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 role in 4-hydroxyproline synthesis, but there is some controversy as to whether secretion of type IV collagen is similarly affected. This question was examined in differentiated F9 cells, which produce only type IV collagen, by labeling proteins with [14C]proline and measuring collagen synthesis and secretion. Hydroxylation of proline residues in collagen was inhibited to a greater extent in cells treated with the iron chelator alpha,alpha'-dipyridyl (97.7%) than in cells incubated without ascorbate (63.1%), but both conditions completely inhibited the rate of collagen secretion after 2-4 h, respectively. Neither treatment affected laminin secretion. Collagen synthesis was not stimulated by ascorbate even after treatment for 2 days. On SDS polyacrylamide gels, collagen produced by alpha,alpha'-dipyridyl-treated cells consisted mainly of a single band that migrated faster than either fully (+ ascorbate) 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 contrast, the partially hydroxylated molecule contained interchain disulfide bonds and it was cleaved by pepsin to collagenous fragments similar in size to those obtained from the fully hydroxylated molecule, but at a faster rate. Kinetic experiments and monensin treatment suggested that completely unhydroxylated type IV collagen was degraded intracellularly in the endoplasmic reticulum or cis Golgi. These studies indicate that partial hydroxylation of type IV collagen confers sufficient helical structure to allow interchain disulfide bond formation and resistance to pepsin and intracellular degradation, but not sufficient for optimal secretion.
Kim
Y R
YR
Laboratory of Biochemistry, National Cancer Institute, Bethesda, Maryland 20892, USA.
Peterkofsky
B
B
D015082:2,2'-Dipyridyl / Q000494:pharmacology*; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D001216:Asparagine / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D003094:Collagen / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D017364:Collagenases; D004220:Disulfides / Q000032:analysis; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D006900:Hydroxylation; D007502:Iron Chelating Agents / Q000494:pharmacology*; D007797:Laminin / Q000032:analysis; D051379:Mice; D008985:Monensin / Q000494:pharmacology; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D010434:Pepsin A; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D014407:Tumor Cells, Cultured
D004220:Disulfides; D007502:Iron Chelating Agents; D007797:Laminin; D015082:2,2'-Dipyridyl; D001216:Asparagine; D003094:Collagen; D008985:Monensin; D011392:Proline; D010434:Pepsin A; D017364:Collagenases; D001205:Ascorbic Acid