SYNTHESIS OF SYNTHESIS OF GLYCOPROTEINSGLYCOPROTEINS

Dr. Nasim APDr. Nasim AP

BIOCHEMISTRYBIOCHEMISTRY

GLYCOPROTEINSGLYCOPROTEINSIntroduction Introduction

GlycoproteinsGlycoproteins

Glycoproteins are proteins that Glycoproteins are proteins that contain oligosaccharide contain oligosaccharide (glycan) (glycan) chains covalently attached to their chains covalently attached to their polypeptide backbones.polypeptide backbones.

Almost all the Almost all the plasma proteins plasma proteins of of humans—except albumin—are humans—except albumin—are glycoproteins.glycoproteins.

Many Many proteins of cellular membranes proteins of cellular membranes contain substantial amounts of contain substantial amounts of carbohydrate.carbohydrate.

A number of the A number of the blood group blood group substances are glycoproteins.substances are glycoproteins.

Certain Certain hormones hormones (eg, chorionic (eg, chorionic gonadotropin) are glycoproteins.gonadotropin) are glycoproteins.

They differ from proteoglycans:They differ from proteoglycans:

1.1.Length of the chain is relatively short Length of the chain is relatively short (usually 2-10 sugar residues) very long in (usually 2-10 sugar residues) very long in GAGs.GAGs.

2.2.Do not have repeating disaccharide units.Do not have repeating disaccharide units.

3.3.They are branched.They are branched.

4.4.May or may not be negatively charged.May or may not be negatively charged.

Glycoproteins occur in most organisms, Glycoproteins occur in most organisms, from bacteria to humans. from bacteria to humans.

Their carbohydrate content ranges from Their carbohydrate content ranges from 1% to over 85% by weight.1% to over 85% by weight.

They perform the following functions:They perform the following functions:

FunctionFunction ExampleExampleStructural roleStructural role CollagenCollagenTransport roleTransport role TransferrinTransferrinImmunologic roleImmunologic role ImmunoglobulinsImmunoglobulinsCell-to-cell Cell-to-cell communicationcommunication

SelectinsSelectinsProteins in Proteins in fertilizationfertilizationCell adhesion Cell adhesion moleculesmolecules

Cell signallingCell signalling Many receptorsMany receptorsClottingClotting Plasma proteinsPlasma proteins

LipoproteinsLipoproteinsLubricationLubrication MucinsMucins

Hormones HCG, TSHHormones HCG, TSH

Anti freeze cold water fishAnti freeze cold water fish

Some of the processes involving glycoproteins:

inflammation blood clotting peptic ulcers AIDS (HIV)

influenza fertilization cancer cystic fibrosis arthritis

The predominant sugars found in The predominant sugars found in glycoproteins are: glycoproteins are:

glucose (Glc), glucose (Glc), galactose (Gal), galactose (Gal), mannose (Man), mannose (Man), fucose (Fuc), fucose (Fuc),

NN-acetylgalactosamine (GalNAc), -acetylgalactosamine (GalNAc), NN-acetylglucosamine (GlcNAc) -acetylglucosamine (GlcNAc) and and NN-acetylneuraminic acid (NANA). -acetylneuraminic acid (NANA).

NANA is also called sialic acid.NANA is also called sialic acid.

The distinction between The distinction between proteoglycans proteoglycans and and glycoproteinsglycoproteins resides in the level resides in the level and types of carbohydrate modification.and types of carbohydrate modification.

Proteoglycans also contain the sugar Proteoglycans also contain the sugar glucuronic acid glucuronic acid (GlcA). (GlcA).

The carbohydrate modifications found in The carbohydrate modifications found in glycoproteins are rarely as complex as glycoproteins are rarely as complex as that of proteoglycans. that of proteoglycans.

The carbohydrates of glycoproteins are The carbohydrates of glycoproteins are linked to the protein component through linked to the protein component through either either OO-glycosidic-glycosidic or or NN-glycosidic-glycosidic bonds.bonds.

The The NN-glycosidic -glycosidic linkage is through the linkage is through the amide group of asparagine (Asn, N). amide group of asparagine (Asn, N).

The The OO-glycosidic-glycosidic linkage is to the linkage is to the hydroxyl of serine (Ser, S), threonine hydroxyl of serine (Ser, S), threonine (Thr, T) or hydroxylysine (hLys). (Thr, T) or hydroxylysine (hLys).

OO-linked sugars:-linked sugars: May be membrane glycoprotein May be membrane glycoprotein

componentscomponents Or extracellular glycoproteins. Or extracellular glycoproteins.

When attached to Ser or Thr, the sugar When attached to Ser or Thr, the sugar of of OO-linked glycoproteins is most often -linked glycoproteins is most often GalNAc.GalNAc.

NN-linked sugars:-linked sugars: The predominant The predominant carbohydrate attachment in glycoproteins carbohydrate attachment in glycoproteins of mammalian cells is via of mammalian cells is via NN-glycosidic -glycosidic linkage. linkage.

They are of two types:They are of two types:

1.1.Complex oligosaccharidesComplex oligosaccharides

2.2.High mannose oligosaccharidesHigh mannose oligosaccharides

Their core pentasaccharide is the same.Their core pentasaccharide is the same.

In the complex form additional sugar In the complex form additional sugar residues are present:residues are present:

NN-acetylglucosamine (GlcNAc) -acetylglucosamine (GlcNAc)

and and NN-acetylneuraminic acid (NANA). -acetylneuraminic acid (NANA).

FucoseFucose

High mannose contain only mannose High mannose contain only mannose residuesresidues

Most proteins that are secreted, or bound Most proteins that are secreted, or bound to the plasma membrane, are modified to the plasma membrane, are modified by carbohydrate attachment. by carbohydrate attachment.

The part that is modified, in plasma The part that is modified, in plasma membrane-bound proteins, is the membrane-bound proteins, is the extracellular portion of the protein. extracellular portion of the protein.

Intracellular proteins are less frequently Intracellular proteins are less frequently modified by carbohydrate attachment. modified by carbohydrate attachment. However, the attachment of carbohydrate However, the attachment of carbohydrate to intracellular proteins confers unique to intracellular proteins confers unique functional activities on these proteinsfunctional activities on these proteins

I – cell diseaseI – cell disease Cancers Cancers Metastasis Metastasis

Structure of GlycoproteinStructure of Glycoprotein

The oligosaccharide components of The oligosaccharide components of glycoproteins glycoproteins is branched is branched heteropolymers heteropolymers composed of composed of D-D-hexoseshexoses, with the addition in some , with the addition in some cases of cases of neuraminic acidneuraminic acid, and of , and of L-L-fucosefucose (6-deoxyhexose) (6-deoxyhexose)

N- and O-linked N- and O-linked oligosaccharidesoligosaccharides

O-Linked oligosaccharidesO-Linked oligosaccharides: found in : found in extracellular glycoproteins or as extracellular glycoproteins or as membrane glycoprotein componentsmembrane glycoprotein components. .

For example, O-linked oligosaccharides For example, O-linked oligosaccharides on the surface of RBCs help provide the on the surface of RBCs help provide the ABO blood ABO blood group determinantsgroup determinants

N-linked oligosaccharidesN-linked oligosaccharides: : two broad classes: two broad classes: 1. 1. complex oligosaccharides complex oligosaccharides 2. 2. high-mannose oligosaccharideshigh-mannose oligosaccharides..

Both contain the Both contain the same core same core pentasaccharide pentasaccharide but the but the complex complex oligosaccharides oligosaccharides contain additional contain additional sugars, sugars, for example; for example; N-acetylglucosamine (GlcNAc), N-acetylglucosamine (GlcNAc),

L-fucose (Fuc), and N-acetylneuraminic acid L-fucose (Fuc), and N-acetylneuraminic acid (NANA)(NANA)

The The high-mannosehigh-mannose oligosaccharides oligosaccharides contain contain mannosemannose

Synthesis of O-linked Synthesis of O-linked glycosidesglycosides

The protein to which the oligosaccharides The protein to which the oligosaccharides are to be attached is synthesized on the are to be attached is synthesized on the RER, and extruded into its lumen. RER, and extruded into its lumen.

Glycosylation begins with the transfer of Glycosylation begins with the transfer of an N-acetylgalactosamine an N-acetylgalactosamine (from UDP-(from UDP-N-acetylgalactosamine) onto a specific N-acetylgalactosamine) onto a specific seryl or threonyl R-groupseryl or threonyl R-group

The The glycosyl-transferasesglycosyl-transferases responsible responsible for the stepwise synthesis of the for the stepwise synthesis of the oligosaccharides are bound to the oligosaccharides are bound to the membranes of the Golgi apparatus.membranes of the Golgi apparatus.

Synthesis of the N-linked Synthesis of the N-linked glycosidesglycosides

First, as with the O-linked glycosides, First, as with the O-linked glycosides, protein is synthesized on the RER and protein is synthesized on the RER and enters its lumen.enters its lumen.

The protein itself does not become The protein itself does not become glycosylated with individual sugars at this glycosylated with individual sugars at this stage of glycoprotein synthesis, but stage of glycoprotein synthesis, but rather a rather a lipid-linked oligosaccharide lipid-linked oligosaccharide is is first constructedfirst constructed

This consists of This consists of dolicholdolichol (an ER (an ER membrane lipid 80 to 100 carbons long) membrane lipid 80 to 100 carbons long) attached through a pyrophosphate attached through a pyrophosphate linkage to an oligosaccharide containing linkage to an oligosaccharide containing N-acetylglucosamineN-acetylglucosamine, , mannose,mannose, and and glucoseglucose..

The sugars to be added to the dolichol by The sugars to be added to the dolichol by the membrane-bound the membrane-bound glycosyltransferases are glycosyltransferases are first N-first N-acetylglucosamine, followed by acetylglucosamine, followed by mannose and glucose mannose and glucose

The oligosaccharide is transferred from The oligosaccharide is transferred from the dolichol to an the dolichol to an asparagine side asparagine side group group of the protein by a protein-of the protein by a protein-oligosaccharide oligosaccharide transferase transferase present in present in the ER.the ER.

Final processing of N-Final processing of N-linked oligosaccharideslinked oligosaccharides

After incorporation into the protein, the N-After incorporation into the protein, the N-linked oligosaccharide linked oligosaccharide is processed by is processed by the removal of specific mannosyl and the removal of specific mannosyl and glucosyl residues glucosyl residues as the glycoprotein as the glycoprotein moves through the ER.moves through the ER.

Finally, the oligosaccharide chains are Finally, the oligosaccharide chains are completed in the Golgi by addition of a completed in the Golgi by addition of a variety of sugars (for example, N-variety of sugars (for example, N-acetylglucosamine, N-acetylglucosamine, N-acetylgalactosamine, and additional acetylgalactosamine, and additional mannoses, and then fucose or NANA as mannoses, and then fucose or NANA as terminal groupsterminal groups

The ultimate fate of N-linked The ultimate fate of N-linked glycoproteins is the same as that of the glycoproteins is the same as that of the O-linked, for example, they can be O-linked, for example, they can be released by the cell, or become part of a released by the cell, or become part of a cell membrane. In addition N-linked cell membrane. In addition N-linked glycoproteins can be translocated to the glycoproteins can be translocated to the lysosomes.lysosomes.

Enzymes destined for Enzymes destined for lysosomeslysosomes

N-linked glycoproteins being processed N-linked glycoproteins being processed through the Golgi can be through the Golgi can be phosphorylated phosphorylated at one or more at one or more specific specific mannosyl residuesmannosyl residues. .

Mannose 6-P receptorsMannose 6-P receptors, located in the , located in the Golgi apparatus, bind the mannose 6-P Golgi apparatus, bind the mannose 6-P residues of these targeted enzymes, residues of these targeted enzymes, resulting in their translocation to the resulting in their translocation to the lysosomeslysosomes

II-cell disease: -cell disease: rare syndromerare syndrome acid hydrolase enzymes normally found in acid hydrolase enzymes normally found in

lysosomes are absent, lysosomes are absent, results in an accumulation of substrates results in an accumulation of substrates

normally degraded by lysosomal enzymes normally degraded by lysosomal enzymes within these vesicles.within these vesicles.

Individuals with I-cell disease:Individuals with I-cell disease: are lacking the enzymic ability to are lacking the enzymic ability to

phosphorylatephosphorylate the mannose residues of the mannose residues of potential lysosomal enzymes, causing an potential lysosomal enzymes, causing an incorrect targeting incorrect targeting of these proteins to of these proteins to extracellular sites, rather than lysosomal extracellular sites, rather than lysosomal vesiclesvesicles

I-cell disease is characterized by;I-cell disease is characterized by; skeletal abnormalitiesskeletal abnormalities restricted joint movementrestricted joint movement coarse facial featurescoarse facial features severe psychomotor impairmentsevere psychomotor impairment. . Death usually occurs by eight years of age. Death usually occurs by eight years of age.

[Note: I-cell disease is considered to be a [Note: I-cell disease is considered to be a glycoprotein storage disease glycoprotein storage disease

Glyco-proteins Glyco-proteins