Production of Human Type I Collagen in Yeast Reveals Unexpected New Insights into the Molecular Assembly of Collagen Trimers

Substantial evidence supports the role of the procollagen C-propeptide in the initial association of procollagen polypeptides and for triple helix formation. To evaluate the role of the propeptide domains on triple helix formation, human recombinant type I procollagen, pN-collagen (procollagen witho...

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Bibliographic Details
Published in:The Journal of biological chemistry 2001-06, Vol.276 (26), p.24038-24043
Main Authors: Olsen, David R., Leigh, Scott D., Chang, Robert, McMullin, Hugh, Ong, Winson, Tai, Ernest, Chisholm, George, Birk, David E., Berg, Richard A., Hitzeman, Ronald A., Toman, P. David
Format: Article
Language:English
Subjects:
Circular Dichroism
Collagen - chemistry
Collagen - genetics
Collagen - metabolism
Collagenases - chemistry
Endopeptidases - chemistry
Humans
Microscopy, Electron
Procollagen - genetics
prolyl hydroxylase
Protein Folding
Protein Structure, Quaternary
Saccharomyces cerevisiae
Saccharomyces cerevisiae - genetics
Sequence Deletion
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Summary:Substantial evidence supports the role of the procollagen C-propeptide in the initial association of procollagen polypeptides and for triple helix formation. To evaluate the role of the propeptide domains on triple helix formation, human recombinant type I procollagen, pN-collagen (procollagen without the C-propeptides), pC-collagen (procollagen without the N-propeptides), and collagen (minus both propeptide domains) heterotrimers were expressed in Saccharomyces cerevisiae. Deletion of the N- or C-propeptide, or both propeptide domains, from both proα-chains resulted in correctly aligned triple helical type I collagen. Protease digestion assays demonstrated folding of the triple helix in the absence of the N- and C-propeptides from both proα-chains. This result suggests that sequences required for folding of the triple helix are located in the helical/telopeptide domains of the collagen molecule. Using a strain that does not contain prolyl hydroxylase, the same folding mechanism was shown to be operative in the absence of prolyl hydroxylase. Normal collagen fibrils were generated showing the characteristic banding pattern using this recombinant collagen. This system offers new opportunities for the study of collagen expression and maturation.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M101613200