No abstract

Short-chain collagen was isolated from bovine and from human placenta after limited pepsin digestion. By reductive cleavage of disulfide bonds under non-denaturing conditions, mainly non-collagenous domains were cleaved off yielding a uniform component composed of three polypeptide chains with molecular masses between 37 kDa and 48 kDa in a nearly equimolar ratio. The chains (SCI *, SC2* and SC3*) were isolated and characterized.By comparison of peptide patterns obtained after various cleavage procedures, they could be identified as more or less shortened forms of SCI, SC2 and SC3, the isolation of which from bovine short-chain collagen has been described [Jander et al. (1981) Eur. J . Biochem. 114,[17][18][19][20][21][22][23][24][25].The peptide patterns; as well as N-terminal sequence determination, give evidence for the genetic individuality of the three chains; the data so far available suggest that together they form one triple-helical structure which represents the collagenous domain of the basic molecular unit of short-chain collagen.A high-molecular-mass disulfide-bonded collagenous protein was first described by Chung et al. [I], who isolated it from aortic walls after limited pepsin digestion. Other authors used human [2,3] or bovine [4] placenta or calf skin [5] as a source for isolation. It represents a new and unique collagen type which, after reduction of disulfide bonds, releases three polypeptide chains of 40 -55 kDa, about half the size of the a chains of interstitial collagen types. We therefore introduced the term 'short-chain collagen'; because of its accumulated, though not exclusive, presence in the intima of vessel walls, it is also referred to as 'intima collagen'. Several chemical characteristics (amino acid composition, sugar content) demonstrate some similarity to typeIV and V collagens. With respect to the unusually high cysteine content, it closely resembles 7s collagen which has recently been identified as the cross-linking region of type IV collagen [6].In a previous paper [4] we described the isolation of three polypeptide chains SCI, SC2 and SC3 and some of their characteristic features. After the native aggregate has been reduced and alkylated under non-denaturing conditions, a second pepsin treatment leads to the loss of a larger noncollagenous portion of the molecule. Three shortened chains can be isolated from the remaining collagenous part. The characterization of these chains, described in this paper, provides evidence that three individual polypeptide chains are involved in the formation of short-chain collagen.

The 140 000-dalton collagenous glycoprotein (CGP) from calf aorta and ligament characterized by Gibson & Cleary (1982) [Gibson, M.A., & Cleary, E.G. (1982) Biochem. Biophys. Res. Commun. 105, 1288-1295] has been studied. In the electron microscope, rotary-shadowed CGP molecules appear similar to the dimers of type VI collagen (short-chain collagen, intima collagen) described by other authors [Furthmayr, H., Wiedemann, H., Timpl, R., Odermatt, E., & Engel, J. (1983) Biochem. J. 211, 303-311] except that they have larger globular domains. As shown by gel electrophoresis, pepsin treatment of CGP at 4 degrees C either before or after reduction releases polypeptide chains corresponding in size to those of type VI collagen. Electron microscopic examination shows that pepsin digestion of nonreduced CGP removes the outer globular domains, reduces the size of the inner ones, and separates the paired central strands. The residual structures look like type VI collagen dimers. When intact CGP is reduced, monomers with two large globular ends are obtained. Pepsin digestion of monomers removes most or all of both globular domains. In immunoblots, CGP and its pepsin-derived fragments react with antibodies directed against type VI collagen. The results indicate that type VI collagen is an integral component of CGP.

The distribution of type-VI collagen in the human iris and ciliary body was investigated by means of immunohistochemical techniques and compared with that of type-IV collagen, fibronectin and laminin. As has been described for other tissues, type-VI collagen surrounds type-I and -III collagen fibers. The aggregated form of type-VI collagen (the "long-spacing" or "curly" collagen), which has already been described in the trabecular meshwork and sclera, was also observed at the ciliary muscle tips surrounding the anterior elastic tendons of this muscle. In addition, staining for type-VI collagen was seen directly adjacent to the basement membranes of the ciliary muscle cells, the iris muscles, the uveal vascular endothelia and nerves, but not adjacent to the epithelial basement membranes. The staining did not form a discrete line like the immunoreaction for type-IV collagen, but bundles of marked fibrils extended into the surrounding connective tissue. We assume that type-VI collagen similar to type-VII collagen forms part of an anchoring system for these tissues. As type-VII collagen has been described only in connection with epithelial basement membranes, both type-VI and type-VII collagens may represent anchoring fibrils, however for different tissue components.