Low molecular weight fibroblast collagen: structure, secretion, and differential expression as a function of fetal and cellular age

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Abstract
A unique low molecular weight collagen that was highly resistant to proteolytic degradation was originally isoltaed from fetal calf ligamentum nuchae fibroblasts and hence termed FCL-1 [Sage, H., Mecham, R., Johnson, C., and Bornstein, P. (1983) J. Cell Biol. 97, 1933-1938]. The differential expression of this protein was studied as a function both of fetal (donor) age and of subcultivation in vitro. Concomitant isolation, subculture, and metabolic radiolabeling experiments performed on cell strains from fetal calf ligament (FCL) and fetal bovine skin (FBS) representing different gestational ages (85-270 days in utero) showed that (a) FCL-1 was synthesized preferentially by fibroblasts from younger animals and (b) expression of FCL-1 diminished as a function of increased passage in culture. Levels of FCL-1, measured as percent of total radiolabeled cultured medium protein that precipitated in a concentration range of 20-50% ammonium sulfate, ranged from 22% in FCL 85 cells to 7.7% in FCL 270 (term) cells. FBS fibroblasts at passages 6-10 secreted from 13% to 6% FCL-1, respectively. When cells from an 85-day fetal ligament were allowed to accumulate copious extracellular matrix in vitro, the production of FCL-1 was increased to 32%. FCL-1 was not immunoreactive with polyclonal antibodies directed toward most of the sequences of the interstitial type I and type III procollagens. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent molecular weight of FCL-1 was 13,000 (on the basis of collagen peptide standards) and approximately 30,000 (on the basis of globular protein standards). Incubation with bacterial collagenase produced a stable cleavage product of Mr 8000 (by collagen standards) or 17,000 (by globular standards). In contrast, pepsin removed a small peptide of approximately 1000-2000 in molecular weight from FCL-1, and a gradual but progressive proteolysis of the collagen was observed over a period of 1-6 h. Pulse-chase studies revealed a secretion time of approximately 60 min for FCL-1, without the appearance of any processed, intermediate forms. These studies confirm that FCL-1 represents a novel member of the collagen gene family that manifests differential expression as a function of development.

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