Isolation and partial characterization of precursors to minor cartilage collagens

Abstract

Suspension cultures of cartilage cells were prepared from 17-day chick embryo sterna and radiolabeled with [14C]-proline under conditions which sought to minimize proteolytic conversion of procollagen to collagen. Collagenous proteins were isolated from the culture medium and cell fraction, were purified in their native state by (NH4)2SO4 precipitation and DEAE-cellulose chromatography, and were characterized by protease susceptibility, SDS-gel-filtration and SDS-polyacrylamide gel electrophoresis. Qualitatively, the precursor components present in the medium were similar to those in the cell extract; quantitatively, it appeared that the minor cartilage collagen precursor components derived from 1 alpha, 2 alpha, 3 alpha and type IX collagens were more prevalent in the cell extract. SDS-PAGE of unreduced samples showed that precursors to both of these collagens migrated as distinct high-molecular-weight aggregates. After chymotrypsin digestion, unreduced type IX collagen migrated as two disulfide-bonded aggregates--a large one (Mr approximately 210K) and a small one (Mr approximately 43K); whereas 1 alpha, 2 alpha, 3 alpha chains migrated identically whether reduced or unreduced. Reduction of undigested type IX aggregate yielded two components of Mr approximately 97K and 78K; whereas reduction of the chymotrypsin resistant 210K and 43 K aggregates gave a single component of Mr approximately 61K and a component which migrated at the dye front, respectively. The molecular origin of these components was confirmed by differential NaCl precipitation. It was concluded that this culture system synthesized precursors to 1 alpha, 2 alpha, 3 alpha and type IX collagens in addition to type II; type X collagen was not detected even though the 17-day sternum contained a population of cells morphologically similar to hypertrophic chondrocytes. The precursor chains to 1 alpha, 2 alpha, 3 alpha collagen had an apparent Mr greater than pro-alpha (II) and could be isolated as a disulfide-bonded aggregate(s); the precursor chains to type IX collagen had an apparent Mr less than pro alpha (II) and could also be isolated as a disulfide-bonded aggregate. All of the cartilage collagen precursors had protease-susceptible regions, but those in type IX appeared to be more sensitive to pepsin than to chymotrypsin.