lecture 32-34 - glycan binding proteins - lectins

Question 1

what do lectins bind? with what affinity?

Answer 1

• tend to bind monosaccarides and oligosaccarides
• N-glycans, O-glycans, glycolipids
• few recognize sulfated GAGs
• low affinity

Question 2

describe N-glycans

Answer 2

• oligosaccarides

- connected to proteins through N-acetyl glucosamine that is N-linked to an Asn res

Question 3

describe O-glycans

Answer 3

-oligosaccarides
-connected to proteins through an O-linkage to Ser/Thr
(typically connected through N-acetyl glucosamine, but other sugars have been found as well)

Question 4

glycolipids

Answer 4

• oligosaccarides

- O-linked to lipid chains through glucose or galactose

Question 5

are lectins branched or unbranched structures? is this the same or the opposite of GAGs?

Answer 5

• branched

- opp of GAGs

Question 6

where do lectins typically bind glycans?

Answer 6

• terminal grps (minimal binding epitopes=what they bind)

- di, tri, tetra-saccarides displayed at the ends of glycans

Question 7

are lectins evolutionarily related?

Answer 7

yes

Question 8

how are lectins classified into groups?

Answer 8

grouped into evolutionarily related families based on carbohydrate-recognition domains (CRDs)

Question 9

what do Galectins (fam of lectins) bind?

Answer 9

Beta-galactose containing glycans

Question 10

explain prototypical galectins

Answer 10

• single CRD

- can associate as homodimers

Question 11

explain chimera galectins

Answer 11

• single CRD
• CRD has a terminal polypeptide tail (usually rich in pro, gly, tyr)
• tail can help form oligomers

Question 12

explain tandem galectins

Answer 12

• 2 CRDs
• CRDs connected through polypep-linker
• each CRD can have diff galectin binding properties

Question 13

what are C-type lectins?

Answer 13

• calcium dependent GBPs
• share primary and secondary homology in their CRDs
• are some exceptions to these

Question 14

explain the CRDs for c-type lectins

Answer 14

• most have a single CRDs

- most are transmembrane proteins

Question 15

what can c-type lectins do to affect bidning affinity? what is the effect?

Answer 15

• can cluster together

- increase binding affinity

Question 16

what do p-type lectins bind?

Answer 16

monnose-6-phosphate on N-glycans

Question 17

what are the actions of p-type lectins?

Answer 17

selective delivery of lysosomal enzymes to lysosomes

Question 18

what family to I-type lectins belong to? why? what does this exclude?

Answer 18

• immunoglobulin super family (bc have similar folds)
• excludes antibodies and t-cell receptors
• most are transmembrane proteins

Question 19

give an example of a major family of I-lectins and their recognition site

Answer 19

-siglecs (sialic acid binding immunoglobulin type lectins)
-have two types of the immunoglobulin folds:
Cset - constant like domain
Vset - variable like domain = carb recognition domain

Question 20

how can the affinity of lectin binding be enhanced?

Answer 20

by multivalency (multiple copies of the carb epitope interact with multiple copies of the lectin/CRD

Question 21

how can multivalency occur?

Answer 21

multivalency can occur w/in glycan/glycoprotein structures OR by clustering (of CRDs)

Question 22

describe monovalent interacitons

Answer 22

• one protein binds one CRD from the lectin to form a glycan

- have part of the glycan unbinded

Question 23

describe multivalent interactions

Answer 23

• multiple binding epitopes on a single glycan bind multiple CRDs
• forms a branched glycan

Question 24

describe multiple glycan binding

Answer 24

• multiple glycans on a single protein scaffold bind multiple CRDs
• have extra branches

Question 25

describe single glycan w multiple binding epitopes

Answer 25

causes clustering of CRD containing proteins

Question 26

describe multiple glycans w single binding epitope

Answer 26

- causes clustering of CRD containing proteins - MOST COMMON - have extra brances

Question 27

CRD recognition sites are selective for what?

Answer 27

specific glycan structures or epitopes

Question 28

what blocks/prevents epitope binding?

Answer 28

- adding sugar residues to epitopes (Fuc, NeuSAC) | - secondary interactions can block OR enhance binding

Question 29

describe the CRD site for C-type lectins

Answer 29

recall: bind Ca and gylcans, CRD has: - 2 (long) loops - where bind Ca (typically bind 2-4) - 2-stranded antiparallel beta-sheet connected by 2-alpha-helicies and a 3-stranded antiparallel beta sheer - 2 conserved disulfide bonds (alpha1-beta5 and beta3-beta5)

Question 30

describe the interactions that occur at the CRD for c-type lectins

Answer 30

- the Ca that binds glycans/sugar is located btwn B4 and the loop - commonly found surrounding aa = asn, asp, glu - carbonyl side chains (c=o) from both the CRD and the sugar coordiate their interaction - H-bonding also occurs (H from aa to O from sugar) ( OH* from sugar to N/O from aa)

Question 31

describe the CRD site for I-type lectins (siglecs specifically)

Answer 31

- recall: CRD = vset domain & likes to bind sialic acids - CRD is on one face of the beta sandwich (typically btwn sheets A and G) - usually a larger, flat surface w two types of residues: - residues at the top = typically hydrophobic (tyr, trp, phe) - other = always ARG (basic/positively charged) = very important for recognition!!

Question 32

describe the struture of a siglec (what are the key motifs?)

Answer 32

6 carbon ring (chair) - c4 = glycerol side chain - c3 =actually an O - c2 = carboxylate and the -O-sugar - c6 = OH - c5 = acetymide (NHAc)

Question 33

which parts of the siglec interact with the aromatic groups in the CRD? what type of interaction is this?

Answer 33

- methyl group of the NHAc and the glycerol side chain | - hydrophobic interactions

Question 34

how is the glycerol side chain able to interact with the aromatic group despite the fact that it has many hydroxyl groups?

Answer 34

its flexible - able to create a hydrophobic (to interact w hydrophobic aromatics) and a hydrophilic face

Question 35

why is the arg residue so important for I-type CRD interactions? what type of interaction is this?

Answer 35

- forms a salt bridge btwn the guanalyle group from the Arg (pos) and the carboxylate group from the siglec (neg) - have ionic (salt bridge) and hydrogen bonding

Question 36

is the arg residue highly conserved throughout I-type CRD? why or why not?

Answer 36

- yes | - responsible for recognizing and binding siglecs

Question 37

what two modifications to the siglec would decrease binding to its CRD?

Answer 37

(1) add OH to the NHAc group (makes more hydrophilic - less hydrophobic interactions) (2) cleave glycerol side chain (also decreases hydrophobic interactions)