Bioc 312: Protein Glycosylation Final

Front 1

What is a glycan?

Back 1

Proteins with sugars attached to them

Front 2

What happens in ptn glycosylation?

Back 2

One or more glycans are attached to a polypeptide backbone

Front 3

What is ptn glycosylation?

Back 3

Major POST-translational modification of:
-most mb bound secreted ptns
-a number of nuclear and cytoplasmic ptns
-carbohydrate can represent 1-90% of the ptn

Front 4

What are the physiological roles of ptn glycosylation?

Back 4

-Range from subtle to essential for development, growth, function and survival
-Vary based on tissue developmental stage and environmental context

Front 5

What are the specific roles of ptn glycosylation? (2)

Back 5

1) Structural and modulatory
2) Specific recognition by other molecules

Front 6

What are the structural roles of protein G?

Back 6

-Physical barrier (glycocalyx)
-Maintenance of tissue structure, porosity and integrity

Front 7

What is the glycocalyx?

Back 7

Sugar layer that surrounds most of the ptns/cells
Protects cells from the extracellular environment
Part is attached to the glycoptns

Front 8

What are the modulatory roles of ptn G?

Back 8

-Protection from degradation (stability): G ptn is less acessible to proteases
-Folding, solubility, conformation
-Modulation of interactions
-"Storage depot": can store a hormone bound to the glycan; doesn't need to be synthesized, just needs to be released from the glycan

Front 9

What are the specific roles for specific recognition by other molecules?

Back 9

-Glycan binding proteins (lectins)
-Glycan-glycan interactions
-Intrinsic/extrinsic
--> Intrinsic: cell-cell interactions, recognition of glycans on the same cell, recognition of extracellular molecules
-->Extrinsic: pathogenic microbial adhesins, agglutinins, toxins or mediation of symbiotic relationships (glycan part can be rec'z by something outsie of the cell-> pathogen can rec'z glycan extracellularly)
**Glycans have a huge number of roles**

Front 10

What fctnal aspect of ptn G is likely to be involved in the dramatic changes that occur during development and cancer?

Back 10

Cell-cell interactions: regulates these processes through the control of proliferation

Front 11

How would you determine if a protein of interest is glycosylated?

Back 11

1) Electrophoresis: G changes the mobility of a ptn.
->can run on a gel, use an enz that digests (glycosidase), expect to see a decrease in MW (should confirm by chem/enz removal of the glycan, changes mobility)
2) MASS spect and NMR: but expensive, so not the best option
3) Fluorescently tag/radiolabel glycan sugars: purify your ptn, look for radioactivity/chemical label
4) See if a lectin binds to your ptn or use an anti-glycan Ab

Front 12

How do you know if G plays a fctnal role in your ptn of interest?

Back 12

Clone ptn lacking G sites
KO/knockdown ptn of interest and express in non-G mutant
-> if the cell is non fctnal, then the G of that ptn was important

Front 13

Why is it important to investigate the role of glycosylation in a whole organism in addition to cultured cells?

Back 13

-maybe G of a ptn has a role in the extrinsic fct of a cell
-there are things in an organism that aren't in your single cell layer: if involved in cell-cell adhesion, etc, need to see in an organism

Front 14

Why would we want to determine the sequence of the glycan chains?

Back 14

Want to know which specific monosac are present
Can sequence the glycan through Mass Spec, NMR
Chain seq determines the Fct (particularly the terminal residues)

Front 15

What are some methods that could be used to determine the glycan sequence?

Back 15

Glycan isolation and partial/complete digestion (chem/enz)
Followed by: HPLC, GLC, Mass Spec, NMR : determines which monosac are present (but not as exact as determining the seq of a ptn)

Front 16

What are the common monosac found on euk glycoptns?

Back 16

Hexoses
Hexosamines
Deoxyhexoses
Hexuroic acids
Pentoses
Sialic acids

Front 17

What are hexoses?

Back 17

6C sugars
All in the D-form (all are enantiomers (stereoisomers))
Either α or β on C1 (C1=aldehyde/ketone)
α: OH on C1 is pointing down
β: OH on C1 is pointing up

Front 18

Which anomeric confirmations are the following in?
Glucose
Galactose
Mannose

Back 18

Glucose: α
Galactose: β
Mannose: α

Front 19

What are hexosamines?

Back 19

Hexoses with an amino group at C2
With or without substituent
->commonly N-acetylated

Front 20

What are examples of hexosamines?

Back 20

N-acetyl-D-glucosamines (GlcNAc): glucose + amino gp on C2 (acetylated)
N-acetyl-D-galactosamine (GalNAc): galactose + amino gp on C2 (acetylated)

Front 21

What is deoxyhexose?

Back 21

Methylpentose
Missing an OH on C6
->L-fucose is usually terminal
-plays a role in recognition

Front 22

What are hexuronic acids?

Back 22

Oxidation of hexoses at C6 to form COOH
Mostly found in proteoglycans
ex: D-glucoronic acid (GlcA)
L-iduronic acid (IdoA)

Front 23

What are pentoses?

Back 23

Only has 5 C
D-xylose (Xyl) is found in animal proteoglycans

Front 24

What are sialic acids?

Back 24

Family of releated C9 monosac
Most common: N-acetylneuraminic acid (NeuAc)

Front 25

Describe NeuAc.

Back 25

-Formed through condensation of N-acetylmannosamine with pyruvate (from PEP)
-Ketose with the reducing gp at C2 and COOH at C1
-Ketone at C2 involved in the formation of ring structure
-N-acetyl gp at C5
-3C gp derived from PEP at C6 (R-gp)
-often found as a terminal sugar

Front 26

What is a glycosidic bond?

Back 26

Linkage between the anomeric C of one sugar and an O group of another

Front 27

Describe structure of Gal(β1-4)GlcNAcα

Back 27

a β-D-galactose with its C1 attached to the C4 of an N-acetyl-α-D-glucosamine, through a β-glycosidic bond

Front 28

How can hydroxyl gps be modified?

Back 28

G, P, S etc
Different linkages: 1,3 or 1,6 etc

Front 29

What determines the specific roles of glycoptns?

Back 29

Terminal glycosyl residues
Unusual structures
Modifications
->these regions also serve as likely targets for pathogens

Front 30

What are N- and O- glycosylated ptns?

Back 30

Sites of glycan attachment

Front 31

What are N-linked glycosylated ptns?

Back 31

Sugars attached to the AMINO gp of Asn (Asparagine)

Front 32

What are O-linked glycosylated ptns?

Back 32

Sugars attached to HYDROXYL gp on Ser/Thr

Front 33

What are the classes of G ptns?

Back 33

-Proteoglycans
-Glycosylphosphatidylinositol (GPI)-anhored glycoptns
-O-GlcNAc glycoptns

Front 34

Describe proteoglycans.

Back 34

1+ glycosaminoglycan (GAG) chains
-O-linked to serine through a core region ending in a xylose (Pentose) residue

Front 35

Describe GPI-anchored glycoptns.

Back 35

Glycan bridge between PI and PE
PE is attached to the C-T of a ptn via an amide linkage
Acts as an anchor to the lipid bilayer (links glycoptn to cell mb)

Front 36

Describe O-GlcNAc glycoptns.

Back 36

Nuclear/cytoplasmic glycoptns
Not elongated or modified
Frequently attached or removed (transient attachment, just the one residue hat is modified/G)

Front 37

Where does ptn G take place?

Back 37

Nuclear/cytosolic
Ass't secretory path

Front 38

Describe nuclear/cytosolic ptn G.

Back 38

Ptns G in the nucleus/cytoplasm tend to stay there
-Nuclear/cytosolic ptns
-O-GlcNAc (not extended or modified)
-Dynamic modification (similar to P, changes when removed/added to the base)
-May compete with P
-TF, cytoskeletal ptns, oncogens, etc

Front 39

What happens in ptn G ass't with the secretory path?

Back 39

-Mb bound/secreted ptns
-Mediated by sequential action of enz (glycosyltransferases: add sugars, glycosidases: remove sugars)in the ER/golgi
-Ptns glycosylated in the secretory path stay in the path or go to the extracellular env't
-No transfer from ecretory path to the cytosol

Front 40

What kind of ptn are glycosyltransferases/glycosidases?

Back 40

Transmembrane proteins

Front 41

What do glycosyltransferases do?

Back 41

Catalyze the transfer of monosac to different acceptors

Front 42

What do glycosidases do?

Back 42

catalyze the removal of monosac from a glycan chain

Front 43

What determines glycan sequence/formation?

Back 43

Specificity: depends on the glycan, aa seq
Expression level, localization and sequential activity

Front 44

How can you determine the sequential activity/localization of glycosyltransferases in teh secretory system?

Back 44

1) Subcellular fractionation: enz activity assays/western blot
->fractionate and look for activity in different fractions (see which will G a specific substrate)
2) Immunofluorescence/immunoelectron microscopy (label a ptn and see what it does)
3)Sequential activity: cna enzyme A act in the absence of enz B

Front 45

What is G when the glycosyltransferase is at the beginning of the secretory path? At the end?

Back 45

Beginning: G a residue at the beginning of the chain
End: G near terminal residues

Front 46

How are glycan sequences variable?

Back 46

Expressin level and activity of glycosyltransferases varies with cell type, developmental stage, environmental conditions, etc
-Enzymes compete for substrate (same substrate can be G by different enz)

Front 47

What is required for the biosynthesis of glycoptns?

Back 47

Activation of monosac to a high E donor form
->These activated sugars are then transferred to the oligosac/polypep chain by glycosyltransferases

Front 48

How are all donor sugars activated (except sialic acid: NeuAc)?

Back 48

Activated through linkage to a nucleoside DI-phosphate via C1 (anomeric C1)

Front 49

How is sialic acid activated?

Back 49

Linked to a nucleoside MONO-P (CMP) via a C2 (anomeric C)

Front 50

What are the activated forms of the following sugar donors?
Glucose, Galactose, GlcNAc, GalNAc, Glucuronic acid and xylose

Back 50

UDP-sugar

Front 51

What is the activated form of mannose and funcose?

Back 51

GDP-sugar

Front 52

What is the activated form of the sugar donor sialic acid?

Back 52

CMP-Sia

Front 53

Where are each of these activated sugar forms found?

Back 53

UDP-sugar: cytosol
GDP-sugar: cytosol
CMP-Sia: Nucleus

Front 54

How is the transport of nuc sugar into the ER/golgi accomplised?

Back 54

Transport of the nuc-di-P into the golgi
Transport of a nuc-mono-P out of golgi
->activated forms of sugars made in the cytoplasm or nucleus
->formation of glycan chain is in the secretory system (ER/golgi)
-> Activated Sugar goes into secretory system
->Sugar is removed and attached to a glycan chain
->PO4 is removed then nuc-mono-PO4 is brought back out
This process is electro neutral because the complex going in and the one going out both hve a -2 charge

Front 55

Are glycans gene products?

Back 55

No becuase you can't predict there sequence or if they'll be G (unlike with ptns)

Front 56

What is microheterogeneity?

Back 56

The same glycosylation site on the same ptn can carry different glycans
->extent of microgeneity varies btw glycosylation sites, ptns nd cell types

Front 57

What are the factors that affect microheterogeneity?

Back 57

-Ptn sequence/conformation, location of G site
-Nucleotide sugar metabolism (availability of sugars)
- Transport rate of the ptn through the ER/golgi (if very fast rate, not enough time for the enz to act .: less G)
-Localization/concentration of specific glycosyltransferase/glycosidases within the secretory pathway; competition
-Environmental conditions/developmental stage/cell or tissue type

Front 58

Why do G ptns appear as smears on an SDS-PAGE gel?

Back 58

There are differen G forms, consequence of microheterogeneity

Front 59

How can the formation of this smear be minimized?

Back 59

Take cells from the same dev'l stage, same tissue type, same cell cycle stage
-can do this by affnity/column chromatography: use a lectin to bind the glycan
->terminal residues can be the same, but underlying polypeptide chain can be very different
->however, don't usually look for just one type .: if they have the same end terminus, it shouldn't really be a problem

Front 60

What are some experimental limitations that preclude elimination of this phenomenon?

Back 60

Glycoforms can vary even within one cell type
-> Not often possible to isolate one glycoptn

Front 61

What is the most common N-linked oligosaccharide linkage?

Back 61

GlcNAcβ1-Asn

Front 62

What is the consensus sequence of N-linked oligosac?

Back 62

Asn-X-Ser/Thr
-can sometimes be Asn-X-Cys
**Just because one of these seq are present doesn't necessarily mean that G will occur, but it is possible

Front 63

What are the 3 types of N-linked oligosac?

Back 63

1) Oligomannose: 2 branches extended by mannose
2) Complex: 2 branches of sugars other than mannoses
3)Hybrid: 1 mannose branch & 1 branch of non-mannose sugars

Front 64

What is the invariable core structure of N-linked oligosac?

Back 64

Always have the same 5 sugars:
3 mannose sugars branched with α1-3 and α1-6 linkages attached to 2-GlcNAc structures

Front 65

How can N-linked oligos be characterized?

Back 65

With peptidases
N-glycosidase F
Endoglcosidase

Front 66

What does a peptide N-glycosidase F do?

Back 66

(PNGaseF, N-glycanase): release complex, hybrid and high-mannose N-glycans from Asn
->When N-glycan released, Asn is converted to Aspartate
->can see where ptn was glycosylated by looking at the chain seq before and after treatment with the glycosidase

Front 67

What do endoglycosidases do?

Back 67

Leave one GlcNAc attached to an Asn
->endoglycosidase H: releases oligomannose and hybrid N-glycans (NOT complex N glycans)
If the N-glycan was not released, it was a complex N-glycan

Front 68

How does N-glycan biosynthesis begin?

Back 68

Transfer of a common oligosac precursor to the nascent polypeptide
->Precursor is formed in the cytosol on a lipid (Dolichol)

Front 69

What is dolichol?

Back 69

Polyisoprenol consisting of repeating isoprene units

Front 70

How is the precursor linked tothe dolichol?

Back 70

Through pyrophosphate

Front 71

What are the steps to the biosynthesis of Dol-PP-oligosaccharide?

Back 71

1)Transfer of GlcNAc-P from UDP-Glc-NAc to mb-bound Dol-P
2) Transfer of the second GlcNAc (from UDP-GlcNAc) and 5 Man (from GDP-Man) residues to the precursor
3)Translocation across the ER mb (will be attached to a ptn in the ER)
->gets flipped across the ER mb with a flippase
4) Extension of the Man5GlcNAc2 by 4 Man and 3 Glc residues
->Dol-P-Man and Dol-P-Glc act as donors
-active monosac transferred from UDP to Dol-P. Dol-P then flips the monosac from the cytosol to ER where it can be attached to growing oligosac
-By end of step 4, precursor is complete
5)14 sugar chain is tansferred to Asn-X-Ser/Thr by oligosaccharyltransferase (OST)
-co-translationsl: transfer done as its being synthesized

Front 72

What is the OST complex?

Back 72

Enz complex that transfers oligosac precursor from dolichol to nascent polypeptide chain

Front 73

What does the transfer of each sugar depend on?

Back 73

Relies on a specific glycosyltransferase

Front 74

What does processing and maturation of N-glycans result in?

Back 74

Oligomannose
Complex
Hybrid

Front 75

What are the steps of processng and maturation of N-glycans?

Back 75

Precursor is transferred to nascent polypeptide chain, then:
1)Sequential removal of Glc and one of the Man residues un the ER (α-glucosidases I and II, ER mannosidase I)
2) If ptn is properly folded: trimming continues in the cis-Golgi (α1-2 mannosidases IA/B/C) (remove additiona mannoses)
3) If trimming is not complete: high mannose N-glycans (no further modification)
4) Further modifications lead to formation of hybrid and complex N-glycans

Front 76

What happens in the targetting of lysosomal enz?

Back 76

If ptn no properly folded, it enters the ER degradation path
-If ptn misfolded, it is retrotranslcated translated from the ER and degraded by the proteasome

Front 77

What are the steps to glycoptn folding and ER quality control?

Back 77

1) Nascent ptns are N-glycosylated by OST
2) Removal of 2 Glc residues
3) If ptn not yet properly folded, it ass't with a complex containing calnexin (cnx) or calreticulin (crt) and Erp57
->cnx/crt: lectins that rec'z monoglucosylated N-glycans
->Erp57: thiol oxidoreductase, helps with Di-S bond formation
4) The cnx/crt-N-glycan complex diss't when last glucose removed by GII and is reformed by α-glucosyltransferase (UGGT)
5) The cycle continues unti ptns acquire their native conformation and are transported to the Golgi (6)
OR
7) ptns are rec'z as terminally misfolded and are retrotranslocated to the cytosol and degraded by the proteasome

Front 78

What are te congenital disorders of glycosylation (CDG)? (inherited disorders in N-glycosylation)

Back 78

Type I: defects in the biosynthesis of the lipid-linked oligosac and in the transfer to the nascent polypeptide chain (**complete absence of N-glycans is lethal, these mutations create hypomorphic alleles, no comlpete KO) (probably problem with OST)
Type 2: defects in subsequent processeing steps (after transfer of precursor to nascent polypeptide chain)

Front 79

What are the clinical features of CDGs?

Back 79

Broad, but involve dev'l of certain regions of the brain and fct of the GI, hepatic, visual and immune systems

Front 80

How would you determine if a newly identified ptn is a glycosyl transferase?

Back 80

Do an in vitro glycosylation assay

Front 81

How are general N-Glycosylation functions determined?

Back 81

Inhibition of early steps in path (ex: tunicamycin: inhibits transfer of GlcNAc-P to Dol-P)
-Use G mutants
-CDG models (use cell lines from these patients)
-Transgenic animal mdels

Front 82

How are specific glycosyltransferase fcts determines?

Back 82

KO/knockdown: see what happens to the phenotype
-> good idea to rescue mutation by reintroducing the WT gene and see if it rescues the phenotype

Front 83

What is the most common O-linked glycosylation?

Back 83

Mucins

Front 84

What are mucins?

Back 84

Heavily glycosylated secreted or cell surface transmembrane ptns
-are a major constituent of mucus secretion s on cell surfaces and in body fluids
-hydrate and protect eyes, respiratory, GI and genitourinary tracts

Front 85

What are the fcts of mucins?

Back 85

1) Provide protection vs infection
->trap bacteria using specific receptor sites
->Mask underlying receptors/Ag (cuz they form a layer btw the cell and the env't, since they're heavily G)
2) Involved in signal transduction and cell-cell adhesion
3) Often antigeni
4) Protect the underlying ptn from degradation
5) Affect ptn stability/expression lvls

Front 86

What is the structure of mucin?

Back 86

α-linkage of GalNAc to -OH of Ser/Thr
-occurs at variable number of tandem repeat (VNTR) seq, regions that are rich in Ser/Thr O-glycan receptor sites
-100s of glycan chains can be attached in these regions
The carbohydrate moiety can be divided into a core, peripheral and backbone region

Front 87

What are the 4 most common mucin core structures?

Back 87

Core 1: not branched, Gal in β1-3 with GlcNAc and α-linked Ser/Thr
Core 2: Gal β1-3 to GalNAc, GlcNAc β6 to GalNAc and α-link to Ser/Thr
Core 3: not branched, GlcNAc to GalNAc, α-linked to Ser/Thr
Core 4: 2 GlcNAc linked to first GalNAc, α-link to Ser/Thr

Front 88

Describe the backbone region.

Back 88

Links core and peripheral regions
-Extremely variable
-Provides a scaffold for the attachment of additional sugars or fctnal gps
->Type 1 repeats: Galβ1-3GlcNAc
-> Tye 2 repeats: Galβ1-4GlcNAc

Front 89

Desribe the peripheral region.

Back 89

Region at end of glycan cain, can react with the external environment
-Carbohydrate structure with a variety of biological recognition properties
->Antigenic determinants (elicit immune response)
-> cellular recognition, cell-cell adhesion
-> binding of pathogens

Front 90

What does mucin biosynthesis depend on?

Back 90

Glycosyltransferase dependent transfer of monosaccharides from a nucleotide sugar donor to a peptide/oligosac chain
->occurs in the ER/golgi (secretory system, like as in N-glycosylation)

Front 91

What are some differences btw mucin and N-glycan synthesis?

Back 91

-Does NOT involve lipid linked intermediates or glycosidases (no Dol-P)
-Is NOT co-translational (happens after the ptn is translated)

Front 92

What is the 1st step of mucin biosynthesis?

Back 92

Transfer of GalNAc to Ser/Thr (highly conserved step)
-Cata;yzed by a polypep-N-acetylgalactosaminetrannsferase (ppGalNAcT)
-Preferentially glycosylates sites found at β-turns in proline-rich regions
-May preferentially G either Ser or Thr residues
-Gat one site is often dependnt on prior G at an adjacent site
-Members of this family are found throughout the golgi

Front 93

How nis mucin biosynthesis controlled?

Back 93

-Chain extension is catalyzed by variousglcosyltransferases present in the golgi
-Regulation/glycan sequence determination:
->assembly line arrangement of glycosyltransferases: ones at beginning of golgi will G near the polypep region, ones near the end will G near the peripheral regions
->Acceptor specificity: if adjacent site requires G
->Relative activities: high rate of activity G more
->Underlying peptide: proline/β-turns, presence of Ser/Thr
-Heterogeneity:
->Multiple enz may compete for the same substrate (throughout the whole secretory system, which enz G depends on cell ycle, env't etc)
-> enz overlap with respect to both substrates and localization: 2 enz for the same substrate can be in the same region