent with different evolutionary lineages. It appears that closely related species may not necessarily share close similarities in their glycan diversity, and that more derived species may have simpler as well as more complex structures. Intraspecies diversity can also be quite extensive, often without obvious functional relevance.

So is the evidence a problem for evolution? No, of course not. For as the paper explains:

Here we discuss the significance of this remarkable diversity, mindful of the oft-repeated adage of Dobzhansky's that “nothing in biology makes sense, except in the light of evolution.”

And so we are back to that “another story” again. This non scientific claim is, for evolutionists, the gift that just keeps on giving. It seems any evidential problem is easily disposed of with this handy truism. It is like a chant for evolutionists. Say it enough times and evolution is, as they say, a fact, in spite of the evidence. Here is how another, slightly more self-conscious, paper put it:

While we would certainly agree with the statement that “nothing in glycobiology makes sense, except in the light of evolution”, we must also realize that evolution only occurred once and that evolution does not follow well-defined rules. This situation is somewhat alleviated by the fact that after lineages diverge, more often than not they remain separated for good and, thus provide researchers with large numbers of iterations (“pseudo samples”) for which evolutionary processes have occurred independently. The study of these divergent lineages provides a good opportunity to elucidate evolutionary mechanisms.

Even in the worst of circumstances this favorite tenet of evolutionary thought is serviceable. It can always do the heavy lifting when necessary.

The cell is not only profoundly complex in the inside, its outer surface is also incredible with its wide array of molecular machines and entities. One important type of molecule on the cell’s surface is the glycans—long carbohydrate molecules that come in phenomenal variety. In fact, this variety is not only tremendous, the trade secret is that it violates every rule of evolution. For instance, though the term “lineage-specific biology,” which is the exact opposite of evolutionary expectations, has been popular in recent years, it could have been used half a century ago for glycans when J.B.S.Haldane observed that “every species of mammal and bird so far investigated has shown quite a surprising biochemical diversity by serological tests. The antigens concerned seem to be proteins to which polysaccharides are attached.” In fact, as one recent paper explains, glycans show “remarkably discontinuous distribution across evolutionary lineages,” for they “occur in a discontinuous and puzzling distribution across evolutionary lineages.” This dizzying array of glycans can be (i) specific to a particular lineage, (i) similar in very distant lineages, (iii) and conspicuously absent from very restricted taxa only. The contradictions are so common even evolutionists are now issuing their own disclaimers.

Glycan Evolution in Response to Collaboration, Conflict, and
Constraint 1

Glycans, oligo- and polysaccharides secreted or attached to
proteins and lipids, cover the surfaces of all cells and have a
regulatory capacity and structural diversity beyond any other
class of biological molecule. Glycans may have evolved these
properties because they mediate cellular interactions and often
face pressure to evolve new functions rapidly. We approach this
idea two ways. First, we discuss evolutionary innovation. Glycan
synthesis, regulation, and mode of chemical interaction influence
the spectrum of new forms presented to evolution. Second,
we describe the evolutionary conflicts that arise when alleles
and individuals interact. Glycan regulation and diversity are
integral to these biological negotiations. Glycans are tasked with
such an amazing diversity of functions that no study of cellular
interaction can begin without considering them. We propose
that glycans predominate the cell surface because their physical
and chemical properties allow the rapid innovation required of
molecules on the frontlines of evolutionary conflict.

All cellular organisms are coated by a glycocalyx, a “sugar
shell” that defines the molecular frontier of cells, tissues, and
whole organisms





Glycans are oligo- or polysaccharide
molecules that can be secreted or attached to proteins or
lipids, forming glycoconjugates. Glycans form one of the four
classes of biomolecules together with nucleic acids, lipids, and
proteins


50% of all human proteins are glycosylated. The extracellular polysaccharides
of plants, fungi, and arthropods (cellulose and chitin) are the
most abundant biomolecules on earth. Glycan synthesis is not
template-driven; genes do not encode them directly. Rather,
glycans are assembled by synthetic enzymes in the endoplasmic
reticulum Golgi or during direct secretion

Glycans contain a variety of monosaccharide units and
linkages joining them in structures that can be branched
and further chemically modified. Sizes range from the single
GalNAc monosaccharide of the Tn antigen to glycosaminoglycan
polymers thousands of units long . Glycan functions
include physical and structural roles (adding rigidity or
charge), extracellular matrix formation and morphogenesis
(glycosaminoglycans), protein folding, transcriptional regulation
(by O-GlcNAcylation), and information exchange between
cells (stage-specific embryonic antigens)

There are striking differences
between proteins and glycans. Proteins are direct
products of alleles encoded in DNA, whereas glycans take a
variety of forms that reflect variation in their synthesis and
attachment. Foreign proteins trigger adaptive immune reactions
due to sampling and presentation by immune cells. Foreign
glycans typically trigger less specific innate immune reactions.
Modes of glycan and protein evolution can also differ.

From the book : The sugar Code, fundamentals of glycosciences

The book of life is written with a molecular alphabet that is not limited to the four letters of the genetic code. This fact is being increasingly recognized not only by chemists but also by biologists and physicians, who have termed emerging fields 'molecular biology, molecular genetics, molecular medicine, molecular cardiology, etc'. For glycosciences, this is also the 'age of the molecule'. Modern views largely accept that the co- and post-translational glycosylation of proteins has a function in molecular recognition. Protein-carbohydrate and carbohydrate-carbohydrate interactions control salient aspects of intra- and intercellular communication and trafficking, and are at the basis of a variety of essential biological phenomena, such as clearance of glycoproteins from the circulatory system, adhesion of infectious agents to host cells, and cell adhesion in the immune system, malignancy and metastasis.

To encode this complex biomolecular recognition ability, Nature has at its disposal a very powerful information tool, the sugar code. The role of reading the sugar-encoded messages is mainly played by a class of carbohydrate-binding proteins called lectins, which, together with their synthetic analogues and the complementary glycomimetics, have attracted the attention of many scientists with a different cultural background and training, coming from organic, medicinal and pharmaceutical chemistry, biochemistry, biology, medicine and even material science.

The modern concepts of glycosciences are not covered in the currently available organic chemistry, biochemistry and medicine textbooks, the former dealing mainly with the synthetic and conformational properties of carbohydrates and the last two with the biosynthesis of polysaccharides such as glycogen and the role of sugars as biochemical fuel in energy metabolism.

from Stephen C. Meyers book : Darwin's doubt :

The Sugar Code

further readings :

http://theskepticalzone.com/wp/?p=28360

http://www.nature.com/nrm/journal/v13/n5/full/nrm3334.html


from Stephen C. Meyers book : Darwin's doubt : The Sugar Code

http://reasonandscience.heavenforum.org/t2071-carbohydrates-and-glycobiology-the-3rd-alphabet-of-life-after-dna-and-proteins#3626

 in at least the case of the sugar molecules on the cell surface, gene products play no direct role. Genetic information produces proteins and RNA molecules, not sugars and carbohydrates. Of course, important glycoproteins and glycolipids (sugar-protein and sugar-fat composite molecules) are modified as the result of biosynthetic pathways involving networks of proteins. Nevertheless, the genetic information that generates the proteins in these pathways only determines the function and structure of the individual proteins; it does not specify the coordinated interaction between the  proteins in the pathways that result in the modification of sugars.  More important, the location of specific sugar molecules on the exterior surface of embryonic cells plays a critical role in the function that these sugar molecules play in intercellular communication and arrangement. Yet their location is not determined by the genes that code for the proteins to which these sugar molecules might be attached.

Many cellular structures are built from proteins, but proteins find their way to correct locations in part because of preexisting three-dimensional patterns and organization inherent in cellular structures. Neither structural proteins nor the genes that code for them can alone determine the three-dimensional shape and structure of the entities they build. Gene products provide necessary, but not sufficient, conditions for the development of three-dimensional  structure within cells, organs, and body plans. If this is so, then natural selection acting on genetic  variation and mutations alone cannot produce the new forms that arise in the history of life. 


One of the biological dogmas is that self-replicating nucleic acids (the first revolution in evolution) provided the basis for development of early life through translation of nucleotide sequence into a sequence of amino acids to create the main effectors of life at the cellular level – the proteins (the second revolution in evolution). The integration of different cells into a complex multicellular organism required additional layers of complexity and the invention of glycans (the third revolution in evolution) through its inherent ability to create novel structures without the need to mutate the genetic information, but to alter it by epigenetic modifications instead, thus providing multicellular organisms with a mechanism to compensate for their longer generation time by using their higher complexity for rapid adaptation to various environmental challenges.

Plant Glycobiology—a diverse world of lectins, glycoproteins, glycolipids and glycans 1

Glycosylation is essential for the growth, development or survival of every organism . Defects in glycan signaling often lead to abnormal development and severe diseases.  Defects in glycan signaling often lead to abnormal development and severe diseases. Glycosylation is ubiquitous and the tremendous structural complexity of glycans makes it quite impossible to predict the biological importance of individual structures. Nowadays, glycans are no longer regarded solely as an energy reservoir, but are associated with storage and transfer of biological information as part of a highly complicated multidimensional coding system. Plants synthesize a wide variety of unique glycan structures and glycan-binding proteins which play pivotal roles during their life cycle.

Some proteins bind to complex carbohydrates in a carbohydrate-specific fashion, including enzymes, lectins and antibodies. These carbohydrates, assembled by sequential glycosyl transferases, also carry biological information, the other side of which is a binding protein that recognizes a specific sugar monosaccharides, sequence, anomerity, linkage, ring size, branching and substitution. It is the latter 7 parameters, however, that give carbohydrates a very large potential for information-carrying capacity in a short sequence. An exponentially growing body of knowledge exists in this aspect of carbohydrate function. 2


Carbohydrates contain an evolutionary potential of information content several orders of magnitude higher in a short sequence than any other biological oligomer. This is due to monomers capable of more than one linkage position, anomerity and branching. This high potential for information capacity exists in biological recognition systems comprised of complex carbohydrate ligands on the one hand which are recognized for targeted activities, on the other hand, by hapten specific protein receptors, such as lectins or antibodies. Evidence is accumulating that carbohydrate-carbohydrate interactions play roles in some recognition systems. Certainly this is true in self-association of polymer chains such as cellulose and chitin.

Bittersweet memories: linking metabolism to epigenetics through O-GlcNAcylation 3

O-GlcNAcylation, which is a nutrient-sensitive sugar modification, participates in the epigenetic regulation of gene expression. The enzymes involved in O-linked β-D-N-acetylglucosamine (O-GlcNAc) cycling — O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) — target key transcriptional and epigenetic regulators including RNA polymerase II, histones, histone deacetylase complexes and members of the Polycomb and Trithorax groups. Thus, O-GlcNAc cycling may serve as a homeostatic mechanism linking nutrient availability to higher-order chromatin organization. In response to nutrient availability, O-GlcNAcylation is poised to influence X chromosome inactivation and genetic imprinting, as well as embryonic development. The wide range of physiological functions regulated by O-GlcNAc cycling suggests an unexplored nexus between epigenetic regulation in disease and nutrient availability.

A little sugar goes a long way: The cell biology of O-GlcNAc 4
Unlike the complex glycans decorating the cell surface, the O-linked β-N-acetyl glucosamine (O-GlcNAc) modification is a simple intracellular Ser/Thr-linked monosaccharide that is important for disease-relevant signaling and enzyme regulation. O-GlcNAcylation requires uridine diphosphate–GlcNAc, a precursor responsive to nutrient status and other environmental cues. Alternative splicing of the genes encoding the O-GlcNAc cycling enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) yields isoforms targeted to discrete sites in the nucleus, cytoplasm, and mitochondria. OGT and OGA also partner with cellular effectors and act in tandem with other posttranslational modifications. The enzymes of O-GlcNAc cycling act preferentially on intrinsically disordered domains of target proteins impacting transcription, metabolism, apoptosis, organelle biogenesis, and transport.


1) file:///E:/Downloads/fpls-05-00604.pdf
2) http://pac.iupac.org/publications/pac/pdf/1997/pdf/6909x1867.pdf
3) http://www.nature.com/nrm/journal/v13/n5/full/nrm3334.html
4) http://jcb.rupress.org/content/208/7/869.full

N-Linked Glycans 1

N-linked glycosylation, modification, and degradation are involved in a wide variety of processes in all organisms from archaea to eukaryotes. It is the most common covalent protein modification in eukaryotic cells. No other post-translational protein modification is as chemically complex or serves as many diverse functions.



Cleaved high-mannose glycans serve as substrates in the Golgi where additional modification and diversification occurs. High-mannose structures are trimmed by the action of mannosidases, removing the mannose extensions and making the glycan available for conversion to hybrid and complex glycans by subsequent addition of GlcNAc sugars (“antennae”) by the actions of N-acetylglucosyl transferases. The mammalian gene Mgat1 codes for the enzyme N-acetylglucosyl transferase-I (GlcNAcT-I), which is responsible for the addition of one GlcNAc in the β(1→2) linkage. The subsequently modified glycan becomes a substrate for α-mannosidase II which removes the α(1→3) and α(1→6) mannose residues and results in a molecular structure that is subject to glucosyl group donation by GlcNAcT-II (N-acetylglucosyl transferase-II), encoded by the Mgat2 gene. Catalysis by GlcNAcT-II converts the hybrid glycans to the complex forms by attaching additional GlcNAc moieties to the hybrid structure.
Fucosylation at the core GlcNAc residue following GlcNAcT-I modification in both hybrid and complex N-linked synthesis is also a common occurrence. In vertebrates, fucose is added in an α(1→6) linkage, while in plants and invertebrates, fucose is added in an α(1→3) linkage.

Inhibition or elimination of glycosylation in the study of N-linked glycans can be brought about by a number of compounds. N-glycosylation is strongly inhibited in the presence of compactin, coenzyme Q, or exogenous cholesterol. Treatment with tunicamycin completely blocks glycosylation as tunicamycin inhibits GlcNAc C-1‑phosphotransferase, an enzyme that is critical in the formation of the dolichol precursor.

α-Mannosidase, β-mannosidase, sialidase and α-fucosidase are the primary exoglycosidases involved in N-linked glycan trimming and degradation. Insect cells express an N-acetylglucosminidase that cleaves terminal GlcNAc residues from N-linked glycans.

Functions

During development, the intermediate structures of a glycoprotein perform specific functions. In the early phases of glycoprotein evolution, different core oligosaccharide structures are necessary for proper protein folding and functional group orientation. Improperly folded proteins are either reglycosylated and refolded, or deglycosylated and degraded. In later phases, the oligosaccharide moiety is required for intracellular transport and targeting of the glycoprotein in the endoplasmatic reticulum, Golgi complex, and trans-Golgi network. In the final phase, the N-linked glycan undergoes extensive modification in the Golgi complex, resulting in a mature glycoprotein.
The protein moiety and the attached hydrophilic N-linked glycans are relatively independent, despite being covalently linked, i.e. glycans can be modified without significantly affecting the protein structure and function. Proteins may contain multiple glycosylation sites that are modified with any of the three classes of N-linked glycans. Vertebrates have been found to possess a diverse compliment of complex and hybrid glycoproteins due to the broad variety of glycosidases and glycosyltransferases coded within the genome. While these three classes of Nlinked glycoproteins are also present in lower organisms, there is less diversity of structure than is found in vertebrate glycoproteins.
In addition to a specific glycoprotein being able to contain multiple glycan structures, different molecules of the same glycoprotein may have different glycan structures attached to the identical substitution site. Modifications in the glycan structures provide identity characteristics to different cell types and to the same cell type at stages of development, differentiation, transformation, maintenance, and aging. This variation in protein glycosylation is known as microheterogeneity and contributes to the difficulty in identifying and isolating specific glycoproteins.

The congenital disorders of glycosylation (CDG) are a series of diseases associated with errors of metabolism due to enzyme deficiencies. The majority of identified CDGs are due to failures in the biosynthesis or degradation of N-glycans because of absence of one of the enzymes involved in N-glycosylation, primarily the exoglycosidic enzymes α-mannosidase, β-mannosidase, sialidase, or α-fucosidase.

1) http://www.sigmaaldrich.com/technical-documents/articles/biology/glycobiology/n-glycans.html#sthash.tZX2I7M4.dpuf

N-linked glycosylation 1

N-linked glycosylation, is the attachment of the sugar molecule oligosaccharide known as glycan to a nitrogen atom (amide nitrogen of asparagine (Asn) residue of a protein), in a process called N-glycosylation, studied in biochemistry.[1] This type of linkage is important for both the structure [2] and function [3] of some eukaryotic proteins. The N-linked glycosylation process occurs in eukaryotes and widely in archaea, but very rarely in eubacteria. The nature of N-linked glycans attached to a glycoprotein is determined by the protein and the cell in which it is expressed.[4] It also varies across species. Different species synthesize different types of N-linked glycan.


The different types of glycans produced in different organisms.


Pathway of N-linked glycan biosynthesis[edit]

Biosynthesis pathway of N-linked glycoproteins:

The synthesis of N-linked glycan starts in the endoplasmic reticulum, continues in the Golgi and ends at the plasma membrane, where the N-linked glycoproteins are either secreted or becomes embedded in the plasma membrane.

The biosynthesis of N-linked glycans occurs via 3 major steps:

Synthesis of dolichol-linked precursor oligosaccharide
En bloc transfer of precursor oligosaccharide to protein
Processing of the oligosaccharide

Synthesis, en bloc transfer and initial trimming of precursor oligosaccharide occurs in the endoplasmic reticulum (ER). Subsequent processing and modification of the oligosaccharide chain is carried out in the Golgi apparatus.
The synthesis of glycoproteins is thus spatially separated in different cellular compartments. Therefore, the type of N-glycan synthesised, depends on its accessibility to the different enzymes present within these cellular compartments.
However, in spite of the diversity, all N-glycans are synthesised through a common pathway with a common core glycan structure. The core glycan structure is essentially made up of two N-acetyl glucosamine and three mannose residues. This core glycan is then elaborated and modified further, resulting in a diverse range of N-glycan structures.

Biosynthesis pathway of N-linked glycoproteins: The synthesis of N-linked glycan starts in the endoplasmic reticulum, continues in the Golgi and ends at the plasma membrane, where the N-linked glycoproteins are either secreted or becomes embedded in the plasma membrane.



Synthesis of precursor oligosaccharide

The process of N-linked glycosylation starts with the formation of dolichol-linked GlcNAc sugar. Dolichol is a lipid molecule composed of repeating isoprene units. This molecule is found attached to the membrane of the ER. Sugar molecules are attached to the dolichol through a pyrophosphate linkage[4] (one phosphate was originally linked to dolichol, and the second phosphate came from the nucleotide sugar). The oligosaccharide chain is then extended through the addition of various sugar molecules in a step-wise manner to form a precursor oligosaccharide.
The assembly of this precursor oligosaccharide occurs in two phases: Phase I and II. Phase I takes place on the cytoplasmic side of the ER and Phase II takes place on the luminal side of the ER.
The precursor molecule, ready to be transferred to a protein, consist of 2 GlcNAc, 9 mannose and 3 glucose molecules.






1) https://en.wikipedia.org/wiki/N-linked_glycosylation

Why are glycans so important? For instance, they provide a eukaryotic organism with a strategy to compete with bacterial and viral pathogens. Bacteria and viruses produce a large number of slightly different offspring in only several hours. Those slight differences arise as a result of mutations in their genetic material and represent a potential for adaptation. Prokaryotes thus seem to outcompete eukaryotic host organisms in their never-ending struggle for survival.

Eukaryotic organisms cannot afford such an expensive strategy due to their longer and more complex life cycles. Instead, they evolved sophisticated mechanisms in order to preserve a small number of highly valuable individuals, which is even more pronounced in high-density populations such as the human population. The mammalian immune system is often not efficient enough to mount an immediate counterattack against novel pathogens. The molecules at the surface of a pathogen represent its “face” by which it is recognized by the immune system. Any variation in the pathogen’s surface could make it resistant to our preexisting immunity. 1

On the other hand, successful infection requires specific recognition of certain features on the surface molecules of the host organism, and one of the mechanisms that higher organisms use to limit transmission of pathogens is the presentation of different structures on their cell surface. This is mostly achieved by variation in cell surface glycoconjugates

The epigenetic modification of glyco-genes, i.e. the genes involved in the synthesis of glycans, offers a way of creating new complex structural features which could evade recognition by a specific virus or bacteria and thus make the organism resistant to infection by these pathogens. Instead of waiting for generations for a mutation to occur, adaptation by tuning gene expression and thus surface presentation, largely by means of epigenetically controlled changes in glycosylation pattern, could provide higher eukaryotes with a powerful way of competing with microorganisms, giving them the edge in the constant struggle for survival.

One of the biological dogmas is that self-replicating nucleic acids (the first revolution in evolution) provided the basis for development of early life through translation of nucleotide sequence into a sequence of amino acids to create the main effectors of life at the cellular level – the proteins (the second revolution in evolution). The integration of different cells into a complex multicellular organism required additional layers of complexity and the invention of glycans (the third revolution in evolution) through its inherent ability to create novel structures without the need to mutate the genetic information, but to alter it by epigenetic modifications instead, thus providing multicellular organisms with a mechanism to compensate for their longer generation time by using their higher complexity for rapid adaptation to various environmental challenges.

The greatest evolutionary advantage that glycans confer to higher eukaryotes is the ability to create new structures without introducing changes into the precious genetic heritage. Glycans contribute up to 50% of the total protein mass and even more to the molecular volume of many proteins. Thus, a large part of glycoproteins is not directly hardwired in the genome but is rather a platform for rapid and extensive epiproteomic adaptation.



1) http://blogs.biomedcentral.com/on-biology/2015/03/18/glycans-the-sweet-difference/

Heparan sulfate proteoglycans: a sugar code for vertebrate development? 1

Vertebrate development – from a fertilized egg to a whole organism – is a complex process orchestrated by many signaling pathways. The generation of various cell types and organs from a single cell is achieved through successive developmental programs that require the spatial and temporal coordination of developmental signaling molecules, including morphogens and growth factors. Evidence accumulated over the past twenty years has shown that most, if not all, of these developmental factors are regulated by heparan sulfate proteoglycans (HSPGs).

HSPGs are composed of a core protein to which long linear glycosaminoglycan heparan sulfate (HS) chains are covalently linked. Syndecans (Sdc) and glypicans (Gpc), which are among the main classes of core protein, are anchored to the cell surface by a transmembrane domain or a glycosylphosphatidylinositol anchor, respectively, and can be released into the extracellular space following cleavage by various enzymes (Fig. 1). 


Examples of key cell surface and extracellular HSPGs. Glypicans (Gpcs) are attached to the cell surface via a glycosylphosphatidylinositol (GPI) anchor. They possess a large globular domain stabilized by conserved di-sulfide (S-S) bonds, and HS chains (represented by chains of pink, blue and purple circles) in their C-terminal part. They can be released into the extracellular matrix following cleavage of their GPI anchor by the lipase Notum. A furin-like convertase can also cleave Gpcs at the C-terminal end of their globular domain, leading to the formation of two subunits that remain attached to each other by disulfide bonds. Syndecans (Sdcs) are single-pass transmembrane proteins with HS chains attached to their N-terminal part. Their intracellular region interacts with many different partners through two conserved domains, constant 1 (C1) and constant 2 (C2), that are separated by a more variable region (V). Like Gpcs, Sdcs can be shed into the extracellular environment after cleavage by proteases such as matrix metalloproteases and a disintegrin and metalloproteinase (ADAM) disintegrins. Agrin and perlecan are large multidomain proteoglycans that carry several HS chains and are secreted as different isoforms generated by alternative splicing. The cleavage of the C-terminal region of perlecan by metalloproteases releases endorepellin, an angiogenesis inhibitor. C-ter, C-terminus; N-ter, N-terminus.


Other HSPGs, including agrin, collagen XVIII and perlecan, are directly secreted into the extracellular matrix (ECM). Core proteins have specific numbers of HS chain addition sites, and some also harbor chondroitin sulfate (CS) as another type of glycosaminoglycan .HS chains are synthesized in the Golgi apparatus in a multi-step process that involves several enzymes (Fig. 2; Table S2). Exostosin (Ext) enzymes elongate HS chains by adding alternating glucuronic acid and N-acetylglucosamine residues, whereas N-deacetylase/N-sulfotransferase (Ndst) enzymes, C5 epimerase and 2-O-, 3-O-, and 6-O-sulfotransferases modify them by catalyzing deacetylation, epimerization and sulfations at different positions (for nomenclature, see Lawrence et al., 2008). These modifications do not occur uniformly along the HS chains but instead are concentrated in different sub-regions, creating domains with variably modified disaccharides that can interact with other proteins in distinct manners.

HSPGs have been shown in cell culture studies to regulate a wide range of cell signaling pathways, cell-cell interactions, extracellular matrix formation, and cellular behaviors implicated in developmental mechanisms. Most recent studies have confirmed that HSPGs do indeed play major roles, many of them developmental, in vivo. However, there remain a number of major challenges in the field.

1. http://dev.biologists.org/content/142/20/3456

Functions of heparan sulfate proteoglycans in cell signaling during development 1

Heparan sulfate proteoglycans (HSPGs) are cell-surface and extracellular matrix macromolecules that are composed of a core protein decorated with covalently linked glycosaminoglycan (GAG) chains. In vitro studies have demonstrated the roles of these molecules in many cellular functions, and recent in vivo studies have begun to clarify their essential functions in development. In particular, HSPGs play crucial roles in regulating key developmental signaling pathways, such as the Wnt, Hedgehog, transforming growth factor-β, and fibroblast growth factor pathways. This review highlights recent findings regarding the functions of HSPGs in these signaling pathways during development.

Introduction

During metazoan development, the formation of complex body structures and patterns is governed by several secreted signaling molecules, including members of the Wnt/Wingless (Wg), Hedgehog (Hh), transforming growth factor-β (TGFβ) and fibroblast growth factor (FGF) families. Over the past decades, intensive biochemical and genetic studies have elucidated the central components of the signaling pathways that these molecules function in. More recently, attention has shifted to understanding the mechanisms by which the distributions of these signaling molecules are regulated in morphogenetic fields. Studies in Drosophila and vertebrates have demonstrated the crucial roles of heparan sulfate proteoglycans (HSPGs) in these signaling pathways during development. This review focuses on recent insights into the functions of HSPGs in regulating the activities and distributions of these signaling molecules. For detailed reviews of previous biochemical and genetic studies on the HSPGs, please see (Belting, 2003; Bernfield et al., 1999; Esko and Selleck, 2002; Lin and Perrimon, 2003;Nybakken and Perrimon, 2002; Princivalle and de Agostini, 2002; Selleck, 2001; Song and Filmus, 2002).

HSPG biochemistry

HSPGs are cell-surface and extracellular matrix (ECM) macromolecules that comprise a core protein to which heparan sulfate (HS) glycosaminoglycan (GAG) chains are attached (Bernfield et al., 1999; Esko and Selleck, 2002). HSPGs are classified into several families based on their core protein structure (Fig. 1).




The three main classes of cell-surface heparan sulfate proteoglycans (HSPGs). 
(A) Syndecan core proteins are transmembrane proteins that contain a highly conserved C-terminal cytoplasmic domain. Heparan sulfate (HS) chains attach to serine residues distal from the plasma membrane. Some syndecans also contain a chondroitin sulfate (CS) chain(s) that attaches to a serine residue(s) near the membrane. 
(B) The glypican core proteins are disulphide-stabilized globular core proteins that are linked to the plasma membrane by a glycosylphosphatidylinositol (GPI) linkage. HS chains link to serine residues adjacent to the plasma membrane. 
(C) Perlecans are secreted HSPGs that carry HS chains.

Glypicans and Syndecans are two major cell surface HSPGs, and are linked to the plasma membrane by a glycosylphosphatidylinositol (GPI) linkage or a transmembrane domain, respectively. Perlecans are secreted HSPGs that are mainly distributed in the ECM. Although Glypicans and Perlecans exclusively bear HS GAG chains, Syndecans are decorated with both HS and chondroitin sulfate (CS). All three families of HSPGs are evolutionarily conserved from vertebrates to Drosophila and C. elegans (Esko and Selleck, 2002; Nybakken and Perrimon, 2002). The Drosophila genome encodes four HSPG homologs: a single Syndecan (Sdc) (Johnson et al., 2004; Spring et al., 1994; Steigemann et al., 2004), two Glypicans [Division abnormally delayed (Dally) and Dally-like protein (Dlp) (Baeg et al., 2001; Khare and Baumgartner, 2000; Nakato et al., 1995)], and a Perlecan [Terribly reduced optic lobes (Trol) (Datta, 1995; Voigt et al., 2002)].

To date, most HSPG studies have demonstrated the importance of their HS chains. HS chains are polysaccharides synthesized in the Golgi apparatus and contain repeating disaccharide units of uronic acid linked to glucosamine (Esko and Selleck, 2002). HS chain biosynthesis is initiated at the GAG attachment site(s) of the core protein, which contains two to four Ser-Gly sequences. As depicted in Fig. 2, various glycosyltransferases and modification enzymes are involved in the polymerization and modification processes of HSPG biosynthesis. These enzymes are conserved in Drosophila and vertebrates (Esko and Selleck, 2002; Lin and Perrimon, 2003; Nybakken and Perrimon, 2002). In recent years, some of these enzymes have been genetically and biochemically characterized in Drosophila (see Table 1).




Heparan sulfate chain biosynthesis. 
Heparan sulfate (HS) glycosaminoglycan (GAG) chains are synthesized on a core protein by the sequential action of individual glycosyltransferases and modification enzymes, in a three-step process involving chain initiation, polymerization and modification. HS chain synthesis begins with the assembly of a linkage tetrasaccharide on serine residues in the core polypeptide. This process is catalyzed by four enzymes (Xyl transferase, Gal transferase I-II and GlcA transferase I), which add individual sugar residues sequentially to the non-reducing end of the growing chain. After the assembly of the linkage region, one or more α-GlcNAc transferases add a single α1,4-linked GlcNAc unit to the chain, which initiates the HS polymerization process. HS chain polymerization then takes place by the addition of alternating GlcA and GlcNAc residues, which is catalyzed by the EXT family proteins. As the chain polymerizes, it undergoes a series of modifications that include GlcNAc N-deacetylation and N-sulfation, C5 epimerization of GlcA to IdoA, and variable O-sulfation at C2 of IdoA and GlcA, at C6 of GlcNAc and GlcNS units, and, occasionally, at C3 of GlcN residues. The HS GAG chains are ∼100 or more sugar units long and have numerous structural heterogeneities. Four Drosophila enzymes, including Botv, Ttv, Sotv and Sfl, which are homologs of vertebrate EXTL3, EXT1, EXT2 and N-deacetylase/N-sulfotransferase, respectively, are highlighted in red. Gal, galactose; GlcNAc, N-acetylglucosamine; GlcA, glucuronic acid; GlcNS, N-sulfoglucosamine; IdoA, iduronic acid.

HSPGs in Hh distribution and signaling

Like Wnts, Hh signaling molecules act as key mediators in many developmental processes and essentially require HSPGs for their proper distribution and signaling activity (Ingham and McMahon, 2001; Lin and Perrimon, 2003).

Functions of HSPGs in Hh distribution

The first evidence that HSPGs function in Hh distribution came from the genetic analysis of ttv (Bellaiche et al., 1998) (see Box 3 and Table 1). In the wing disc, Hh acts as a morphogen that forms a concentration gradient in an anterior strip of cells near the anteroposterior (AP) border of the wing disc (see Box 2). In wing discs containing ttv mutant clones, Hh can only be detected in the posterior-most ttv mutant cells that lie adjacent to wild-type cells. Furthermore, Hh can diffuse through a ptc mutant clone in the wing disc, but not through ptc-ttv double mutant cells (Bellaiche et al., 1998). Mainly based on these data, Bellaiche et al. proposed that a Ttv-modified HSPG is required for Hh to move from the cells where it is expressed to the anterior Hh-receiving cells (Bellaiche et al., 1998). Several recent studies have also shown similar defects in Hh distribution in wing clones mutant for sfl, sotv or botv(Han et al., 2004a; Takei et al., 2004). Importantly, Hh protein accumulates abnormally in the posterior compartment when the ttv-botv double mutant clones are made in the anterior compartment along the AP boundary, further suggesting that Hh fails to move into the HSPG mutant cells (Takei et al., 2004).

HSPGs are also required for Hh movement in the embryonic epidermis. The mature form of Hh, HhNp (see Box 4) is distributed as large punctate particles between Hh-expressing cells (Gallet et al., 2003). Cholesterol modification is required for HhNp to form these punctate particles (Gallet et al., 2003). In ttv mutant embryos, these punctate particles are not distributed between Hh-expressing cells (Gallet et al., 2003; The et al., 1999). Although Ttv is required for HhNp movement, it is not required for that of HhN (see Box 4) (Gallet et al., 2003; The et al., 1999). Together, these studies support the notion that HSPGs are required for the movement of cholesterol-modified HhNp.

The HSPG core proteins Dally and Dlp are also involved in Hh movement in the embryonic epidermis (Han et al., 2004b). dlp mutant embryos exhibit virtually identical defects to those of hh mutant embryos, and in their epidermis, Hh punctate particles can only be detected in Hh-expressing cells, and not in adjacent cells (Han et al., 2004b). However, in the wing disc, both Dally and Dlp act partially redundantly in Hh movement (Han et al., 2004b).

HSPGs might also control the stability of Hh by protecting it from degradation. Interestingly, Hh levels are reduced in Hh-producing cells mutant for sotv or ttv (Bornemann et al., 2004). Bornemann et al. argue that, by extension, Hh ligand instability could also contribute to reduced Hh levels and signaling in Hh-receiving cells lacking HSPGs (Bornemann et al., 2004). Although the evidence of Hh accumulation in front of HSPG-defective cells implicates HSPGs in Hh movement (Takei et al., 2004), current data suggest that HSPGs are likely to be involved in both Hh movement and stability.

Mechanism(s) of HSPG-mediated Hh movement

Both vertebrate sonic hedgehog (Shh) and Drosophila Hh are secreted from cells as multimeric and monomeric forms (Chen et al., 2004; Zeng et al., 2001). The soluble Shh multimeric form is freely diffusive (Zeng et al., 2001) and is responsible for activating Shh-target genes (Chen et al., 2004). Do secreted Hh proteins freely diffuse to receiving cells through extracellular spaces, and what is the role of HSPGs in Hh movement? Han et al. have demonstrated that a narrow strip of sfl or ttv mutant cells in the fly wing disc is sufficient to completely block Hh signaling in the anterior wild-type cells adjacent to mutant cells, suggesting that Hh fails to move across these HSPG-deficient cells (Han et al., 2004a) (Fig. 3). Similar results are observed in clones mutant for both dally and dlp (Han et al., 2004a). Han et al. further showed that HSPG-mediated Hh movement is independent of dynamin-mediated endocytosis (Han et al., 2004a). On the basis of these and other data, Han et al. proposed that Hh movement is mediated by restricted diffusion involving Dally and Dlp (Han et al., 2004b) (Fig. 3A).

Roles of HSPGs in Hh signaling

HSPGs are also required for Hh signaling. In tissue culture experiments, Dlp, but not Dally, Sdc or Trol, is required for Hh signaling (Lum et al., 2003). As mentioned earlier,  Dlp is specifically required for Hh signaling in the embryonic epidermis (Desbordes and Sanson, 2003). Although there are some arguments regarding whether Dlp is also involved in Wg signaling (Perrimon et al., 2004), the reduced bagpipe expression, which is normally activated by Hh signalling but is inhibited by Wg signalling in mesodermal cells of dlp null embryos, resemble that of hh mutant embryos, providing further evidence that Hh signaling requires Dlp during fly embryogenesis (Han et al., 2004a). Importantly, although previous studies have shown that ectopic expression of Hh can rescue the cuticle defects associated with HS GAG mutants, such as those seen in sgl, sfl, frc andslalom mutants (Luders et al., 2003; Perrimon et al., 2004; Selva et al., 2001), ectopic expression of Hh fails to restore Hh signaling activity, as assayed by wg expression, in dly RNAi embryos (Desbordes and Sanson, 2003). These results suggest that the core protein of Dly is crucially required for Hh signaling, whereas the attached HS chains are required for optimal Hh signaling activity. Alternatively, the Dly core protein may be required for Hh processing in the embryonic epidermis. It is also important to note that although Dlp is required for Hh signaling during embryogenesis, Dlp is functionally redundant with Dally in Hh signaling in the wing disc (Han et al., 2004b), suggesting that the specificity of HSPG involvement in Hh signaling depends on the developmental context.

Dlp might regulate Hh signaling in several ways. It might modulate Hh levels at the cell surface and indirectly control the interaction of Hh with its receptor Ptc. Alternatively, it might act as a co-receptor, perhaps by transferring Hh to its receptor Ptc, or by forming a Hh-Dlp-Ptc ternary complex in which Dlp may function to facilitate the Hh-Ptc interaction or to stabilize a Hh-Ptc complex, as in the case of FGF signaling (Ornitz, 2000; Pellegrini, 2001). On the one hand, in tissue culture experiments, Lum et al. showed that Dlp acts cell autonomously upstream or at the level of Ptc to activate the expression of an Hh responsive-reporter, indicating that Dlp might deliver Hh to its receptor. However, the knockdown of Dlp did not block Hh signaling when Hh was expressed in responding cells (Lum et al., 2003). These results suggest that Dlp is not absolutely required for Hh signaling in the presence of relatively large amounts of Hh. On the other hand, ectopic expression of Hh or HhN fails to rescue Hh signaling defects in dlp-RNAi embryos (Desbordes and Sanson, 2003), and thus these data suggest that Dlp acts as a co-receptor for Hh signaling (Desbordes and Sanson, 2003). Studies in the dlp null mutant should help to resolve this issue.
Hh signaling might also be regulated by other HSPG core proteins in different tissues or developmental processes. For example, mutations in the gene encoding Trol, the Drosophila Perlecan that forms a complex with Hh, causes neuroblasts to undergo cell cycle arrest in the larval brain (Datta, 1995; Park et al., 2003; Voigt et al., 2002). Genetic interaction experiments also indicate that trol is required for Hh signaling during neuroblast division (Park et al., 2003).

1. http://dev.biologists.org/content/131/24/6009

The lipids are just the beginning. Protein–lipid complexes are the required passive transport sites and active pumps for the passage of ions and molecules through bilayer membranes, often with high specificity. Some allow passage for substrates into the compartment, and others their exit. The complexity increases further because all lipid bilayers have vast numbers of polysaccharide (sugar) appendages, known as glycans, and the sugars are no joke. These are important for nanosystem and microsystem regulation. The inherent complexity of these saccharides is daunting. Six repeat units of the saccharide D-pyranose can form more than one trillion different hexasaccharides through branching (constitutional) and glycosidic (stereochemical) diversity.8 Imagine the breadth of the library!

Polysaccharides are the most abundant organic molecules on the planet. Their importance is reflected in the fact that they are produced by and are essential to all natural systems. Every cell membrane is coated with a complex array of polysaccharides, and all cell-to-cell interactions take place through saccharide participation on the lipid bilayer membrane surface. Eliminating any class of saccharides from an organism results in its death, and every cellular dysfunction involves saccharides.

In a report entitled “Transforming Glycoscience,” the US National Research Council recently noted that,

very little is known about glycan diversification during evolution. Over three billion years of evolution has failed to generate any kind of living cell that is not covered with a dense and complex array of glycans.9
What is more, Vlatka Zoldoš, Tomislav Horvat, and Gordan Lauc observed: “A peculiarity of glycan moieties of glycoproteins is that they are not synthesized using a direct genetic template. Instead, they result from the activity of several hundreds of enzymes organized in complex pathways.”10

Polysaccharides are the most abundant organic molecules on the planet. Their importance is reflected in the fact that they are produced by and are essential to all natural systems. Every cell membrane is coated with a complex array of polysaccharides, and all cell-to-cell interactions take place through saccharide participation on the lipid bilayer membrane surface. Eliminating any class of saccharides from an organism results in its death, and every cellular dysfunction involves saccharides.
In a report entitled “Transforming Glycoscience,” the US National Research Council recently noted that,

very little is known about glycan diversification during evolution. Over three billion years of evolution has failed to generate any kind of living cell that is not covered with a dense and complex array of glycans.⁹

What is more, Vlatka Zoldoš, Tomislav Horvat, and Gordan Lauc observed: “A peculiarity of glycan moieties of glycoproteins is that they are not synthesized using a direct genetic template. Instead, they result from the activity of several hundreds of enzymes organized in complex pathways.”¹⁰
Saccharides are information-rich molecules. Glycosyl transferases encode information into glycans and saccharide binding proteins decode the information stored in the glycan structures. This process is repeated according to polysaccharide branching and coupling patterns.¹¹Saccharides encode and transfer information long after their initial enzymatic construction.¹² Polysaccharides carry more potential information than any other macromolecule, including DNA and RNA. For this reason, lipid-associated polysaccharides are proving enigmatic.¹³

http://inference-review.com/article/an-open-letter-to-my-colleagues

The Sugar Code - the third alphabet of life

https://reasonandscience.catsboard.com/t2071-carbohydrates-and-glycobiology-the-3rd-alphabet-of-life-after-dna-and-proteins#6077

Imagine the Amazon forest. Millions of trees. each tree has not a random structure, but its branches, every leaf, has a specific complex instruction why it is there and can be interpreted and understood, and upon its structure and arrangement, a specific signal is emitted  to neighboring trees and branches, and this neighbouring tree can understand the signal and message, a specific action happens. Now imagine biological Cell walls. Each cell wall has membrane proteins that have a structure similar to trees with tree branches. they are glycosylated. That is polysaccharides sugars form a tree-like structure annexed to these proteins, and these structures form an informational code, which can be interpreted, understood by neighboring Cells, and in that way, signals and information can be transmitted. Got the picture? 

Do Carbohydrates Provide a Structural Code?
The surprisingly low number of genes in the genomes of complex multicellular organisms has led biochemists to consider other explanations for biological complexity and diversity. Oligosaccharides and polysaccharides, endowed with an unsurpassed variability of structures, are information carriers, and glycoconjugates—complexes of proteins and lipids with oligosaccharides and polysaccharides—are the mediators of information transfer by these carbohydrate structures. Individual sugar units are the “letters” of the sugar code, and the “words” and “sentences” of this code are synthesized by glycosyltransferases, glycosidases, and other enzymes. The total number of permutations for a six-unit polymer formed from an alphabet of 20 hexose monosaccharides is a staggering 1.44x10^15, whereas only 6.4 x 10^7 hexamers can be formed from 20 amino acids and only 4096 hexanucleotides can be formed from the four nucleotides of DNA.

 The covalent addition of glycans to proteins and lipids represents not only the most abundant post-translational modification of proteins, but also the most structurally diverse. Glycoconjugates respond to and control metabolic states and developmental stages of cells, and they are directly involved in almost every biological process and play a major role in disease. The vast array of possible glycan structures adds a glycomic dimension to the genomic complexity achieved by protein expression in organisms. The “glycome” is the complete set of glycans and glycoconjugates that are made by a cell or organism under specific conditions, and “glycomics” refers to studies that attempt to define or quantify the glycome of a cell, tissue, or organism.

The cell surface is the platform for presentation of biochemical signals that are required for intercellular communication. Their profile necessarily needs to be responsive to internal and external factors in a highly dynamic manner. The structural features of the signals must meet the criterion of high-density information coding in a minimum of space. Thus, only biomolecules that can generate many different oligomers (‘words’) from few building blocks (‘letters’) qualify to meet this challenge. Examining the respective properties of common biocompounds that form natural oligo- and polymers comparatively, starting with nucleotides and amino acids (the first and second alphabets of life), comes up with sugars as clear frontrunner. The enzymatic machinery for the biosynthesis of sugar chains can indeed link monosaccharides, the letters of the third alphabet of life, in a manner to reach an unsurpassed number of oligomers (complex carbohydrates or glycans).

 Fittingly, the resulting glycome of a cell can be likened to a fingerprint. Conjugates of glycans with proteins and sphingolipids (glycoproteins and glycolipids) are ubiquitous in Nature. Coding in this sense means that a biochemical message is written by using a molecular alphabet with the aim to obtain a distinct cellular response after a molecular recognition event between the signal and its reader.  Ubiquitous presence of a compound class used as signal in Nature can then be interpreted as a sign of broad physiological significance, and this is not only the case for nucleic acids and proteins. Beyond these two classes of biopolymers, the family of complex carbohydrates constitutes an enormous biomass, cellulose (a glucose polymer) and chitin (an N-acetylglucosamine polymer) being the two most abundant compounds on Earth Glycans attached to scaffolds are a “multipurpose tool”. Their presentation at these sites intimates that their sugar chains can be highly versatile signals for the cells’ communication. This assumption warrants to examine the chemical properties of carbohydrates, the building blocks of the glycan chains, from the perspective of coding capacity. If a sugar code is operative, then we can expect to find an enormous potential for structural variability by closely looking at carbohydrates.  2

Writers of sugar-encoded information
A group of proteins with different activities is required to establish carbohydrate-based signals. First, the letters are synthesized, if not supplied by food, then they are activated by reaction with the appropriate nucleotide triphosphate and the product, for example, CMP-N-acetylneuraminic acid prepared in the nucleus, will then often need a ride with a transporter to the site of glycan assembly. In this process cascade, glycosyltransferases move the carbohydrate from its donor, the conjugate with a nucleotide such as CMP, GDP or UDP, to the acceptor, i.e. a scaffold (protein or sphingolipid) to initiate glycoconjugate synthesis or a growing glycan chain. Obviously, all enzymes that are involved in preparing and using the activated substrates as well as the transport proteins to have the substrates available at sites of glycan production constitute the set of so-called glycogenes necessary to write the sugar-based signal. In addition, enzymes for placing position-specific substitutions such as sulfate groups.

The emerging results from glycome analysis (the equivalent of genomics and proteomics) are making clear that glycans truly are “ideal for generating compact units with explicit informational properties”. 

A group of proteins with different activities is required to establish carbohydrate-based signals. First, the letters are synthesized, if not supplied by food, then they are activated by reaction with the appropriate nucleotide triphosphate and the product, for example CMP-N-acetylneuraminic acid prepared in the nucleus, will then often need a ride with a transporter to the site of glycan assembly. In this process cascade, glycosyltransferases move the carbohydrate from its donor, the conjugate with a nucleotide such as CMP, GDP or UDP, to the acceptor, i.e. a scaffold (protein or sphingolipid) to initiate glycoconjugate synthesis or a growing glycan chain. Obviously, all enzymes that are involved in preparing and using the activated substrates as well as the transport proteins to have the substrates available at sites of glycan production constitute the set of so-called glycogenes necessary to write the sugar-based signal.

Evolution, or design ? 




The human knee joint is a marvel of nature’s ( or the creators ) engineering. 
Articular cartilage slides with extremely low friction against the cushioning meniscus when the knee bends. Articular cartilage is 68%–85% water, adsorbed in a network of collagen fibers (10%–20%) and proteoglycans (5%–10%). The meniscus is also mostly water (70%). The meniscus’s dry weight is 75% collagen and 25% proteoglycan. Hyaluronate forms the backbone of proteoglycan structures, such as those found in cartilage. The proteoglycan subunits consist of a core protein containing numerous O-linked and N-linked glycosaminoglycans. The glycosaminoglycans in the meniscus proteoglycan include chondroitin sulfate (50%–60%), dermatan sulfate (20%–30%), and keratan sulfate (15%). In cartilage, these highly hydrated proteoglycan structures are enmeshed in a network of collagen fibers. Release (and subsequent reabsorption) of water by these structures during compression accounts for the shock-absorbing qualities of cartilaginous tissue.

Lectins Translate the Sugar Code
The processes of cell migration, cell–cell interaction, immune response, and blood clotting, along with many other biological processes, depend on information transfer modulated by glycoconjugates. Many of the proteins involved in glycoconjugate formation belong to the lectins—a class of proteins that bind carbohydrates with high specificity and affinity. Lectins are the translators of the sugar code. Table below describes a few of the many known lectins, their carbohydrate affinities, and their functions. 






1

A few examples of lectin–carbohydrate complexes and their roles in biological information transfer will illustrate the nature of these important and complex interactions.

1. https://www.genome.jp/kegg/catalog/codes2.html
2. http://sci-hub.tw/https://www.sciencedirect.com/science/article/pii/S0303264717301983

Previous chapters have described how glycans are not directly encoded by genes and that they are biosynthetically and structurally complex.

https://www.ncbi.nlm.nih.gov/books/NBK453097/