Quantitation of type I, III, and V collagens in human tissue samples by high-performance liquid chromatography of selected cyanogen bromide peptides.

A method to determine the proportions of the major fiber-forming collagens (types I, III, and V) in noncartilaginous human tissues is presented. The procedure relies on direct solubilization of tissue collagen as cyanogen bromide peptides. The peptides are subjected to cation exchange chromatography followed by gel permeation chromatography in a manner consistent with the rapid resolution and quantitation of relatively low-molecular-weight marker peptides for each collagen. The marker peptides utilized for type I, III, and V collagens are alpha 1 (I)-CB2, alpha 1 (III)-CB2, and alpha 1 (V)-CB1, respectively. Quantitation of the peptides is attained as a function of ultraviolet absorbance during gel permeation chromatography. The nature of the marker peptides, the use of high-performance liquid chromatography techniques, and quantitation of the peptides by ultraviolet absorbance renders the method suitably rapid, sensitive, and accurate for routine evaluations of collagen composition. The utility of the method is illustrated in the presentation of analyses on specimens of placental membranes and blood vessel walls.

Immunolocalization of types V and XI collagen in cartilage using monoclonal antibodies.

Monoclonal antibodies produced against pepsin-solubilized newborn rat skin type V collagen [alpha 1(V)]2 alpha 2(V), and chondrosarcoma type XI collagen [alpha 1(XI) alpha 2(XI) alpha 3(XI)] are used to localize the collagens in sections of the chondrosarcoma as well as the normal rat knee joint by indirect immunofluorescence. Immunostaining for type V collagen shows strong cellular staining of chondrocytes; while the interstitial matrix as well as the lacunae are not stained. In contrast, antitype XI stains not only chondrocytes, but the extracellular compartments as well. In ELISA, rat anti-type XI collagen reacts with its native antigen, but does not cross-react with native types I, II, III, or V collagen from rat. The distinct locations of type V and XI collagens in cartilaginous tissue suggest varied functional roles for these constituents in the tissue.

Characterization of mammalian type IX collagen fragments from limited pepsin digests of a transplantable swarm rat chondrosarcoma.

Collagenous fragments from type IX molecules have been solubilized by limited pepsin proteolysis of a transplantable rat chondrosarcoma and isolated by selective salt precipitation. Chromatography of the solubilized precipitate on CM-cellulose under nondenaturing conditions yielded three fractions. When examined by polarimetry, the material in all three fractions revealed native collagen helical structure with melting points which ranged from 31-37 degrees C. When the fractions were denatured and rechromatographed on a column of agarose beads, the most acidic fraction eluted as 13-kDa polypeptides with and without prior reduction and alkylation. In contrast, the second and third fractions eluted as 100-kDa and 30-kDa polypeptides prior to reduction, but on reduction and alkylation produced reducible products of 34 kDa and 10 kDa, respectively. The general compositional features of the three fractions closely resemble comparable collagenous fragments of type IX collagen from other species. The denaturation products of the 13-kDa nonreducible, the 30-kDa reducible, and the 100-kDa reducible fractions were sequentially purified by CM-cellulose and reversed-phase chromatography to resolve the chain constituents. The isolated 10-kDa, 13-kDa, and 34-kDa chains were cleaved with CNBr, and the cleavage products identified by gel-permeation chromatography. Two 13-kDa polypeptides, 13K2 and 13K3, which did not contain any methionyl residues and were not cleaved with CNBr, were digested with trypsin, and the peptide digests were resolved by reversed-phase chromatography. Comparisons of the CNBr and tryptic cleavage products demonstrate that the three major collagenous fragments are composed of three unique polypeptides. A partial amino acid sequence of an 8-kDa CNBr peptide derived from a purified 10-kDa peptide (10K1) matches identically the amino acid sequence derived from a cDNA sequence in the rat alpha 1(IX) chain (Kimura et al., 1989). These studies, then, present convenient procedures useful in the isolation of mammalian type IX collagen fragments and describe features of the rat molecule, indicating that it is similar to the avian counterpart with respect to chain composition and general molecular structure.

Isolation and characterization of the cyanogen bromide peptides of the collagen 2 alpha chain from a transplantable rat chondrosarcoma.

The rat collagen 2 alpha chain was isolated from a transplantable Swarm chondrosarcoma following limited pepsin proteolysis. The chromatographically purified 2 alpha chain when cleaved with cyanogen bromide yields nine peptides which have been isolated and characterized with respect to their molecular weight and their amino acid composition. Eight of these peptides can be rapidly separated by gel-permeation high-performance liquid chromatography and retrieved for further purification by ion-exchange chromatography. The nine peptides are recovered in equimolar amounts and together account for a total of 1,014 amino acid residues. The features of the isolated cyanogen bromide peptides of the 2 alpha chain clearly differentiate it from other known collagen polypeptides. The possible homologies between the 2 alpha chain and other collagen alpha chains are noted. Comparison of the cyanogen bromide peptides indicates a close relationship of the 2 alpha to the alpha 1(V) chain.

Different levels of glycosylation contribute to the heterogeneity of alpha 1(II) collagen chains derived from a transplantable rat chondrosarcoma.

Three collagen fractions, each of which contain molecules composed of alpha 1(II) chains, have been isolated from pepsin-solubilized rat chondrosarcoma collagen. One fraction could be selectively precipitated from the pepsin digest at 0.7 M NaCl. Two additional fractions were obtained on chromatography of the collagen precipitating at 1.2 M NaCl on carboxymethyl cellulose under nondenaturing conditions. When chromatographed on carboxymethyl cellulose under denaturing conditions, each fraction contained components eluting in the position expected for alpha 1(II) chains. One of the fractions precipitating at 1.2 M NaCl contained the recently described 1 alpha and 2 alpha chains in addition to material eluting as alpha 1(II) chains. Comparison of the chains eluting as alpha 1(II) chains in the various fractions with respect to amino acid composition, carbohydrate content, and cyanogen bromide-cleavage products showed that they differed only in the number of glycosylated hydroxylysyl residues. In this regard, alpha 1(II) chains obtained from collagens precipitated at 1.2 M NaCl exhibited significantly higher levels of glucosylgalactosylhydroxylysyl residues than alpha 1(II) chains precipitated at 0.7 M NaCl. These results indicate that molecules composed of alpha 1(II) chains are heterogeneous with respect to levels of hydroxylysine-linked carbohydrate moieties and that the more highly glycosylated molecules require higher salt concentrations for precipitation from acidic solutions. The data also indicate that a proportion of the more highly glycosylated alpha 1(II) chains are involved in the formation of one or more molecular species with 1 alpha and 2 alpha chains.

Identification of collagen chains as a function of cyanogen bromide peptide patterns using gel permeation high-performance liquid chromatography.

A gel permeation high-performance liquid chromatography system utilizing commercially-available silica-based gels has been developed for evaluation of the cyanogen bromide cleavage products derived from collagen a chains. The high efficiency and precision of the system permits unequivocal identification of various chains by inspection of the peptide elution pattern following a single run requiring 40 minutes. The system is sufficiently sensitive to permit analyses to be performed with as little 1.0 microgram of sample, although the columns utilized may accommodate samples as large as 1.0 mg making the system useful for preparative purposes as well.

Isolation of a unique collagenous fraction from limited pepsin digests of human placental tissue. Characterization of one of the constituent polypeptide chains.

Fractionation of the highly soluble collagens released during limited pepsin digestion of whole human placenta has resulted in the isolation of a unique collagenous fraction comprised exclusively of high molecular weight aggregates. On reduction and alkylation, the aggregates dissociated yielding collagen-like polypeptides with an apparent Mr = 40,000 as well as a heterogeneous mixture of much smaller noncollagenous peptides. Ion exchange chromatography of the collagen-like, 40,000-dalton subunits results in recovery of a single relatively acidic component and a mixture of at least two relatively basic components. Both the basic and acidic components contain large amounts of cysteine, hydroxylysine glycosides, and glucosamine, and exhibit compositional features indicative of the presence of collagenous and noncollagenous domains. These unusual features have been verified for the more acidic component through isolation of its major cyanogen bromide cleavage products. Characterization of the cyanogen bromide fragments further indicated that this particular subunit is characterized by alternating collagenous and noncollagenous domains as opposed to a lengthy collagenous domain which is either preceded or followed by noncollagenous sequences. The molecular organization and possible derivation of these unique pepsin-resistant collagenous components is discussed.