Procollagen biosynthesis in embryonic chick arteries.

Abstract

Collagen is initially synthesized as a precursor form called procollagen (for reviews, see Schofield & Prockop, 1973; Grant et al., 1974). By using freshly isolated cells, obtained by enzymic digestion of embryonic chick tendon (Dehm & Prockop, 1971), cartilage (Dehm & Prockop, 1973) and lens (Grant et al., 1972), it has been possible to study the synthesis and secretion of the different types of procollagen from each of these tissues. Here we report on the synthesis and secretion of procollagen by freshly isolated cells from the major arteries of chick embryos. The major arteries of 17-day chick embryos were dissected (Murphy et al., 1972) and the cells were isolated essentially as described by Dehm & Prockop (1971, 1973); 5 x 106-7 x lo6 cells were obtained/embryo. The cells were incubated in modified Krebs medium at a density of 1 x lo7 cells/ml. Continuous labelling of the freshly isolated cells with [I4C]proline indicated that the synthesis of '4C-labelled polypeptides, the synthesis of non-diffusible peptide-bound hydr~xy['~C]proline and the secretion of peptide-bound hydr~xy['~C]proline were linear for up to 6h. In two separate experiments a lag of about 7min was observed between the time at which the incorporation of I4C into polypeptides became linear and the time at which the synthesis of peptide-bound hydr~xy['~C]proline became linear (Fig. 1). This compares with a lag of 2-3min in both tendon and cartilage cells (Dehm & Prockop, 1972,1973) and it may indicate that, in the artery cells, the synthesis of collagen hydroxyproline may be largely a post-ribosomal event, whereas in tendon and cartilage cells it occurs mainly on nascent chains (see Grant et al., 1974). There was also a considerable lag (about 31 min in 2expts.) between the time at which synthesis of peptide-bound hydroxy[C]proline became linear and the time at which its secretion became linear (Fig. 1). This is similar to the lag of about 33 min seen in cartilage cells (Dehm & Prockop, 1973), but it contrasts with that of about 18min seen in tendon cells (Dehm & Prockop, 1972). This observation is of interest since the artery cells appear to be synthesizing type I procollagen (see below), as do tendon cells, and the difference in lag times for these two cell types synthesizing the same type of procollagen may have important implications in terms of the intracellular processing of procollagen polypeptide before their secretion. Chromatography of reduced I4C-labelled polypeptides from the medium on 6% agarose indicated that the bulk of the hydr~xy[~~C]proline was in pro a chains of procollagen. When reduction was omitted, essentially all of the ['*C]procollagen waseluted as a high-molecular-weight aggregate (>300000) in which the pro a chains were apparently linked by interchain disulphide bonds. In a parallel experiment in which the medium 14C-labelled polypeptides were treated with pepsin at 15C, the hydr~xy['~C]prolinecontaining polypeptides were now eluted from the agarose column with collagen a chains indicating that the procollagen was in a triple-helical conformation. In experiments to determine what type of procollagen was being synthesized by the artery cells the hydr~xy['~C]proline-containing polypeptides in the medium were first precipitated by using 30 %-satd. (NH4)2S04 (Dehm & Prockop, 1973); about W%of the medium hydr~xy['~C]proline was precipitated by this procedure. The precipitated 14Clabelled polypeptides were examined, after reduction, by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, and two radioactive bands, corresponding to pro a1 and pro a2, were observed. The ratio of radioactivity in the pro a1 band to that in the pro a2 band was about 2 in several experiments, which is the value expected for a type I procollagen. Procollagen from the medium of tendon cells, which appears to be a type I procollagen as judged by these criteria (seeschofieldetal., 1974). gave similar results to those observed for the artery procollagen when it was electrophoresed in parallel gels. These observations indicate that the artery cells from 17-day embryo synthesize and secrete a type I procollagen. This is somewhat surprising in view