Properties of the collagen type XVII ectodomain - Evidence for N- to C-terminal triple helix folding

Collagen XVII is a transmembrane component of hemidesmosomal cells with imp ortant functions in epithelial-basement membrane interactions. Here we repo rt on properties of the extracellular ectodomain of collagen XVII, which ha rbors multiple collagenous stretches. We have recombinantly produced subdom ains of the collagen XVII ectodomain in a mammalian expression system. rCol XVII-A spans the entire ectodomain from delta NC16a to NC1, rColXVII-B is s imilar but lacks the NC1 domain, a small N-terminal polypeptide rColXVII-C encompasses domains delta NC16a to C15, and a small C-terminal polypeptide rColXVII-D comprises domains NC6 to NC1, Amino acid analysis of rColXVII-A and -C demonstrated prolyl and lysyl hydroxylation with ratios for hydroxyp roline/proline of 0.4 and for hydroxylysine/lysine of 0,5, A small proporti on of the hydroxylysyl residues in rColXVII-C (similar to3.3%) was glycosyl ated. Limited pepsin and trypsin degradation assays and analyses of circula r dichroism spectra clearly demonstrated a triple-helical conformation for rColXVII-A, -B, and -C, whereas the C-terminal rColXVII-D did not adopt a t riple-helical fold. These results were further substantiated by electron mi croscope analyses, which revealed extended molecules for rColXVII-A and -C, whereas rColXVII-D appeared globular, Thermal denaturation experiments rev ealed melting temperatures of 41 degreesC (rCol/XVII-A), 39 degreesC (rColX VII-B), and 35 degreesC (rColXVII-C), In summary, our data suggest that tri ple helix formation in the ectodomain of ColXVII occurs with an N- to C-ter minal directionality.