Glycobiology Flashcards
Glycan
A general term for sugars found in glycoproteins or glycolipids
Glycoconjugate
A protein or lipid with sugars attached
Glycoprotein
A protein containing covalently linked sugar molecules that are added as co-translational or post-translational modifications
Glycome
The total complement of glycans found in a cell, tissue or organism
Glycosyltransferase
An enzyme that catalyzes the transfer of a sugar from a
donor molecule to an acceptor molecule to form a glycosidic link
Glycosidase
An enzyme that hydrolyzes a glycosidic linkage
Lectin
A glycan binding protein. Enzymes with sugar substrates and sugarbinding antibodies are usually not classed as lectins
Carbohydrate-recognition domain (CRD)
The part of a glycan-binding protein (lectin) that contains the sugar-binding site. Lectins are classified into different families based on similarities in sequence of their CRDs
O-linked oligosaccharide/glycan
Linked through an oxygen atom and attached to serine or threonin residues
N-linked oligosaccharide/glycan
Linked through the amide nitrogen of an asparagine residue
Isomers
same molecular formular, different structure
Constitunal isomers
differ in order of attachement of atoms (Glycerinaldehyde, Dihydroxyaceton)
Stereoisomer
same order, different arragnement
Enantiomer
nichtdeckungsgleiche Spiegelbilder (D-glyceraldehyde, L-Glyceraldedhyd) Gehören zu Stereoisomeren
Diastereoisomere
Isomers that are no mirror images (D-Aldose, D-Glucose)
Gehören zu Stereoisomeren
Epimers
Differ at one several asymertric carbon atom (D-Glucose, D-Mannose)
Gehören zu Diastereoisomers
Anomers
Differ in new asymetric forms in ring closure (alpha - D Glucose, beta D-Glucose)
Gehören zu Diastereoisomers
Which conformation is prefered for hexoses?
4C1-chair conformation preferred with all OH-groups in equatorial position (in contrast to boat conformation)
What means desoxyribose in contrast to ribose?
2nd Carbon: no Oxygen
Neu5Gc and cancer progression
Association between red meat consumption and disease risk: Neu5Gc trace amounts incorporated Anti-Neu5Gc antibody response (first „xenoautoantigen“)
• Auto-reactive antibodies are tumor-promoting (induction of inflammatory processes?)
• anti-Neu5Gc antibodies are of broad and variable specificities
AB0 blood groups are formed by different terminal
sugar building blocks on glycan structures of red blood cells
Karl Landsteiner (1868-1943):
• Studies with Emil Fischer in Würzburg
• Discovery of AB0 blood group system, of poliovirus and the typhus pathogen
• 1930 nobel prize for medicine
Glycosyltransferases build up glycan structures
• Transferase-Gene are inhereted and effect synthesis of different blood group antigens
Disaccharides
Two monosaccharide building blocks, linked via O-glycosidic bond
• Reducing sugars: Aldehyde or keto group is conserved on one end (i.e. lactose, maltose)
• Non-reducing sugars: OH-group at position C1 of one of the both partners has condensed to an acetal, no keto or aldehyde function present in the molecule (i.e. saccharose)
Polysaccharides
Cellulose: Most abundant organic compound of the biosphere • 1015 kg produced per year • β-1,4-linkage produces fibres with high tensile strength
• Starch and glycogen built helical structures
• Increased solubility in water to provide a rapidly accessible glucose storage
Glycosyltransferases
Glycosyltransferases need activated sugars
• Activation via nucleotide coupling
• Highly specific enzymes! („One linkage, one enzyme“)
High structural diversity in glycans
2 amino acids = 400 dipeptides
2 hexoses = 64 106 disaccharides
Donor binds first, loop ordering for water exclusion, then acceptor binding
Glycoproteins
- 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
N-linked Glycans
- 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
O-linked glycosylation
- 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
Summary I
- Carbohydrates occur as mono-, oligo- oder polysaccharides
- Hexoses and ketoses are semiacetals or semiketals in pyranose and furanose rings and form α- und β-anomers.
- Acetal formation results in glycosidic bonds.
- Glucose α-1,4-linked in starch and glycogen.
- Glucose β-1,4-linked in cellulose, GlcNAc β-1,4-linked in chitin.
- Glycosyltransferases specifically catalyze the synthesis of defined glycosidic bonds from nucleotide-activated precursors.
- 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.
Glycobiology Toolbox - Glycosidases
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
Glycobiology Toolbox – Mass spectrometry
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
Glycobiology Toolbox - NMR
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)
Glycobiology Toolbox – Glycan arrays
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
Databases for glycobiology
Glycan structures and analysis: CFG and SweetDB
Glycan biosynthesis: CFG, KEGG, CAZy
Glycan binding receptos: Animal binding receptors, CFG
Siglecs and the immune system
- 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
CD22 („Siglec-2“)
• 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
Glycans in cell-cell recognition: Fertilization
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
O-GlcNAc
- 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
UDP-GlcNAc is
sensor for the metabolic state of the cell
O-GlcNAc in cancer
- 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
Summary II
- Glycan analyis relies on a combination of high resolution mass spectrometry and biochemical techniques, specificity determinations in glycan arrays and conformational studies with NMR
- Sialic acid recognizing lectins play important roles in mammalian cell signalling and the immune system (siglecs, sialoadhesins, fertilization).
- O-GlcNAc modification of proteins has an important role in signalling and adds complexity to phosphorylation-mediated signalling pathways.
- O-GlcNAc modification is coupled to glucose metabolism and may play a role in cancer progression.
