A single amino acid substitution enables canine VWFpp to store human Δpro VWF.

Canine VWFpp's containing single human amino acid substitutions were coexpressed in trans with human Δpro in AtT-20 cells. Panels A, D, G, and J show cells stained for VWFpp (green). Panels B, E, H, and K show cells stained for VWF (red). The merges of VWFpp and VWF staining are shown in panels C, F, I, and L. Colocalization of VWFpp and VWF produces granules (yellow). In panels A-C, a canine VWFpp with a valine-to-isoleucine substitution at amino acid 425 (C-VWFpp-Val425Ile) coexpressed with human Δpro showed a granular staining pattern for the mutated canine VWFpp (A), but the human VWF was not cotrafficked to storage (B-C). Similarly, a canine VWFpp with an isoleucine-to-leucine substitution at amino acid 473 (C-VWFpp-Ile473Leu) also did not cotraffic human Δpro (E-F), although normal storage of the mutated VWFpp was maintained (D). A single, nonconservative substitution in canine VWFpp at amino acid 416, glutamine to arginine (C-VWFpp-Gln416Arg), resulted in a granular staining pattern for both the mutated VWFpp (G) and human VWF (H). The 2 proteins were colocalized in granules (I). The converse, an arginine-to-glutamine substitution at amino acid 416 in human VWFpp, resulted in a loss of human VWF storage (K-L), with a normal granular staining pattern for the mutated human VWFpp (J). A single amino acid, 416, is important for VWFpp/VWF association and costorage. Bar, 10 μm.