Glycobiology Flashcards

Question

Glycoproteins

Answer 1

* Glycoproteins carry covalently linked carbohydrate structures („Glycans“) * Part of cellular membranes for Signalling * Glycosylation stabilizes proteins * Glycan linked to Asparagin: Nglykosidic bond * Glycan linked to Serin or Threonin: O-glycosidic bond * Glycosylation of proteins in the endoplasmic reticulum * Processing of complex glycans in the Golgi-apparatus

Answer 2

* Cornerstone for understanding mammalian glycobiology ! * Generated through a common pathway in the endoplasmatic reticulum and linked to Asn * Differential action of glycosidases and glycosyltransferases (GTs) generates high Man, complex and hybrid type * Particular structures found on proteins depend on GTs and their expression level * Specific terminal elaborations added to core structures mediate specific functions * Key objective is to understand how GT expression, activity and regulation influences glycosylation

Answer 3

* Diverse in structure and function * Lack a common biosynthetic pathway * Attached to Ser, Thr, Hydroxylysine * O-GlcNAc common in cytoplasm and nucleus * O-GalNAc found in mucins that hold water * IgA is O-glycosylated in the hinge region * Proteoglycans are heavily Oglycosylated proteins that strengthen the extracellular matrix

Answer 4

1. Carbohydrates occur as mono-, oligo- oder polysaccharides 2. Hexoses and ketoses are semiacetals or semiketals in pyranose and furanose rings and form α- und β-anomers. 3. Acetal formation results in glycosidic bonds. 4. Glucose α-1,4-linked in starch and glycogen. 5. Glucose β-1,4-linked in cellulose, GlcNAc β-1,4-linked in chitin. 6. Glycosyltransferases specifically catalyze the synthesis of defined glycosidic bonds from nucleotide-activated precursors. 7. Glycoproteins are important carriers of glycan structures (O-glycosylations at Ser or Thr, N-Glycosylations at Asn) that play major roles in cell-cell signaling.

Answer 5

Sequential protocol: 1. Glycan release (PNGase F) 2. Reducing end labelling 3. Sequential degradation with specific glycosidases (“restriction enzymes”) 4. Monitoring by chromatography of digestion products

Answer 6

MS is an indispensable glycomics tool! • Resolve components of a glycan mixture (glycan profiling) • Derive information about structure of individual glycans • Accurate masses plus fragmentation experiments to derive exact structures (tandem mass spectrometry) • Combine with sequential degradation with enzymes • Detect modifications (phosphorylations, sulfatations) • Analysis of glycopeptides to identify glycosylation sites

Answer 7

Ultimate source of definitive information on glycan structures! • Used to determine structures of a large number of typical oligosaccharides • Substantial amount of material required (~mg), not well adapted for mixtures • Unique patterns for equatorial and axial protons • Anomeric protons well resolved and deshielded • Information obtained about stereochemistry of sugars (monosaccharide and linkage configurations)

Answer 8

Functional glycomics: Studies on glycan arrays are leading to better understanding of biological roles of glycans • Determine parts of glycans that form epitopes recognized by receptors • Screening of glycan-binding proteins

Answer 9

Glycan structures and analysis: CFG and SweetDB Glycan biosynthesis: CFG, KEGG, CAZy Glycan binding receptos: Animal binding receptors, CFG

Answer 10

* Cell surface receptors containing Ig-like domains that bind carbohydrates (Itype lectins) * Sialic acid binding group: Siglecs, 14 forms in humans * Related to variable (V-set) and constant (C-set) Ig-like domains * Sialic acid binding via V-set linked via disulfide bonds to C-set as spacer domains * Present on cells of the immune system

Answer 11

• Found on B-cells, contains multiple tyrosinebased sequence signalling motifs (activating and inhibiting) • Preferentially binds to alpha-2,6-linked sialic acid. • Inhibitory function predominant, knock-out mice are more sensitive to LPS and have tendency to produce auto-antibodies for B cell activation

Answer 12

Mammalian sperm-egg binding mediated by eggbinding protein on sperm plasma membrane with glycans on glycoproteins of egg‘s zona pellucida (ZP) • Fucoidan inhibits binding • Overlapping binding specificity with selectins? • Glycan characterization on ZP challenging: ZP from 195 unfertilized human oocytes isolated

Answer 13

* Intracellular glycosylation: nucleus and cytoplasm (dogma: N- and O-linked glycosylation restricted to ER and Golgi) * Not elongated or modified, only GlcNAc * Cycling rapidly at different rates * similar to phosphorylation in signaling * occurs in many organisms * essential for viability at the single-cell level in mammalian cells * Difficult to detect with conventional proteomics methods due to uncharged character * High levels of hydrolases destroy the O-GlcNAc modification upon cell lysis * often lost during ionization in MS

Answer 14

sensor for the metabolic state of the cell

Answer 15

* Warburg effect: cancer cells use glycolysis instead of oxidative phosphorylation even at high O2-concentrations * enhanced Glc uptake results in increased O-GlcNAc levels in all human malignancies examined * elevated OGT-levels * O-GlcNAc contributes to cancer progression * OGT may be a selective target in cancer cells

Answer 16

1. Glycan analyis relies on a combination of high resolution mass spectrometry and biochemical techniques, specificity determinations in glycan arrays and conformational studies with NMR 2. Sialic acid recognizing lectins play important roles in mammalian cell signalling and the immune system (siglecs, sialoadhesins, fertilization). 3. O-GlcNAc modification of proteins has an important role in signalling and adds complexity to phosphorylation-mediated signalling pathways. 4. O-GlcNAc modification is coupled to glucose metabolism and may play a role in cancer progression.