Chinese hamster lung cells synthesize and confine to the cellular domain a collagen composed solely of B chains.

Abstract

The acid-soluble collagen extracted from cultured Chinese hamster lung (CHL) cell layers has been isolated after limited pepsin digestion and differential salt fractionation. Polyacrylamide gel electrophoresis of this material under denaturing conditions showed the presence of collagen chains with an apparent molecular mass of 120,000 daltons both before and after reduction, indicating the absence of interchain disulfide bonds in the native molecule. When chromatographed on CM-cellulose under denaturing conditions, the majority (>90%) of the CHL cell layer collagen chains eluted as relatively basic components slightly before the human a2(I) chain and coincident with the human B chain. In addition, the CM-cellulose elution profiles of the cyanogen bromide peptides derived from the human B chain and from the CHL cell ayer chain were essentially identical. Examination of CHL cells in culture by using affinity-purified antibody to human B chain revealed this collaren to localized in an extracellular matrix surrounding the cells. Furthermore, analysis of the culture medium indicated the absence of any comparable collagen chain. These data provide additional evidence for the existence of a molecular form of collagen composed solely of B chains and suggest that this molecular form o collagen has an unusual affinity for the cell layer in this system.The class of proteins collectively referred to as collagen represents various distinct gene products. The synthesis of these proteins is somewhat tissue specific and is altered in pathological conditions (1, 2). At present, at least nine genetically distinct chains have been described as the primary constituents of various collagen molecules. Four collagen chains-the al(I), a2(I), al(II), and al(III) chains-comprise the interstitial collagens (1). Recent evidence indicates that five additional unique collagen chains are present in collagen molecules in basement membrane-like structures and in basement membranes. These are the aA and aB chains (3-6) and aC chain (7) isolated from highly vascularized tissue, and two additional chains obtained from lens capsule (8-10) and human placenta (11-13). However, little is currently known about the mechanisms that control the differential expression of the collagen genes. This paucity of information reflects, at least in part, the limited number of systems available for studying the regulation and dissecting the genetics of mammalian procollagen biosynthesis.This (5, 16). The origin of the CHL cell line as well as the clone (HT1) used in these studies has been described (14,17).Growth of Cells in Culture and Metabolic Labeling. CHL cells were maintained and grown in Dulbecco's modified Eagle's minimal essential medium supplemented with 10% fetal bovine serum as described (14,15). For immunological studies, cells were grown in 15 X 60 mm dishes at 370C under a 10% C02/90% air atmosphere to t90% confluency. The medium was removed, the cell layer was washed with three 5-ml portions of cold phosphate-buffered saline, and the cells...

Phenotypic differences in subclones and long‐term cultures of clonally derived rat bone cell lines

“…However, the apparent loss of type I collagen expression in RCJ 3.2.4.1 cells and both type I and 111 collagens in the RCJ 3.2.4.4 cells, both of which are subclones of RCJ 3.2.4, were the most obvious differences observed. The synthesis of type V collagen as the principal collagen type for RCJ 3.2.4.4 has also been reported in a Chinese hamster lung fibroblast (CHL) clone [34], in a rhabdomyosarcoma cell line [35], and in Chinese hamster ovary collagen. Since it is recognized that certain specialized cells have the capacity to express multipotentiality and, in some cases, perhaps also totipotentiality [43], the synthesis of type IV collagen, laminin, and basement membrane proteoglycan in these cells may result from the activation of genes that would otherwise be dormant in connective tissue cells.…”

Diversity in the processing events at the N‐terminus of type‐V collagen

“…The excess of al(V) chains is likely to occur as an [al(V)I3 homotrimer, which has already been described in cell and tissue cultures (Haralson et al, 1980;Fessler et al, 1981a, b;Kumamoto and Fessler, 1980). The fact that the long form of the al(V) chain does not undergo full processing may indicate that its Nproteinase cleavage site is in an inappropriate conformation for cleavage to occur.…”

Homozygosity and Heterozygosity for Null Col5a2 Alleles Produce Embryonic Lethality and a Novel Classic Ehlers-Danlos Syndrome–Related Phenotype

“…Because a2(V) chains are thought to be unstable in the absence of a1(V) chains with which to combine, 8,28 the phenotype of Col5a1 À/À embryos likely results from a complete absence of both a1(V) and a2(V) chains and thus a lack of collagen V. However, in the absence of a2(V) chains with which to combine, a1(V) chains form stable a1(V) 3 homotrimers. 28,29 Thus, the 2 additional days of embryonic survival of Col5a2 À/À , compared with Col5a1 À/À embryos, suggests that a1(V) 3 homotrimers can functionally compensate, in part, for the loss of a1(V) 2 a2(V) heterotrimers.…”