Interchain Disulphide Bonding in Procollagen from Embryonic Chick Tendon Cells and the Formation of the Triple-Helical Structure

Abstract

Earlier work from this laboratory showed that up to 60 % of the procollagen (for a review of procollagen, see Schofield & Prockop, 1973) secreted by embryonic chick tendon cells was in the form of a high-molecular-weight aggregate, in which the polypeptide chains were linked by interchain disulphide bonds (Dehm et a[., 1972). Similar conclusions have been reached in subsequent studies with other biological systems (Burgeson et al., 1972; Goldberg et al., 1972; Smith et al., 1972; Monson & Bornstein, 1973). In this report we present further evidence to show that procollagen secreted by matrixfree tendon cells is intramolecularly cross-linked by interchain disulphide bonds in the amino-terminal extensions, and we present data concerning the stage at which the interchain disulphide bonds and the triple-helical structure of the molecule form during the intracellular biosynthesis of this protein. Unreduced procollagen from the medium of matrix-free tendon cells (Dehm & Prockop, 1971), when chromatographed on 6% agarose in the presence of sodium dodecyl sulphate (Jimenez et al., 1971), was eluted in two major peaks. The first, near the void volume (fraction 25), contained about 75 % of the procollagen and the second (fractions 31-32) contained about 25 % (Fig. la). Upon reduction, all of the procollagen was recovered in the second peak (pro-a chains). This was a consistent result and is somewhat at variance with previous results with these cells (Dehm et a[., 1972), as in that study only 30-60 % of the unreduced procollagen was aggregated. This may be due to the use of more carefully controlled conditions during isolation of the cells leading to a more complete removal of extracellular proteases. When fractions from various positions in the void-volume peak (Fig. la) were reduced and rerun, only material eluted in the position of pro-u chains was observed. Analysis of the unreduced procollagen by polyacrylamidegel electrophoresis (Weber et al., 1972) showed that the major component had a mobility slightly smaller than that of y chains from acid-soluble bovine skin collagen. Chromatography of the unreduced procollagen, on 6% agarose in the presence of sodium dodecyl sulphate, after digestion with purified bacterial collagenase, which leaves the aminoterminal extensions intact (Dehm & Prockop, 1973), gave chromatograms as shown in Fig. l(b). Three peaks were seen; the main one (fractions 36-37) contained no hydroxyproline, whereas the other peaks did. Only the first and last peaks contained cystine. Upon reduction and rechromatography of fractions 3&37, all the radioactivity was transferred to a peak in fractions 42-43. If the original digest was reduced directly, then the major peak now occurred in fractions 42-43 and the other two peaks were unaffected. The peak in fractions 36-37 apparently contains the three amino-terminal extensions linked by interchain disulphide bonds. We conclude that procollagen is secreted from these cells in the form of an aggregate, in which the three pro-a chains are linked to each other by disulphide bonds in the aminoterminal-extension sequences. When cells were pulse-labelled for 4min with [14C]proline and the unreduced cell proteins examined by gel filtration, as above, essentially none of the radioactivity was elu