AN

ASSIGNMENT

ON

GLYCOPROTEINS IN FISH

( COURSE FNB 506) TINCY VARGHESE FNB 44

Introduction Glycoproteins are type of proteins containing carbohydrate moieties (glycans) which are covalently attached to their polypeptide side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification in endoplasmic reticulam of the cell. Carbohydrates are a much smaller percentage of the weight of glycoproteins than of proteoglycans Glycoproteins were discovered abundantly in living organisms which perform diverse functions in relation with different biological processes. Like other vertebrates fish also contains several types of glycoproteins. Because carbohydrates and proteins by themselves serve in a vast number of biological functions, it should not be surprising that linking the two together results in a macromolecule with an extremely large number of functions.any glycoproteins have structural functions such as collagen. They are also found in gastrointestinal mucus secretions. Glycoproteins are used as protective agents and lubricants. They are also found abundantly in the blood plasma where they serve many functions. Many of the hormonal receiptors are also glycoproteins. There can be broad and fine structural differences which account for the large diversity of functions among glycoproteins. Regulation of glycoproteins is achieved through synthesis and degradation processes controlled by very specific enzymes. Structure Structurally, glycoproteins consist of a polypeptide covalently bonded to a carbohydrate moiety. The carbohydrate can make up anywhere from less than one percent to more than 80 percent of the total protein mass. The saccharide chains, referred to as glycans, can be linked to the polypeptide in two major ways: Nglycosylation and O-glycosylation O-glycosylation In O-glycosylation, the addition of sugar chains can happen on the hydroxyl oxygen on the side chain of hydroxylysine, hydroxyproline, serine, or threonine. Thus, potential glycosylation sites can be detected within amino acid sequences. However,the glycosylation in the potential sites is actually depends on other aspects of the protein structure and on the cell type in which the protein is expressed. The glycan chain usually contain an N-acetylgalactosamine which is attached through a glycosidic bond to the O-terminus of either threonine or serine.. Nacetylgalactosamine is simply a galactose molecule with an amine group covalently bonded to the second carbon. This amine group is bonded to a carboxyl group. Nacetylgalactosamine attaches to the carboxyl group of the amino acid through the hydroxyl group of its anomeric carbon. Another type of O-linked glycan consists of a galactose or a glucosyl-galactose disaccharide linked to the hydroxyl of hydroxylysine. Yet another type of O-linkage involves the binding of arabinose to the hydroxyl of hydroxyproline. In all of the 0-linked glycans, there can be a variety

of different monosaccharide or polysaccharide chains attached to the sugar that is bonded to the amino acid . N-Glycosylation In N-glycosylation ,the addition of sugar chains can happen at the amide nitrogen on the side chain of the asparagine. These involve a glycosidic bond between N-acetylglucosamine and the N-terminus (amide nitrogen)of an asparagine residue. N-acetylglucosamine is simply a glucose molecule which is bonded to an amine group. This amine group, in turn, is bonded to a hydroxyl group. The Nacetylglucosamine is bonded to the asparagine through its anomeric carbon. The asparagine must be surrounded by a specific amino acid sequence, or sequon. This sequence is -X-Asn-X-Thr; the X can be any amino acid. A large variety of polysaccharide side chains can be linked to the N-acetylglucosamine. A typical polysaccharide chain is Man2 a(1-6)-Man B(1-4)-GlcNAc B(1-4)-GlcNAc B(1-N) Asn. Adding on to this structure can create many different N-linked glycans. Site of Glycosylation Usually protein glycosylation takes place inside the lumen of the endoplasmic reticulum (ER) and the Golgi complex, organelles that play central roles in protein trafficking. There is a signal sequence, which directs the protein through a channel in the ER membrane, is cleaved from the protein in the transport process into the ER. After the protein has entered the ER, the glycosylation process begins. The N-linked glycosylation begins in the ER i.e. core glycosylation, and continues in the Golgi complex, i.e. terminal glycosylation .The O-linked glycosylation takes place exclusively in the Golgi complex. Process of glycosylation In N linked glycoproteins the oligosaccharide is first attached to dolichol phosphate, a specialized lipid molecule containing as many as 20 isoprene (C5) units.Then it enters the ER and additional sugars are added by enzymes in the ER lumen. The monosaccharides activated by attachment to dolichol phosphate. Dolichol pyrophosphate released in the transfer of the oligosaccharide to the protein is recycled to dolichol phosphate by the action of a phosphatase. Before a glycoprotein leaves the ER, two glucosidases cleave the three glucose residues of the oligosaccharide in a step-by-step fashion. The unfolded proteins are again attached by a glucose residue by the glucosyl transferase. These glucose residues are binded by 2 proteins namely calnexin and calreticulin which wont allow these proteins to leave ER before the complete folding of protein. Transport Vesicles Carry Proteins from the Endoplasmic Reticulum to the Golgi Complex for Further Glycosylation and Sorting. The glycosyltransferases present in the Golgi compartment mediates further glycosylation: usually some of the mannose residues are removed. Many hydrolytic enzymes are directed by a marker ( Mannose 6-phosphate) from the Golgi complex to lysosomes.

Regulation/Control of glycoprotein synthesis Addition of any other sugar can be in any possible combination, according to the desired resulting functions. The specificity of the enzymes is very important in the synthesis process. Every sugar added, is catalyzed by a different enzyme. These group of enzymes are called glycosyltransferases. In N glycosylation the first addition of GlcNAc to dolichol phosphate is catalyzed by the specific enzyme that cleave peptide bonds, and glycosidases . enzymes that remove sugars one at a time from the end of an oligosaccharide chain. Both of these groups are contained in lysozomes. The lysosome attaches to a phagocyte, which has engulfed a substance that needs to be broken down, and releases its enzymes in it). Next, these enzymes begin their catalytic action. Thus the glycosylalation is specified by specific enzymes. Functions The carbohydrate chains can assist in protein folding or improve proteins' stability. Thus they play a role in the structure of the polypeptide. Because of this and their biologically ubiquitous nature, the best way to go about exploring glycoprotein function is to break it down into categories that are fairly general. Type of glycoproteins based on functions Structural molecule Collagens Lubricant and protective Mucins agent Transport molecule Transferrin, ceruloplasmin Immunologic molecule Immunoglobins, histocompatibility antigens Human chorionic gonadotropin (HCG), thyroidHormone stimulating hormone (TSH) Glycoprotein enzymes are of three types. These are Enzymes oxidoreductases, transferases, and hydrolases eg, alkaline phosphatase Cell attachment- Various proteins involved in cell-cell (eg, sperm-oocyte), recognition site virus-cell, bacterium-cell, and hormone cell interactions Antifreeze Certain plasma proteins of coldwater fish Interact with specific Lectins, selectins (cell adhesion lectins), antibodies carbohydrates Receptor Various proteins involved in hormone and drug action Affect folding of certain Calnexin, calreticulin proteins Regulation development Notch and its analogs, key proteins in development Hemostasis(and Specific glycoproteins on the surface membranes of thrombosis) platelets

Egg and spermatozoal To increase the chance of fertilization and avoid glycoproteins polyspermy Glycoproteins associated with vision

Analysis Some important methods used to study glycoproteins Method Use Detects glycoproteins as pink bands after Periodic acid-Schiff stain electrophoretic separation. Incubation of cultured cells Leads to detection of a radioactive sugar after with glycoproteins as electrophoretic separation. radioactive decay bands Resultant shifts in electrophoretic migration help Treatment with appropriate distinguish among proteins with N-glycan, O-glycan, endo- or exoglycosidase or or GPI linkages and also between high mannose and phospholipases complex N-glycans. Agarose-lectin column To purify glycoproteins or glycopeptides that bind the chromatography particular lectin used. Compositional analysis Identifies sugars that the glycoprotein contains and following acid hydrolysis their stoichiometry. Provides information on molecular mass, Mass spectrometry composition, sequence, and sometimes branching of a glycan chain. To identify specific sugars, their sequence, linkages, NMR spectroscopy and the anomeric nature of glycosidic chain. Measures the mechanisms underlying the Dual Polarisation biomolecular interactions, including reaction rates, Interferometry affinities and associated conformational changes. Methylation (linkage) analysis To determine linkage between sugars. Amino acid or cDNA Determination of amino acid sequence. sequencing

TYPES OF GLYCOPROTEINS IN FISH Glycoprotein Hormones These are heterodimeric glycoproteins composed of a common subunit and a hormone-specific subunit. The N-linked oligosaccharides of these hormones are necessary for proper folding, assembly, secretion, metabolic clearance and biological activity. The free subunit, which is shown to have a physiological function, is maintained in the uncombined form due to its glycan structures. The Nglycans of the glycoprotein hormones contain a variety of terminal residues, which are responsible for the differential targeting and clearance of the hormones. Glycosylation of these hormones is regulated by a variety of physiological and pathological conditions, leading to subtle alterations in their bioactivities. Recent studies on the structures and specific functions of different glycans of natural and recombinant glycoprotein hormones have provided valuable insight into the glycobiology of these hormones

Glycoprotein hormone in fishes include:

GTH I(FSH) GTH II (LH) Thyroid-stimulating hormone(TSH) POMC with N-terminal peptides

POMC proipio melano cortin. GtH I (FSH) and GtH II (LH) are secreted from the Gonadotropes while TSH is produced by thyrotrphs of the Anterior Pituitary. POMC is produced by ACTH and MSH cells in the pituitary .N-POMC, ACTH, b-LPH -are produced in the posttranslational processing by Proprotein Convertase. Features of Glycoprotein hormones 1.All glycoproteins are composed of two subunits ;namely and These two subunits that are joined by noncovalent forces. The -subunit is similar in all hormones while the -subunit is specific for each hormone. Both the and subunits are highly glycosylated .All the subunits have two N-linked sugar chains and the subunits consist of one or two N linked sugars depending on the hormone. LH, TSH contain one N-linked oligosaccharide chain while in FSH it contain two N-linked oligosaccharide chains. The subunits are coded on separate genes and each has a propeptide sequence. The free subunit found in the pituitary and placental cells contains an additional 0-glycosidic linkage which is considered abnormal because it does not

combine with the subunit. However, removal of the 0-linked sugars restores its ability to recombine and regain hormone function to the - complex.

2. High degree of disulfide bridging is found in each subunit of the glycoprotein hormones.There are 10 half-cystines in the subunit whose positions are highly

conserved from one species to another while there are 12 half-cystines in all of the subunits, and they are highly conserved not only between species, but also among the different hormones. There are no free sulfhydryl groups due to the extensive sulphide linkages. The task of differenciating the disulfide bonds is difficult because of clustering of haif-cystine residues and the lack of enzymatic cleavage points within these clusters .Also there are problems of disulfide interchange between the hormone subunits.Several disulfide bridges are known in each subunit with a reasonable degree of certainty: The examples are 7-31,1 lO-32, 93-100, 26-110, and 23-72. Other disulfide bonds that have been proposed, but are not yet certain, are 9-90, 34-88, and 38-57. 3.The existence of two types of carbohydrate side chains has been depicted in glycoprotein hormones. There are asparagine (N)-linked chains on all and subunits .In addition, there are serine (0)-linked chains on the carboxy terminal extension of hCG . The 0-linked chains are relatively simple structures and they are not required for biological activity because they are absent from the LH molecule. The N-linked carbohydrates are complex biantennary (hCG) or triantennary (FSH) structures with a central mannose core and branches that terminate with galactose-sialic acid or galactose-sulfate depending on the species and tissue of origin 4. There are conserved amino acid regions that may be important in the maintenance of secondary and tertiary structure andsubunit interactions. These sites are also required for biological activity.The conservation of the 10 half-cystine residues in the subunit was mentioned above. In addition, residues 27 to 67 are highly conserved in the subunit, being identical in six species. A second conserved region in the subunit is between residues 82 and 92, the COOHterminus 5. subunit :Analysis of human subunits of hCG, LH, FSH, and TSH indicate two constant regions, 16-38 and 56-100, which are good candidates for contact surfaces for interaction with the subunits Of the remaining variable domains there are some regions that show commonality among hormones with LH activity 1-7, 40-55, and 93-100. These regions are all candidates for receptor binding sites. 6.Carbohydrate moieties on the glycoprotein hormones affect their half life in the circulation and thus their in vivo biological potency. Carbohydrate facilitates proper disulphide bond formation during folding of the nascent peptide chains and prevents proteolysis and aggregation of subunits.I n general they indicate that removal of Nlinked carbohydrates.It does not interfere with receptor binding but markedly reduces biological responses such as stimulation of the adenylate cyclase enzyme

and steroidogenesis. Removal of the carbohydrate from the a subunit enhances receptor binding when it is recombined with an intact subunit. The data indicate that the carbohydrates are needed on both subunits for full expression of biological activity 7.Glycans of hormone units: All -subunits have carbohydrate attached at Asn-56 and -82 .Also all -subunits have carbohydrate attached at Asn-13 and/or -30 with both sites glycosylated in follitropin- Only Asn-30 is glycosylated in thyrotropin- . The C-terminal extensions of human and equine choriogonadotropin and equine lutropin also contain at least four glycans linked to serine or threonine residues.

8.Type of sugar chains The asparagine-linked sugar chains can be classified into three subgroups. The high-mannose type glycans contain only mannose and Nacetylglucosamine residues attached to a common heptasaccharide core. Complex type sugar chains contain a pentasaccharide core and structural variations occur through the number and structure of the outer chain moieties attached to the a-mannosyl core residues.

Hybrid type oligosaccharides have structures characteristic of high-mannose and complex type sugar chains.

Signal transduction in glycoprotein hormones The hormone (-dimer) binds to high-affinity receptor site (or sites) R to make an effective coupling to the G, protein, leading to adenylate cyclase activation and subsequent hormone response.

After a productive H-R interaction, the signal is amplified at each of the subsequent steps for full hormone response. When the hormone is deglycosylated ,binding occurs more rapidly and tightly Thus the H-R complex will tilt away from the G proteins, so as not to permit effector coupling.For this reason, adenylate cyclase activation and hormone response are greatly reduced. The two important phases of glycoprotein hormone action are receptor binding and cellular activation are associated with hormone glycosylation. Overwhelming evidence demonstrates that correct and complete N-glycosylation of the common subunit of glycoprotein hormones, in particular that of the first N glycosylation site (from the NH2 terminus), is critical for hormone signal transduction. Hormones in which either one or both subunits are deglycosylated by chemical means, enzymes, or site-directed mutagenesis show excellent receptor binding, but cellular activation depends on which subunit is glycosylated. Lack of carbohydrate in the subunit particularly site1 of two sites transforms an agonistic hormone into one of antagonistic nature. As glycosylation is of vital importance to hormone function, it could be subject to endocrine regulation. Glycoprotein Structure and Steroid Hormone Function Glycoproteins play an important part in hormone function. The action of hormones depends on the initial binding of the hormone to a protein receptor molecule. In many cases this molecule is a glycoprotein. Many hormones bind to receptors in the cell membrane; these hormones never actually enter the cell. Steroids, on the other hand, bind to an intracellular protein receptor. There is still controversy over whether the steroid hormone receptor is found in the nucleus or the cytosol. However, it is clear that after the steroid binds, the hormone-receptor complex moves to the nucleus. The hormone binding domain of the receptor is found at the C-terminus. The amino acid sequence of this region is highly diverse; it is not conserved from one protein to the next. Binding of the hormone stimulates a conformational change in the hormone receptor. This change allows the hormonereceptor complex to bind to the DNA. The DNA binding domain is highly conserved and is found within the central core of the protein. The central core contains a very basic amino acid sequence. In the cellular environment, these bases will tend to pick up a hydrogen and become positively charged. The positive charge will attract the hormone-receptor complex to the negatively charged DNA. Binding of the complex to the DNA stimulates transcription (Evans 890-891 and Mathews 808-809

Antifreeze proteins (AFPs) in Fish

AFPs or ice structuring proteins (ISPs) are a class of polypeptides produced by certain vertebrates, plants, fungi and bacteria that permit their survival in subzero environments. The Antactic ice fishes produce AFPs , which are also a type of glycoproteins.They bind to small ice crystals to inhibit growth and

recrystallization of ice that would otherwise be fatal.. Thus it suggests that the glycoproteins are also involved in cold acclimatization in the form of AFPs Properties They act as an antifreeze at concentrations 300-500 times lower than other dissolved solutes in a non colligative manner This minimizes their effect on osmotic pressure The unusual capabilities of AFPs are attributed to their binding ability at specific ice crystal surfaces. AFPs create a difference between the melting point and freezing point known as thermal hysteresis. They act as interface between ice and water by attaching on ice surface. .The maximum level of thermal hysteresis shown by fish AFP is approximately 1.5 C (2.7 F Fish AFPs

The three faces of Type I AFP Antifreeze glycoproteins or AFGPs are found in Antarctic notothenioids and northern cod. They are 2.6-3.3 kD Type I AFPs are found in winter flounder and shorthorn sculpin. They are the best documented AFP because it was the first to have its 3D structure determined. Type I AFPs consist of a single, long, amphipathic alpha helix. They are approximately 3.3-4.5 kD in size. There are three faces to the 3D structure: the hydrophobic, hydrophilic, and Thr-Asx face Type I-hyp AFP (where hyp stands for hyperactive) are found in several righteye flounders. It is approximately 32 kD (two 17 kD dimeric molecule). The protein was isolated from the blood plasma of winter flounder. It is considerably better at depressing freezing temperature than most fish AFPs Type II AFPs are found in sea raven, smelt and herring. They are cysteine-rich globular proteins containing five disulfide bond.

Type III AFPs are found in Antarctic eelpout. They exhibit similar overall hydrophobicity at ice binding surfaces to type I AFPs. They are approximately 6kD in size. Type IV AFPs are found in longhorn sculpins. They are alpha helical proteins rich in glutamate and glutamine. This protein is approximately 12KDa in size and consists of a 4 helix bundle. Its only post-translational modification is a pyroglutamate residue, a cyclized glutamine residue at its N-terminal. Scientists at the University of Guelph in Canada are currently examining the role of this pyroglutamine residue the antifreeze activity of type IV AFP from the longhorn sculpin. Mechanism of AFP type I The presence of AFPs exposes different faces of the ice crystals.. The ice surface 2021 is the preferred binding surface of AFP type I. Through studies on type I AFP, It was initially thought that ice and AFP interacted through hydrogen bonding. However, recent data suggests that hydrophobic interactions could be the main contributor. Glycosylation in Antifreeze glycoproteins Four distinct macromolecular antifreezes have been isolated and characterized from different marine fish. These include the glycoprotein antifreezes (Mr 2.5-33 K), which are made up of a repeating tripeptide (Ala-Ala-Thr)n with a disaccharide attached to the threonyl residues, and three antifreeze protein (AFP) types. Type I is an alanine-rich, amphiphilic, alpha-helix (Mr 3-5 K); type II is a larger protein (Mr 14 K) with a high content of reverse turns and five disulfide bridges; and type III is intermediate in size (Mr 6-7 K) with no distinguishing features of secondary structure or amino acid composition. Despite their marked structural differences, all four antifreeze types appear to function in the same way by binding to the prism faces of ice crystals and inhibiting growth along the a-axes. It is suggested that type I AFP binds preferentially to the prism faces as a result of interactions between the helix macrodipole and the dipoles on the water molecules in the ice lattice. Binding is stabilized by y hydrogen bonding, and the amphiphilic character of the helix results in the hydrophobic phase of the helix being exposed to the solvent. When the solution temperature is lowered further, ice crystal growth occurs primarily on the uncoated, unordered basal plane resulting in bipyramidalshaped crystals. The structural features of type I AFP that could contribute to this mechanism of action are reviewed. Current challenges lie in solving the other antifreeze structures and interpreting them in light of what appears to be a common mechanism of action Glycoprotein receiptors.

Many glycoproteins are components of cell membranes, where they play a variety of roles in processes such as cell adhesion. Also most of the peptide hormonal receiptors are glycoproteins Peptide hormone receptors are often transmembrane proteins. They include 1.G-protein-coupled receptors, sensory receptors or ionotropic receptors. These receptors generally function via intracellular second messengers, including cyclic AMP (cAMP), inositol 1,4,5trisphosphate (IP3) and the calcium (Ca2+)-calmodulin system 2.Tyrosine kinase receiptors, which affect neuronal survival and differentiation through several signal cascades. However, the activation of these receptors also has significant effects on functional properties of neurons. Mucoproteins Mucins are another important types of glycoproteins found in the body of many animals including fish, which are secreted in the mucus of the respiratory and digestive tracts. The sugars attached to mucins give them considerable waterholding capacity and also make them resistant to proteolysis by digestive enzymes. They are high molecular weight polymers and found on internal epithelial surfaces. They form a highly viscous gel that protects epithelium form chemical, physical, and microbial disturbances. Examples of mucin sites are the digestive tract, urinary tract, and respiratory tracts. "Cervical mucin" is a glycoprotein found in the cervix of animals that regulates access of spermatozoa to the upper reproductive tract. Mucins are also found on the outer body surfaces of fish to protect the skin. Not only does mucin serve the function of protection, but it also acts as a lubricant. Altered glycosylation patterns change adhesion properties in mucins or mucoproteins present in the intestine of fish. It means that the glycosylation is essential to maintain normal functioning of mucoproteins. Immunoglobulins Many immunoglobulins are actually glycoproteins Immunoglobulins interact directly with antigens.The major immunoglobulin in fish is IgM. It is a tetramer.Each monomer contain 2 heavy chains and two light chains.Each heavy and light chain have variable and constant regions.The carbohydrate moiety of cod serum IgM was analysed using oligosaccharide sequencing techniques. The carbohydrate moiety constituted about 10% of the molecular weight of cod IgM, was associated with the constant region of the heavy chains (Fc), and was composed of N-linked complex type oligosaccharides. Considerable heterogeneity was observed. Sixteen different glycan structures were identified, over 60% were sialylated and 40% contained core fucose. The carbohydrate moiety of cod IgM was shown to provide protection against protease digestion, and partial deglycosylation abolished the antigen binding property of natural cod anti-TNP-BSA antibody. molecules of the major histocompatibility complex (or MHC), which are expressed on the surface of cells and interact with T cells as part of the adaptive

immune response. Soluble immune mediators such as helper, suppressor, and activator cell have been shown to bind to glycoproteins found on the surface of their target cells. B and T cells contain surface glycoproteins that attract bacteria to these sites and bind them. In much the same manner, glycoproteins can direct phagocytosis Glycoproteins related to nerve cell adhesion The interactions between cells is mediated by the glycoproteins on those cell's surfaces. In different domains of the body, different glycoproteins act to unite cells. For example, nerve cells recognize and bind to one another via the glycoprotein N-CAM (nerve cell adhesion molecule). N-CAM is also found on muscle cells indicating a role in the formation of myoneural junctions. With cellsubstratum adhesion, glycoproteins serve as cell surface receptors for certain adhesion ligands that mediate and coordinate the interaction of cells. Substrates with the appropriate receptor will bind to the cell related to that receptor. For example, a substrate containing the glycoprotein fibronectin will be recognized and adhered to by fibroblasts. The fibroblasts will then secrete adhesion molecules and continue to spread, producing a pericellular matrix Cortical alveolar Glycoproteins There are several glycoproteins which are isolated from fish eggs. Cortical vesicles are specialized Golgi derived secretory organelles found in the peripheral cytoplasm of mature eggs of all animals including fish. Upon fertilization they release their content to perivitelline space. This wil help the normal development. These vesicles are ten times larger in fish egg than other animals (2-40m) which are called cortical granules. These granules contain several glycoproteins. There are some glycoprotein components in the zona pellucida, which surrounds the oocyte, and is important for sperm-egg interaction. Hyposhorins These are group of glycoproteins which are ubiquitously found in cortical alveoli of fish eggs. These proteins have high carbohydrate content(80-90% w/w). Apohyposhorins comprises tandom repeats of peptide sequences.protein is completely cleaved into repeating units when the dormant unfertilized eggs commence development in response to sperm fusion or parthenogenetic stimulus. PSGP(Phosphosialoglycoprotein) It is found in mature unfertilized egg of rainbow trout. It contain more than 5% in weight of sialic acid.(NeuSGc) In thePSGP all glycan chains are O glycosidically linked. The major carbohydrate moiety is Galactose (Gal) and galactosamine(GalNAC). Sialic acid occurs usually as a single nonreducing terminal residue in glycoproteins. i.e. polysialic acid residue is not common in glycoproteins. Aminoacid sequence of L-PSGP of rainbow trout

Asp-Asp-Ala-Thr *-Ser *Glu-Ala-Ala-Thr*-Gly-Pro-Ser*-Gly * indicates O glycosylation.. H-PSGP is a polymerized form of L-PSGp.It is found in unfertilized eggs/immature oocyte Biological function of PSGP Formation and maturation of Golgi derived secretory bodies. It takes part in the fertilization and embryonic development.

Mucin type glycoproteins in fish eggs These type of glycoproteins are present in viteline envelope and ovarian fluid. .In fish the egg coat has different layers which forms the vitelline envelope after fertilization by reformation of various molecules. These glycoproteins assist in species specific fertilization, prevent polyspermy, Gives protection to eggs. Fix embryo to substrate These glycoproteins contain more than 50% of carbohydrates, designated as KDN(2-Keto-3-deoxy-d-glycero-d-galacto-nononic Acid ) glycoproteins. These are without sialic acid. In rainbow trout egg Kdn glycoproteins ar with 15% protein and 855 carbohydrate which is O linked at serine and Threonine. ApoKDN Glycoprotein contain 40% Threonine and 27% Alanine. Another type of glycoprotein is Sia-Glycoproteins.It contains sialic acid Instead of KDN glycoproteins. Functions Among Mucin type glycoproteins KDN group have strong Ca 2+ binding capacity. It may control Ca2+ and H+ causes in ovarian fluid. They facilitate movement of cells also protect the cells from proteolytic degradation and bacterial invasion. The anionic carbohydrate chain contribute to this type of resistance. Glycoproteins related to vitellogenesis Vitellogenin ,the protein responsible for vitellogenesis is synthesized in liver under hormonal .Then it is transported to blood and sequestred through receiptor mediated transport to oocytes. Vitellogenin have a 200KDa molecular weight .It is glycosylated and phosphorylated at the site of synthesis. When vitellogenin is incorporated to oocyte it is cleaved to lipovitellins and phosphovitins. Phosphovitins are phosphoylated and glycosylated .Phosphovitins are small proteins(2-30KDa). Lysosomal system is involved in sequestration and processing of fish vitellogenin to yolk.

Complex free sialoglycans are found in unfertilized mature eggs of 25 species of fishes including plecoglossus altivelis,Tribolodon haleonensis .These have free Nglycans which may be derived from vitellogenin. It is found that the enzyme Acid PNGase(protein N glycanase) participate in deglycosylation of vitellogenin during vitellogenesis. Structural glycoproteins This include several proteoglycans such as hyaluronate, chondriotin sulphate and heparan sulphate. Shark bones contain considerable amount of chondriotin sulphate which is isolated because of its high medicinal value. Hyaluronate act as an intracellular cementing substance present in the body of fishes and an important component of egg membrane. Collagen This occur in connective tissue. These help bind together the fibers, cells, and ground substance of connective tissue. They may also help components of the tissue bind to inorganic substances, such as calcium in bone. In fish skin and intramuscular connective tissue, type I and type V collagens have been identified as major and minor collagen respectively. In fish collagen the fibril diameter is shown to increase progressively during subsequent fish life. This increase of fibril diameter can be achieved by two well-known processes: accretion of new collagen molecules deposited by the fibroblasts and/or fusion of existing fibrils. Glycosylation is an important factor which involve in the regulation of the fibril diameter of collagen (Ibrahim et Harding, 1990. Other structural glycoproteins In addition to collagen many glycoproteins are found throughout matrices. They act as receptors on cell surfaces that bring other cells and proteins (collagen) together giving strength and support to a matrix Proteoglycan-linking glycoproteins cross links proteoglycan molecules and is involved in the formation of the ordered structure within cartilage tissue. In nerve tissue glycoproteins are abundant in gray matter and appear to be associated with synaptosomes, axons, and microsomes. Prothrombin, thrombin, and fibrinogen are all glycoproteins that play an intricate role in the blood clotting mechanism . Iron transport Glycoproteins This include transferrin and ceruloplasmin. Glycoproteins in ammonia excretion

Ammonia excretion from the gill in teleost fish is essential for nitrogen elimination. Although numerous physiological studies have measured ammonia excretion, the mechanism of ammonia movement through the membranes of gill epithelial cells is still unknown. Mammalian Rh glycoproteins are members of a family of proteins that mediate ammonia transport in bacteria, yeast, and plants. Out of glycoprotein homologs, fRhag, fRhbg, fRhcg1, and fRhcg2, of the pufferfish, Rh glycoprotein homologue fRhag was present in red blood cells and the hematological organs (spleen and kidney) in fish. All four pufferfish Rh glycoproteins are specifically localized in the gill and line the pillar cells, pavement cells, and the mitochondrion-rich cells.. The Rh glycoproteins were shown to have ammonia transport activity when expressed in Saccharomyces cerevisiae and Xenopus oocyte heterologous systems These results suggest that pufferfish Rh glycoproteins are involved in ammonia excretion from the gill. Thus glycoproteins are involved in ammonia excretion, and specifically in the fish gill, where passive diffusion through the plasma membrane is generally thought to be the mechanism of transport. REFERENCES 1.Jean montreuil, j. F. G. Vliegenthart, harry ,1997 - science, glycoproteinsII, Pages 143-165. 2. Jeremy m berg, john l tymoczko, and lubert stryer,johns hopkins university school of medicine. Carleton college .. Stryer biochemistry, 5th edition 3.David .H. Evans ,1998,2nd edition , The physiology of fishes ,CRC press LLC 4.http://www.cs.stedwards.edu/chem/Chemistry/CHEM43/CHEM43/Glycoproteins /Glycoproteins.HTML 5.http://www.science.nd.edu/chemistry/bretthauer.html 6.http://www.fasebj.org/cgi/content/full/21/4/1067#SEC3 7.http://www.wikipedia.org 8.Ivatt, Raymond J. The Biology of Glycoproteins. Plenum Press: New York, 1984. Kornfeld Rosaline, and Stuart Kornfeld. "Assembly of Asparagine-Linked Oligosaccharide." Annual Review of Biochemistry 54 (1985): 631-664 . 9.Ruddock & Molinari (2006) Journal of Cell Science 119, 4373-4380 Anne Dell, Howard R Morris: "Glycoprotein structure determination by mass spectrometry", Science 291(5512), 2351-2356 (2001), Review 10. Nakada, T., Westhoff, C. M., Kato, A., Hirose, S. Ammonia secretion from fish gill depends on a set of Rh glycoproteins.

11.Neuhaus henner ; Van der marel marian ; Caspari nancy ; Meyer wilfried ; enss marie-luise ; Steinhagen dieter biochemical and histochemical effects of perorally applied endotoxin on intestinal mucin glycoproteins of the common carp cyprinus carpio.