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 trimming at a basic cleavage motif indicating a similar release in vivo. Domain NC11 showed a globular shape after rotary shadowing and was resistant to neutral proteases. Specific antibodies could be raised against recombinant NC11 and were used for the analysis of other cell clones transfected with the full-length alpha 1 (XVI) chain. Immunoprecipitation of detergent extracts of metabolically labelled cells demonstrated the presence of disulfide-bonded 200-kDa polypeptides possessing NC11 epitopes. This material was partially resistant to pepsin, indicating the formation of alpha 1 (XVI) chain homotrimers with a triple-helical conformation. Yet a substantial proportion of these homotrimers was degraded to fragments of variable size (35-150 kDa) when secreted into the culture medium. Several of these fragments could be obtained on a semi-preparative scale from cells grown in hollow fiber cassettes and showed substantial hydroxylation of proline, consistent with triple-helix formation. Edman degradation demonstrated the origin of some from the N-terminal and of one from a more C-terminal position of collagen 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 process also occurs in situ remains to be shown.
Tillet
E
E
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Mann
K
K
Nischt
R
R
Pan
T C
TC
Chu
M L
ML
Timpl
R
R
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis* / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014162:Transfection
D010446:Peptide Fragments; D011994:Recombinant Proteins; D003094:Collagen