Search

Fibrils of embryonic cartilage are heterotypic alloys formed by collagens II, IX, and XI and have a uniform diameter of approximately 20 nm. The molecular basis of this lateral growth control is poorly understood. Collagen II subjected to fibril formation in vitro produced short and tapered tactoids with strong D-periodic banding. The maximal width of these tactoids varied over a broad range. By contrast, authentic mixtures of collagens II, IX, and XI yielded long and weakly banded fibrils, which, strikingly, had a uniform width of about 20 nm. The same was true for mixtures of collagens II and XI lacking collagen IX as long as the molar excess of collagen II was less than 8-fold. At higher ratios, the proteins assembled into tactoids coexisting with cartilage-like fibrils. Therefore, diameter control is an inherent property of appropriate mixtures of collagens II and XI. Collagen IX is not essential for this feature but strongly increases the efficiency of fibril formation. Therefore, this protein may be an important stabilizing factor of cartilage fibrils.

Type V collagen was prepared from acetic acid extracts of lathyritic chick bone. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the extracted material demonstrated two collagenous bands of slower mobility than pepsin-extracted alpha 1(V) and alpha 2(V) chains. Cyanogen bromide peptide maps of these protein bands identified them as forms of alpha 1(V) and alpha 2(V). Segment long spacing (SLS) crystallite banding patterns of the acid-extracted Type V were identical within the triple-helical domain to the SLS banding patterns of pepsin-extracted Type V collagen, supporting the identification of this material. A globular domain at one end of the triple helix of the acid-extracted Type V was visualized by both rotary shadowing and negative staining of SLS crystallites. The molecular weights of the globular terminal peptides were 18,000 and 29,000, respectively, for alpha 1(V) and alpha 2(V), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after bacterial collagenase digestion of the isolated alpha chains. The results presented here indicate that fully processed Type V collagen in chick bone exists as a higher molecular weight form than that from pepsin extracts and retains a globular domain at one end of the triple helix. This is in contrast to the interstitial collagens in which only very small non-triple-helical domains (telopeptides) are retained in the fully processed molecules. In vitro aggregation studies demonstrated the intact fully processed form of Type V collagen forms uniform small-diameter fibrous structures. These results suggest that Type V collagen may be present in fibrous structures within tissues.

Five types of collagen with triple-helical regions approximately 300 nm in length were found in lamprey tissues which show characteristic D-periodic collagen fibrils. These collagens are members of the fibril forming family of this primitive vertebrate. Lamprey collagens were characterized with respect to solubility, mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, carboxylmethyl-cellulose chromatography, peptide digestion patterns, composition, susceptibility to vertebrate collagenase, thermal stability, and segment long spacing-banding pattern. Comparison with fibril-forming collagens in higher vertebrates (types I, II, III, V, and XI) identified three lamprey collagens as types II, V, and XI. Both lamprey dermis and major body wall collagens had properties similar to type I but not the typical heterotrimer composition. Dermis molecules had only alpha 1(I)-like chains, while body wall molecules had alpha 2(I)-like chains combined with chains resembling lamprey type II. Neither collagen exhibited the interchain disulfide linkages or solubility properties of type III. The conservation of fibril organization in type II/type XI tissues in contrast to the major developments in type I and type III tissues after the divergence of lamprey and higher vertebrates is consistent with these results. The presence of type II and type I-like molecules as major collagens and types V and XI as minor collagens in the lamprey, and the differential susceptibility of these molecules to vertebrate collagenase is analogous to the findings in higher vertebrates.

Incubation of tail tendon from a young rat in solutions containing D-ribose resulted in attachment of the monosaccharide to collagen and subsequent cross-link formation at a rate much faster than found for glucose. The collagen rapidly became resistant to solubilization and showed increasing fluorescence. Ribose bound to all major CNBr peptides of collagen, with some preference for the alpha 2-CB3,5 peptide and the triple-helical region of alpha 1-CB6, and was incorporated into higher molecular weight material. Extensive pepsin digestion permitted isolation of dimers of alpha chains cross-linked in triple-helical regions as a result of incubation with ribose. The dimers were identified as beta 11, beta 12, and beta 22 components, and the limited degree of heterogeneity of these components indicated that cross-linking occurred at several sites, some of which must be intermolecular. Isolated beta components were strongly fluorescent with a spectrum similar to that of collagen in aged tissues. Fluorescent dimers with similar characteristics were found in pepsin digests of tail tendons from older rats.