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How do short polypeptide chains serve as identifiable antigens?

How do short polypeptide chains serve as identifiable antigens?


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My understanding is that MHC class I and II bind to relatively short polypeptide chains (~ 20ish amino acids). I'm surprised that a non-self protein fragment that short would be distinctive enough to target for an immune response. Also, how does expressing one polypeptide chain help the immune system identify the source? Isn't it quite likely that the presented polypeptide is in the interior of a protein and would never actually be exposed to the environment until after it's been lysed? Or is it hydrophilic peptides that bind?


Glycoproteins

Sugar chain structures of glycoprotein hormones

The structural information of the glycoprotein hormones was first obtained from human chorionic gonadotropin (hCG). As shown in Fig. 1 , both subunits of hCG contain two N-linked sugar chains. The β-subunit of hCG (hCGβ) also contains four O-linked sugar chains. It was reported that sialylated N5, N6, and N8 in Fig. 2 are included in hCG samples purified from the urine of healthy pregnant women. All sialic acids are linked at the C-3 positions of the galactose residues. The structure of O-linked sugar chains of hCG was found to be Oa (shown in Fig. 3 ). Afterward, Ob in Fig. 3 and sialylated N9 and N10 in Fig. 2 were added as minor O- and N-linked sugar chains of hCG.

Figure 2 . Structures of desialylated N-linked sugar chains isolated from various hCG preparations. All sialic acid residues are exclusively linked at the C-3 position of the galactose residues. Gal, galactose GlcNAc, N-acetylglucosamine Man, mannose Fuc, L-fucose.

Figure 3 . Structures of O-linked sugar chains found in hCG. Neu5Ac, N-acetylneuraminic acid GalNAc, N-acetylgalactosamine. Other abbreviations are the same as in Fig. 2 .

Detection of various N-linked sugar chains in hCG could be considered as a typical case of microheterogeneity, which is widely observed in the sugar chains of glycoproteins . However, comparative study of the N-linked sugar chains of hCGα and hCGβ revealed that sialylated N5 is distributed mainly in hCGβ and that sialylated N8 is distributed mainly in hCGα, whereas sialylated N6 is detected as a major sugar chain of both subunits. The specific distribution of different N-linked sugar chains at the four N-glycosylation sites of hCG molecule cannot be explained by our current knowledge of the biosynthetic mechanism of the N-linked sugar chains. An unknown control mechanism involving the steric effects of the polypeptide moiety may play a role in the formation of N-linked sugar chains of hCG. This assumption was supported by the study of the N-linked sugar chains of the free α-subunit.

A small amount of α-subunit occurs in free form in the urine of pregnant women. Interestingly, this free α-subunit cannot bind to hCGβ, in contrast to hCGα dissociated from hCG heterodimer. Because the free α-subunit has the same amino acid sequence as does hCGα, it was assumed that the free α-subunit contains different sugar chains from hCGα. Structural studies of the sugar chains of the free α-subunit revealed that it contains sialylated N5 as its major sugar chains. This evidence indicated that bulky sialylated N5 on the free α-subunit may sterically inhibit its association with hCGβ. Maturation of the N-linked sugar chains of the free α-subunit to larger complex-type sugar chains might be induced because the subunit did not bind to β-subunit. Therefore, uneven distribution of the N-linked sugar chains at the four N-glycosylation sites of hCG may be produced only when the two subunits are associated before the N-linked sugar chains start maturation at the Golgi apparatus.

Later on, the structures of the N-linked sugar chains of LH and TSH of various mammals, including human (hLH and hTSH), were elucidated (as shown in Fig. 4 ), as were those of human FSH (hFSH) (as shown in Fig. 5 ). In contrast to hCG, the three pituitary glycoprotein hormones contain triantennary and tetraantennary complex-type sugar chains, and their sialic acids are linked at the C-3 and C-6 positions of galactose residues. Occurrence of bisected sugar chains was also found. The most interesting evidence is that a part of the N-linked sugar chains of hLH and hTSH contain the SO44GalNAcβ14GlcNAc group as their outer chains.

Figure 4 . Structures of N-linked sugar chains found in LH and TSH. R represents the Neu5Acα23 or 6Galβ1 group, and R′ represents the SO44GalNAcβ1 group. Abbreviations of the sugar units are the same as in Figs. 2 and 3 .

Figure 5 . Structures of the N-linked sugar chains found in hFSH. Abbreviations of the sugar units are the same as in Figs. 2 and 3 .

An N-acetylgalactosaminyltransferase, which forms the GalNAcβ14GlcNAc group, was found to occur in the pituitary but not in the placenta. This enzyme requires presence of the ProXArg motif in the polypeptide portion of the substrate glycoprotein. Such sequence is present in hLH and hTSH but not in hFSH, explaining the absence of N-acetylgalactosamine residue in hFSH. The enzyme responsible for sulfation of the sugar chains of pituitary glycoprotein hormones was detected in the Golgi membrane preparation of pituitary gland by the transfer of sulfate residues from 3′-phosphoadenosine 5′-phosphosulfate. This 4-O-N-acetylgalactosamine sulfotransferase does not require any specific peptide motif and can transfer sulfate even to the trisaccharide: GalNAcβ14GlcNAcβ12Man. Therefore, addition of a β-N-acetylgalactosamine residue to the sugar chains is a prerequisite for the sulfation of the sugar chains.

Both FSH and LH are synthesized by gonadotrophs in the anterior pituitary. Although they share the same α-subunit polypeptide, their sugar chains differ in branching and terminal modification. Therefore, assembly of the two subunits should control the maturation of the sugar chains, as discussed previously for the site-directed maturation of the sugar chains of hCG. Actually, assembly of the two subunits of glycoprotein hormones was found to occur when the N-linked sugar chains are still in the state of high mannose types, which are sensitive to endo-β-N-acetylglucosaminidase H digestion. In further processing and maturation steps to lead to complex-type sugar chains, association to different β-subunits will result in different sugar chain formation.


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.

  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

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.

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.

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.

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.

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.

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.

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.

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-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.

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.

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.

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.


Ionic Bonds

Sodium has a single electron in its outermost orbital shell, and it is thermodynamically more stable if it gives up this electron. This loss of a negative electron results in a positively charged sodium ion, abbreviated Na + . Chlorine, on the other hand, has seven electrons in its outermost orbital shell, and it is more thermodynamically stable if it acquires an extra electron to complete the outer orbital shell. This results in a negatively charged chloride ion, abbreviated Na+. The positively charged sodium ions and the negatively charged chloride ions attract each other and result in the formation of an ionic bond. In the absence of water, sodium and chloride form a crystal lattice because of the attraction of negative and positive ions.

However, if sodium chloride crystals are placed in water, the polar water molecules will "hydrate" the sodium and chloride atoms because the water molecules are polar. In the illustration below the darker blue V-shaped figures represent water molecules, which are polar. The positive ends of the water molecules are attracted to the negatively charged chloride ions, while the negative pole of the water molecule is attracted to the positive sodium ions. As a result, the ions are hydrated and the crystal lattice dissolves into the aqueous solution. This is exactly what happens when you add crystalline table salt to a glass of water.

The video below provides an animated explanation of how salts like NaCl dissolve in water.

More Complex Biological Molecules


3-7. Antibodies bind antigens via contacts with amino acids in CDRs, but the details of binding depend upon the size and shape of the antigen

In early investigations of antigen binding to antibodies, the only available sources of large quantities of a single type of antibody molecule were tumors of antibody-secreting cells. The antigen specificities of the tumor-derived antibodies were unknown, so many compounds had to be screened to identify ligands that could be used to study antigen binding. In general, the substances found to bind to these antibodies were haptens (see Section 3-4) such as phosphorylcholine or vitamin K1. Structural analysis of complexes of antibodies with their hapten ligands provided the first direct evidence that the hypervariable regions form the antigen-binding site, and demonstrated the structural basis of specificity for the hapten. Subsequently, with the discovery of methods of generating monoclonal antibodies (see Appendix I, Section A-12), it became possible to make large amounts of pure antibodies specific for many different antigens. This has provided a more general picture of how antibodies interact with their antigens, confirming and extending the view of antibody-antigen interactions derived from the study of haptens.

The surface of the antibody molecule formed by the juxtaposition of the CDRs of the heavy and light chains creates the site to which an antigen binds. Clearly, as the amino acid sequences of the CDRs are different in different antibodies, so are the shapes of the surfaces created by these CDRs. As a general principle, antibodies bind ligands whose surfaces are complementary to that of the antibody. A small antigen, such as a hapten or a short peptide, generally binds in a pocket or groove lying between the heavy- and light-chain V domains (Fig. 3.8, left and center panels). Other antigens, such as a protein molecule, can be of the same size as, or larger than, the antibody molecule itself, and cannot fit into a groove or pocket. In these cases, the interface between the two molecules is often an extended surface that involves all of the CDRs and, in some cases, part of the framework region of the antibody (Fig. 3.8, right panel). This surface need not be concave but can be flat, undulating, or even convex.

Figure 3.8

Antigens can bind in pockets or grooves, or on extended surfaces in the binding sites of antibodies. The panels in the top row show schematic representations of the different types of binding site in a Fab fragment of an antibody: left, pocket center, (more. )


Conjugation Route Regulates the Abundance of Protein Antigen Epitopes Presented by Dendritic Cells Post KLH-Protein Antigen Conjugates Pulsing

This abstract is from the Experimental Biology 2016 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract

Owing mainly to its large size, and abundant and complex carbohydrate structures on its peptide chains, Keyhole Limpet Hemocyanin (KLH) has been widely used for decades as an antigen carrier to generate antibodies or to build vaccines for immunotherapy. In both circumstances, an antigen is conjugated to KLH. Depending on the nature of the antigens, there are many conjugation methods utilized in both academia and industry. At Stellar, we are also exploring the conjugation methods used to conjugate various types of antigens to KLH. We chose ovalbumin (OVA) as a model antigen to build KLH-protein antigen conjugates. We conjugated OVA to KLH, both the 400 KDa monomer (subunit KLH) and the 8 MDa didecamer (high molecular weight KLH), via disulfide bonds or primary amine groups on OVA polypeptide chains. We also conjugated OVA to KLH via the carbohydrate structures on OVA or the carbohydrate structures on KLH. For each of these methods, we utilized different ratios of KLH to the linker, KLH to OVA. The KLH-OVA conjugates were used to pulse mouse bone marrow-derived primary dendritic cells (DCs) and immature mouse DC line JAWSII cells. After being pulsed with conjugates, DCs were stained with fluorescence tag conjugated antibodies that recognized and bound to the OVA epitopes presented by MHC II molecules on the surface of the DCs. The abundance of OVA epitopes on DC surface was analyzed with flow cytometry. We found that the conjugation route affected the capability of KLH to serve as a carrier for protein antigens. We also found that, for certain KLH samples, conjugating a protein antigen via carbohydrate structures (instead of disulfide bonds or primary amines) did not interfere with protein antigen processing and even increased the amount of protein antigens to be successfully presented by DCs.


O-Linked and N-Linked Glycoproteins

Glycoproteins are categorized according to the attachment site of the carbohydrate to an amino acid in the protein.

  • O-linked glycoproteins are ones in which the carbohydrate bonds to the oxygen atom (O) of the hydroxyl group (-OH) of the R group of either the amino acid threonine or serine. O-linked carbohydrates may also bond to hydroxylysine or hydroxyproline. The process is termed O-glycosylation. O-linked glycoproteins are bound to sugar within the Golgi complex.
  • N-linked glycoproteins have a carbohydrate bonded to the nitrogen (N) of the amino group (-NH2) of the R group of the amino acid asparagine. The R group is usually the amide side chain of asparagine. The bonding process is called N-glycosylation. N-linked glycoproteins gain their sugar from the endoplasmic reticulum membrane and then are transported to the Golgi complex for modification.

While O-linked and N-linked glycoproteins are the most common forms, other connections are also possible:

  • P-glycosylation occurs when the sugar attaches to the phosphorus of phosphoserine.
  • C-glycosylation is when the sugar attaches to the carbon atom of an amino acid. An example is when the sugar mannose bonds to the carbon in tryptophan.
  • Glypiation is when a glycophosphatidylinositol (GPI) glycolipid attaches to the carbon terminus of a polypeptide.

Contents

Glycosylation is the process by which a carbohydrate is covalently attached to a target macromolecule, typically proteins and lipids. This modification serves various functions. [4] For instance, some proteins do not fold correctly unless they are glycosylated. [1] In other cases, proteins are not stable unless they contain oligosaccharides linked at the amide nitrogen of certain asparagine residues. The influence of glycosylation on the folding and stability of glycoprotein is twofold. Firstly, the highly soluble glycans may have a direct physicochemical stabilisation effect. Secondly, N-linked glycans mediate a critical quality control check point in glycoprotein folding in the endoplasmic reticulum. [5] Glycosylation also plays a role in cell-to-cell adhesion (a mechanism employed by cells of the immune system) via sugar-binding proteins called lectins, which recognize specific carbohydrate moieties. [1] Glycosylation is an important parameter in the optimization of many glycoprotein-based drugs such as monoclonal antibodies. [5] Glycosylation also underpins the ABO blood group system. It is the presence or absence of glycosyltransferases which dictates which blood group antigens are presented and hence what antibody specificities are exhibited. This immunological role may well have driven the diversification of glycan heterogeneity and creates a barrier to zoonotic transmission of viruses. [6] In addition, glycosylation is often used by viruses to shield the underlying viral protein from immune recognition. A significant example is the dense glycan shield of the envelope spike of the human immunodeficiency virus. [7]

Overall, glycosylation needs to be understood by the likely evolutionary selection pressures that have shaped it. In one model, diversification can be considered purely as a result of endogenous functionality (such as cell trafficking). However, it is more likely that diversification is driven by evasion of pathogen infection mechanism (e.g. Helicobacter attachment to terminal saccharide residues) and that diversity within the multicellular organism is then exploited endogenously.

Glycosylation can also module the thermodynamic and kinetic stability of the proteins. [8]

Glycosylation increases diversity in the proteome, because almost every aspect of glycosylation can be modified, including:

    —the site of glycan linkage
  • Glycan composition—the types of sugars that are linked to a given protein
  • Glycan structure—can be unbranched or branched chains of sugars
  • Glycan length—can be short- or long-chain oligosaccharides

There are various mechanisms for glycosylation, although most share several common features: [1]

  • Glycosylation, unlike glycation, is an enzymatic process. Indeed, glycosylation is thought to be the most complex post-translational modification, because of the large number of enzymatic steps involved. [9]
  • The donor molecule is often an activated nucleotide sugar.
  • The process is non-templated (unlike DNA transcription or protein translation) instead, the cell relies on segregating enzymes into different cellular compartments (e.g., endoplasmic reticulum, cisternae in Golgi apparatus). Therefore, glycosylation is a site-specific modification.

N-linked glycosylation Edit

N-linked glycosylation is a very prevalent form of glycosylation and is important for the folding of many eukaryotic glycoproteins and for cell–cell and cell–extracellular matrix attachment. The N-linked glycosylation process occurs in eukaryotes in the lumen of the endoplasmic reticulum and widely in archaea, but very rarely in bacteria. In addition to their function in protein folding and cellular attachment, the N-linked glycans of a protein can modulate a protein's function, in some cases acting as an on/off switch.

O-linked glycosylation Edit

O-linked glycosylation is a form of glycosylation that occurs in eukaryotes in the Golgi apparatus, [10] but also occurs in archaea and bacteria.

Phosphoserine glycosylation Edit

Xylose, fucose, mannose, and GlcNAc phosphoserine glycans have been reported in the literature. Fucose and GlcNAc have been found only in Dictyostelium discoideum, mannose in Leishmania mexicana, and xylose in Trypanosoma cruzi. Mannose has recently been reported in a vertebrate, the mouse, Mus musculus, on the cell-surface laminin receptor alpha dystroglycan 4 . It has been suggested this rare finding may be linked to the fact that alpha dystroglycan is highly conserved from lower vertebrates to mammals. [11]

C-mannosylation Edit

A mannose sugar is added to the first tryptophan residue in the sequence W–X–X–W (W indicates tryptophan X is any amino acid). A C-C bond is formed between the first carbon of the alpha-mannose and the second carbon of the tryptophan. [12] However, not all the sequences that have this pattern are mannosylated. It has been established that, in fact, only two thirds are and that there is a clear preference for the second amino acid to be one of the polar ones (Ser, Ala, Gly and Thr) in order for mannosylation to occur. Recently there has been a breakthrough in the technique of predicting whether or not the sequence will have a mannosylation site that provides an accuracy of 93% opposed to the 67% accuracy if we just consider the WXXW motif. [13]

Thrombospondins are one of the proteins most commonly modified in this way. However, there is another group of proteins that undergo C-mannosylation, type I cytokine receptors. [14] C-mannosylation is unusual because the sugar is linked to a carbon rather than a reactive atom such as nitrogen or oxygen. In 2011, the first crystal structure of a protein containing this type of glycosylation was determined—that of human complement component 8. [15] Currently it is established that 18% of human proteins, secreted and transmembrane undergo the process of C-mannosylation. [13] Numerous studies have shown that this process plays an important role in the secretion of Trombospondin type 1 containing proteins which are retained in the endoplasmic reticulum if they do not undergo C-mannosylation [13] This explains why a type of cytokine receptors, erythropoietin receptor remained in the endoplasmic reticulum if it lacked C-mannosylation sites. [16]

Formation of GPI anchors (glypiation) Edit

Glypiation is a special form of glycosylation that features the formation of a GPI anchor. In this kind of glycosylation a protein is attached to a lipid anchor, via a glycan chain. (See also prenylation.)

Chemical glycosylation Edit

Glycosylation can also be effected using the tools of synthetic organic chemistry. Unlike the biochemical processes, synthetic glycochemistry relies heavily on protecting groups [17] (e.g. the 4,6-O-benzylidene) in order to achieve desired regioselectivity. The other challenge of chemical glycosylation is the stereoselectivity that each glycosidic linkage has two stereo-outcomes, α/β or cis/trans. Generally, the α- or cis-glycoside is more challenging to synthesis. [18] New methods have been developed based on solvent participation or the formation of bicyclic sulfonium ions as chiral-auxiliary groups. [19]

Non-enzymatic glycosylation Edit

The non-enzymatic glycosylation is also known as glycation or non-enzymatic glycation. It is a spontaneous reaction and a type of post-translational modification of proteins meaning it alters their structure and biological activity. It is the covalent attachment between the carbonil group of a reducing sugar (mainly glucose and fructose) and the amino acid side chain of the protein. In this process the intervention of an enzyme is not needed. It takes place across and close to the water channels and the protruding tubules. [20]

At first, the reaction forms temporary molecules which later undergo different reactions (Amadori rearrangements, Schiff base reactions, Maillard reactions, crosslinkings. ) and form permanent residues known as Advanced Glycation end-products (AGEs).

AGEs accumulate in long-lived extracellular proteins such as collagen [21] which is the most glycated and structurally abundant protein, especially in humans. Also, some studies have shown lysine may trigger spontaneous non-enzymatic glycosylation. [22]

Role of AGEs Edit

AGEs are responsible for many things. These molecules play an important role especially in nutrition, they are responsible for the brownish color and the aromas and flavors of some foods. It is demonstrated that cooking at high temperature results in various food products having high levels of AGEs. [23]

Having elevated levels of AGEs in the body has a direct impact on the development of many diseases. It has a direct implication in diabetes mellitus type 2 that can lead to many complications such as: cataracts, renal failure, heart damage. [24] And, if they are present at a decreased level, skin elasticity is reduced which is an important symptom of aging. [21]

They are also the precursors of many hormones and regulate and modify their receptor mechanisms at the DNA level. [21]

There are different enzymes to remove the glycans from the proteins or remove some part of the sugar chain.

    (from Arthrobacter ureafaciens): cleaves all non-reducing terminal branched and unbranched sialic acids. (from Streptococcus pneumoniae): releases only β1,4-linked, nonreducing terminal galactose from complex carbohydrates and glycoproteins. (from Streptococcus pneumoniae): cleaves all non-reducing terminal β-linked N-acetylglucosamine residues from complex carbohydrates and glycoproteins. (O-glycosidase from Streptococcus pneumoniae): removes O-glycosylation. This enzyme cleaves serine- or threonine-linked unsubstituted Galβ1,3GalNAc : cleaves asparagine-linked oligosaccharides unless α1,3-core fucosylated.

Notch signalling is a cell signalling pathway whose role is, among many others, to control the cell differentiation process in equivalent precursor cells. [25] This means it is crucial in embryonic development, to the point that it has been tested on mice that the removal of glycans in Notch proteins can result in embryonic death or malformations of vital organs like the heart. [26]

Some of the specific modulators that control this process are glycosyltransferases located in the Endoplasmic reticulum and the Golgi apparatus. [27] The Notch proteins go through these organelles in their maturation process and can be subject to different types of glycosylation: N-linked glycosylation and O-linked glycosylation (more specifically: O-linked glucose and O-linked fucose). [25]

All of the Notch proteins are modified by an O-fucose, because they share a common trait: O-fucosylation consensus sequences. [25] One of the modulators that intervene in this process is the Fringe, a glycosyltransferase that modifies the O-fucose to activate or deactivate parts of the signalling, acting as a positive or negative regulator, respectively. [27]

There are three types of glycosylation disorders sorted by the type of alterations that are made to the glycosylation process: congenital alterations, acquired alterations and non-enzymatic acquired alterations.

  • Congenital alterations: Over 40 congenital disorders of glycosylation (CGDs) have been reported in humans. [28] These can be divided into four groups: disorders of protein N-glycosylation, disorders of protein O-glycosylation, disorders of lipid glycosylation and disorders of other glycosylation pathways and of multiple glycosylation pathways. No effective treatment is known for any of these disorders. 80% of these affect the nervous system. [citation needed]
  • Acquired alterations: In this second group the main disorders are infectious diseases, autoimmune illnesses or cancer. In these cases, the changes in glycosylation are the cause of certain biological events. For example, in Rheumatoid Arthritis (RA), the body of the patient produces antibodies against the enzyme lymphocytes galactosyltransferase which inhibits the glycosylation of IgG. Therefore, the changes in the N-glycosylation produce the immunodeficiency involved in this illness. In this second group we can also find disorders caused by mutations on the enzymes that control the glycosylation of Notch proteins, such as Alagille syndrome. [27]
  • Non-enzymatic acquired alterations: Non-enzymatic disorders, are also acquired, but they are due to the lack of enzymes that attach oligosaccharides to the protein. In this group the illnesses that stand out are Alzheimer's disease and diabetes. [29]

All these diseases are difficult to diagnose because they do not only affect one organ, they affect many of them and in different ways. As a consequence, they are also hard to treat. However, thanks to the many advances that have been made in next-generation sequencing, scientists can now understand better these disorders and have discovered new CDGs. [30]

Effects on therapeutic efficacy Edit

It has been reported that mammalian glycosylation can improve the therapeutic efficacy of biotherapeutics. For example, therapeutic efficacy of recombinant human interferon gamma, expressed in HEK 293 platform, was improved against drug-resistant ovarian cancer cell lines. [31]


Advances in Carbohydrate Chemistry and Biochemistry

II Mucins: Background

Mucins are heavily O-glycosylated linear glycoproteins that are secreted by higher organisms to protect and lubricate epithelial cell surfaces. Mucin and mucin-like domains are also involved in modulating immune response, inflammation, adhesion, and tumorigenesis. (cf.30–32) The tandem repeat domains of mucins and mucin-like glycoproteins contain high contents of clustered Ser and Thr residues, with many of these residues O-linked to a variety of “core” oligosaccharide structures, including linear and branched-chain blood-group determinants. 32 The oligosaccharide chains on mucins have been shown to be important for a variety of their biological properties including their interactions with such animal lectins as the selectins and galectins, as well as their physical properties including their extended linear structures. 33

In addition to the variety of O-linked carbohydrates found on mucins, there are at least 17 mucin gene products (MUC1– MUC17 ). 31 These gene products represent two structurally and functionally distinct classes of mucins: secreted gel-forming mucins and transmembrane mucins, although there are a few mucin gene products that do not appear to fit into either category. The transmembrane mucins include MUC1 whose structure includes a cytoplasmic domain, in addition to the extracellular O-glycosylated polypeptide tandem repeat domains. 34 Evidence indicates that the C-terminal cytoplasmic domain of MUC1 is involved in signal transduction mechanisms, including T-cell activation and inhibition, and adhesion signaling responses. 35,36

Mucins are also useful in the diagnosis of a variety of diseases. 33 In particular, the level of expression of mucin peptide antigens and type of carbohydrate chains of mucins have proved to be useful diagnostic markers for a variety of cancers. 31,37 For example, MUC1 expression as detected immunologically is increased in colon cancers and is associated with a poor diagnosis. 31 Colon cancer-associated mucins also have differences associated with their core carbohydrate structures, often presenting shorter chain versions of normal mucins. Colon cancer mucins often have increased expression of the αGalNAcThr/Ser (Tn-antigen), βGal3GalNAc (T or TF antigen) and αNeuAc6GalNAc (sialyl Tn-antigen). 31 Importantly, recent studies have shown that binding of galectin-3, an endogenous Gal-specific animal lectin, to cancer-associated MUC1 causes increased endothelial cancer cell adhesion. 38 Thus, the molecular recognition properties of cancer related mucins, including their truncated carbohydrates, are important in terms of gaining insight into their structure–activity properties.

Porcine submaxillary mucin (PSM) is a physically well-characterized mucin, and the subject of studies of the regulation of O-glycosylation with glycosyltransferases (cf.39) and of binding interactions with lectins. 28 The cDNA sequence of PSM has been determined, 40 and the 81 amino acid tandem repeat domain that is present in 100 copies is shown in Fig. 2A . The structures of the carbohydrate chains were determined by chemical 41 and NMR techniques. 42 Gerken and Jentoft 42 isolated the O-glycosylated domain of PSM that possesses a molecular mass of

10 6 Da and is fully decorated with naturally occurring carbohydrates (Fd-PSM) ( Fig. 2B ). The O-glycosylated domain of PSM possessing only αGalNAc residues (Tn-PSM) ( Fig. 2C ) was also obtained using chemical and enzymatic treatments. 43 The αGalNAc1-O-Ser/Thr residue(s) in Tn-PSM is the pancarcinoma carbohydrate antigen Tn that is aberrantly expressed in such mucins as MUC1 in adenocarcinomas. 44 The 81-mer tandem repeat domain of Tn-PSM (81-mer Tn-PSM) ( Fig. 2D ) and the 38/40-mer digest of this domain (38/40-mer Tn-PSM) ( Fig. 2E ) also have been obtained using enzymatic digests. 43

Fig. 2 . Structural representations of (A) the amino acid sequence of the 100-repeat 81-residue polypeptide O-glycosylation domain of intact PSM (B) the fully carbohydrate-decorated form (described in the text) of the 100-repeat 81-residue polypeptide O-glycosylation domain of PSM (Fd-PSM) (C) the 100-repeat 81-residue polypeptide O-glycosylation domain of PSM containing only peptide-linked αGalNAc residues (Tn-PSM) (D) the single 81-residue polypeptide O-glycosylation domain of PSM containing peptide-linked αGalNAc residues (81-mer Tn-PSM) (E) the 38/40-residue polypeptide(s) derived from the 81-residue polypeptide O-glycosylation domain of PSM containing peptide-linked αGalNAc residues (38/40-mer Tn-PSM). The number of glycan chains in Fd-PSM and Tn-PSM is

2300. The number of αGalNAc residues in 81-mer Tn-PSM is

23, while the number of αGalNAc residues in 38/40-mer Tn-PSM is

The binding and crosslinking of cell-surface mucins and mucin-like glycoproteins by lectins is known to lead to signal transduction effects, including cell growth and cell death. 16,45 For example, galectin-1 crosslinking of CD43, a transmembrane mucin-type glycoprotein receptor that possesses approximately 80 O-linked chains with terminal LacNAc epitopes, 46 along with CD45 induces apoptosis in susceptible T cells. 47 However, details of the energetics and mechanisms of lectin binding and crosslinking of mucins and mucin-type receptors has been lacking.


Lipid and Polysaccharide Antigens

Lipid Antigens

  • Lipid antigens are presented to T cells by cell-surface molecules designated CD1 ("cluster of differentiation" 1).
  • Antigen-presenting cells express several different forms of CD1 at their surface. Each is probably specialized to bind a particular type of lipid antigen (e.g. lipopeptide vs glycolipid).
  • The exposed surface of CD1 molecules forms an antigen-binding groove much like that of MHC molecules except that
  • the amino acids in the groove are more hydrophobic than those in MHC molecules.
  • Like protein antigens, lipid antigens are also presented as fragments, i.e., as a "hot dog in a bun".

Polysaccharide Antigens

Some bacterial polysaccharides ingested by APCs

  • can be degraded in their lysosomes
  • and presented to T cells by MHC class II molecules.

The binding of a T cell to an antigen-presenting cell (APC) is by itself not enough to activate the T cell and turn it into an effector cell: one able to, for examples,

  • kill the APC (CD8 + cytotoxic T lymphocytes [CTLs])
  • carry out cell-mediated immune reactions (CD4 + Th1 cells)
  • provide help to B cells (CD4 + Th2 cells)

In order to become activated, the T cell must not only bind to the epitope (MHC-peptide) with its TCR but also receive a second signal from the APC. The receipt of this second signal is called costimulation. Among the most important of these costimulators are molecules on the APC designated B7 and their ligand on the T cell designated CD28. The binding of CD28 to B7 provides the second signal needed to activate the T cell.

Although T cells may encounter self antigens in body tissues, they will not respond unless they receive a second signal. In fact, binding of their TCR ("signal one") without "signal two" causes them to self-destruct by apoptosis. Most of the time, the cells presenting the body's own antigens either

  • fail to provide signal two or
  • transmit an as-yet-unidentified second signal that turns the T cell into a regulatory T cell (Treg) that suppresses immune responses.

In either case, self-tolerance results.