Abstract
Human embryonic kidney cells (293-EBNA) have been transfected with the full-length human alpha1 chain of collagen V using an episomal vector. High yields (15 microgram/ml) of recombinant collagen were secreted in the culture medium. In presence of ascorbate, the alpha1(V) collagen is correctly folded into a stable triple helix as shown by electron microscopy and pepsin resistance. Circular dichroism data confirm the triple-helix conformation and indicate a melting temperature of 37.5 degrees C for the recombinant homotrimer. The major secreted form is a 250-kDa polypeptide (alpha1FL). N-terminal sequencing and collagenase digestion indicate that alpha1FL retains the complete N-propeptide but lacks the C-propeptide. However, alpha1FL might undergo a further N-terminal trimming into a form (alpha1TH) corresponding to the main triple-helix domain plus the major part of the NC2 domain. This processing is different from the one of the heterotrimeric (alpha1(V))2alpha2(V) and could have some physiological relevance. Analysis of cell homogenates indicates the presence of a 280-kDa polypeptide that is disulfide-linked through its C-terminal globular domain. This C-propeptide is rapidly cleaved after secretion in the medium, giving the first evidence of a C-terminal processing of recombinant fibrillar collagens. Rotary shadowing observations not only confirm the presence of a globular domain at the N-terminal end of the molecule but reveal the presence of a kink within the triple helix in a region poor in iminoacids. This region could represent a target for proteases. Together with the thermal stability data, these results might explain the low amount of (alpha1(V))3 recovered from tissues.
Highlights
Human embryonic kidney cells (293-EBNA) have been transfected with the full-length human ␣1 chain of collagen V using an episomal vector
In addition to the importance of the Npropeptide retention, heterotypic fibrils were shown to be thinner in tissues where the amount of collagen V is high [7,8,9,10,11], and a reduction in the proportion of collagen V molecules alters the regulation of fibrillogenesis [12]
Expression of Recombinant ␣1(V) Chains—Electrophoresis analysis of serum-free medium from ␣1(V)-transfected 293EBNA cells demonstrated an additional 250-kDa protein band referred to ␣1FL, which is absent in nontransfected cell medium (Fig. 1A)
Summary
Human embryonic kidney cells (293-EBNA) have been transfected with the full-length human ␣1 chain of collagen V using an episomal vector. ␣1FL might undergo a further N-terminal trimming into a form (␣1TH) corresponding to the main triple-helix domain plus the major part of the NC2 domain This processing is different from the one of the heterotrimeric (␣1(V))2␣2(V) and could have some physiological relevance. Analysis of cell homogenates indicates the presence of a 280-kDa polypeptide that is disulfide-linked through its C-terminal globular domain This C-propeptide is rapidly cleaved after secretion in the medium, giving the first evidence of a C-terminal processing of recombinant fibrillar collagens. Rotary shadowing observations confirm the presence of a globular domain at the N-terminal end of the molecule but reveal the presence of a kink within the triple helix in a region poor in iminoacids This region could represent a target for proteases. Whereas collagens I, II, and III undergo a processing that reduces the molecule mainly to the triple-helix domain, collagen V retains a large part of the
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