The use of monoclonal antibodies to fragments of chicken type IV collagen in structural and localization studies

In previous experiments, three pepsin-resistant fragments of type IV collagen were isolated from chicken gizzards and designated 7S, F3, and (F1)2F2 (Mayne, R., and Zettergren, J. G. (1980) Biochemistry 19, 4065-4072). In the present experiments, a series of monoclonal antibodies to type IV collagen were prepared, each one of which recognized an epitope present in only one of the three fragments. A high molecular weight fraction of type IV collagen (designated 7S + arms (215 nm)) was isolated after agarose gel filtration and characterized by electron microscopy after rotary shadowing and by gel electrophoresis. Analysis of 7S + arms (215 nm) by inhibition enzyme-linked immunosorbent assay demonstrated the presence of the epitopes for 7S and F3 but not for (F1)2F2. This result, therefore, provides additional evidence that the order of the pepsin-resistant fragments of chicken type IV collagen is 7S-F3-(F1)2F2.     

Monoclonal antibodies against chicken type IV and V collagens: electron microscopic mapping of the epitopes after rotary shadowing

The location of the epitopes for monoclonal antibodies against chicken type IV and type V collagens were directly determined in the electron microscope after rotary shadowing of antibody/collagen mixtures. Three monoclonal antibodies against type IV collagen were examined, each one of which was previously demonstrated to be specific for only one of the three pepsin-resistant fragments of the molecule. The three native fragments were designated (F1)2F2, F3, and 7S, and the antibodies that specifically recognize each fragment were called, respectively, IA8 , IIB12 , and ID2 . By electron microscopy, monoclonal antibody IA8 recognized an epitope located in the center of fragment (F1)2F2 and in tetramers of type IV collagen at a distance of 288 nm from the 7S domain, the region of overlap of four type IV molecules. Monoclonal antibody IIB12 , in contrast, recognized an epitope located only 73 nm from the 7S domain. This result therefore provides direct visual evidence that the F3 fragment is located closest to the 7S domain and the order of the fragments must be 7S-F3-(F1)2F2. The epitope for antibody ID2 was located in the overlap region of the 7S domain, and often several antibody molecules were observed to binding to a single 7S domain. The high frequency with which antibody molecules were observed to bind to fragments of type IV collagen suggests that there is a single population of type IV molecules of chain organization [alpha 1(IV)]2 alpha 2(IV), and that four identical molecules must form a tetramer that is joined in an antiparallel manner at the 7S domain. The monoclonal antibodies against type V collagen, called AB12 and DH2 , were both found to recognize epitopes close to one another, the epitopes being located 45-48 nm from one end of the type V collagen molecule. The significance of this result still remains uncertain, but suggests that this site is probably highly immunoreactive. It may also be related to the specific cleavage site of type V collagen by selected metalloproteinases and by alpha-thrombin. This cleavage site is also known to be located close to one end of the type V molecule.     

Mapping of domains in human laminin using monoclonal antibodies: localization of the neurite-promoting site

Monoclonal antibodies were made against a truncated form of human laminin isolated from placenta. 12 antibodies were isolated and characterized. All antibodies stained basement membranes in placenta and immunoprecipitated laminin from media of cultured choriocarcinoma cells. Three antibodies, 3E5, 4C7, and 4E10, partially blocked the neurite-promoting activity of laminin. Addition of a second antibody, goat anti-mouse IgG, caused more complete blocking of the activity. Two of the blocking antibodies, 4C7 and 4E10, reacted with epitopes within the globular domain at the end of the long arm of laminin, and the third one, 3E5, reacted at the end of the rod-like portion of the long arm adjacent to the globular domain, as shown by electron microscopy after rotary shadowing. Five nonblocking antibodies used in the same test reacted with epitopes in other domains of the molecule. Blocking antibodies 3E5 and 4E10 could be used in immunoblotting and both antibodies reacted with the same polypeptides in pepsin fragments of human laminin, the predominant polypeptides being approximately 400 kD. When a crude extract of human amnion was used as a source of intact laminin, the 4E10 antibody detected a single polypeptide of approximately 400 kD. A nonblocking antibody, 2E8, which reacted at the center of the laminin cross, reacted predominantly with a 200-kD polypeptide in human laminin fragments and exclusively with a 200-kD polypeptide in amnion extract and in rat laminin. Our results with human laminin match the results by Edgar, D., R. Timpl, and H. Thoenen, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:1463-1468, in which the neurite-promoting activity of mouse laminin resides at the end of the long arm, which is also the site for heparin binding. However, since the active fragments of human laminin did not bind to heparin, the neurite-promoting site should be different from the heparin-binding site. Our results further suggest that the neurite-promoting site may be contained in or close to the 400-kD component of laminin.     

Drosophila basement membrane procollagen alpha 1(IV). II. Complete cDNA sequence, genomic structure, and general implications for supramolecular assemblies

A Drosophila melanogaster gene for a basement membrane procollagen chain was recently identified from the sequence homology of the carboxyl (NC1) end of the polypeptide that it encodes with the corresponding domain of human and murine collagens IV (Blumberg, B., MacKrell, A. J., Olson, P. F., Kurkinen, M., Monson, J. M., Natzle, J. E., and Fessler, J. H. (1987) J. Biol. Chem. 262, 5947-5950). This gene is at chromosome location 25C. Here we report the complete 6-kilobase cDNA sequence coding for a chain of 1775 amino acids, as well as the genomic structure. The gene is composed of nine relatively large exons separated by eight relatively small introns. This organization is different from the multiple small exons separated by large introns reported for mouse and human type IV collagens (Kurkinen, M., Bernard, M. P., Barlow, D. P., and Chow, L. T. (1985) Nature 317, 177-179. Sakurai, Y., Sullivan, M., and Yamada, Y. (1986) J. Biol. Chem. 261, 6654-6657. Soininen, R., Tikka, L., Chow, L., Pihlajaniemi, T., Kurkinen, M., Prockop, D. J., Boyd, C. D., and Tryggvason, K. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1568-1572). Drosophila and human alpha 1(IV) procollagen chains share not only polypeptide domains near their amino and carboxyl ends for making specialized, intermolecular junctional complexes, but also 11 of 21 sites of imperfections of the collagen triple helix. However, neither the number nor the nature of the amino acids in these imperfections appear to have been conserved. These imperfections of the helical sequence may be important for the supramolecular assembly of basement membrane collagen. The 9 cysteine residues of the Drosophila collagen thread domain are arranged as several variations of a motif found in vertebrate collagens IV only near their amino ends, in their "7 S" junctional domains. The relative positions of these cysteine residues provide numerous opportunities for disulfide bonding between molecules in both parallel and antiparallel arrays. There is a pseudorepeat of one-third of the thread length, and there are numerous possibilities for disulfide-linked microfibrils and networks. We propose that collagen microfibrils, stabilized by disulfide segment junctions, are a versatile ancestral form from which specialized collagen fibers and networks arose.     

Characterisation of a monoclonal antibody against native human type I collagen

A monoclonal antibody against a pepsin-soluble mammalian type I collagen has been produced. This antibody, subclass IgG1, kappa, was specific for type I collagen and did not cross-react with a range of other collagen types or connective tissue proteins. The epitope recognized by the antibody was dependent upon an intact triple-helical structure for the collagen, and was shown by rotary shadowing and by immunoblotting of collagenase-derived fragments to be near the C-terminal of the pepsin-soluble collagen. Although the antibody had a low affinity, with Kd = 4 x 10(-7) M, it could be used for immunohistology of tissue sections and for studies of collagen produced by cells in culture. The antibody, which was raised against human collagen, also recognized type I collagens from certain other species, including calf, pig, sheep, goat and dog.     

Purification and characterization of native type XIV collagen.

A new molecule, type XIV collagen, with domains homologous to type IX and XII collagens has been recently discovered in pepsin extracts of fetal bovine tissues (Dublet, B., and van der Rest, M. (1991) J. Biol. Chem. 266, 6853-6858). In the present study, we describe the purification and the characterization of the intact native form of this newly discovered collagen. By using only two chromatographic steps we were able to obtain pure type XIV collagen. Furthermore, minor modifications of the protocol allowed us to perform the simultaneous large scale purification of type XII and type XIV collagens from the same tissue. Intact type XIV collagen migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as two bands of 220 and 290 kDa (reducing conditions). After collagenase treatment, a single band of 190 kDa is observed, which represents the large non-collagenous domain of the molecule (NC3). Rotary shadowing electron micrographs of intact type XIV collagen show a cross-shaped structure formed by a thin tail attached through a central globule to three identical "fingers." These properties are similar to those previously described for intact chicken type XII collagen (Dublet, B., Oh, S., Sugrue, S. P., Gordon, M. K., Gerecke, D. R., Olsen, B. R., and van der Rest, M. (1989) J. Biol. Chem. 264, 13150-13156), but the two molecules are different gene products and have charge and glycosylation differences. Finally, we show that the three chains of purified type XIV collagen have an apparent molecular mass of approximately 220 kDa and are not cross-linked to each other by bonds other than disulfide bridges. The same observation was made for type XII collagen. In both cases, the 290-kDa migrating band in SDS-PAGE is due to incomplete denaturation in electrophoresis sample buffer in the absence of urea.     

Binding of laminin to type IV collagen: a morphological study

A mixture of laminin and type IV collagen was analyzed by rotary shadowing using carbon/platinum and electron microscopy. Laminin was found to form distinct complexes with type IV collagen: one site of interaction is located 140 nm from the COOH-terminal, noncollagenous (NC1) domain and the other is located within the NH2-terminal region. The isolated NC1 fragment of type IV collagen does not appear to interact with laminin, while pepsin-treated type IV collagen, which lacks the NC1 domain, retains its ability to form complexes with laminin. Analysis of the laminin-type IV complexes indicates that laminin binds to type IV collagen via the globular regions of either of its four arms. This finding is supported by experiments using fragment P1 of laminin which lacks the globular regions and which does not bind to type IV collagen in a specific way. In addition, after heat-denaturation of laminin no specific binding is observed.     

Structural and immunological characterization of type IV collagen isolated from chicken tissues

Previously, a type IV collagen fraction was isolated from chicken gizzard and further fractionated into three components called F1, F2 and F3 [Mayne, R. and Zettergren, J.G. (1980) Biochemistry, 19, 4065-4072]. F1 and F2 were consistently isolated in a 2:1 proportion, and the existence of a small native fragment of structure (F1)2F2 was proposed. In the present series of experiments, a type IV collagen fraction was isolated from the chicken kidney and shown to consist almost entirely of F1 and F2 which were again present in a 2:1 proportion. Identical one-dimensional peptide maps for F1 and F2 from both sources were obtained by polyacrylamide gel electrophoresis of peptides obtained after cleavage with cyanogen bromide or Staphylococcus aureus V8 protease. The denaturation temperature of a preparation containing F1 and F2 in native form was determined by optical rotatory dispersion and a single melting curve was observed with a melting temperature of 33 degrees C. This result provides further supportive evidence that F1 and F2 exist as a native fragment (F1)2F2. Antibodies were prepared in rabbits against a type IV collagen fraction isolated from chicken gizzard, and immunofluorescent staining of a wide variety of basement membranes was demonstrated. Experiments were performed in which various type IV collagen fractions were observed in the electron microscope after rotary shadowing. The lengths of (F1)2F2 and F3 were 147 nm and 174 nm respectively, the sum of these lengths (321 nm) corresponding closely to the length of the major triple-helical domain of type IV collagen (326-328 nm). A specific cleavage site was located at a distance of 215 nm from the 7-S domain which, together with the length of (F1)2F2, gives a total length of 362 nm. This value corresponds closely to the maximum length of the arms which originate from the 7-S domain (355 nm) when type IV collagen was solubilized with a low concentration of pepsin. The results suggest that (a) type IV collagen isolated from the chicken gizzard is closely related, if not identical, to type IV collagen isolated from other tissues; (b) there is a single type IV collagen molecule of chain organization[alpha 1(IV)]2 alpha2(IV); (c) the order of the pepsin-resistant fragments within a type IV molecule is 7S-F3-(F1)2F2.     

Detection of anti-collagen antibodies by a solid phase radioimmunologic technic

A solid-phase radioimmunoassay using 125I-protein A is described for the detection of antibodies to collagens of different types. The optimal conditions for the adsorption of collagen onto polystyrene microplates, then the incubations with the antiserum and finally with the 125I-protein A have been evaluated. The technique was applied successfully to antisera raised in rabbit, goat, guinea pig and mouse against human type I, II, III, IV, V and bovine type I, II, 1 alpha 2 alpha 3 alpha, X1-X7 collagens.     

Isolation and sequencing of cDNAs and genomic DNAs encoding the alpha 4 chain of basement membrane collagen type IV and assignment of the gene to the distal long arm of human chromosome 2.

We cloned three overlapping cDNAs covering 2,452 base pairs encoding a new basement membrane collagen chain, alpha 4(IV), from rabbit corneal endothelial cell RNA. Nucleotide sequence analysis demonstrated that the clones encoded a triple-helical domain of 392 1/3 amino acid residues and a carboxyl non-triple-helical (NC1) domain of 231 residues. We also isolated a genomic DNA fragment for the human alpha 4(IV) chain, which contained two exons encoding from the carboxyl end of the triple-helical domain to the amino end of the NC1 domain. Identification of the clones was based on the amino acid sequence identity between the cDNA-deduced amino acid sequence and the reported amino acid sequence obtained from a fragment of the alpha 4(IV) collagen polypeptide M28+ (Butkowski, R. J., Shen, G.-Q., Wieslander, J., Michael, A. F., and Fish, A. J. (1990) J. Lab. Clin. Med. 115, 365-373). When compared with four other type IV collagen chains, the NC1 domain contained 12 cysteinyl residues in positions identical to those of the residues in those chains. The domain demonstrated 61, 70, 55, and 60% amino acid similarity with human alpha 1, human alpha 2, bovine alpha 3, and human alpha 5 chains, respectively. The human genomic DNA fragment allowed us to map the alpha 4(IV) gene (COL4A4) to the 2q35-2q37.1 region of the human genome.     

Antigenic determinants of the cytoplasmic domain of band 3 from bovine erythrocyte membrane

Rabbit antibodies were prepared against the cytoplasmic 38K-Da fragment of bovine band 3 and the immunological cross-reactivity with human, murine, rat, and chicken band 3 was examined. The antibodies cross-reacted with human and rodent band 3, indicating that there is an antigenic determinant(s) common to primate and nonprimate species. However, the antibodies did not recognize chicken band 3. Antigenic sites on the 38K-Da fragment were determined via amino acid sequence and immunoblotting analyses of proteolytic peptides of the fragment. Positions of antigenic determinants which were assumed to be common to primate and nonprimate species were mapped to the areas of residues 127-160 and of residues 259-304 in the primary structure of human band 3. Another epitope(s), which is absent in human band 3, existed in a region having a bovine-specific amino acid sequence. In addition, comparison of sequence data from different species showed that a proposed hinge region and a tryptophan-rich region on the cytoplasmic domain of band 3 [P. S. Low et al. (1984) J. Biol. Chem. 259, 13,070-13,076; R. R. Kopito and H. F. Lodish (1985) Nature (London) 316, 234-238] are also conserved in the bovine case.     

Conformational epitopes on interstitial collagens.

The antigenic response to the helical domain of collagens is normally very low, with the nature of the epitopes recognized by antibodies being dependent on the species of origin. Thus, in certain species, for example rabbit, sequential determinants on single alpha-chains are found, whereas in other species such as mouse, conformational epitopes are predominant. A variety of techniques for identification of epitopes, including rotary shadowing, examination of specific fragments and chemical modification reactions are discussed. The application of these techniques is illustrated using a range of monoclonal antibodies to interstitial collagens. These antibodies show that epitopes are distributed over the length of the collagen molecule.     

Degradation of basement membranes by human matrix metalloproteinase 3 (stromelysin).

Connective tissue cells synthesize and secrete a group of matrix metalloproteinases (MMPs), all of which are capable of degrading the extracellular-matrix components. One of them, MMP-3 (stromelysin) has been shown to degrade purified basement-membrane components, collagen IV and laminin [Okada, Y., Nagase, H. & Harris, E. D., Jr. (1986) J. Biol. Chem. 261, 14245-14255]. Here we report that MMP-3 degrades collagen IV and laminin in intact basement membranes from bovine glomeruli (GBM) and bovine anterior-lens capsules (LBM). Degradation products were analysed by SDS/polyacrylamide-gel electrophoresis to determine the number and sizes of polypeptide fragments. Immunoblotting techniques were used to identify the origins of the fragments, i.e. collagen IV or laminin. The fragments of collagen IV were further mapped using specific antibodies that recognize the N-terminal (7 S) domain, the C-terminal (NC-1) domain, or the major triple-helical region between the terminal domains. Degradation of collagen IV was extensive; many fragments were found, from both GBM and LBM, in the Mr range 25,000-380,000. A large fragment of laminin (Mr greater than 380,000) was found in the GBM digests without reduction, but it dissociated into 220,000-Mr chains upon reduction. The results suggest that MMP-3 plays an important role in the catabolism of basement membranes.     

Monoclonal antibodies as structural probes of surface residues in the regulatory subunit of cAMP-dependent protein kinase II from porcine heart

Two murine monoclonal antibodies (H5 and B6) generated against bovine heart type II regulatory subunit of cAMP-dependent protein kinase were shown to cross-react equally well with the homologous subunit from porcine heart. The antibodies demonstrated specificity for only the type II regulatory subunit and showed negligible cross-reactivity with the type I regulatory subunit, the catalytic subunit, and cGMP-dependent protein kinase. Following limited proteolysis of type II regulatory subunit with chymotrypsin, the H5 monoclonal antibody was shown to cross-react with the Mr = 37,000 cAMP-binding domain corresponding to the COOH-terminal region of the polypeptide chain. To more specifically localize the antigenic sites, the porcine type II regulatory subunit was carboxymethylated and cleaved with cyanogen bromide. Both monoclonal antibodies cross-reacted with the NH2-terminal CNBr peptide, and this peptide demonstrated affinities similar to native bovine type II regulatory subunit in competitive displacement radioimmunoassays. Tryptic cleavage of this CNBr fragment destroyed all antigenicity for both monoclonal antibodies, whereas antigenicity was retained following chymotryptic digestion. A single major immunoreactive chymotryptic fragment that cross-reacted with H5 was isolated by gel filtration and reverse phase high performance liquid chromatography. this peptide retained the complete antigenic site and had the following sequence: Asn-Pro-Asp-Glu-Glu-Glu-Glu-Asp-Thr-Asp-Pro-Arg-Val-Ile-His-Pro-Lys-Thr-Asp-Gl n. This antigenic site was localized just beyond the major site of autophosphorylation, approximately a third of the distance from the NH2-terminal end of the polypeptide chain.     

Self-assembly of basement membrane collagen

The in vitro self-assembly of murine type IV collagen was examined by using biochemical and morphological techniques. Dimeric collagen undergoes a rapid and reversible thermal gelation at neutral pH without an appreciable lag period. The process is seen to be concentration dependent and inhibited by 2 M urea. The formed complex can be visualized by electron microscopy rotary shadowing as an irregular polygonal lattice network with extensive side by side associations within the collagenous triple-helical part of the molecules, two and three strands thick. Measurements on the matrix suggest a median stagger dimension of 170 nm, one-fifth the length of a dimer. The conversion of pepsin-generated monomers into N-terminally bound tetramers can also be demonstrated in vitro. This process is also concentration dependent and inhibited and reversed by 2 M urea but is thermally irreversible and occurs at a slow rate relative to the lateral associations. These tetramers can be seen by rotary shadowing as four-armed "spider" structures. It is proposed that lateral associations, by virtue of their faster rate of formation, precede 7S bond formation, and several models for the assembly of basement membrane collagen are discussed.     

Type IV and V collagens in von Recklinghausen's neurofibromas. An immunohistochemical and electrophoretical study

Type IV and V collagens were localized in neurofibromas from six patients with von Recklinghausen's neurofibromatosis (NF) using the peroxidase anti-peroxidase (PAP) technique. The collagens were also isolated from neurofibromas by pepsin digestion and fractionating salt precipitations and demonstrated with sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Staining reactions for both collagens were detected in most of the cells in the disorganized NF tumor tissue. These cells also were S-100 protein-positive and were considered to be of Schwannian cell origin, while the type IV collagen-negative cells showed fibroblastoid, mast cell and histiocytic characteristics. Type IV collagen detection was also used to study the structure of a neurofibroma after 3 weeks in tissue culture. The proportion of fibroblastoid, type IV collagen-negative cells increased significantly in the cultured neurofibromas and "buds" containing solely fibroblastoid cells were seen at the periphery of the tumor fragments. Cultured 6th passage tumor cells produced type V but no type IV collagen as estimated with SDS-PAGE. Further, two malignant Schwannomas from a patient with NF were stained with antibodies to type IV collagen. A positive staining reaction was associated only with the vascular walls in the malignant Schwannomas suggesting that type IV collagen expression is linked with cell differentiation. The present data show that the detection of type IV collagen using the PAP-method is useful in studying the organization of tumors with mixed cell populations such as neurofibromas. Large neurofibromas might also serve as a source for the isolation of human type IV and V collagens.     

Binding of nidogen and the laminin-nidogen complex to basement membrane collagen type IV

The laminin-nidogen complex and purified nidogen both bind collagen IV but not other collagens, as shown by solid-state ligand-binding and inhibition assays. Laminin purified from the dissociated complex and a variety of laminin proteolytic fragments failed to bind collagen IV. Complexes formed in solution between nidogen or laminin-nidogen and collagen IV were visualized by rotary shadowing which identified one major binding site about 80 nm away from the C-terminus of the collagen triple helix. A second, weaker binding site may exist closer to its N-terminus. Binding sites of nidogen were assigned to its C-terminal globular domain which also possesses laminin-binding structures. A more diverse collagen-IV-binding pattern was observed for the laminin nidogen complex, whereby interactions may involve both nidogen and short-arm structures of laminin.     

An analysis by rotary shadowing of the structure of the mammalian vitreous humor and zonular apparatus

An electron microscopic analysis of human and bovine vitreous humor after rotary shadowing showed the presence of both collagen fibrils and an extensive loose network of hyaluronan molecules. No interaction between the collagen fibrils and the hyaluronan molecules was observed under the conditions used for rotary shadowing. Periodic "struts" were present on the surface of the collagen fibrils. These struts showed an organization the same as that previously observed for type IX collagen on the surface of collagen fibrils from chicken cartilage and vitreous. However, the knob of the noncollagenous NC4 domain of cartilage type IX collagen was not observed at the ends of the struts in a manner identical to that of chicken vitreous humor. Zonular fibrils were dissected out from bovine eyes and shown by rotary shadowing to contain a beaded fibril which is similar in morphology to the "elastin-associated" microfibrils of many connective tissues. Experiments in which the zonular fibrils were stretched and fixed prior to rotary shadowing showed that the distance between each bead is variable and can be accounted for by the bowing out of overlapping filaments which connect each bead.     

New pepsin-solubilized low molecular weight collagenous component possibly unique to periodontal ligament

Limited pepsin digestion of bovine periodontal ligament releases genetic types I, III, and V collagen and a high cystine containing low molecular weight collagenous component. Salt fractionation and molecular sieve chromatography allowed the isolation of the latter as an apparently pure homogeneous moiety which had an approximate molecular mass of 30 000 daltons. Reduction with mercaptoethanol yielded a single 10 000-dalton band on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This led us to conclude that the newly isolated low molecular weight collagen fragment consists of three similar molecular weight chains. Unreduced collagen-like glycoprotein (CGP) [Jander, R., Troyer, D., & Rauterberg, J. (1984) Biochemistry 23, 3675-3681] after extraction from tissues with collagen denaturing solvents yields the GP140 glycoprotein upon reduction and does not release any collagen fragment below 90 000 daltons upon mild or vigorous pepsin digestion. The GP140 glycoprotein [Heller-Harrison, R. A., & Carter, W. G. (1984) J. Biol. Chem. 259, 6858-6864] isolated by extraction under reducing and collagen denaturing solvent conditions did not yield a collagen fragment below 40 000 daltons after pepsin treatment. It was clearly shown that both CGP and GP140 yield type VI collagen fragments in the above-cited reports. Since this report demonstrates that the Mr 30 000 collagen fragment is only released by pepsin treatment of nondenaturing solvent treated periodontal ligament and that only very small peptides are found in denaturing solvent treated tissue after pepsin digestion, it is concluded that the newly isolated Mr 30 000 collagen fragment reported here is not derived from type VI collagen.(ABSTRACT TRUNCATED AT 250 WORDS)     

p-HMW-collagen, a minor collagen obtained from chick embryo cartilage without proteolytic treatment of the tissue

The fragments of minor collagens of cartilages, called HMW and LMW, were isolated after pepsin treatment of sternal cartilages of young chickens and were shown to be entirely triple-helical molecules as judged by their circular dichroic spectra. Studies on renaturation kinetics of HMW suggested that the interchain disulfide bonds in HMW reside at one of the ends of the so-called long arm. Polyclonal antibodies against HMW were raised and affinity purified. These antibodies did not cross-react with type II collagen nor with other minor collagens such as LMW and 1 alpha, 2 alpha, 3 alpha collagen in native or denatured structure. The antibodies were used to identify HMW-related molecules which were synthesized by embryonic chick cartilages in vitro. Some of these molecules were secreted into the organ culture medium and could be recovered from it by ammonium sulfate precipitation. Polyacrylamide gel electrophoresis of this precipitate gave one band of high molecular weight which could be reduced to two bands migrating slightly faster than the alpha 1(II) chain when identified by immunoblotting. These bands could also be identified among about six radiolabelled polypeptides present in the ammonium sulfate precipitate of medium proteins when analysed by polyacrylamide gel electrophoresis followed by fluorography. The same polypeptides could be recovered from the medium by immunoprecipitation with anti-HMW antibodies. Their presence in cartilage tissue was shown by immunoblotting of material extracted from cartilage tissue and separated on polyacrylamide gels. We suggest that the protein containing these polypeptide chains represents the parent molecule of the peptic fragment HMW as it is synthesized in vivo and have designated it p-HMW-collagen.