BIOL3013 Richard Meek: Lectures 6-9 Glycans

Define glycan

Any sugar/assembly of sugars in free form or attached to another molecules. Term used interchangeably in this module with saccharide or carbohydrate

What are the three types of projections for glycans? and what are there positives and negatives

Fisher: linear projection of monosaccharides

• not representative of true structure

• shows hydroxyl position & orientation well

Haworth: depicts sugar as planar rings

• not representative of true structure

• better for stereochemistry evaluation & showing cyclic structure

Chair: most accurate projection by showing its actual conformation

• representative of true structure

• shows equatorial & axial hydroxyl groups

• harder to interpret than other projections

What is the angle between substituents & saturated ring of glycans & how is this angle altered?

Optimal angle is 60^o, and preferentially has this

Conformational transitions require catalysts to overcome the higher energy barrier to alter these angles

What is the anomeric carbon?

The carbon that was originally part of the carbonyl group, it becomes a special carbon in the ring, usually C1. Can have 2 conformations= equatorial or axial OH

Explain the nomenclature of Beta-D-glucopyranose

Beta: refers to OH position on the anomeric carbon. If equatorial & matching C5 = Beta, if axial & not matching C5= alpha

D= refers to which enantiomer the molecule is, mirror image. most sugars are D except fucose notably

pyranose= refers to ring size, in this case a 6 membered ring

Explain alpha & beta conformations of glycosidic bonds

bond between 2 monosaccharides, causing expulsion of H20, fixes conformation to either alpha or beta depending on orientation of hydroxyl groups

Alpha= OH group on anomeric carbon is axial & below plant of ring

Beta= OH group on anomeric carbon is equatorial & above plane of ring

What are the overarching functions of glycans

Helps enable protein folding through quality control & chaperone recruitment; cell signalling & communication, acting as receptors or ligands, mediating cell-cell interaction & signal transduction; & immune system modulation (pathogen recognition, immune cell activation & inflammation regulation

What are the key steps in synthesis & transfer of glycan from dolichol-P-PGlcNAc2Man9Glc3

1. Glycan assembly on dolichol phosphate: sugars are sequentially added early on cytosolic face of ER before being flipped into the ER lumen

2. ALG proteins: add specific sugars to glyan within ER lumen

3. Transfer to protein: by oligosaccharyl transferases, transfer en bloc to the N residue on the protein co-translationally

At what residue are N glycans added?

N= Asparagine, specifically on motif N-X-S/T

What are the two types of glycoside hydroxylase do?

Exoglycosidase= only removes the terminal sugar

Endoglycosidase= cleaves from an internal glycosidic link to release terminal saccharide + non-terminal saccharide

How are subsites on glycans numbered in relation to glycoside hydroxylases?

-1 position refers to the sugar that is having its glycosidic bond broken by catalysis, sits close to the terminal end of the sugar (non-reducing end) &

+1 position refers to the sugar on the other side of catalysis that avoids removal (towards reducing end)

Difference between a-Glu I & a-Glu II

A-Glu I: removes terminal glucose from 3→2

A-Glu II: removes the terminal glucose from 2→1

What do the alpha & beta subunit in A-Glu II do?

Alpha= catalytic section where the sugar binds & is removed

B-subunit: contains a KDEL sequence for ER localisation & has a mannose-6-phosphate receptor homology domain which can recognise & bind high mannose type N-glycans for terminal glucose removal

Difference between A-Glu I & A-Glu II

A-Glu I is membrane bound whilst A-Glu II is soluble in ER lumen

A-Glu I removes terminal glucose from 3→2 whilst A-Glu II removes terminal glucose from 2→1 and 1→0 after the CXN/CRT cycle

How is specificity of glucosidases possible?

Use of specific reisudesin specific orientations determine exactly how a sugar molecule must be oriented to fit into the active site, for example, between a-glucosidase I & II they have tryptophans at the same residue but in different conformations, accommodating different terminal glucose residues only

LIst the names of the sugars in the D1 arm of the main N-glycan for protein quality control

Glc(3), Glc(2), Glc(1), Man(D1), Man(C), Man(4) etc….

What specific N-glycan do calnexin & calreticulin recognise

Glc1Man9GlcNAc2

What domain selects & binds glycans?

The lectin domain of CXN & CRT

Similarities between CXN & CRT?

• Lectin activity & glycan specificity: both recognise 1 terminal glucose on the N-glycan, binding is mediated by alpha 1,2-linked glucose

• Shared domains: lectin domain, P domain

• Protein recruitment: both recruit accessory proteins to assist in protein folding = Cyclophilin B & ERp57

• Calcium binding: both have for this for stabilisation of lectin domain, easy due to high abundance of calcium in Er relative to cytoplasm

Differences between CXN & CRT?

• CXN is a type I single pass transmembrane protein anchored in the ER membrane, CRT is a soluble protein in the ER lumen

• ER retention mechanism: CXN has transmembrane domain, CRT has a KDEL sequence in its highly negatively charged coil region

• Substrate preference: CXN primarily interacts with membrane bound glycoroteins, CRT with mainly soluble as a result of their localisation

Why can calreticulin not bind to any other terminal sugar residues

Why not Man4? glucose is essential at the non-reducing end as the hydroxyl group at C2 of a mannose would clash with Met131. There are also glucose H-bonds with Gly124 & Lys111

Why not Glc2Man2? Glucose cannot be in 2nd position due to linkage changing from alpha 1,2→1,3= a loss of H-bonds & Man4 would intersect the protein surface

What sugar residues do CXN/CRT bind to?

GlcMan3

What is function of the P-domain in CXN & CRT?

Recruits protein binding factors like CYclophilin B & ERp57

What is the function of Cyclophilin B & ERp57

Cyclophilin B: converts proline from cis to trans conformation and vice versa, crucial for changing direction of the polypeptide chain for proper folding

ERp57: breaks, forms & rearranges disulphide bonds, a PDI

What do the domains within ERp57 do?

4 domains total

A & A’: catalytic subunits with the CXXC motif. Allowing them to bind free cysteine residues & rearrange disulphide bond, 2 the 2 C residues means they can self-rescue by forming a disulphide bond with itself to release it from the substrate (TRXL domain)

B & B’: have no catalytic activity, thought to contribute to substrate binding & stability

What is the name of the enzyme that can reinstall Glc(1) if the protein is still not properly folded after the CXN/CRT cycle

UGGT= UDP-glucose glycosyltransferase

UDP= Uridine diphosphate

What are the key domains within UGGT? and what are their functions?

TRXL domain: recognises specific exposed hydrophobic residues as it is an indicator of improper folding

CAZy GT24 domain: a glycosyl transferase domain that adds glucose back onto Man(D1) at the end of the N-glycan

What is the species that has a fully solves structure of UGGT?

It is UGGT1 of the species Chaetomium thermophilum, a thermophilic fungus. Human version hasn’t been solved due to issues during crystallisation

What does the TRXL domain do?

It is a folding sensor region, recognising hydrophobic residues that shouldn’t be exposed

• TRRXL3 is present in ERp57. Has a hydrophobic cavity that is exposed upon displacement of a short helix

• Has a CXXC motif like ERp57 that enables them to catalyse the formation & breakage of disulphide bonds, this is absent from the UGGT TRXL domains

What motif localises UGGT to the ER?

THe motif REEL, unlike other proteins (A-Glu II & CRT) that use the KDEl sequence

What are the proteins involved in the exit cycle for unfolded & misfolded proteins after successive failures to fold in the CXN/CRT cycle?

EDEMs= ER-degradation enhancing alpha mannosidase-like proteins

ERADs= ER-associated degradation proteins

Explain the action of EDEMs and which specific EDEMs do what

They remove 1 mannose residue from the D2 & the D3 arm of the N-glycan to signal degradation by ERADs and then the UPS

EDEM1: cleaves mannose(D3)

EDEM2: cleaves mannose(D2)

EDEM3: cleaves mannose(D3)

Give some details about EDEMs (speed, activity, domains)

Slow enzymes, allow for time for folding in the CXN/CRT cycle

Activity is dependent on the folding state of the substrate, acting preferentially on misfolded proteins compared to completely unfolded ones

TRXL domains

What is the homology of EDEMs in yeast? What is it stabilised by? What part binds to proteins?

Mnl1, in Saccharomyces cerevisiae, 40% sequence homology with EDEMs

• Stabilised by interactions with Pdi1 (PDI) via disulphide bonds, blocking Pd1 function at the same time

• C-terminal domain is most likely what binds hydrophobic regions of proteins through a hydrophobic groove, its deletion = lower activity

What is unclear about mammalian EDEMs?

EDEM1 & 2 lack CTDs, makes it unclear how they bind unfolded proteins

EDEM3 has C a CTD, but it appears unrelated to Mnl1

All three EDEMs have been found to bind PDIs, but unsure if the mechanism is shared with Mnl1

What are the localised versions of ERADs called?

ERAD-L: for folding defects in the luminal domain

ERAD-M: for folding defects in the membrane domain

ERAD-C: for folding defects in the cytoplasmic domain

What is the function and structure of the lectin OS-9

Part of the ERAD complex

• Flattened B-barrel structure with 3 S-S bonds

• binds to truncated N-glycans that are cleaved by EDEMs to guide them through Hrd9 or other E3 ubiquitin ligases

• has a mannose-6 phosphate receptor homology domain that enables binding to the truncated D3 arm

• it is common in ERAD-L this particular lectin

What residues are important for Lectin OS-9 binding to truncated N-glycans?

W117 & W118 tryptophan residues on OS-9 are key for forming favourable H-bond formation with Mannose(B) attributing to the specificity pocket

W117 & W188 tryptophan residues are important for recognising the linkage of mannose residues, specifically the presence of an alpha 1-6 linkage. If other links like alpha 1-2 from pre-mannose cleavage are present there would not be correct binding to OS-9

What is the function of Hrd9 in ERADs?

It is an E3 ubiquitin ligase that facilitates the retrotranslocation of the misfolded protein (with its mannose residues trimmed) out of the ER and into the cytoplasm

• others do exist