RECOMBINANT ANALYSIS OF HUMAN ALPHA-1(XVI) COLLAGEN - EVIDENCE FOR PROCESSING OF THE N-TERMINAL GLOBULAR DOMAIN

The N-terminal non-collagenous domain NC11 of the human collagen alpha 1(XVI) chain was obtained as a recombinant 35-kDa protein from stably transfected kidney cell clones. This form had undergone proteolytic t rimming at a basic cleavage motif indicating a similar release in vivo . Domain NC11 showed a globular shape after rotary shadowing and was r esistant to neutral proteases. Specific antibodies could be raised aga inst recombinant NC11 and were used for the analysis of other cell clo nes transfected with the full-length alpha 1(XVI) chain. Immunoprecipi tation of detergent extracts of metabolically labelled cells demonstra ted the presence of disulfide-bonded 200-kDa polypeptides possessing N C11 epitopes. This material was partially resistant to pepsin, indicat ing the formation of alpha 1(XVI) chain homotrimers with a triple-heli cal conformation. Yet a substantial proportion of these homotrimers wa s degraded to fragments of variable size (35-150 kDa) when secreted in to the culture medium. Several of these fragments could be obtained on a semi-preparative scale from cells grown in hollow fiber cassettes a nd showed substantial hydroxylation of proline, consistent with triple -helix formation. Edman degradation demonstrated the origin of some fr om the N-terminal and of one from a more C-terminal position of collag en XVI. This extensive degradation may be explained by the release of NC11 and by further cleavages within some of the nine interruptions of the triple-helical domain of the alpha 1(XVI) chain. Whether this pro cess also occurs in situ remains to be shown.