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67 Cards in this Set
- Front
- Back
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Structural roles of protein glycosylation: (2)
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Physical barrier
Maintenance of tissue structure, porosity, integrity |
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Glycoalyx
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Physical barrier surrounding all cells
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Modulatory (4)
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Protection from degradation (stability)
Folding, solubility, conformation Modulation of interactions Storage depot |
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Recognition
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Glycan binding proteins (lectin)
Glycan-glycan interactions Intrinsic cell interactions Extrinsic cell interactions |
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How would you go about determining whether a protein is glycosylated?
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glycosidase and few if the electrophoretic shift has altered
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Name three methods of examining (experimentally) glycosylation
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Electrophoresis (glycosidase)
Anti-glycan Ab Radiolabelling of sugar residues |
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Experimental view of glycosylation function on development. (2)
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Knock out protein of interest in vivo.
In vitro, clone the protein lacking a glycosylation site. |
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Alpha?
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OH pointing down
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Beta?
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OH in cis form. (middle)
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Deoxyhexose
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Missing OH on the 5' C
also known as methylpentose |
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Hexuronic acids
Where is it found (what protein)? |
oxidation of hexoses at C-6 to form COOH
found in proteoglycans |
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Pentoses are found where?
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In animal proteoglycans
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Sialic acids size.
Most common form? |
C9 monosaccharides
N-acetylneuraminic acid |
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NeuAc (2)
Biosynthetic reaction: Where is it found? |
Condensation of N-acetylmannosamine with pyruvate (from PEP)
Often found as the terminal sugar |
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Glycosidic bond between what molecules
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Anomeric carbon of one sugar is attached to the OH group of another.
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On which OH groups of the sugar can a glycosidic bond be formed?
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1-, 4-, 6-, 3-
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How are the specific roles of glycoproteins determined by? (3)
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Terminal glycosyl residues
Unusual structures Modifications |
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While proteins have evolved to use glycosylation to protect itself from bacteria. Why is this not necessarily a good thing?
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Bacteria and viruses can now target those structures for viral entry.
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N-linked glycosylated proteins are attached to which aminoacid residue?
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Asparagine
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O-linked glycosylated proteins are attached to which amino acids?
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Serine and Threonine
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Three common classes of glycosylated proteins:
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Proteoglycans
Glycosylphosphatidylinositol (GPI)-anchored glycoproteins Glycoproteins |
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Proteoglycans: (2)
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One/more glycosaminoglycan (GAG)
O-linked to serine through a core region ending in a xylose residue |
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Glycosylphosphatidylinositol (GPI)-anchored glycoproteins (3)`
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Glycan bridge between PI and PE
PE attached to C-T of a protein via an amide linkage Anchor to the lipid bilayer. |
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O-GlcNAc
Glycoproteins: (3) |
Nuclear/cytoplasmic glycoproteins
not elongated or modified Frequently attached/removed |
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Where are glycosylated proteins found within a cellular context?
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Within the secretory pathway or extracellularly
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Determining the sequential activty of glycosyltransferases within the secretory system? (3)
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Subcellular fractionation (enzyme activity assay/western blot)
Immunofluorescence/ Immunoelectron microscopy Sequential activity |
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Synthesis of glycoproteins requires what modification on a monosaccharide?
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Activation to a high energy donor form.
linkage to a nucleoside diphosphate via C1 (anomeric carbon) |
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Exception to C'1 modification of an activated monosaccharide
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Sialic acid: linked to a nucleoside MONOPHOSPHATE via C2.
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Sialic acid: (3)
What is it linked to? How is it linked to? Where is the activated form found? |
Monophosphate linkage via C2.
Found in the nucleus! CMP |
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Transport of nucleotide sugars into the ER/Golgi.
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Antiport: simultaneous exit of phosphosugar and entry of nucleotide between ER and cytoplasm.
Process is electroneutral |
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Microheterogeneity:
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Same glycosylation site on the same protein can carry different glycans.
Since glycans are not gene products-->extremely variable. |
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Factors affecting microheterogeneity: (5)
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1) Protein sequence/conformation, locatin of glycosylation site
2) Nucleotide sugar metabolism 3) Transport rate of protein through the ER/Golgi 4) Localiation/concentration of specific glycosyltransferases/glycosidases within the secretory pathway: competition 5) Environmental conditions/developmental stage/cell or tissue type |
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Cause of smear on SDS-PAGE gel:
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Microheterogeneity
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Minimize smearing: (2)
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Take cells from same tissue type or cell type.
Synchronize cell stages. |
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N-linked oligosaccharides consensus sequence:
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Asn-X-Ser/Thr sometimes Asn-X-Cys
does not mean it wil actually occur |
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Three types of N-linked Oligosaccharides:
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Oligomannose
Complex Hybrid |
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Invariable common core structure of N-linked oligosaccharides: (3)
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1)- Three branched mannose with alpha 1-3 and alpha 1-6
2)- Two N-acetyl-glucosamine 3)- Asparagine residue |
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Oligomannose:
Complex: Hybrid: |
Oligomannose, multiple mannose branches
Complex; different glycosyl branches Hybrid; complex branch and mannose branches |
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Two N-linked oligosaccharidases:
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Peptide N-glycosidase F
Endoglycosidases |
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Peptide N-gllycosidase F: (2)
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Removes all branches and converts asparagine into aspartate.
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Endoglycosidase: (2)
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Leaves one GlcNAc attached to Asn
Does not release complex N-glycans |
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Beginning of biosynthesis of N-glycan
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Transfer of a common oligosaccharide precursor to the nascent polypeptide
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Precursor: formed where? on what?
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Formed on a lipid called dolichol.
formation occurs in the cytosol. |
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Precursor consists of what?
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Polyisoprenol; repeating isoprene units with a phosphate attached to the end.
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How is the precursor linked to dolichol?
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Through pyrophosphate bridge
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Biosynthesis of Dol-PP-oligosaccharide: (5)
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1)- Transfer of GlcNAc-P from UDP-GlcNac to membrane-bound Dol-P
2) Transfer of the second GlcNAc and 5 mannose residues. Mannose residues are attached to GDP 3)- Translocation across ER membrane 4)- Extension by 4 mannose and 3 Glc residues. Dol-P-Man/Glc act as donors 5)- 14 sugar chain is transferred to Asn-X-Ser/Thr by oligosacchryltransferase (co-translational!) |
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Processing and maturation of N-glycans (3)
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Sequential removal of Glc and one Man residues in ER (glucosidases and mannosidases)
Proper folding (trimming continues in cis-Golgi No trimming complete (no further modifications- oligomannose!) Further modifications lead to formation of hybrid and complex N-glycans |
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Improper folded protein- Receptor: and function
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Calnexin or calreticulin: lectins that recognize monoglucosylated N-glycans
Erp57: thiol oxidoreductase |
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When bound to these receptors, what occurs?
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Constant removal and addition of glucose/mannose residues until protein has adopted proper conformation. glycosidases and glucosyltransferase
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Congenital disorders of N-glycosylation: (2)
Clinical features |
1)- Defects in the biosynthesis of lipid-linked oligosacchraide, and transfer to the nascent polypetide chain
2)- Defects in subsequent processing steps Features: development issues. |
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Tunicamycin
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inhibits GlcNAc-P onto Dol-P
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O-linked glycosylation is also known as:
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Mucins
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Mucin cellular description:
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heavily glycosylated secreted or cell surface transmembrane proteins
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Functions of mucin: (5)
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Provide protection against infection
Involved in signal transduction and cell-cell adhesion Often anti-genic Protect the underlying protein from degradation Affect protein stability/expression levels. |
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Mucin structure:
What type of linkage (i.e. what type of linkage of the sugar residue to the a.a.) |
Alpha linkage to GalNAc to -OH of Ser/Thr
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Where does the mucin structure occur?
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At variable number of tandem repeat sequences (regions rich in Ser/Thr O-glycan acceptor sites.
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Division of carbohydrate moiety of O-linked oligosaccharides: (3)
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Core region
Backbone Region Peripheral region |
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Most common mucin core structures: (4)
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Not branched: GalNAc with Gal
Branched: Gal or GlcNAc attached in multiples (like complex) |
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Backbone region consists of:
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Gal(B1-3) GlcNAc
Gal(B1-4)GlcNAc |
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Peripheral region function: (3)
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Antigenic determinants
Cellular recognition, cell-cell adhesion Binding of pathogens. |
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Mucin biosynthesis unlike N-linked glycans: (2)
like: (1) |
-Does not involve lipid linked intermediates or glycosidases
-Is not co-translational + is dependent on transfer of monosaccharides from a nucleotide sugar to a peptide chain |
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Transfer of GalNAc to Ser/Thr (2)
Catalysed by? preferentially glycosylates which sites? |
Catalysed by polypeptide-N-acetylgalactosaminetransferase (ppGalNAcT)
Glycosylates beta turns in proline-rich regions |
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Control of mucin biosynthesis:
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Assembly line of glycosyltransferases
Acceptor specificity |
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Heterogeneity involves:
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Enzymes overlap with respect to substrates and localization.
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N-linked oligosaccharides have their first GlcNAc residues attached by what type of glycosidic bond?
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beta
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O-linked oligosaccharides have their first GalNAc attached by what form of glycosidic bond
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alpha
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Once transferance of the dolichol-oligosaccharide has occured onto the a.a. what occurs?(3- proper protein maturation)
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Removal of two glucose residues.
Check if proper folding, Removal of glucose residues further (GII) Addition of glucose from alpha-glucosyltransferase |
