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PRESENTOR: SARANYA.S, JIPMER.

MODERATOR:Dr. NANDEESHA. H, JIPMER.

Protein glycosylation and its associated disorders

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2Content :Introduction Protein Glycosylation Synthesis Biological FunctionDisorders Method Of AnalysisInhibitors Summary

OUTLINE OF PROTEINS SYNTHESIS

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WHAT IS POST TRANSLATIONAL MODIFICATION ???

Post translational modification (PTM) is the chemical modification of a protein after its translation.

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Why PTM is necessary???Stability of protein

Biochemical activity (activity regulation)

Protein targeting (protein localization)

Protein signaling (protein - protein interaction, cascade amplification)

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Post-translational modification

Modification Involving Peptide Bonds

Modification of amino acids

Subunit aggregation

Protein folding and chaperones

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Protein glycosylation : The attachment of sugar moieties to proteins to form glycoprotein.

Glycosylation is a critical function of the biosynthetic-secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus.

Approximately half of all proteins typically expressed in a cell undergo this modification.

Secreted proteins, surface receptors and ligands and organelle-resident proteins.

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Function:In the ER, it is used to monitor the status of protein folding.Facilitate their delivery to the correct destination.It act as ligands for receptors on the cell surface to mediate cell attachment or stimulate signal transduction pathways.It can affect protein-protein interactions by either facilitating or preventing proteins from binding to cognate interaction domains. Because they are hydrophilic, they can also alter the solubility of a protein.8

Glycoprotein Proteoglycan

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Glycoprotein differ from Proteoglycan:Protein with oligosaccharide chains (glycans).Carbohydrates chain is relatively short. The chains are often branched instead of linearN-linked or O-linked saccharides. Found on cell surfaces

Consists of a core protein with glycosaminoglycan (GAG) chain(s).The chains are long, linear and are negatively charged due to the occurrence of sulfate and uronic acid groups.Categorized depending upon the nature of their GAG chains.Found mainly in connective tissues

GlycoproteinProteoglycan10

Types of glycosylation:N Linked

O Linked

Glypiation

C Linked

PhosphoglycosylationGlycan bind to the amino group of asparagine in the ER

Monosaccharides bind to the hydroxyl group of serine or threonine in the ER, Golgi, cystosol and nucleus

Glycan core links a phospholipid and a protein

Mannose binds to the indole ring of tryptophan

Glycan binds to serine via phosphodiester bond

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Where this Protein Glycosylation occurs :

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Begins in ER:

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15Directing proteins with the signals to the ER :

N linked glycosylation :Synthesis of dolichol-linked precursor oligosaccharide (ER)

En bloc transfer of precursor oligosaccharide to protein (ER)

Processing of the oligosaccharide (Golgi)

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Dolichol phosphateIt is an isoprenoid compound (90-100 carbons total) made fromdolichol by phosphorylation catalyzed by a kinase that usesCTPas the energy and phosphate source.

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18Synthesis of precursor oligosaccharide and en bloc transfer:

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Calnexin cycle:

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21Transport vesicles are key players in intracellular protein traffic :

22Glycan Maturation In The Golgi

All N-linked glycoproteins have the same precursor glycan structureTrimming and adding sugars to diversify the glycans on individual glycoproteins.This maturation pathway to generate diverse oligosaccharides is highly ordered, such that each step is dependent upon the previous step.The Golgi segregates specific enzymes into different cisternae to facilitate this step-wise process

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Complex oligosaccharides contain multiple sugar typesHigh-mannose oligosaccharides multiple mannose residuesHybrid branches of both high mannose and complex common core stage become resistant to glycan removal by endoglycosidase H (endo H)

25O linked glycosylation :This type of glycosylation is essential in the biosynthesis of mucins.

O-glycosylation is also critical for the formation of proteoglycan core proteins that are used to make extracellular matrix components.

Antibodies are often heavily O-glycosylated.

O-glycosylation occurs post-translationally on serine and threonine side chains in the Golgi apparatus.

26Continues. . .Involves glycosyltransferase acting in stepwise manner. Each transferase is specifically for particular type of linkage.

The enzyme involved are located in various compartment of golgi apparatus.

Each glycosylation involves the appropriate nucleotide sugar.

Dolichol p p oligosaccharide is not involved and the reaction is not inhibited by tunicamycin.

27Mechanism:Proteins trafficked into the Golgi are most often O-glycosylated by N-acetylgalactosamine (GalNAc) transferase.

Transfers a single GalNAc residue to the -OH group of serine or threonine.

Some proteins are O-glycosylated with GlcNAc, fucose, xylose, galactose or mannose, depending on the cell and species.

Sugar nucleotides are used as monosaccharide donors for O-glycosylation.

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29Glypiation :The covalent attachment of a glycosylphosphatidylinositol (GPI) anchor is a common post-translational modification that localizes proteins to cell membranes.

GPI anchors consist of :Phosphoethanolamine linker that binds to the C-terminus of target proteinsGlycan core structurePhospholipid tail that anchors the structure in membrane

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31Some GPI linked proteins :Acetylcholinesterase (red cell membrane)

Alkaline phosphatase (intestinal, placental)

Decay accelerating factors (red cell membrane)

5 Nucleotidase (T lymphocyte, other cells)

Thy 1 antigen (brain, T lymphocyte)

32Post-Glycosylation ModificationsSulfation at Man and GlcNAc residues in the production of glycosaminoglycans (GAGs), which are components of proteoglycans in the extracellular matrix.

Acetylation of sialic acid to facilitate protein-protein interactions.

Phosphorylation, such as with Man residues on precursor lysosomal proteins (mannose 6-phosphate) to ensure trafficking to lysosomes by binding to mannose 6-phosphate receptor (M6PR) in the Golgi.

33Glycoproteins are involved in many biological process . . .

34Fertilization

Zona pellucida: ZP 1-3

ZP 3 O linked glycoprotein act as receptor for sperm.

Sperm surface contain galactosy transferase.

Induce the acrosomal reaction.

35Erythropoietin is a vital hormone :Secreated by kidney and stimulate production of RBC.165 amino acid, N glycosylated to 3 asp residue & O glycosylated on serine residue.Mature EPO is 40% carbohydrate by weight and enhance stability.Unglycosylated protein has only about 10% of bioactivity.

36Blood groups are based on protein glycosylation pattern :Common oligosaccharide : O antigen.

A antigen contain extra N acetylgalactosamine by type A transferase.

B antigen contain galactose by type B transferase.

37Lectin ligand interactions in lymphocyte movement :

38Some factors affecting the glycoprotein processing enzymes :Factor Comment Previous enzymeCertain glycosyltransferase act only on oligosaccharide chain if it has been acted upon by another processing enzyme.Development The cellular profile of processing enzyme may change during development of their gene turned on or off.Intracellular locationFor eg, if an enzyme inserted in to the membrane of ER it may never encounter golgi located processing enzymes.Protein conformationDifferences in conformation of different protein may facilitate or hinder access of processing enzyme to identical oligosaccharide chain.Species Same cells eg: fibroblast from different species may exhibit different pattern of processing enzymes.Cancer Cancer cell may exhibit processing enzymes different from those of corresponding normal cell.

39Disorders Associated With Impaired Glycosylation . . .

40Abnormalities in biosynthesis of glycoprotein Disease Cancer Congenital disorders of glycosylationCongenital dyserythropoetic anemia type II Leukocyte adhesion deficiency type IIParoxysmal nocturnal hemoglobinuria (PNH)I cell disease

41Cancer :Loss of normal topology and polarisation of epthelial tissue in cancer result in mucins secretion in to bloodstream.Tumor cells invading the tissues and bloodstream also present such mucins on their cell surface.

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43Congenital disorders of glycosylation :

Autosomal recessive disorder.Generally affect the central nervous system.Result in psychomotor retardation.Type I disorder mutation in gene coding for phosphomannomutase 2 ( involved in synthesis of dolichol P P oligosaccharide)Type II disorder mutation in gene coding for GlcNAc transferase 2 ( involved in process of N glycan chain.Atleast 15 distinct disorders have been recognised.

44Congenital Dyserythropoietic Anemia Type II :Maturation ofN-glycans from oligomannose to complex-type structures- essential role in cell adhesion and recognition events in metazoan organisms.-glucosidases and -mannosidases in the endoplasmic reticulum (ER) and Golgi complex to result in the Man3GlcNAc2core structure necessary for conversion to complex-typeN-glycans

45Leukocyte adhesion deficiency type IIRareautosomalrecessivedisorder characterized byimmunodeficiency resulting in recurrentinfections.Recurrentbacterialinfections, defects inneutrophiladhesion.Resulting from a general deficiency of fucose possibily due to defect in transformation of GDP mannose in to GDP fucose.

46Paroxysmal nocturnal hemoglobinuria (PNH)Acquired mutations in the PIG A gene of certain hematopoietic cellsDefective synthesis of the GlcNH2 PI linkage of GPI anchorsDecreased amounts in the RBC membrane of GPI anchored proteins, with decay accelerating factor and CD59 being special importanceCertain components of the complement system are not opposed by DAF and CD59, resulting in complement mediated lysis of red cell.

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48I cell diseasePart of thelysosomal storage diseaseDefectivephosphotransferase (an enzyme of theGolgi apparatus)The proteins are instead excreted outside the cell -- the default pathway for proteins moving through the Golgi apparatus.Lysosomes cannot function without these proteinsA buildup of these substances occurs within lysosomes because they cannot be degraded, resulting in the characteristic "I cells," or "inclusion cells."

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50Other disorders related to glycosylation:

51Analysis Of Glycoprotein :

Mass Spectrometry.

Glycoprotein Gel Stain.

X Ray Crystallography.

NMR Spectroscopy.

52Inhibitors of enzyme involved in N Glycosylation Inhibitor Site of actionTunicamycin Inhibits GlcNAc-P transferase, the enzyme catalysing addition of GlcNAc to dolichol-P.Deoxynojirimycin Inhibitor of glucosidases I and IISwainsonine Inhibitor of mannosidase II

53Summary :Glycosylation is one of the most important posttranslational modification of proteins.

There are various types of carbohydrateprotein glycosidic linkage and a great variety of structures of proteinlinked oligosaccharides (glycans).

At least certain of the oligosaccharide chain of glycoprotein encode biological information.

Glycosidases hydrolyze specific linkages in oligosaccharides and used to explore both the structural and functions of glycoprotein.

Lectins are carbohydrate binding proteins involved in cell adhesion and many other biological process.

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The major class of glycoproteis are O linked, N linked and GPI linked.

The endoplasmic reticulum and golgi apparatus plays a major role in glycosylation reactions involved in the biosynthesis of glycoproteins.

The oligosaccharide chain of O linked glycoproteins are synthesized in stepwise addition of sugars donated by nucleotide sugars.

The synthesis of N linked glycoproteins involves specific dolichol p p oligosaccharide and various enzymes. It can synthesis complex, hybrid or high mannose type.

55Continues . . .Alterations of glycosylation in many diseases are used for diagnostic and prognostic purposes.

Geneticallybased defects in protein glycosylation are the reason of severe diseases that is a direct evidence for the importance of glycosylation.

Various glycosylation pathways are studied as potential targets for therapeutic purposes.

56References :Cox. MM, Nelson. DL; Lehninger Principle of Biochemistry; 5th edition.Berg JM, Tymoczko JL, Stryer L; Biochemistry; 7th edition.Murray RK, Bender DA, Botham KM, Kennelly PJ et al; Harper Illustrated Biochemistry; 29th edition.Schwarz F, Aebi M; Mechanisms and principles of N-linked protein glycosylation; Current Opinion in Structural Biology 2011, 21:576582.Spiro R. G. Protein glycosylation: Nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds. Glycobiology. 12, 43R-56R.Varki A, Cummings RD, Esko JD, et al., Essentials ofGlycobiology. 2nd edition.Chapman NR, Christopher LR; The role of carbohydrate in sperm-ZP3 adhesion; Molecular Human Reproduction vol.2 no.10.

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