DIFFERENTIAL-EFFECTS OF ASCORBATE DEPLETION AND ALPHA,ALPHA'-DIPYRIDYL TREATMENT ON THE STABILITY, BUT NOT ON THE SECRETION, OF TYPE-IV COLLAGEN IN DIFFERENTIATED F9 CELLS

Ascorbic acid stimulates secretion of type I collagen because of its r ole in 4-hydroxyproline synthesis, but there is some controversy as to whether secretion of type IV collagen is similarly affected. This que stion was examined in differentiated F9 cells, which produce only type IV collagen, by labeling proteins with [C-14]proline and measuring co llagen synthesis and secretion. Hydroxylation of proline residues in c ollagen was inhibited to a greater extent in cells treated with the ir on chelator alpha,alpha'-dipyridyl (97.7%) than in cells incubated wit hout ascorbate (63.1%), but both conditions completely inhibited the r ate of collagen secretion after 2-4 h, respectively Neither treatment affected laminin secretion. Collagen synthesis was not stimulated by a scorbate even after treatment for 2 days. On SDS polyacrylamide gels, collagen produced by alpha,alpha'-dipyridyl-treated cells consisted ma inly of a single band that migrated faster than either fully (+ ascorb ate) or partially (- ascorbate) hydroxylated alpha 1(IV) or alpha 2(IV ) chains. It did not contain interchain disulfide bonds or asn-linked glycosyl groups, and was completely digested by pepsin at 15 degrees C . These results suggested that it was a degraded product lacking the 7 S domain and that it could not form a triple helical structure. In co ntrast, the partially hydroxylated molecule contained interchain disul fide bonds and it was cleaved by pepsin to collagenous fragments simil ar in size to those obtained from the fully hydroxylated molecule, but at a faster rate. Kinetic experiments and monensin treatment suggeste d that completely unhydroxylated type IV collagen was degraded intrace llularly in the endoplasmic reticulum or cis Golgi. These studies indi cate that partial hydroxylation of type IV collagen confers sufficient helical structure to allow interchain disulfide bond formation and re sistance to pepsin and intracellular degradation, but not sufficient f or optimal secretion. (C) 1997 Wiley-Liss, Inc.