Biosynthesis of A,B procollagen.

Abstract

Interest in cell-associated collagens led others to isolate A and B collagen chains, also known as type V collagen, from many tissues, but only after pepsin cleavage. Soluble precursors of these chains are synthesized in vitro by crop, a chicken embryo muscle tissue, and are converted by at least two processing steps from procollagens via intermediates to final forms which are larger than the pepsin-derived A and B chains. Heterotrimeric, disulfide-bridged procollagen molecules corresponding to B2A exist. Components were separated by ion exchange chromatography, velocity sedimentation, and electrophoresis, and the relationships between them were establis ed by sequential radioactive labeling and comparison of peptides generated by protease cleavage. Collagens form a family of related gene products composed of two general groups: the interstitial and the basement membrane collagens. Two (4,5) reported different ratios of these chains and also pepsin-resistant B chains, indicating 13 trimers. We find proaA chains disulfide-linked to proaB chains, which establishes the heterotrimer [(proaB)2proaA]. However, an excess of B chains suggests that homotrimer B3 also exists. Because type V collagens have been located immunologically in muscle tissue (6, 7), have been found in chicken embryo tissues (7-9), and have been shown to be made by cultured smooth muscle (10) and chicken cells (7, 11), we chose a rapidly growing muscle tissue of the chicken embryo, the crop. Because this thin-walled diverticulum of the esophagus does not produce digestive enzymes, accidental proteolysis was minimized; furthermore, other studies support our conclusion of stepwise processing.t MATERIALS AND METHODS Typically, crops from 50 19-day chicken embryos were incubated in 15 ml of Dulbecco's modified Eagle's medium (DME medium) by described procedures (12) with the modifications given below. The medium lacked lactalbumin hydrolysate and the amino acids used for labeling; it contained 2-aminopro- 40C. Crops were homogenized in 1 M NaCl/50 mM Tris/10 mM EDTA, pH 7.5 (buffer H), and centrifuged (40,000 rpm, 15 min). The pellet was rehomogenized in 0.5 M acetic acid and the suspension was digested with pepsin (100 gg/ml; Worthington) for 18 hr at 40C. The supernatant was diluted with 1 vol of 4 M urea/0.2% Triton X-100/50 mM Tris, pH 7.5, and, after dialysis against 0.5 M acetic acid/0.2 M NaCl/0.1% Triton X-100 (Buffer P), it was digested with pepsin as above. Further purification of A and B chains was as described (2, 7) except that NaCl was added to 5% by dialysis and type I and III carrier collagens (0.1 mg/ml) were added to help precipitate material from the buffer H extract. CNBr cleavage and peptide separation were as described (2).Extraction of Type V Precursors, DEAE-Cellulose Chromatography, and Velocity Sedimentation. Tissue was homogenized in buffer I [buffer H plus 1250 Ag N-ethylmaleimide (MaINEt) and 150 ,g of phenylmethylsulfonyl fluoride (PheMeSO2F) per ml] and centrifuged (40,000, rpm, 15 min). The supernatant was ...

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.…”

“…The [al(V)12a2(V) stoichiometry was previously reported in many tissues (Bentz et al, 1978;Kumamoto and Fessler, 1980;Fessler et al, 1981a) while the al(V)a2(V)-a3(V) assembly was only described for placental tissues (Niyibizi et a]., 1984) and for granulation tissue (Hashimoto et al, 1988). Collagen V was shown to occur in an [a1(V)I3 stoichiometry in Chinese hamster lung cell cultures (Haralson et a]., 1980) and in small amounts in cultures of chick embryo crop and blood vessels (Kumamoto and Fessler, 1980;Fessler et al, 1981b). However, such homotrimeric molecules have not been isolated from tissues.…”

Control of Heterotypic Fibril Formation by Collagen V Is Determined by Chain Stoichiometry

“…The heterotrimeric form, ␣1(V)␣2(V)␣3(V), only found in placenta (9), remains poorly studied, although the recent determination of the primary structure of the human ␣3(V) chain will facilitate further research (10). The homotrimeric form [␣1(V)] 3 was first observed in cell cultures and is likely to be present in embryonic tissues, although in too small amounts to be purified (11)(12)(13). Recently, however, using recombinant technology, we have reported the large-scale production and characterization of the collagen V homotrimer (14).…”

The Pro-α3(V) Collagen Chain

“…However, other forms of type V collagen include an ␣1(V) 3 homotrimer that is secreted by a line of Chinese hamster cells (14) and which may also exist in normal tissues (15,16), and a poorly characterized ␣1(V)␣2(V)␣3(V) heterotrimer, isolated primarily from placenta (17, 18), but also reported in uterus, skin, and synovial membranes (12, 19 -21). Type XI collagen, in the form of an ␣1(XI)␣2(XI)␣3(XI) heterotrimer (22), was first characterized as a minor collagen of cartilage.…”