The propeptide, vW AgII, trafficks vWF to granular storage in AtT-20 cells.

vW AgII functions as an intracellular chaperone. AtT-20 cells were transiently transfected with human propeptide alone, vW-AgII, human mature vWF alone, or Hu-Δpro or were cotransfected with Hu-vW-AgII + Hu-Δpro. After 72 hours, cells were fixed, permeabilized, dual stained with a mix of monoclonal antibodies to vW AgII and a polyclonal antibody to vWF, and detected with Texas Red-conjugated donkey antimouse IgG and FITC-conjugated donkey antirabbit IgG by confocal microscopy. (A-C) Cells stained for vW AgII. (D-F) vWF staining. The merges of vW AgII stain and vWF stain are shown in G to I. Colocalization of vW AgII and vWF is shown in yellow. Expression of vW AgII alone resulted in granular storage of vW AgII, as shown in A, indicating that vW AgII contains the epitope or conformation necessary for sorting to regulated storage; no vWF was detected (D). Expression of mature vWF alone (B, E, H) showed only a cytoplasmic staining pattern of vWF; no granular storage was observed. Expression of vW AgII in trans with mature vWF resulted in granular storage of both vW AgII and vWF (C, F, I). Furthermore, the 2 proteins were colocalized in storage granules, as shown in I. This suggests that vW AgII contains the necessary signal(s) for sorting to storage, and only through interaction with vW AgII is vWF sorted to storage. Total magnification, 1410×.