CHARACTERIZATION OF HUMAN TYPE-X PROCOLLAGEN AND ITS NC-1 DOMAIN EXPRESSED AS RECOMBINANT PROTEINS IN HEK293 CELLS

Type X collagen is a short-chain, network-forming collagen found in hy pertrophic cartilage in the growth zones of long bones, vertebrae, and ribs. To obtain information about the structure and assembly of mamma lian type X collagen, we generated recombinant human type collagen X b y stable expression of full-length human alpha 1(X) cDNA in the human embryonal kidney cell line HEK293 and the fibrosarcoma cell line HT108 0, Stable clones were obtained secreting recombinant human type X coll agen (hrColX) in amounts of 50 mu g/ml with alpha 1(X)-chains of appar ent molecular mass of 75 kDa, Pepsin digestion converted the native pr otein to a molecule migrating as one band at 65 kDa, while bands of 55 and 43 kDa were generated by trypsin digestion, Polyclonal antibodies prepared against purified hrColX reacted specifically with type X col lagen in sections of human fetal growth cartilage, Circular dichroism spectra and trypsin/chymotrypsin digestion experiments of hrColX at in creasing temperatures indicated triple helical molecules with a reduce d melting temperature of 31 degrees C as a result of partial underhydr oxylation. Ultrastructural analysis of hrColX by rotary shadowing demo nstrated rodlike molecules with a length of 130 nm, assembling into ag gregates via the globular noncollagenous (NC)-1 domains as reported fo r chick type X collagen, NC-1 domains generated by collagenase digesti on of hrColX migrated as multimers of apparent mass of 40 kDa on SDS p olyacrylamide gel electrophoresis, even after reduction and heat denat uration, and gave rise to monomers of 18-20 kDa after treatment with t richloroacetic acid, The NC-1 domains prepared by collagenase digestio n comigrated with NC-1 domains prepared as recombinant protein in HEK2 93 cells, both in the multimeric and monomeric form. These studies dem onstrate the potential of the pCMVsis expression system to produce rec ombinant triple helical type X collagens in amounts sufficient for fur ther studies on its structural and functional domains.