Structure and manipulation of the VWF A2 domain N-linked glycans. (A) Crystal structure of the VWF A2 domain (3ZQK, chain C) is shown with the 2 N-linked glycosylation attachment sites (N1515 and N1574), scissile bond (Y1605-M1606), vicinal disulfide bond (C1669-C1670) and calcium binding site (Ca²⁺) residues in black. (B) Basic representation of N-linked glycan structure with asparagine backbone attachment motif and carbohydrate structure shown in black. Enzymatic cleavage sites (PNGase and EndoH) and inhibition of complex sugars (GnT^−/−) shown by gray dashed lines with approximate MWs displayed. (C) VWF A2 domain fragments with full glycosylation (WT), mutated asparagine attachment site(s) (N1515, N1574Q, and N1515/N1574Q), or truncated glycan structures (GnT^−/−, EndoH, and PNGase) after expression with or without enzymatic treatment and purification were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on a 4% to 12% Bis-Tris gel followed by silver staining to determine changes in MW/glycan structure.