The Nature of the Collagen Synthesized by Cultured Human Fibroblasts

Abstract

The hydroxyproline-containing proteins (hyproproteins) synthesized by cultured human fibroblasts have been partially characterized. The hyproprotein extracted from the cell layer was found to be similar to the collagen extracted from skin in the ratio of hydroxyproline to proline, chain composition, solubility, and resistance to proteolytic digestion. In earlier studies, the synthesis of collagen by fibroblasts and other cells in vitro was followed by measuring nondialyzable hydroxyproline, a relatively unique marker for collagen, and by observing extracellular collagen fibrils with the electron microscope (1-6). It has been reported that only about half of the nondialyzable hydroxyproline formed by human fibroblasts was deposited in the cell layer, while the remainder was found in the medium (4). We have observed that most of the hydroxyproline-containing protein (hyproprotein) associated with the cell layer was insoluble in cold neutral salt solution. However, when hyproproteins were synthesized in the presence of 13-aminopropionitrile, a specific inhibitor of collagen cross-linking, a hyproprotein could be readily extracted and characterized. Its properties have been compared with the hyproproteins secreted into the medium. MATERIALS AND METHODSFibroblasts were obtained from biopsies of normal human skin by standard techniques and grown in plastic culture flasks containing 20 ml of Dulbecco-Vogt medium supplemented with 300 Mig/ml of glutamine, 50 ug/ml of ascorbic acid, 50 units/ml of penicillin, 50 /Ag/ml of streptomycin, and 10% fetal calf serum. Cultures of confluent fibroblasts were labeled with [U-14C ]iproline (2 /ACi/ml) or both [U-14C ]irproline (1 0Ci/ml) and [U-14C ]glycine (2,4Ci/ml) in 15 ml of Eagle's basal medium. In addition, certain flasks received 50 ,ug/ml of f3-aminopropionitrile-HCl (BAPN) to block the cross-linking of collagen (7). After 24 hr, the medium was decanted from the cell layer and dialyzed exhaustively against the appropriate buffers used for chromatography. The cell layer was rinsed with unlabeled medium, removed from the flask surface in 20 ml of 1 M NaCl-0.05 M Tris (pH 7.4), and extracted for 2 days at 40C. The soluble proteins were separated from the insoluble material by centrifugation at 10,000 rpm for 10 min, then dialyzed against the appropriate buffers used for chromatography. Carrier collagen, (20 mg) prepared from the skin of lathyritic rats (8), was added to the medium or cell-extract samples to serve as an internal standard for measuring the recoveries of the radioactive al and a2 chains, as well as to mark their position on elution. In certain experiments, medium or cell extract samples were brought to 0.5 M acetic acid and incubated with pepsin (9) (100 ,ug/ml) for 6 hr at 15'C. The pH of the samples was adjusted to 8.0 with 1 M NaOH and allowed to stand for at least 20 min to irreversibly inactivate pepsin (10). CM-cellulose chromatography was performed on the heat-denatured proteins as described by Piez et al. (8). Molecular-sieve chromatography was p...

Conformation of underhydroxylated collagen synthesized by 3T6 fibroblasts in culture

“…It has been well established that pepsin at 15°C does not digest the triple helical portion of the collagen molecule but only the non-helical moiety [20,21]. Denaturated chains are completely degraded.…”

The Phenotypic Complexity of Myogenic Clones

“…"Chondrogenic clone" was as described in Chacko et al (15). Cells in the ultimate compartment of the fibrogenic lineage were mononucleated and synthesized (al),a2 collagen subunits (16)(17)(18), hyaluronic acid, and various amounts of chondroitin sulftate (19,20 (21,22). Occasionally, clones with myotubes during the second week were lacking in myotubes during the third week; either the myotubes degenerated or they were obscured by overgrowth of replicating mononucleated cells.…”

The structure and assembly of procollagen – a review

“…Clearcut experimental evidence for the existence of a higher molecular weight biosynthetic precursor of collagen has come from several sources: 1) studies o f the synthesis and structure of the hydroxyproline-containing material secreted into the medlum by cultured fibroblasts (17)(18)(19)(20)(21)(22)(23); 2) studies o f the synthesis and secretion of procollagen by freshly isolated embryonic chick cells obtained from tendon, cartilage, or lens capsule (24-29); 3) an investigation of a heritable disorder of cattle and sheep (dermatosparaxis), in which an enzymatic defect in the conversion of procollagen t o collagen was found (30)(31)(32)(33)(34)(35); an analogous defect has been identified as a form of the Ehlers-Danlos syndrome in man (36); 4) studies of procollagen synthesis and structure by cranial bone explants (13,(37)(38)(39)(40)(41)(42); and 5) isolation of derivatives of procollagen from tissues of normal animals (43)(44)(45)(46).…”