L5: Proteoglycans and Signalling

Question 1

PG signalling: How do PGs act and why are they so diverse in effect?

Answer 1

• Signalling pathways still function in absence of PGs -> co-receptors
• PGs can influence a vast range of signalling pathways because their side chains (GAGs) are capable of huge diversity in structure

Question 2

GAGs: Structure, examples

Answer 2

• Glycosaminoglycans
• Repeating disaccharide of around 80 sugar residues
• Each residue, apart from those of HA, may be sulphated
• e.g. chrondroitin / dermatan sulphate, keratan sulphate, hyaluronan, heparan sulphate (HS / heparin

Question 3

How are PGs structured?

Answer 3

• Core protein
• One or more GAGs attached

Question 4

Examples of cell-surface processes of PGs (beyond co-receptor activity):

Answer 4

• Dimerisation
• Two-dimensional sliding
• Surface transport
• Transcellular transport

Question 4

Families of membrane PGs (x2)

Answer 4

• Syndecans (transmembrane with core protein with HS and CS GAGs)
• Glypicans (anchored to PM via GPI, core globular domain with HS GAGs); major part of the extracellular matrix
• 4 mammalian syndecans exist, with differential tissue expression etc
• 6 mammalian glypicans exist
• In both cases, heparan sulfate is able to bind E-C ligands and modulate their interactions with cell surface receptors

Question 5

What is dally?

Answer 5

• Glypican found in drosophila (discovered as a mutation in which glypican is not being synthesised correctly, producing only a weak signal (signalling still occurs in part)
• Dally is involved in diffusion of Dpp; abnormally delays cell division -> in dally mutant the shape of the morphogen gradient is altered (shortened, doesn’t reach as far from source in short periods)
• • Dally-like is needed for Hh signalling in the developing wing
• • Dpp: Growth-promoting morphogen in TGFb family

Question 5

Types of concentration responses in cell signalling (x2):

Answer 5

• Cliff-edge like (defined thresholds)
• Gradient (response observed even in very small concentrations)

Question 6

Potential defects in PGs: (x3)

Answer 6

• Incorrect synthesis of core or lack of sulphation -> weak signal
• Overactive PG which does not release morphogens once bound -> weak signal
• Overactive notum -> PGs cleaved in excess, unable to localise morphogen properly -> weak signal

Question 7

Further examples of PG mutants:

Answer 7

• Sugarless (UDP glucose dehydrogenase)
• Sulphateless (sulfotransferase)

Question 8

Origin of HS diversity:

Answer 8

• Different esters on the disaccharides can be sulfated -> increasing or decreasing negative charge
• e.g. Sulfation or lack thereof at a particular position is essential for binding of certain FGFs
• e.g. specifically, HGF binding necessitates L-iduronic acid to be unsulphated

Question 9

What are Sulfs?

Answer 9

• e.g. Qsulf
• Qsulf enzymes remove sulphates -> post translational modification of PGs
• Discovered in quail
• Known to have major roles in development and cancer -> explore further?

Question 10

+ Expression of GPC3 in cancer

Answer 10

• GPC3 is overexpressed in >70% of hepatocellular carcinomas
• Not in normal adult tissues

Question 11

+ Role of Wnt in healthy adult tissues:

Answer 11

• Wnt signalling promotes tissue renewal and regeneration
• Highly hydrophobic

Question 12

+ What 4 groups of molecules do glypicans typically interact with?

Answer 12

• Morphogens
• Cytokines
• Chemokines
• Growth factors

Question 13

+ Disease associated with dysfunction of glypicans (human)

Answer 13

• SGBS (Simpson Golabi Behmel Syndrome)
• X-linked overgrowth syndrome mainly affecting males, also causes susceptibility to certain malignancies
• Associated with loss of function of GPC3
  *

Question 14

+ SULF2 in human cancers:

Answer 14

• SULF2 is upregulated in >60% of human cancers
• Wnt is thus released from GPC3 (2-O and 6-O sulfation found to be essential for binding; SULF2 is a 6-O-desulfatase)

Question 15

+ What tissues are Syndecans 1-3 associated with?

Answer 15

• 1: Epithelial
• 2: Mesenchymal
• 3: Neuronal tissue and cartilage

Question 16

+ In what context may syndecans be released from the membrane

Answer 16

• They are typically transmembrane proteins
• However, through the action of sheddases they can become soluble active ligands (e.g. MMPs, disintegrin) -> possibly operating using sRNAs (specifically miRNA)
• Termed the shedding process

Question 18

+ Which 3 glypicans are associated with cancer?

Answer 18

• GPC1
• GPC3
• GPC5

Question 19

+ How does GPC1 influence carcinogenesis?

Answer 19

• Shows increased expression in human gliomas and glioma-derived cell lines
• Enhances FGF basic signalling and mitogenesis
• Also overproduced in pancreatic and breast cancer cell lines

Question 20

+ Evidence for role of GPC1 in pancreatic cancer progression:

Answer 20

• Antisense depletion of GPC1 in pancreatic cancer cells reduces their ability to form tumours in vivo

Question 21

+ Name the cartilage CS proteoglycan and its key GAG chain:

Answer 21

• Aggrecan
• Hyaluronan

Question 22

+ What are the two components of GAG disaccharide building blocks?

Answer 22

• Amino sugar (which can be N-acetylated or N-sulfated)
• Uronic acid (glucuronic or iduronic acid) or galactose

Question 23

+ Additional example of cell surface PGs beyond syndecans and glypicans which is found on stem cells:

Answer 23

• NG2: Surface marker expressed on stem cell populations, cartilage chondroblasts, myoblasts etc

Question 24

+ What are slit proteins? What are their receptor and co-receptor?

Answer 24

• Large secreted glycoproteins characterised by unusual tandem of 4 LRR domains
• Various functions including guiding neural development, vasculature and immune system
• Receptors: Robos via Syndecans

Question 25

+ What is a ‘part-time’ PG? How is it different from syndecan and glypican?

Answer 25

• Syndecan and glypican bear HS chains at all times
• Part-time PGs have HS chains under certain conditions
• e.g. CD-44, betaglycan

Question 26

+ What is the function of the protein cores of PGs?

Answer 26

• Proffer HS chains to the E-C environment at defined distances and volumes
• Also influence rate and mechanisms of HS turnover

Question 27

+ What roles do syndecans 1 and 3 play in HIV infection?

Answer 27

• Syndecan-3: Binding interaction of initial pathogen to host cell -> internalisation to macrophage etc (readily express HSPGs)
• Syndecan-1: Promotes lymphoma migration when HIV expresses Tat protein