Characterization of recombinant human type IX collagen - Association of alpha chains into homotrimeric and heterotrimeric molecules

As type IX collagen is a minor cartilage component, it is difficult to puri fy sufficient amounts of it from tissues or cultured cells to study its str ucture and function. Also, the conventional pepsin digestion used for fibri llar collagens cannot be utilized for purifying type IX collagen, because i t contains several interruptions in its collagenous triple helix. A baculov irus expression system was used here to produce recombinant human type IX c ollagen by coinfecting insect cells with three viruses containing full-leng th cDNAs for the alpha 1(IX), alpha 2(IX), and alpha 3(IX) collagen chains together with a double promoter virus for the alpha and beta subunits of hu man prolyl 4-hydroxylase, Correctly folded recombinant type IX collagen was secreted, consisting of the three alpha chains in a 1:1:1 ratio and showin g the expected biphasic thermal melting profile. When the individual alpha chains were expressed, disulfide-bonded homotrimers and homodimers of the a lpha chains were observed, When the cells were coinfected with the viruses for all three alpha chains, heterotrimers of alpha 1(IX), alpha 2(IX), and alpha 3(IX) were detected in cell culture medium, and the other possible co mbinations were less prominent. When any two of the alpha chains were co-ex pressed, in addition to the homodimers and homotrimers, only alpha 1(IX) an d alpha 3(IX) chains were disulfide-bonded. The results thus suggest that t he most favored molecular species is an alpha 1(IX)alpha 2(IX)alpha 3(IX) h eterotrimer, but the chains are also able to form disulfide-bonded heterotr imers of (alpha 1(IX) and alpha 3(IX) chains and (alpha 1(IX))(3), (alpha 2 (IX))(3), and (alpha 3(IX))(3) homotrimers.