Electron-microscopical approach to a structural model of intima collagen.

Intima collagen was studied by electron microscopy (rotary shadowing and negative staining) and by analytical ultracentrifugation. It was found that the monomeric unit (Mr 170 000) consists of a 105 nm-long triple helix terminated by a small globular domain (Mr about 30 000) at one end and a large globular domain (Mr about 40 000) at the other end. The monomer was produced by selective reduction of interchain disulphide bridges. Before reduction, dimers, tetramers and larger filamentous structures were found. Dimers are lateral staggered aggregates of two monomers aligned in an anti-parallel fashion. This gives rise to an inner 75 nm-long region of two slightly intertwisted triple helices flanked by the large globular domains. The outer triple-helical segments (length 30 nm) with the small globular domains at their ends emerge at both sides of this structure. Interchain disulphide bridges are probably located in the vicinity of the large domains. Only the outer segments could be degraded by bacterial collagenase. In tetramers the outer segments of two dimers are covalently linked, forming a scissors-like structure. In the fibrous forms several tetramers are assembled end-to-end with an overlap between the outer segments. The molecular masses and sedimentation coefficients were calculated for these various forms from the electron-microscopically observed dimensions and agreed with results obtained by ultracentrifugation. The unique structure of intima collagen suggests that it originates from a microfibrillar component and that it can be considered a unique collagenous protein, for which we propose the designation type VI collagen.     

Characterization of the amino-terminal segment in type III procollagen.

Native type III collagen and procollagen were prepared from fetal bovine skin. Examination of the cleavage products produced by digestion with tadpole collagenase demonstrated that the three palpha1(III) chains of type III procollagen were linked together by disulfide bonds occurring at both the amino-terminal and carboxy-terminal portions of the molecule. Type III collagen contained interchain disulfide bonds only in the carboxy-terminal region of the molecule. After digestion of procollagen with bacterial collagenase an amino-terminal, triple-stranded peptide fragment was isolated. The reduced and alkylated chain constituents of this fragment had molecular weights of about 21 000. After digestion of procollagen with cyanogen bromide a related triple-stranded fragment was isolated. The chains of the cyanogen bromide fragment had a molecular weight of about 27 000. When the collagenase-derived peptide was fully reduced and alkylated, it became susceptible to further digestion with bacterial collagenase. This treatment released a fragment of about 97 amino acid residues which contained 12 cystein residues and had an amino acid composition typical for globular proteins. A second, non-helical fragment of about 48 amino acid residues contained three cysteines. This latter fragment is formed from sequences that overlap the amino-terminal region in the collagen alpha1(III) chain by 20 amino acids and possesses an antigenic determinant specific for the alpha1(III) chain. The collagenase-sensitive region exposed by reduction comprised about 33 amino acid residues. It was recovered as a mixture of small peptides. These results indicate that the amino-terminal region of type III procollagen has the same type of structure as the homologous region of type I procollagen. It consists of a globular, a collagen-like and a non-helical domain. Interchain disulfide bonding and the occurrence of cysteines in the non-helical domain are, however, unique for type III procollagen.     

The N-terminal N5 subdomain of the alpha 3(VI) chain is important for collagen VI microfibril formation

Collagen VI assembly is unique within the collagen superfamily in that the alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains associate intracellularly to form triple helical monomers, and then dimers and tetramers, which are secreted from the cell. Secreted tetramers associate end-to-end to form the distinctive extracellular microfibrils that are found in virtually all connective tissues. Although the precise protein interactions involved in this process are unknown, the N-terminal globular regions, which are composed of multiple copies of von Willebrand factor type A-like domains, are likely to play a critical role in microfibril formation, because they are exposed at both ends of the tetramers. To explore the role of these subdomains in collagen VI intracellular and extracellular assembly, alpha 3(VI) cDNA expression constructs with sequential N-terminal deletions were stably transfected into SaOS-2 cells, producing cell lines that express alpha 3(VI) chains with N-terminal globular domains containing modules N9-N1, N6-N1, N5-N1, N4-N1, N3-N1, or N1, as well as the complete triple helix and C-terminal globular domain (C1-C5). All of these transfected alpha 3(VI) chains were able to associate with endogenous alpha 1(VI) and alpha 2(VI) to form collagen VI monomers, dimers, and tetramers, which were secreted. Importantly, cells that expressed alpha 3(VI) chains containing the N5 subdomain, alpha 3(VI) N9-C5, N6-C5, and N5-C5, formed microfibrils and deposited a collagen VI matrix. In contrast, cells that expressed the shorter alpha 3(VI) chains, N4-C5, N3-C5, and N1-C5, were severely compromised in their ability to form end-to-end tetramer assemblies and failed to deposit a collagen VI matrix. These data demonstrate that the alpha 3(VI) N5 module is critical for microfibril formation, thus identifying a functional role for a specific type A subdomain in collagen VI assembly.     

The globular domains of type VI collagen are related to the collagen-binding domains of cartilage matrix protein and von Willebrand factor.

Type VI collagen is a transformation-sensitive glycoprotein of the extracellular matrix of fibroblasts. We have isolated and sequenced several overlapping cDNA clones (4153 bp) which encode the entire alpha 2 subunit of chicken type VI collagen. The deduced amino acid sequence predicts that the alpha 2(VI) polypeptide consists of 1015 amino acid residues that are arranged in four domains: a hydrophobic signal peptide of 20 residues, an amino-terminal globular domain of 228 residues, a collagenous segment of 335 residues and a carboxy-terminal globular domain of 432 residues. The collagenous domain contains seven Arg-Gly-Asp tripeptide units, some of which are likely to be used as cell-binding sites. The globular domains contain three homologous repeats with an average length of 180 amino acid residues. These repeats show a striking similarity to the collagen-binding motifs found in von Willebrand factor and cartilage matrix protein. We therefore speculate that the globular domains of the alpha 2(VI) polypeptide may interact with collagenous structures.     

A collagen-like glycoprotein of the extracellular matrix is the undegraded form of type VI 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 C5 domain of the collagen VI alpha3(VI) chain is critical for extracellular microfibril formation and is present in the extracellular matrix of cultured cells

Collagen VI, a microfibrillar protein found in virtually all connective tissues, is composed of three distinct subunits, alpha1(VI), alpha2(VI), and alpha3(VI), which associate intracellularly to form triple helical heterotrimeric monomers then dimers and tetramers. The secreted tetramers associate end-to-end to form beaded microfibrils. Although the basic steps in assembly and the structure of the tetramers and microfibrils are well defined, details of the interacting protein domains involved in assembly are still poorly understood. To explore the role of the C-terminal globular regions in assembly, alpha3(VI) cDNA expression constructs with C-terminal truncations were stably transfected into SaOS-2 cells. Control alpha3(VI) N6-C5 chains with an intact C-terminal globular region (subdomains C1-C5), and truncated alpha3(VI) N6-C1, N6-C2, N6-C3, and N6-C4 chains, all associated with endogenous alpha1(VI) and alpha2(VI) to form collagen VI monomers, dimers and tetramers, which were secreted. These data demonstrate that subdomains C2-C5 are not required for monomer, dimer or tetramer assembly, and suggest that the important chain selection interactions involve the C1 subdomains. In contrast to tetramers containing control alpha3(VI) N6-C5 chains, tetramers containing truncated alpha3(VI) chains were unable to associate efficiently end-to-end in the medium and did not form a significant extracellular matrix, demonstrating that the alpha3(VI) C5 domain plays a crucial role in collagen VI microfibril assembly. The alpha3(VI) C5 domain is present in the extracellular matrix of SaOS-2 N6-C5 expressing cells and fibroblasts demonstrating that processing of the C-terminal region of the alpha3(VI) chain is not essential for microfibril formation.     

Characterization of the Head-to-Tail Overlap Complexes Formed by Human Lamin A, B1 and B2 “Half-minilamin” Dimers

Half-minilamins, representing amino- and carboxy-terminal fragments of human lamins A, B1 and B2 with a truncated central rod domain, were investigated for their ability to form distinct head-to-tail-type dimer complexes. This mode of interaction represents an essential step in the longitudinal assembly reaction exhibited by full-length lamin dimers. As determined by analytical ultracentrifugation, the amino-terminal fragments were soluble under low ionic strength conditions sedimenting with distinct profiles and s-values (1.6-1.8 S) indicating the formation of coiled-coil dimers. The smaller carboxy-terminal fragments were, except for lamin B2, largely insoluble under these conditions. However, after equimolar amounts of homotypic amino- and carboxy-terminal lamin fragments had been mixed in 4 M urea, upon subsequent renaturation the carboxy-terminal fragments were completely rescued from precipitation and distinct soluble complexes with higher s-values (2.3-2.7 S) were obtained. From this behavior, we conclude that the amino- and carboxy-terminal coiled-coil dimers interact to form distinct oligomers (i.e. tetramers). Furthermore, a corresponding interaction occurred also between heterotypic pairs of A- and B-type lamin fragments. Hence, A-type lamin dimers may interact with B-type lamin dimers head-to-tail to yield linear polymers. These findings indicate that a lamin dimer principally has the freedom for a “combinatorial” head-to-tail association with all types of lamins, a property that might be of significant importance for the assembly of the nuclear lamina. Furthermore, we suggest that the head-to-tail interaction of the rod end domains represents a principal step in the assembly of cytoplasmic intermediate filament proteins too.     

Molecular assembly, secretion, and matrix deposition of type VI collagen

Monoclonal antibodies reactive with the tissue form of type VI collagen were used to isolate the type VI collagen polypeptides from cultured fibroblasts and muscle cells. Two [35S]methionine-labeled polypeptides of 260 and 140 kD were found intracellularly, in the medium, and in the extracellular matrix of metabolically labeled cells. These polypeptides were disulfide cross-linked into very large complexes. The 260- and 140-kD polypeptides were intimately associated and could not be separated from each other by reduction without denaturation. In the absence of ascorbic acid, both polypeptides accumulated inside the cell, and their amounts in the medium and in the matrix were decreased. These results suggest that both the 260- and the 140-kD polypeptides are integral parts of the type VI collagen molecule. Examination of type VI collagen isolated from the intracellular pool by electron microscopy after rotary shadowing revealed structures corresponding to different stages of assembly of type VI collagen. Based on these images, a sequence for the intracellular assembly of type VI collagen could be discerned. Type VI collagen monomers are approximately 125 nm long and are composed of two globules separated by a thin strand. The monomers assemble into dimers and tetramers by lateral association. Only tetramers were present in culture media, whereas both tetramers and multimers were found in extracellular matrix extracts. The multimers appeared to have assembled from tetramers by end-to-end association into filaments that had prominent knobs and a periodicity of approximately 110 nm. These results show that, unlike other collagens, type VI collagen is assembled into tetramers before it is secreted from the cells, and they also suggest an extracellular aggregation mechanism that appears to be unique to this collagen.     

Characterization of the precursor form of type VI collagen

Well characterized monospecific antisera against pepsin-extracted bovine type VI collagen were used to identify and characterize the intact form of type VI collagen. In immunoblotting experiments the antisera reacted with the pepsin-resistant fragments of the alpha 1(VI) and alpha 3(VI) chains, but not with the fragment of the alpha 2(VI) chain. Extracts obtained from uterus and aorta with 6 M guanidine HCl contained two immunoreactive polypeptides of Mr = 190,000 and 180,000 based on globular protein standards. Cleavage of extracts with pepsin generated the previously characterized pepsin-resistant fragments of alpha 1(VI) and alpha 3(VI), indicating that the higher molecular weight polypeptides represent the intact parent chains, alpha 1(VI) and alpha 3(VI). Digestion of extracts with bacterial collagenase released an Mr = 100,000 noncollagenous fragment from the alpha 1(VI) chain. Thus, intact type VI collagen in tissues contains a relatively short triple helical domain and at least one very large globular domain which is sensitive to pepsin but resistant to collagenase digestion. Immunoblotting revealed a polypeptide of Mr = 240,000, which we suggest represents the pro-alpha 1(VI) chain, in the culture medium of bovine fibroblasts. Bands intermediate in molecular weight between 240,000 and 190,000 were identified in cell layers. These findings establish type VI collagen as a protein with very large nontriple helical domains, a property that undoubtedly plays an important role in its function.     

Collagen VI Microfibril Formation Is Abolished by an α2(VI) von Willebrand Factor Type A Domain Mutation in a Patient with Ullrich Congenital Muscular Dystrophy

Collagen VI is an extracellular protein that most often contains the three genetically distinct polypeptide chains, α1(VI), α2(VI), and α3(VI), although three recently identified chains, α4(VI), α5(VI), and α6(VI), may replace α3(VI) in some situations. Each chain has a triple helix flanked by N- and C-terminal globular domains that share homology with the von Willebrand factor type A (VWA) domains. During biosynthesis, the three chains come together to form triple helical monomers, which then assemble into dimers and tetramers. Tetramers are secreted from the cell and align end-to-end to form microfibrils. The precise molecular mechanisms responsible for assembly are unclear. Mutations in the three collagen VI genes can disrupt collagen VI biosynthesis and matrix organization and are the cause of the inherited disorders Bethlem myopathy and Ullrich congenital muscular dystrophy. We have identified a Ullrich congenital muscular dystrophy patient with compound heterozygous mutations in α2(VI). The first mutation causes skipping of exon 24, and the mRNA is degraded by nonsense-mediated decay. The second mutation is a two-amino acid deletion in the C1 VWA domain. Recombinant C1 domains containing the deletion are insoluble and retained intracellularly, indicating that the mutation has detrimental effects on domain folding and structure. Despite this, mutant α2(VI) chains retain the ability to associate into monomers, dimers, and tetramers. However, we show that secreted mutant tetramers containing structurally abnormal C1 VWA domains are unable to associate further into microfibrils, directly demonstrating the critical importance of a correctly folded α2(VI) C1 domain in microfibril formation.     

Covalent and non-covalent interactions of betaig-h3 with collagen VI. Beta ig-h3 is covalently attached to the amino-terminal region of collagen VI in tissue microfibrils

Transforming growth factor-beta induced gene-h3 (betaig-h3) was found to co-purify with collagen VI microfibrils, extracted from developing fetal ligament, after equilibrium density gradient centrifugation under both nondenaturing and denaturing conditions. Analysis of the collagen VI fraction from the non-denaturing gradient by gel electrophoresis under non-reducing conditions revealed the present of a single high molecular weight band that immunostained for both collagen VI and betaig-h3. When the fraction was analyzed under reducing conditions, collagen VI alpha chains and betaig-h3 were the only species evident. The results indicated that betaig-h3 is associated with collagen VI in tissues by reducible covalent bonding, presumably disulfide bridges. Rotary shadowing and immunogold staining of the collagen VI microfibrils and isolated tetramers indicated that betaig-h3 was specifically and periodically associated with the double-beaded region of many of the microfibrils and that this covalent binding site was located in or near the amino-terminal globular domain of the collagen VI molecule. Using solid phase and co-immunoprecipitation assays, recombinant betaig-h3 was found to bind both native and pepsin-treated collagen VI but not individual pepsin-collagen VI alpha chains. Blocking experiments indicated that the major in vitro betaig-h3 binding site was located in the pepsin-resistant region of collagen VI. In contrast to the tissue situation, the in vitro interaction had the characteristics of a reversible non-covalent interaction, and the Kd was measured as 1.63 x 10(-8) m. Rotary shadowing of immunogold-labeled complexes of recombinant betaig-h3 and pepsin-collagen VI indicated that the in vitro betaig-h3 binding site was located close to the amino-terminal end of the collagen VI triple helix. The evidence indicates that collagen VI may contain distinct covalent and non-covalent binding sites for betaig-h3, although the possibility that both interactions use the same binding region is discussed. Overall the study supports the concept that betaig-h3 is extensively associated with collagen VI in some tissues and that it plays an important modulating role in collagen VI microfibril function.     

Type XIV collagen, a new homotrimeric molecule extracted from fetal bovine skin and tendon, with a triple helical disulfide-bonded domain homologous to type IX and type XII collagens

Previously undescribed disulfide-bonded collagenous pepsin-derived fragments have been isolated from fetal calf tendon and skin. One fragment, 10.5 kDa after reduction, was shown to be similar but distinct to the COL1 domain of the recently characterized type XII collagen (64% primary structure identity). The similarity includes important features such as size, location of the cysteine residues, and nature and position of an imperfection of the triple helix. From fetal calf skin, two approximately 34-kDa disulfide-bonded trimeric fragments were isolated in the unreduced form. Amino acid sequencing showed that one fragment contained solely the COL1 domain of type XII collagen while the other one only contained the COL1 domain of the new chain. Like type XII collagen, the new chain is therefore part of a homotrimeric molecule and should thus be considered as a distinct collagen type. We propose to call the molecule from which this fragment is derived, type XIV collagen, with a chain composition (alpha 1 (XIV]3. The presence of a domain similar to the COL1 domain of collagens types IX and XII suggests that type XIV collagen belongs to the group of fibril-associated collagens with interrupted triple helices (FACIT). Two other fragments, 13.5 and 17 kDa after reduction, were also purified. They were shown to contain the same triple helical domain with different pepsin cleavage sites at the amino terminus. Several tryptic peptides were sequenced, and the derived sequences could be aligned with the COL2 domain of type XII collagen or with flanking sequences in the NC2 and NC3 domains (61% sequence identity). These fragments are very likely to be also derived from type XIV collagen.     

Characterization of structural and immunological properties of specific domains of Friend ecotropic and dual-tropic murine leukemia virus gp70s.

A detailed comparison of the gp70 proteins of cloned ecotropic Friend murine leukemia virus (FLV) and dual-tropic Friend mink focus-forming virus (FrMCF) was performed by analyzing the structural and immunological properties of amino- and carboxy-terminal domains of these molecules generated upon controlled trypsinization. The two gp70s gave characteristic fragmentation patterns; the amino-terminal fragments of FrMCF gp70 were smaller than the corresponding fragments of FLV and contained a trypsin site which resulted in a 19,000-dalton amino-terminal fragment not observed for FLV, whereas both molecules yielded an identically sized carboxy-terminal fragment. All amino-terminal fragments of both gp70 molecules contained an endo H-sensitive oligosaccharide chain; for FrMCF, a second endo H-sensitive carbohydrate was present as well at a carboxy-terminal site for approximately 50% of the molecules. Several aspects of the disulfide interactions of the two gp70s were conserved; in both cases the carboxy-terminal fragments were disulfide bonded to p15(E), there were no disulfide bonds between amino- and carboxy-terminal fragments, and the amino-terminal fragments exhibited a significant increase in mobility upon analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Analysis of the immunoreactivity of the different domains of the proteins by immunoprecipitation of the fragments with antisera prepared against xenotropic murine leukemia virus and feline leukemia virus gp70s indicated major differences in antigenicity for the amino-terminal domains of FLV and FrMCF gp70, whereas the carboxy-terminal domains were immunologically conserved. Similar analyses with antibodies specific for p15(E) and Pr15(E) demonstrate that these components are conserved as well. These data provide direct evidence that p15(E) and the C-terminal gp70 domain of FrMCF gp70 are related to the corresponding regions of the ecotropic FLV parent and indicate that the acquisition of MCF-specific properties is due to the replacement of the ecotropic amino-terminal gp70 domain with sequences related to those of xenotropic gp70s.     

Structural basis of type VI collagen dimer formation

We have determined the interactive sites required for dimer formation in type VI collagen. Despite the fact that type VI collagen is a heterotrimer composed of alpha1(VI), alpha2(VI), and alpha3(VI) chains, the formation of dimers is determined principally by interactions of the alpha2(VI) chain. Key components of this interaction are the metal ion-dependent adhesion site (MIDAS) motif of the alpha2C2 A-domain and the GER sequence in the helical domain of another alpha2(VI) chain. Replacement of the alpha2(VI) C2 domain with the alpha3(VI) domain abolished dimer formation, whereas alterations in the alpha2(VI) C1 domain did not disrupt dimer formation. When the helical sequences were investigated, replacement of the alpha2(VI) sequence GSPGERGDQ with the alpha3(VI) sequence GEKGERGDV abolished dimer formation. Mutating the Pro-108 to a Lys-108 in this alpha2(VI) sequence did not influence dimer formation and suggests that, unlike the integrin I-domain/triple-helix interaction, hydroxyproline is not required in collagen VI A-domain/helix interaction. These results demonstrate that the alpha2(VI) chain position in the assembled triple-helical molecule is critical for antiparallel dimer formation and identify the interacting collagenous and MIDAS sequences involved. These interactions underpin the subsequent assembly of type VI collagen.     

The role of the main noncollagenous domain (NC1) in type IV collagen self-assembly

Type IV collagen incubated at elevated temperatures in physiologic buffers self-associates (a) via its carboxy-terminal (NC1) domain, (b) via its amino-terminal (7S) domain, and (c) laterally; and it forms a network. When examined with the technique of rotary shadowing, isolated domain NC1 was found to bind along the length of type IV collagen to four distinct sites located at intervals of approximately 100 nm each. The same 100-nm distance was observed in domain NC1 of intact type IV collagen bound along the length of the collagen molecules during initial steps of network formation and in complete networks. The presence of anti-NC1 Fab fragments in type IV collagen solutions inhibited lateral association and network formation in rotary shadow images. During the process of self-association type IV collagen develops turbidity; addition of isolated domain NC1 inhibited the development of turbidity in a concentration-dependent manner. These findings indicate that domain NC1 of type IV collagen plays an important role in the process of self-association and suggest that alterations in the structure of NC1 may be partially responsible for impaired functions of basement membranes in certain pathological conditions.     

Bethlem myopathy and engineered collagen VI triple helical deletions prevent intracellular multimer assembly and protein secretion.

Mutations in the genes that code for collagen VI subunits, COL6A1, COL6A2, and COL6A3, are the cause of the autosomal dominant disorder, Bethlem myopathy. Although three different collagen VI structural mutations have previously been reported, the effect of these mutations on collagen VI assembly, structure, and function is currently unknown. We have characterized a new Bethlem myopathy mutation that results in skipping of COL6A1 exon 14 during pre-mRNA splicing and the deletion of 18 amino acids from the triple helical domain of the alpha1(VI) chain. Sequencing of genomic DNA identified a G to A transition in the +1 position of the splice donor site of intron 14 in one allele. The mutant alpha1(VI) chains associated intracellularly with alpha2(VI) and alpha3(VI) to form disulfide-bonded monomers, but further assembly into dimers and tetramers was prevented, and molecules containing the mutant chain were not secreted. This triple helical deletion thus resulted in production of half the normal amount of collagen VI. To further explore the biosynthetic consequences of collagen VI triple helical deletions, an alpha3(VI) cDNA expression construct containing a 202-amino acid deletion within the triple helix was produced and stably expressed in SaOS-2 cells. The transfected mutant alpha3(VI) chains associated with endogenous alpha1(VI) and alpha2(VI) to form collagen VI monomers, but dimers and tetramers did not form and the mutant-containing molecules were not secreted. Thus, deletions within the triple helical region of both the alpha1(VI) and alpha3(VI) chains can prevent intracellular dimer and tetramer assembly and secretion. These results provide the first evidence of the biosynthetic consequences of structural collagen VI mutations and suggest that functional protein haploinsufficiency may be a common pathogenic mechanism in Bethlem myopathy.     

Large complex globular domains of type VII procollagen contribute to the structure of anchoring fibrils

Type VII collagen, in the form of an antiparallel dimer, is a major protein component of anchoring fibrils. The ultrastructural appearance of these fibrils suggests that they may serve to anchor the basement membrane zone to the underlying connective tissue matrix. We report here the identification and initial characterization of Type VII procollagen, recovered from the media of epidermoid carcinoma cell cultures. Immunoblotting using monospecific antibodies to Type VII procollagen identifies a single, homogeneous band of at least Mr 320,000 following disulfide bond reduction. This chain contains 170 kDa of collagen triple helix and 150 kDa of non-helical domain at the carboxyl terminus. Pepsin digestion of this material yields Type VII collagen identical to that isolated from whole tissue and a series of quasi-stable peptides derived from the carboxyl-terminal region. Cell extracts contain procollagen chains identical in size to those secreted into the media. There is no evidence for processing of this material in cell culture. Partial purification by velocity sedimentation and transmission electron microscopic observation following rotary shadowing reveals both monomers (426 nm) and dimers (785 nm). Dimers are antiparallel and interact through 60-nm overlap, with amino-terminal globular domains present at the ends of the overlap. The multi-domain carboxyl-terminal region appears as three similar arms originating from a centralized globular region adjacent to the collagen helix. The carboxyl globular domain is present in whole tissue and may participate in the unique fibril form of this collagen. The amino-terminal globule may function in the antiparallel assembly of dimers.     

Factors affecting gamma-chain multimer formation in cross-linked fibrin.

The major covalently linked multimolecular D fragments found in plasmic digests of factor XIIIa cross-linked fibrin formed under physiological pH and ionic strength conditions consist of D dimers, D trimers, and D tetramers. These fragments are linked by epsilon-amino-gamma-glutamyllysine bonds in the carboxy-terminal regions of their gamma chains, which had originated in the cross-linked fibrin as gamma dimers, gamma trimers, and gamma tetramers, respectively. In this study, factors affecting the degree and rate of formation of these three classes of cross-linked gamma chains were determined by analyzing the D-fragment content of plasmic digests of cross-linked fibrin that had been sampled after all gamma-chain monomers had been consumed in the cross-linking process. D trimers and D tetramers, expressed as a proportion of the total D-fragment content, both increased at the expense of the D-dimer population as a function of increasing factor XIII concentration, the time of cross-linking, or the CaCl2 concentration. Their levels decreased as the ionic strength was raised by NaCl addition. However, the ionic strength effect could be reversed by concomitantly raising the CaCl2 concentration. Digests of clots prepared from recalcified fresh citrated plasma also contained each type of cross-linked D fragment, and the proportion of D trimers and D tetramers in the digest increased with increasing clot incubation time. These results indicate that gamma-trimer and gamma-tetramer formation is a dynamic physiological process.(ABSTRACT TRUNCATED AT 250 WORDS)