Specific N-linked glycosylation sites modulate synthesis and secretion of von Willebrand factor

Abstract

We examined the role that N-linked glycans play in the synthesis and expression of von Willebrand Factor (VWF). Blocking the addition of N-linked glycans (NLGs) or inhibiting initial glycan processing prevented secretion of VWF. To determine whether specific glycosylation sites were important, the 16 VWF N-linked glycosylation sites were mutated followed by expression in HEK293T cells. Four NLG mutants affected VWF expression: N99Q (D1 domain), N857Q (D' domain), N2400Q (B1 domain), and N2790Q (CK domain) either abolished or reduced secretion of VWF and this was confirmed by metabolic labeling. Multimer analysis of mutant N2790Q cell lysate revealed an increase in VWF monomers, which was also observed when the isolated CK domain was expressed with N2790 mutated. Immunofluorescence microscopy showed that mutants N99Q, N857Q, and N2790Q were primarily retained within the ER, producing only few pseudo Weibel-Palade bodies over longer time periods compared with wtVWF. All the variants also showed an increase in free thiol reactivity. This was greatest with N857Q and D4-C2 NLG mutants, which had approximately 6-fold and 3-to 4-fold more free thiol reactivity than wtVWF. These data provide further evidence of the critical role that individual N-linked glycans play in determining VWF synthesis and expression. (Blood. 2010;116(4):640-648) Introductionvon Willebrand factor (VWF) is a large multimeric plasma glycoprotein essential for normal hemostasis, acting firstly by supporting platelet adhesion to surfaces at sites of vascular injury and secondly as the carrier molecule for pro-coagulant Factor VIII (FVIII). 1,2 Synthesis of VWF is limited to megakaryocytes and endothelial cells. 3 The pre-pro-VWF molecule comprises a 22 amino acid signal peptide, a 741 amino acid propeptide, and a 2050 amino acid mature subunit. The pro-VWF monomer is composed of 4 types of domains (A-D) arranged as follows: NH 2 -D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK-COOH. 1 VWF multimers are formed by C-and N-terminal intermolecular disulphide bonds, 4,5 with the largest multimers exceeding 2 ϫ 10 4 kDa and having the greatest adhesive activity. 6 During synthesis, VWF undergoes extensive posttranslational modification resulting in the addition of 12 N-linked and 10 O-linked glycosylation sites per mature monomer. 7 Furthermore, the propeptide also contains 4 potential N-linked glycosylation sites although it is not known if these are used. In total, carbohydrate accounts for approximately 20% of the molecular weight of VWF. 8 N-linked glycosylation is one of the most common co/ posttranslational modifications. It is characterized by the addition of a carbohydrate moiety to the protein via a beta-glycosidic linkage between an N-acetylglucosamine residue and the ␦-amide of an asparagine residue in the sequence NXS/T (or in some cases NXC), 9 where X is any amino acid except proline. 10 This takes place after translocation into the endoplasmic reticulum (ER) when the enzyme oligosaccharyltransferase (OST) transfers a preformed 14-saccharide core...

N-linked glycans within the A2 domain of von Willebrand factor modulate macrophage-mediated clearance

“…59 Although the molecular mechanism through which N-linked glycan determinants regulate coagulation glycoprotein clearance remains unknown, the effect may be due to general properties of the complex sugar chains or instead may be attributable to particular carbohydrate structures located at specific N-linked sites. 42,43 Given the role of the A1-A2-A3 domains in modulating the interaction of VWF with macrophages, we further investigated whether the 2 N-linked glycosylation sites located at N1515 and N1574, respectively, might play a particular role in regulating VWF clearance. Importantly, we observed that loss of N-linked glycans following site-directed mutagenesis at either N1515 or N1574 resulted in markedly enhanced clearance of full-length rVWF.…”

“…In keeping with previous studies defining the biological significance of these glycans, [42][43][44] each asparagine residue was mutated to glutamine (N1515Q and N1574Q) to eliminate the N-linked glycans at these positions. Mutations were verified by DNA sequencing to ensure the absence of any other randomly introduced mutations.…”

“…by guest www.bloodjournal.org From ( Figure 3A). 7,43 To determine whether these additional N-linked glycans may also be important in regulating VWF clearance, we expressed a rVWF variant from which the A2 domain was deleted (DA2-VWF) ( Figures 3A-B). Interestingly, in vivo clearance of DA2-VWF was not accelerated following PNGase F digestion ( Figure 3C), suggesting that the N-linked glycans within the A2 domain play a critical role in modulating the enhanced clearance of PNG-VWF.…”

O-linked glycosylation of von Willebrand factor modulates the interaction with platelet receptor glycoprotein Ib under static and shear stress conditions

“…14,15 VWF N-linked glycans have been studied extensively, although the data on the role of VWF OLGs are limited and often contradictory. 16,17 VWF lacking O-glycans is synthesized, multimerized, secreted, and binds heparin and collagen type I in the same way as the fully glycosylated form. 18 We recently published a detailed study of the VWF O-glycome composition showing the presence of unusual disialosyl motifs and ABH-bearing core 2 structures.…”

Lateral self-association of VWF involves the Cys2431-Cys2453 disulfide/dithiol in the C2 domain

“…8,12,13 The mechanism of formation of the VWF strings involves cysteine thiol/disulfide exchange, because the formation of the strings is inhibited by thiol alkylation 12 and the string structure is disrupted by the small thiol N-acetylcysteine. 14 Mature VWF, both purified from plasma and made recombinantly, contains unpaired cysteine thiols 12,[15][16][17] that have been localized to the N-terminal D3 and C-terminal C domains. 12,13,17 Presumably, one or more of these cysteine thiols is involved in VWF self-association.…”