Glycoproteins: Synthesis and Role | Biochemistry

In this article we will discuss about Glycoproteins:- 1. Subject Matter of Glycoproteins 2. Oligosaccharides of Glycoproteins 3. Synthesis of Complex Carbohydrates 4. Lectins can be Used for Purification 5. Blood Group Antigens 6. Role in Fertilization 7. Proteoglycans 8. Tunicamycin.

Contents:

1. Subject Matter of Glycoproteins
2. Oligosaccharides of Glycoproteins
3. Synthesis of Complex Carbohydrates of Glycoproteins
4. Lectins can be Used for Purification of Glycoproteins
5. Blood Group Antigens
6. Role of Glycoproteins in Fertilization
7. Proteoglycans and Glycoproteins
8. Tunicamycin Inhibits N-linked Glycoproteins

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1. Subject Matter of Glycoproteins:

a. Their molecular weight ranges from 15,000 to over 1 million containing 15 or fewer sugar units per chain.

b. Their carbohydrate contents range from 1 to 85 per cent by weights.

c. They are present in plants, bacteria, fungi, viruses and animals. The most membrane proteins and secreted proteins are glycoproteins.

d. They act as structural molecules in cell walls, collagen, elastin, fibrins, and bone matrix; as lubricants and protective agents in mucins, mucus secretions.

e. They are utilized as transport molecules for vitamins, lipids, minerals and trace el­ements; as immunologic molecules for immunoglobins, histocompatibility anti­gens, complement and interferon; as hor­mones in chorionic gonadotropin, thyro­tropin (TSH); as enzymes in proteases, nucleases, glycosidases, hydrolases, and clotting factors; as recognition sites in cell-cell, virus-cell, bacterium-cell, and hor­mone receptors.

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2. Oligosaccharides of Glycoproteins:

a. The oligosaccharide chains contain nine different sugar residues. Glucose (Glc) is found only in collagen, but galactose (Gal) and mannose (Man) are more common and widely distributed. The hexoses are N- acetylgalactosamine (Gal NAC) and N- acetyl glucosamine (Glc NAC). Fucose (Fuc) is a common constituent.

Two pentoses-arabinose (Ara) and Xylose (Xyl) are found and the ninth are the sialic acids (Sial) of which N- acetylneuraminic acid (Nana) is an example. The fucose and Nana residues are more distal in the chain, frequently at terminal sites.

b. The oligosaccharide chains are attached to the polypeptide backbone at one of five amino acid residues-asparagine (Asn), serine (Ser), threonine (Thr), hydro-xylysine (Hyl), or hydroproline (Hyp). Two types of chemical bonds that provide the attachment sites are (a) O-glycosidic links and (b) N-glycosidic links.

(a) O-glycosidic Links:

(i) The O-glycosidic links occur through the free alcoholic groups of Ser or Thr residues of the polypeptide in a tripeptide se­quence of Asn-Y-Ser (Thr), where Y is an amino acid other than aspartic acid.

(ii) Gal NAC is the most common sugar resi­due attached directly to the Ser or Thr resi­due. Six different types of oligosaccha­ride are attached to this Gal NAC-Ser (Thr) linkage.

(iii) The initiation and extension of different types of oligosaccharide chains of glycoproteins occur by the stepwise do­nation of sugar residues from pyrimidine or purine nucleotide sugars.

(iv) Oligosaccharides may be linked to pro­teins via O-glycosidic bonds to Hyl or Hyp which are amino acid residues found in collagens and some fibrous proteins of plants.

(b) N-glycosidic linkage:

(i) The N-linked oligosaccharide consists of a core region with the structure Man-β-1, 4-Glc NAC-β-1,4-Glc NAC-Asn. This core region is of two types—the high mannose (simple) type and the complex type. A sin­gle protein can contain oligosaccharide chains of both high mannose and com­plex types.

(ii) Although all high-mannose oligosaccha­rides are synthesized from nucleotide sug­ars, there exists an important lipid-linked precursor oligosaccharide that is trans­ferred en bloc from a lipid carrier to the Asn of the protein.

(iii) The complex N-linked oligosaccharides also contain the β-man-di-N-acetylchitobiose core structure but consist also of a variable number of outer chains contain­ing Sial, Gal, and Fuc residues linked to the core.

(iv) Complex N-linked oligosaccharide struc­tures are found only in higher animals; whereas the high mannose types are com­mon in primitive organisms.

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3. Synthesis of Complex Carbohydrates of Glycoproteins:

(i) The nucleotide of sialic acid, CMP-Sial, is formed from CTP by sialyltransferases located in the Golgi complex and in the nucleoplasm.

(ii) In animal cells, the sugars are linked to the nucleotides by the alpha-linkage with the exception of the beta-linkage of L-fucose to GDP. The alpha-bridges are con­verted to the beta-bridges and vice-versa during the transfer of the sugar moiety to the oligosaccharide.

(iii) A number of specific glycosyl transferase enzymes catalyze the transfer of the sugar moieties to generate the complex glyco­proteins. These enzymes require Mn⁺⁺.

(iv) A Golgi-localized enzyme, UDP Glc NAC transferase 1, can then denote a Glc NAC to a linear or branched alpha-Man moiety to form Glc NAC-β-1, 2-Man linkages. A second transferase, UDP Glc NAC transferase 11, will denote its Glc NAC moiety only to a branched structure by the trans­ferase 1 enzyme.

(v) Fucosyl-transferases can then act on the products of GLc NAC transferase 1 or transferase 11. The galactosyltansferase enzymes are also located on the Golgi complex and attach a galactosyl residue usually to the end of a chain. The galactosyl residues are linked to Glc NAC by beta-1, 4-linkages but occasionally by beta-1, 6-linkages.

(vi) Four different sialyltransferase enzymes are found in the Golgi complex and use CMP-sialic acid as donor for the sialation of protein-linked oligosaccharides.

(vii) The elongation process generating the complex type oligosaccharides of glycoproteins occurs exclusively in the Golgi complex. Each linkage is carried out by a specific glycosyl-transferase; thus, there seems to be a “one linkage, one glycosyltransferase” synthetic arrange­ment.

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4. Lectins can be Used for Purification of Glycoproteins:

a. Lectins, the sugar-binding protein, that precipitate glycoconjugates. Immunoglo­bulins that react with sugars are not lectins. Lectins contain at least two sugar-bind­ing sites; proteins with a single sugar-bind­ing site will not precipitate glycoconju­gates.

b. Enzymes, toxins, and transport proteins can be classed as lectins if they bind car­bohydrate.

c. Lectins such as concanavalin A (con A) can be attached covalently to inert sup­porting media such as sepharose. The re­sulting sepharose-con A may be used for the purification of glycoproteins.

d. Smaller amounts of certain lectins are re­quired to cause agglutination of tumor cells than of normal cells.

e. When mammalian cells in tissue culture are exposed to appropriate concentrations of certain lectins (e.g., Con A), most are killed, but a few resistant cells survive. Such cells are found to lack certain en­zymes involved in oligosaccharide syn­thesis. The cells are resistant because they do not produce oligosaccharide chains that interact with the lectin used.

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5. Blood Group Antigens:

In 1900, Landsteiner described the ABO blood groups. Today, there are more than twenty blood group systems expressing more than 160 distinct antigens. These erythrocyte antigens are linked to specific membrane proteins by O-glycosidic bonds in which Gal NAC is the most proximal sugar resi­due.

The specific oligosaccharides exist in three forms:

(i) As glycosphingolipids and glycoproteins on the surfaces of erythrocytes and other cells,

(ii) As oligosaccharides in milk and urine, and

(iii) As oligosaccharides attached to mucins secreted in the gastrointestinal, genitourinary, and respiratory tracts.

Four independent gene systems are related to the expressions of these oligosaccharide antigens.

The H Locus:

This codes for a fucosyltransferase that attaches a fucose residue in alpha-1, 2- linkage to a Gal residue, itself attached in beta-1, 4-linkage to an oligosaccharide. Fuc-α-1, 2- Gal-β- R is a precursor for the formation of both the A and B oligosaccharide antigens.

The h allele of the H locus codes for an inactive fucosyltransferase. Therefore, individuals with the hh genotype can­not generate this necessary precursor of the A and B antigens. Hence hh genotypic persons will be type O.

The Secretor Locus:

This controls the appear­ance of the H-specifie Fuctransferase in some se­cretory organs, such as the exocrine glands, but not in the erythrocytes. Accordingly, individuals with the Hh or HH genotype and an Se allele will gener­ate the A and B antigen precursor in the exocrine glands that form saliva.

The individuals who are SeSe or Sese and possess an H allele will be secretors of the A or B antigens (or both), when the A- or B- specific transferase are present. Individuals who are sese genotype will not secrete A or B antigens; but if they possess an H allele and A or B allele, their erythrocytes will express the A, B or both antigens.

The ABO Locus:

These codes for two specific transferases that act to transfer specific Gal moie­ties to the Fuc-α-1, 2-Gal-β-R precursor oligosac­charide formed by the action of the H allele-coded fucosyltransferase.

Persons possessing an A allele will attach a Gal NAC moiety to the precursor gen­erated by the H allele transferase and an individual possessing a B allele will transfer a Gal moiety to the same precursor. Individuals possessing both A and B allele will generate both A and B alleles (00 homozygotes) will not attach either Gal NAC or Gal to the precursor.

When neither Gal NAC nor Gal is at the reducing terminus of this oligosaccha­ride, it will not be recognized by either anti-A or anti-B antisera, and the blood group antigen is said to be type O Individuals with the hh genotype in­capable of attaching the Fuc moiety to the appro­priate Gal-β-R oligosaccharide is incapable of ex­pressing the A or the B antigen determinant and thus is considered to be of the O type blood group.

The Lewis Locys:

The Lewis-dependent fucosyltransferase is not specific about what is not the Gal-1, 3-β group. When no H allele is present (hh), the product of the Lewis α-1, 4-fucosyltrans­ferase is referred to as the Lea antigen which cannot have A or B antigenicity even when the A or B transferases are also present.

When both the H al­lele and the Le allele fucosyltransferases have acted on the Gal-1, 3-R oligosaccharide, the product is referred to as the Le^b antigen. The Le^b antigen may also exist without A antigenicity or B antigenicity on the same molecule. The le allele codes for an inactive Lewis transferase, and thus neither Le^a nor Le^b antigens will be formed in a person with lele genotype.

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6. Role of Glycoproteins in Fertilization:

(a) A sperm has to traverse the zona pellucida (ZP) which contains three glycoproteins ZPI-3, particularly ZP3, (an O-linked glycoprotein that functions as a receptor for the sperm) to reach the plasma mem­brane of an oocyte.

(b) A protein on the sperm surface interacts with oligosaccharide chains of ZP3. This interaction induces the acrosomal reaction in which proteases and hyaluronidase, and other contents of the acrosome of the sperm are released. The liberation of these en­zymes helps the sperm to pass through the zona pellucida and reach the plasma mem­brane of the oocyte.

(c) Another glycoprotein pH-30 is important in binding of the PM of the sperm to the PM of oocyte. These interactions enable the sperm to enter and thus fertilize the oocyte.

(d) It is also possible to inhibit fertilization by developing drugs or antibodies that in­terfere with the normal functions of ZP3 and PH-30 and which thus act as contra­ceptive agents.

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7. Proteoglycans and Glycoproteins:

Each polysaccharide of proteoglycans consists of repeating disaccharide units in which D- glucosamine or D-galactosamine is always present. Each disaccharide contains a uronic acid, glu­curonic acid (G1C UA), L-iduronic acid (ldUA). All polysaccharides contain sulphate groups with the exception of hyaluronic acid.

The linkage of the polysaccharides to their polypeptide chain is one of three types:

(i) An O-glycosidic bond between Xyl and Ser, a bond that is unique to proteoglycans.

(ii) An O-glycosidic bond between Gal NAC and Ser (Thr), present in keratan sulphate II.

(iii) An N-glycosylamine bond between G1C NAC and the amide nitrogen of Asn.

The elongation process of chain involves the nucleotidyl sugars acting as donors. The reactions are performed by the substrate specificities of the specific glycosyltransferases. Thus, “one enzyme, one linkage” relationship holds. The specificity of these reactions is dependent upon the nucleotide sugar donor, the acceptor oligosaccharide.

The polysaccharide chain growth termination results from (i) capping effects of isolation by the specific sialyl transferases, (ii) sulfation, particu­larly at the 4-positions of the sugars, and (iii) the progression of the particular polysaccharide away from the site in the membrane where the catalysis occurs. After formation of the polysaccharide chain, numerous chemical modifications take place.

Inherited defects in the degradation of the polysaccharide chains lead to the group of diseases known as mucopolysaccharidoses and mucolipi­doses.

Seven types of polysaccharides are covalently attached to the proteins of proteoglycans. Six of them contain alternating uronic acid and hex­osamine residues. Except hyaluronic acid all con­tain sulphated sugars. These seven types of polysaccharides are distinguished by their monomer composition, their glycosidic linkage, and the amount and location of their sulphate substituents.

Functions of Glycosaminoglycans and Proteoglycans:

1. Glycosaminoglycans can interact with ex, tracellular macromolecules, plasma pro­teins, cell surface components, and intra­cellular macromolecules.

2. Because of their polyanionic nature the binding of this is generally electrostatic.

3. These with IdU A bind proteins with greater affinities than those containing GlcUA as their only uronic acid constituent.

4. The binding between these and other ex­tracellular macromolecules contributes to the structural organization of connective tissue matrix.

A. Interactions with Extracellular Macremetecules:

(i) All glycosaminoglycan’s except those that lack sulphate groups or carboxyl groups bind to collagen electrostatically at neu­tral pH. Tighter binding is promoted by the presence of IdUA and the proteo­glycans interact more strongly than glycosaminoglycan’s.

(ii) The chondroitin sulphate and keratan sul­phate chains of proteoglycans aggregate with hyaluronic acid.

B. Interactions with Plasma Proteins:

(i) Dermatan sulphate binds plasma lipoproteins and appears to be the major glycosaminoglycan synthesized by arte­rial smooth muscle cells. This dermatan sulphate may play an important role in the development of atherosclerosis.

(ii) Heparin with its high negative charge den­sity (due to the IdUA and sulphate residues) interacts strongly with several plasma com­ponents.

It interacts with antithrombin 111. Heparin sulphate is also capable of accel­erating the action of antithrombin 111, but is much less potent than heparin. Heparin can bind to lipoprotein lipase present in capillary walls and causes a release of that triglyceride-degrading enzyme into the cir­culation. Hepatic lipase also binds heparin but with lower affinity.

C. Cell Surface Molecules:

(i) Heparin associates with blood platelets, arterial endothelial cells, and liver cells. Chondroitin sulphate, dermatan sulphate, and heparan sulphate bind to independ­ent sites on surface of cells such as fibroblasts. At those sites, the glycosaminoglycan’s and proteoglycans are taken up by fibroblasts and degraded.

(ii) Some proteoglycans serve as receptors and carriers for macromolecules. These proteoglycans are involved in the regula­tion of cell growth.

D. Intracellular Macromolecules:

(i) Proteoglycans and their glycosaminoglycan components have effects on protein synthesis and intra-nuclear func­tions. Glycosaminoglycan’s are found in significant quantities in nuclei from dif­ferent cell types.

(ii) The acid hydrolases in lysosomes may be naturally complexed with glycosaminoglycan’s to provide a protected and inac­tive form. Chondroitin sulphates, dermatan sulphates, and heparin can affect the ac­tivities of various lysosomal acid hydro­lase in negative or positive ways.

(iii) Many storage or secretory granules such as the chromaffin granules in adrenal me­dulla, the prolactin secretory granules in the pituitary gland, and the basophilic granules in mast cells contain sulphated glycosaminoglycan’s. The glycosamino- glycan-peptide complexes that occur in these granules play a role in the release of biogenic amines.

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8. Tunicamycin Inhibits N-linked Glycoproteins:

a. Many compounds are involved in inhib­iting various reactions of glycoproteins. Tunicamycin, deoxynojirimycin, and swainsonine are the agents which can be used experimentally to inhibit various stages of glycoprotein biosynthesis. If cells are grown in the presence of tunicamycin, no glycosylation of their normally N-linked glycoproteins will oc­cur.

In certain cases, lack of glycosylation increases the susceptibility of these pro­teins to proteolysis.

b. Inhibition of glycosylation does not have a consistent effect upon the secretion of glycoproteins that are normally secreted.

c. The inhibitors of glycoprotein processing do not affect the biosynthesis of O-linked glycoproteins. The extension of O-linked chains can be prevented by GalNAC- benzyle. This compound competes with natural glycoprotein substrates.