Glycosyltransferases and Glycosidases TEST Next Thursday--only individuals signed up for a letter grade Problem Set Hand Out Today--Will go over in class Tuesday Also Tuesday--1st clinical correlation (Lance presenting) - read papers BEFORE class & have 2 questions to hand in regarding the papers at the BEGINNING of class Rearrangement of presenters 2nd clinical correlation paper will be posted before the weekend Refresher of Tuesday’s lecture

newly synthesized N-linked glycan GlcNAc Mannose Glucose Galactose Sialic acid Fucose

N-linked glycan synthesis: branching via GlcNAc addition branch = antennae GlcNAcT-III ß4 GlcNAcT-I ß2 GlcNAcT-II ß2 GlcNAcT-IV ß4 GlcNAcT-V ß6 GlcNAcT-VI ß4 GlcNAc Mannose Glucose Galactose Sialic acid Fucose

Mechanisms of Glycosyltransferases  Two main catalytic mechanisms can be envisioned for glycosyltransferases : inversion of the anomeric configuration (for instance UDP-glucose -> b-glucoside) or retention of the anomeric configuration (for instance UDP-glucose -> a-glucoside). glycosyltransferases (EC 2.4.x.y) Core 3 GlcNAcT Ser/Thr b3 Ser/Thr UDP-GlcNAc UDP -most have divalent ion requirement and prefer neutral to acidic pH

Mammals utilize only 9 (?) sugar nucleotide donors for glycosyltransferases: UDP-glucose, UDP-galactose, UDP-GlcNAc, UDP-GalNAc, UDP-xylose, UDP-glucuronic acid, GDP-mannose, GDP-fucose, and CMP-sialic acid. Other organisms have an extensive range of sugar nucleotide donors. Many glycosyltransferases in higher and lower organisms use lipid linked glycosyl donors where the lipid is frequently a terpenoid such as dolichol or polyprenol.

STRUCTURE OF GLYCOSYLTRANSFERASES --vast majority fall into 3 types of folds -- CAZy ~ Carbohydrate-Active enZymes glycosyltransferases into over 86 families (structure, mechanism, substrate, acceptor)

Type II Transmembrane Proteins --couple of exceptions in Man-6-P lysosomal targeting pathway

Many Transferases are Expressed in Soluble Forms and/or Processed and Secreted-----Why?????

Strict acceptor substrate requirements One-linkage-one-enzyme paradigm

Exceptions to the Paradigm Specific linkage can be product of several related gene products (2-8 sialytransferase family) Rarely a glycosyltransferase can synthesize 2 different linkages (FucT-III 1-3 and 1-4) Acceptor specificity modulation (GlcNAc to Glc for b4-galactosyltransferase by lactalbumin) Glycosyltransferase that catalze two stepwise reactions (copolymerases of GAG chains)

Glycosidases Two Mechanisms--inverting/retention Very Diverse in Primary and Tertiary Structure Most localized to lysosome

Retention of Configuration (EC 3.2.1.-) http://afmb.cnrs-mrs.fr/CAZY/index.html

Inversion of Configuration

Degradation of N-linked Proteins--both reducing & non-reducing end degradations

Glycosphingolipid Degradation -terminal in lysosomes -some cleavages require SAPs (sphingolipid activator proteins)

Non-reducing end degradation HA Degradation Non-reducing end degradation

Degradation of Heparan Sulfate Non-reducing end degradation

Keratan Sulfate Degradation Non-reducing end breakdown

For the Test Next Week Read Essentials and Go Over Presentations Nomenclature (Three letter glycans, linkage, etc..) Major Types of Glycosylation (N-linked, O-linked, Lipid linked, Proteoglycans)