VL 14 (Jörg Fettke) Flashcards
Carbohydrates
Carbohydrates are present in all kingdoms of life and fulfill various functions apart from building cell walls or in energy metabolism
- Formula (CH2O)n, n => 3
- Oligosaccharides: linked monosaccharides (building blocks)
- Polysaccharides (cellulose, glycogen, starch)
Important definitions
- Glycan: term for sugars in glycoproteins/-lipids
- Glucan: only glucose
- Glycoconjugate: protein/lipid + sugar
- Glycoprotein: protein containing covalently linked sugar molecules (added as co-/post-translational modifications)
- Glycome: total complement of glycans in cell/tissue/organism
- Glycosyltransferase: E; sugar transfer from donor→acceptor→form: glycosidic link
- Glycosidase: E; hydrolyzes glycosidic linkage
- Lectin: glycan binding protein; E with sugar S, sugar-binding Ab are usually not classified as lectins; classified in different families based on CRD-sequence similarities
- Carbohydrate-recognition domain (CRD): part of glycan-binding protein (lectin) that contains sugar-binding site
- O-linked oligosaccharide/glycan: linked through O-atom, attached to Ser/Thr
- N-linked oligosaccharide/glycan: linked through N-atom (Asp)
Stereochemistry of Carbohydrates
D-Aldoses
D-Ketoses
Confirmations of hexoses
- H-atom at C1/4→destabilize boat conformation→stabilize chair conformation
- 4C1-chair conformation preferred: all OH-groups equatorial
Form of Pyranose and Furanose
Common Monosaccharide Building Blocks
Disaccherides and Polysaccherides
Disaccherides:
* Two monosaccharide
* O-glycosidic bond
* Reducing sugars: aldehyde/keto group conserved on one end (i.e. lactose, maltose)
* Non-reducing sugars: OH-group at C1 of one of both partners has condensed
→acetal (no keto/aldehyde function present in molecule (i.e. saccharose))
Monosaccherides:
* Cellulose: insoluble zick-zack layers of carbohydrates
* Starch: helic, double-helix structure
Function of glycans
Intrinsic glycan functions
* providing structural components
–> cell walls
–> ECM
* modifying protein properties
–> solubility
–> stability
Extrinsic functions resulting from glycan-lectin interactions
* directing trafficking of glycoconjugates; mediating, modulating signaling
–> Intra-/extracellular
* Mediating, modulating cell adhesion
–> Cell-cell/matrix interactions
Steps in encoding and decoding information in glycan structures
Comparison of glycans and proteins
Glycosyltransferase
- activation: nucleotide coupling
- need activated sugars
- Glycosyltransferases need
activated sugars - Activation via nucleotide
coupling - Highly specific enzymes! („One
linkage, one enzyme“)
N-linked Glycans
- generated trough pathway in ER; Asn-linked (motive: Asn-X(no Pro)-Ser(Thr), X = aa)
- differential action of glycosidases, glycosyltransferases (GTs) generates high Man, complex, hybrid typ e
- particular structures on proteins depend on GTs + their expression level
- spec. terminal elaborations added to core structures mediate spec. functions
- key objective: how GT expression, activity, regulation influences glycosylation
O-linked glycosylation:
- lack common biosynthetic pathway
- attached: Ser, Thr, Hydroxylysine
- O-GlcNAc in cytoplasm, nucleus
- O-GalNAc in mucins that hold H2O
- IgA: O-glycosylated in hinge region
- Proteoglycans: O-glycosylated proteins → strengthen ECM
O-GlcNAc:
- Not elongated or modified, only GlcNAc
- Cycling rapidly at different rates
- Similar to phosphorylation in signaling
- Essential for viability at the single-cell level in mammalian cells
Glycobiology Toolboxes
Glycosidase:
* Sequential protocol
–> Glycan release (PNGase F)
–> Reducing end labelling
–> Sequential degradation with spec. glycosidases (“restriction E”) o Monitoring by chromatography of digestion products
Mass Spectrometry
MS is an indispensable glycomics tool
* Resolve components of glycan mixture (glycan profiling)
→structure information (masses + glycosylation sites + fragmentation experiments (tandem MS))
* Combine with sequential degradation with E
* Detect modifications (phosphorylations, sulfatations)
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) Taylor/Drickamer, Inroduction to Glycobiology 2nd ed.
glycan arrays:
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 DB for
Databases for glycobiology:
* examples: CFG, SweetDB, KEGG, CAZy, Animal lectin resource
