Human Recombinant α1(V) Collagen Chain

Abstract

Human embryonic kidney cells (293-EBNA) have been transfected with the full-length human α1 chain of collagen V using an episomal vector. High yields (15 μg/ml) of recombinant collagen were secreted in the culture medium. In presence of ascorbate, the α1(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 °C for the recombinant homotrimer. The major secreted farm is a 250-kDa polypeptide (α1FL). N-terminal sequencing and collagenase digestion indicate that α1FL retains the complete N-propeptide but lacks the C-propeptide. However, α1FL might undergo a further N-terminal trimming into a form (α1TH) 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 (α1(V)) 2α2(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 (α1(V)) 3 recovered from tissues.