2 FORMS OF COLLAGEN-XVII IN KERATINOCYTES - A FULL-LENGTH TRANSMEMBRANE PROTEIN AND A SOLUBLE ECTODOMAIN

The cDNA sequence of human collagen XVII predicts an unusual type II t ransmembrane protein, but a biochemical characterization of this struc ture has not been accomplished yet. Using domain-specific antibodies a gainst recombinant collagen XVII fragments, we identified two molecula r forms of the collagen in human skin and epithelial cells. Full-lengt h collagen XVII appeared as a homotrimeric transmembrane molecule of t hree 180-kDa alpha 1(XVII) chains. The globular intracellular domain w as disulfide-linked, and the N-glycosylated extracellular domain of th ree 120-kDa polypeptides was triple-helical at physiological temperatu res. A second, soluble form of collagen XVII in keratinocyte culture m edia was recognized with antibodies to the ectodomain, but not the end odomain, The soluble form exhibited molecular properties of the collag en XVII ectodomain: a triple-helical, N-glycosylated molecule of three 120-kDa polypeptides, Northern blot analysis with probes spanning eit her the distal 5' or the distal 3' end of the collagen XVII cDNA revea led an identical 6-kb mRNA, suggesting that both the 180- and 120-kDa polypeptides were translated from the same mRNA, and that the 120-kDa polypeptide was generated post-translationally, In concert, keratinocy tes harboring a homozygous nonsense mutation in the COL17A1 gene synth esized neither the 180-kDa alpha 1(XVII) chain nor the 120-kDa polypep tide. Finally, treatment of normal keratinocytes with a synthetic inhi bitor of furin proprotein convertases, decanoyl-RVKR-chloromethyl keto ne, prevented the generation of the 120-kDa polypeptide. These data st rongly suggest that the soluble 120-kDa polypeptide represents a speci fically cleaved ectodomain of collagen XVII, generated through furin-m ediated proteolytic processing. Thus, collagen XVII is not only an unu sual type II transmembrane collagen, but the first collagen with a spe cifically processed, soluble triple-helical ectodomain.