glycoproteins in fish

8/3/2019 glycoproteins in fish

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AN

ASSIGNMENT

ON

GLYCOPROTEINS IN FISH

( COURSE FNB 506)

TINCY VARGHESE

FNB 44


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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 muchsmaller 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 othervertebrates 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 glycoproteinshave 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 achievedthrough 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: N-

glycosylation and O-glycosylation

O-glycosylation

In O-glycosylation, the addition of sugar chains can happen on the hydroxyloxygen 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. Theglycan chain usually contain an N-acetylgalactosamine which is attached through a

glycosidic bond to the O-terminus of either threonine or serine.. N-

acetylgalactosamine is simply a galactose molecule with an amine group covalentlybonded to the second carbon. This amine group is bonded to a carboxyl group. N-

acetylgalactosamine attaches to the carboxyl group of the amino acid through the

hydroxyl group of its anomeric carbon. Another type of O-linked glycan consists ofa 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


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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 amidenitrogen 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 anamine group. This amine group, in turn, is bonded to a hydroxyl group. The N-

acetylglucosamine 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 ofGlycosylationUsually 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 processbegins. 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 ERlumen. 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 ofthe 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 theseproteins 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 glycosyltransferasespresent 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.


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Regulation/Control of glycoprotein synthesis

Addition of any other sugar can be in any possible combination, accordingto 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 thefirst 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 containedin 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 thisand 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 protectiveagent

Mucins

Transport molecule Transferrin, ceruloplasmin

Immunologic molecule Immunoglobins, histocompatibility antigens

HormoneHuman chorionic gonadotropin (HCG), thyroid-stimulating hormone (TSH)

EnzymesGlycoprotein enzymes are of three types. These areoxidoreductases, transferases, and hydrolases

eg, alkalinephosphatase

Cell attachment-recognition site

Various proteins involved in cell-cell (eg, sperm-oocyte),virus-cell, bacterium-cell, and hormone cell interactions

Antifreeze Certain plasma proteins of coldwater fish

Interact with specific

carbohydrates

Lectins, selectins(cell adhesion lectins), antibodies

Receptor Various proteins involved in hormone and drug action

Affect folding of certainproteins

Calnexin, calreticulin

Regulation development Notch and its analogs, key proteins in development

Hemostasis(and

thrombosis)

Specific glycoproteins on the surface membranes of

platelets
http://en.wikipedia.org/wiki/Protein_foldinghttp://en.wikipedia.org/wiki/Collagenhttp://en.wikipedia.org/wiki/Mucinhttp://en.wikipedia.org/wiki/Mucinhttp://en.wikipedia.org/wiki/Transferrinhttp://en.wikipedia.org/wiki/Ceruloplasminhttp://en.wikipedia.org/wiki/Antibodyhttp://en.wikipedia.org/wiki/Histocompatibilityhttp://en.wikipedia.org/wiki/Human_chorionic_gonadotropinhttp://en.wikipedia.org/wiki/Phosphatasehttp://en.wikipedia.org/wiki/Spermhttp://en.wikipedia.org/wiki/Oocytehttp://en.wikipedia.org/wiki/Antifreezehttp://en.wikipedia.org/wiki/Lectinhttp://en.wikipedia.org/wiki/Lectinhttp://en.wikipedia.org/wiki/Selectinhttp://en.wikipedia.org/wiki/Selectinhttp://en.wikipedia.org/wiki/Receptor_(biochemistry)http://en.wikipedia.org/wiki/Calnexinhttp://en.wikipedia.org/wiki/Calnexinhttp://en.wikipedia.org/wiki/Calreticulinhttp://en.wikipedia.org/wiki/Notch_signalinghttp://en.wikipedia.org/wiki/Hemostasishttp://en.wikipedia.org/wiki/Thrombosishttp://en.wikipedia.org/wiki/Platelethttp://en.wikipedia.org/wiki/Protein_foldinghttp://en.wikipedia.org/wiki/Collagenhttp://en.wikipedia.org/wiki/Mucinhttp://en.wikipedia.org/wiki/Transferrinhttp://en.wikipedia.org/wiki/Ceruloplasminhttp://en.wikipedia.org/wiki/Antibodyhttp://en.wikipedia.org/wiki/Histocompatibilityhttp://en.wikipedia.org/wiki/Human_chorionic_gonadotropinhttp://en.wikipedia.org/wiki/Phosphatasehttp://en.wikipedia.org/wiki/Spermhttp://en.wikipedia.org/wiki/Oocytehttp://en.wikipedia.org/wiki/Antifreezehttp://en.wikipedia.org/wiki/Lectinhttp://en.wikipedia.org/wiki/Selectinhttp://en.wikipedia.org/wiki/Receptor_(biochemistry)http://en.wikipedia.org/wiki/Calnexinhttp://en.wikipedia.org/wiki/Calreticulinhttp://en.wikipedia.org/wiki/Notch_signalinghttp://en.wikipedia.org/wiki/Hemostasishttp://en.wikipedia.org/wiki/Thrombosishttp://en.wikipedia.org/wiki/Platelet


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Egg and spermatozoal

glycoproteins

To increase the chance of fertilization and avoid

polyspermy

Glycoproteins associated

with vision

.

Analysis

Some important methods used to study glycoproteins

Method Use

Periodic acid-Schiff stainDetects glycoproteins as pink bands after

electrophoretic separation.

Incubation of cultured cells

with glycoproteins asradioactive decay bands

Leads to detection of a radioactive sugar after

electrophoretic separation.

Treatment with appropriateendo- or exoglycosidase or

phospholipases

Resultant shifts in electrophoretic migration help

distinguish among proteins with N-glycan, O-glycan,or GPI linkages and also between high mannose and

complex N-glycans.

Agarose-lectin columnchromatography

To purify glycoproteins or glycopeptides that bind theparticular lectin used.

Compositional analysisfollowing acidhydrolysis

Identifies sugars that the glycoprotein contains andtheir stoichiometry.

Mass spectrometryProvides information on molecular mass,composition, sequence, and sometimes branching of a

glycan chain.

NMR spectroscopyTo identify specific sugars, their sequence, linkages,and the anomeric nature of glycosidic chain.

Dual Polarisation

Interferometry

Measures the mechanisms underlying the biomolecular interactions, including reaction rates,

affinities and associated conformational changes.

Methylation (linkage) analysis To determine linkage between sugars.

Amino acid or cDNA

sequencingDetermination of amino acid sequence.
http://en.wikipedia.org/wiki/Periodic_acid-Schiff_stainhttp://en.wikipedia.org/wiki/Electrophoresishttp://en.wikipedia.org/wiki/Radioactive_decayhttp://en.wikipedia.org/wiki/Endoglycosidasehttp://en.wikipedia.org/wiki/Exoglycosidasehttp://en.wikipedia.org/wiki/Exoglycosidasehttp://en.wikipedia.org/wiki/Phospholipasehttp://en.wikipedia.org/wiki/Mannosehttp://en.wikipedia.org/wiki/Agarosehttp://en.wikipedia.org/wiki/Lectinhttp://en.wikipedia.org/wiki/Column_chromatographyhttp://en.wikipedia.org/wiki/Column_chromatographyhttp://en.wikipedia.org/wiki/Hydrolysishttp://en.wikipedia.org/wiki/Hydrolysishttp://en.wikipedia.org/wiki/Mass_spectrometryhttp://en.wikipedia.org/wiki/Molecular_masshttp://en.wikipedia.org/wiki/Molecular_masshttp://en.wikipedia.org/wiki/Molecular_masshttp://en.wikipedia.org/wiki/NMR_spectroscopyhttp://en.wikipedia.org/wiki/Dual_Polarisation_Interferometryhttp://en.wikipedia.org/wiki/Dual_Polarisation_Interferometryhttp://en.wikipedia.org/wiki/Conformational_changehttp://en.wikipedia.org/wiki/Methylationhttp://en.wikipedia.org/wiki/Amino_acidhttp://en.wikipedia.org/wiki/Complementary_DNAhttp://en.wikipedia.org/wiki/Periodic_acid-Schiff_stainhttp://en.wikipedia.org/wiki/Electrophoresishttp://en.wikipedia.org/wiki/Radioactive_decayhttp://en.wikipedia.org/wiki/Endoglycosidasehttp://en.wikipedia.org/wiki/Exoglycosidasehttp://en.wikipedia.org/wiki/Phospholipasehttp://en.wikipedia.org/wiki/Mannosehttp://en.wikipedia.org/wiki/Agarosehttp://en.wikipedia.org/wiki/Lectinhttp://en.wikipedia.org/wiki/Column_chromatographyhttp://en.wikipedia.org/wiki/Column_chromatographyhttp://en.wikipedia.org/wiki/Hydrolysishttp://en.wikipedia.org/wiki/Mass_spectrometryhttp://en.wikipedia.org/wiki/Molecular_masshttp://en.wikipedia.org/wiki/NMR_spectroscopyhttp://en.wikipedia.org/wiki/Dual_Polarisation_Interferometryhttp://en.wikipedia.org/wiki/Dual_Polarisation_Interferometryhttp://en.wikipedia.org/wiki/Conformational_changehttp://en.wikipedia.org/wiki/Methylationhttp://en.wikipedia.org/wiki/Amino_acidhttp://en.wikipedia.org/wiki/Complementary_DNA


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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

hormonesare necessary for proper folding, assembly, secretion, metabolic clearance and

biological activity. The free subunit, which is shown to have a physiological

function, is maintainedin the uncombined form due to its glycan structures. TheN-glycansof 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 regulatedby a variety of physiological and

pathological conditions, leading to subtle alterations in their bioactivities. Recentstudies 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 ACTHand MSH cells in the pituitary .N-POMC, ACTH, b-LPH -are produced in the post-

translational 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 chainsand 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 separategenes 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
http://en.wikipedia.org/wiki/Thyroid-stimulating_hormonehttp://en.wikipedia.org/wiki/Thyroid-stimulating_hormone


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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


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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 extensivesulphide linkages.

The task of differenciating the disulfide bonds is difficult because of

clustering of haif-cystine residues and the lack of enzymatic cleavage points withinthese 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 withgalactose-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-cystineresidues 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 COOH-terminus

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 contactsurfaces 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 properdisulphide bond formation during folding of the nascent peptide chains and prevents

proteolysis and aggregation of subunits.I n general they indicate that removal of N-

linked carbohydrates.It does not interfere with receptor binding but markedly

reduces biological responses such as stimulation of the adenylate cyclase enzyme


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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 biologicalactivity

7.Glycans of hormone units: All -subunits have carbohydrate attached at Asn-56and -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 humanand 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 residuesattached 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.


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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.


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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 Gproteins, 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 Nglycosylation 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 ofcarbohydrate 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 ofhormones 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 isfound 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 hormone-receptor 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 baseswill 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 Mathews808-809

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 typeof glycoproteins.They bind to small ice crystals to inhibit growth and


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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 otherdissolved 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 asthermal 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 flounderand shorthorn sculpin. They are the best

documented AFP because it was the first to have its 3D structure determined. TypeI 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 righteyeflounders. 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.
http://en.wikipedia.org/wiki/Acclimatizationhttp://en.wikipedia.org/wiki/Osmotic_pressurehttp://en.wikipedia.org/wiki/Osmotic_pressurehttp://en.wikipedia.org/wiki/Glycoproteinshttp://en.wikipedia.org/wiki/Winter_flounderhttp://en.wikipedia.org/wiki/Sculpinhttp://en.wikipedia.org/wiki/Sea_ravenhttp://en.wikipedia.org/wiki/Smelthttp://en.wikipedia.org/wiki/Smelthttp://en.wikipedia.org/wiki/Herringhttp://en.wikipedia.org/wiki/File:AFPnew.svghttp://en.wikipedia.org/wiki/Acclimatizationhttp://en.wikipedia.org/wiki/Osmotic_pressurehttp://en.wikipedia.org/wiki/Osmotic_pressurehttp://en.wikipedia.org/wiki/Glycoproteinshttp://en.wikipedia.org/wiki/Winter_flounderhttp://en.wikipedia.org/wiki/Sculpinhttp://en.wikipedia.org/wiki/Sea_ravenhttp://en.wikipedia.org/wiki/Smelthttp://en.wikipedia.org/wiki/Herring


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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 andconsists of a 4 helix bundle. Its only post-translational modification is a

pyroglutamate residue, a cyclized glutamine residue at its N-terminal. Scientists atthe 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 disulfidebridges; and type III is intermediate in size (Mr 6-7 K) with no distinguishing

features of secondary structure or amino acid composition. Despite their markedstructural differences, all four antifreeze types appear to function in thesame 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 thehelix macrodipole and the dipoles on the water moleculesin the icelattice. Binding is stabilized by y hydrogen bonding, and the amphiphilic

character of the helix results in the hydrophobic phase of the helix beingexposed to

the solvent. When the solution temperature is lowered further, ice crystal growthoccurs primarily on the uncoated, unordered basal plane resulting in bipyramidal-

shaped crystals. The structural features of type I AFP that could contribute to this

mechanism of action are reviewed. Current challenges lie in solving the otherantifreeze structures and interpretingthem in light of what appears to be a common

mechanism of action

Glycoprotein receiptors.
http://en.wikipedia.org/wiki/Eelpouthttp://en.wikipedia.org/wiki/Eelpout


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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 oftentransmembrane 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 1,4,5-trisphosphate (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 onfunctional 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 water-holding capacity and also make them resistant to proteolysis by digestive enzymes.

They are high molecular weight polymers and found on internal epithelialsurfaces. 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 foundin 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 isessential 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 codserum 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 wascomposed 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 wasshown 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 areexpressed on the surface of cells and interact with T cells as part of the adaptive


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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 thesesites 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 thosecell'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 cell-substratum 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 andadhered 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. Corticalvesicles 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 iscompletely 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 rainbowtrout. 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 singlenonreducing terminal residue in glycoproteins. i.e. polysialic acid residue is not

common in glycoproteins.

Aminoacid sequence of L-PSGP of rainbow trout


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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 asKDN(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. ApoKDNGlycoprotein 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 facilitatemovement 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 throughreceiptor 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.


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Complex free sialoglycans are found in unfertilized mature eggs of 25 species of

fishes including plecoglossus altivelis,Tribolodon haleonensis .These have free N-

glycans which may be derived from vitellogenin. It is found that the enzyme AcidPNGase(protein N glycanase) participate in deglycosylation of vitellogenin during

vitellogenesis.

Structural glycoproteins

This include several proteoglycans such as hyaluronate, chondriotinsulphate 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 bodyof 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 thetissue 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. Thisincrease 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 thefibril 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


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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 gillepithelial cells is still unknown. Mammalian Rh glycoproteins are members of a

family of proteinsthat mediate ammonia transport in bacteria, yeast, and plants.

Out of glycoprotein homologs, fRhag, fRhbg, fRhcg1, and fRhcg2, of the

pufferfish, Rh glycoprotein homologue fRhag waspresent in red blood cells and thehematological organs (spleen and kidney) in fish. All four pufferfish Rh

glycoproteins arespecifically localized in the gill and line the pillar cells,pavement

cells, and the mitochondrion-rich cells.. The Rh glycoproteins were shown to haveammonia transport activity when expressed in Saccharomyces cerevisiae and

Xenopus oocyte heterologous systems These results suggest that pufferfish Rh

glycoproteins are involved in ammonia excretionfrom the gill. Thus glycoproteinsare involved in ammonia excretion, and specificallyin the fish gill, where passive

diffusion through the plasmamembrane 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-LinkedOligosaccharide." Annual Review of Biochemistry 54 (1985): 631-664

.

9.Ruddock & Molinari (2006) Journal of Cell Science 119, 4373-4380Anne 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 secretionfrom fishgill depends on a set of Rh glycoproteins.


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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 carpcyprinus carpio.

glycoproteins in fish

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