PTM

Question 1

Function of O linked glycans in vwf

Answer 1

VWF O-linked glycans present ABO blood group sugars and a rare double sialic acid motif
•Sialic acids are negatively charged monosaccharides with diverse function in vWF
•Protection from proteolytic degradation and modulation of protein clearance;
•Presence of sialic acid leads to decrease sensitivity to ADAMTS13 proteolysis
•Therefore, reduced sialylation results in reduced plasma VWF levels and more rapid clearance from blood stream
Sialic acid on OLG also affects GpIb binding, in a state of reduced sialic acid expression there will be increased platelet binding;
•Highlighted by the use of Ristocetin; as Ristocetin levels increase platelet binding is increased,
•At the same levels of, when there is Desialylation of the vWF, there is increased levels of platelet binding (mainly influenced by the OLGs found between A1 and D3.

Question 2

OLG of vwf

Answer 2

While NLG are spaced out, OLG seem to be clustered around A1 (platelet binding) region of vwf. Surrounding the A1 is D’D3 on one side, with 4 OLG sites in this linker region between them and on the other side is A2 with another linker region between them with another 4 OLG sites.

Question 3

NLG in vwf

Answer 3

There are 12 NLG sites on vWF, all providing different functions and properties:

1. Formation of vWF dimers/ multimers:
   •Prevention of NLG results in intracellular retention of VWF monomers
   oUsing ELISA, its shown that the first NLG at the 99th amino acid in the D1 region is vital; if the amino acids is changed from N (asparagine) to glutamine (Q) NLG can no longer take place and vWF is retained within the cell.
   •N-linked glycosylation in the CK knot is required for efficient dimer formation
2. Presenting ABO blood sugars (n linked glycan chains of vwf are decorated with aboh blood sugars) and the type affect vwf levels
   • H transferse attaches a fucose residue to the terminal galactose of the N linked glycan structures. This is blood group O. A transferase adds Galnac and B transferase adds galactose.
   • vWF-NLG present ABO group determinants – this influences vWF plasma levels
   • 25% lower level of VWF in group O individuals
   • Within non-O blood group VWF levels were highest in AB individuals, followed by group B then group A
   • Lowest VWF levels in Bombay blood group (no H-antigen)
3. Disulphide bond formation = maintenance of globular structure of vWF
   •Inhibition of NLG leads to early exposure of binding sites and an increased tendency to clot
4. Protection from proteolysis by ADAMTS13
   •Inhibition of NLG (particularly the N1574 NLG in the A2 domain) results in increased cleavage by ADAMTS13

Question 4

NLG functions (general) (thermal stability)

Answer 4

In general, glycoproteins are more stable than their corresponding unglycosylated counterparts, even when there are no major structural changes associated with glycosylation.
oSerine protease inhibitor with two glycosylation sites at Asn10 and Asn52, was examined in both the glycosylated and non-glycosylated forms. The melting temperature (Tm) of the glycosylated derivative was increased by 4.8°C, corresponding to an increase in the free energy of unfolding. (DeKoster et al.)

Question 5

NLG other functions

Answer 5

Disulphide bond formation- In the extracellular loop of the Na+ coupled transporters, the formation of the first disulfide bond relies on the presence of the second disulfide bond and is affected by N-glycosylation. When the two disulfide bonds are missing, EL-3 adopts an extended conformation highly accessible to protease digestion. (J Biol Chem 2015)
Protection from proteolysis - Mutants lacking Asn78 in HCG are poorly secreted and rapidly degraded in vivo (Barabara Imperiali et al.)
Protein clearance - EPO produced with incompletely processed NLG exhibit normal in vitro activity but is at least 500-fold less effective in stimulating erythropoiesis in vivo. Studies on the survival of bioactive EPO remaining in the circulation, demonstrated that EPO with incomplete NLG exhibisted a sevenfold increased rate of clearance. (Wasley et al. 1991)

Question 6

Generic OLG

Answer 6

Its functions are less well understood than NLG, but it provides stability to flexible regions of proteins and may have roles in clearance and ligand recognition
•The above study by Wasley showed that EPO produced without O-linked carbodyrates exhibit normal in vivo acitivity and clearance. (Wasley et al. 1991)
•O-linked glycosylation confers protease and heat resistance to glycoproteins, for example, to the mucins (Gowda and Davidson, 1994) The decay accelerating factor (DAF; CD55) is another typical example. When the O-linked sugars are removed from DAF, the molecule undergoes proteolysis (Coyne et al, 1992).