Abstract
N-linked glycosylation is an important co-translational protein modification affecting protein folding, secretion, stability & function. We have previously demonstrated that N-linked glycosylation of Von Willebrand Factor (VWF) modulates its interaction with ADAMTS13, the plasma metalloprotease responsible for controlling VWF multimeric size. In the present study we have investigated the role of N-linked glycosylation in modulating ADAMTS13 expression, secretion & function.
Lectin analysis of plasma derived & recombinant ADAMTS13 (rAD13) demonstrated that ADAMTS13 presents sialic acid & galactose residues, indicating the presence of complex glycans structures. Analysis with the lectin GNL, specific for high mannose structures showed that plasma derived ADAMTS13 also contained a small portion of basic high mannose glycans & this was increased in rAD13. Enzymatic modification of rAD13 with PNGase F to remove whole N-linked glycans & neuraminidase to remove terminal sialic residues were completely effective. However, neither modification affected the enzymatic activity of ADAMTS13 towards VWF, indicating that following secretion ADAMTS13 N-linked glycans are expendable.
Using an ADAMTS13 ELISA and western blotting, we showed that expression of rAD13 in the presence of Tunicamycin prevented N-linked glycosylation & prevented secretion, indicating that N-linked glycosylation is essential for secretion of ADAMTS13. Expression in the presence of the glucosidase inhibitor castanospermine, that inhibits binding to the endoplasmic lectin chaperones calnexin & calreticulin, also prevented secretion of ADAMTS13 suggesting that glycan dependent interaction with these chaperones is essential for the correct cellular processing of ADAMTS13
To further explore these observations, 10 individual point mutations encoding asparagine to glutamine changes were generated to prevent glycosylation at each of the 10 N-linked glycans sites in ADAMTS13: N462 & N46Q in the metalloprotease (MP) domain, N552Q in the cysteine rich domain, N579Q, N614Q & N667Q in the spacer domain, N707Q in the second Thrombospondin repeat (TSR), N828Q in the forth TSR and N1235Q and N1354Q in the CUB domains. Mutations N42Q, N579Q, N614Q, N707Q, N828Q and N1354Q caused only a slight to moderate reduction in ADAMTS13 secretion; 57%, 62%, 50%, 84%, 70% and 68% of wild type respectively. Mutations N552Q and N667Q significantly reduced ADAMTS13 secretion to 23% and 25% of wild type respectively indicating that glycosylation of the cysteine rich & spacer domains is required for efficient secretion. Significantly, mutation N46Q in the MP domain abolished secretion resulting in intracellular retention of ADAMTS13.
All the secreted mutants with the exception of N828Q and N1235Q were able to effectively proteolyse the short VWF-115 substrate and full length VWF (FL-VWF). Interestingly, N828Q was demonstrated to have reduced activity towards VWF-115 and FL-VWF under both static and flow conditions. Although binding to immobilised VWF A2 domain and FL-VWF was not altered, a difference was observed in the binding of N828Q to globular FL-VWF. Conversely, mutation of N1235 increased activity of ADAMTS13 towards VWF.
Together these data demonstrate that N-linked glycosylation of ADAMTS13, in particular at N46, is essential for efficient cellular processing & secretion of the enzyme. Following secretion the N-linked glycans are not required for proper function; however glycosylation of TSR 4 and the CUB domains are required for ADAMTS13 to assume a correct conformation for proper enzymatic activity towards VWF.
Frameshift intermediates in homopolymer runs are removed efficiently by yeast mismatch repair proteins.
