Identification of the Goodpasture antigen as the alpha 3(IV) chain of collagen IV

The organizational relationship between the recently identified alpha 3 chain of basement membrane collagen (Butkowski, R.J., Langeveld, J.P.M., Wieslander, J., Hamilton, J., and Hudson, B.G. (1987) J. Biol. Chem. 262, 7874-7877) and collagen IV was determined. This was accomplished by the identification of subunits in hexamers of the NC1 domain of collagen IV that were immunoprecipitated with antibodies prepared against subunits M1, corresponding to alpha 1(IV)NC1 and alpha 2(IV)NC1, and M2, corresponding to alpha 3NC1, and by amino acid sequence analysis. The presence of at least two distinct types of hexamers was revealed, one enriched in M1 and the other enriched in M2, but in both types, M1 and M2 coexist. Evidence was also obtained for the existence of heterodimers comprised of M1 and M2. These results indicate that M2 is an integral component of the NC1 hexamer of collagen IV. The amino acid sequence of the NH2-terminal region of M2 was found to be highly related to the collagenous-NC1 junctional region of the alpha 1 chain of collagen IV. Therefore, M2 is designated alpha 3(IV)NC1 and its parent chain alpha 3(IV). These findings lead to a new concept about the structure of collagen IV: namely, 1) collagen IV is comprised of a third chain (alpha 3) together with the two classical ones (alpha 1 and alpha 2); the alpha 3(IV) chain exists within the same triple-helical molecule together with the alpha 1(IV) and alpha 2(IV) chains and/or within a separate triple-helical molecule, exclusive of alpha 1(IV) and alpha 2(IV) chains, but connected through the NC1 domains to the classical triple-helical molecule comprised of alpha 1(IV) and alpha 2(IV) chains. Additionally, a portion of those triple-helical molecules exclusive of alpha 1(IV) and alpha 2(IV) chains may be connected to each other through their NC1 domains; and 3) the epitope to which the major reactivity of autoantibodies are targeted in glomerular basement membrane in patients with Goodpasture syndrome is localized to the NC1 domain of the alpha 3(IV) chain.     

Glomerular basement membrane. Identification of a fourth chain, alpha 4, of type IV collagen

The noncollagenous domain hexamer of collagen IV from bovine glomerular basement membrane was further investigated to determine the types of collagen chain from which subunits M2*b and M3 are derived. M2*b was shown to be a shorter form, containing 9 fewer residues, of M2*a which was previously established as the noncollagenous domain of a third chain, alpha 3, of collagen IV (Saus, J., Wieslander, J., Langeveld, J.P.M., Quinones, S., and Hudson, B.G. (1988) J. Biol. Chem. 263, 13374-13380). M3 was identified as the noncollagenous domain of a fourth chain, alpha 4, of type IV collagen, on the basis of additional sequence data together with previous findings. A comparison of the collagenous-noncollagenous junction regions of alpha 3(IV) and alpha 4(IV) chains with those of classical alpha 1(IV) and alpha 2(IV) chains reveals structural information which provides a potential strategy for molecular cloning of these novel chains. The results further reveal the complexity of electrophoresis patterns of the hexamer and potential ambiguities in using one-dimensional patterns to determine whether molecular defects of collagen IV occur in pathological processes affecting basement membranes.     

Complete primary structure of the triple-helical region and the carboxyl-terminal domain of a new type IV collagen chain, alpha 5(IV)

We have isolated and characterized overlapping cDNA clones which code for a previously unidentified human collagen chain. Although the cDNA-derived primary structure of this new polypeptide is very similar to the basement membrane collagen alpha 1(IV) and alpha 2(IV) chains, the carboxyl-terminal collagenous/non-collagenous junction sequence does not correspond to the junction sequence in either of the newly described alpha 3(IV) or alpha 4(IV) chains (Butkowski, R.J., Langeveld, J.P.M., Wieslander, J., Hamilton, J., and Hudson, B. G. (1987) J. Biol. Chem. 262, 7874-7877). Thus the protein presented here has been designated the alpha 5 chain of type IV collagen. Four clones encode an open reading frame of 1602 amino acids that cover about 95% of the entire chain including half of the amino-terminal 7S domain and all of the central triple-helical region and carboxyl-terminal NC1 domain. The collagenous region of the alpha 5(IV) chain contains 22 interruptions which are in most cases identical in distribution to those in both the alpha 1(IV) and alpha 2(IV) chains. Despite the relatively low degree of conservation among the amino acids in the triple-helical region of the three type IV collagen chains, analysis of the sequences clearly showed that alpha 5(IV) is more related to alpha 1(IV) than to alpha 2(IV). This similarity between the alpha 5(IV) and alpha 1(IV) chains is particularly evident in the NC1 domains where the two polypeptides are 83% identical in contrast to the alpha 5(IV) and alpha 2(IV) identity of 63%. In addition to greatly increasing the complexity of basement membranes, the alpha 5 chain of type IV collagen may be responsible for specialized functions of some of these extracellular matrices. In this regard, it is important to note that we have recently assigned the alpha 5(IV) gene to the region of the X chromosome containing the locus for a familial type of hereditary nephritis known as Alport syndrome (Myers, J.C., Jones, T.A., Pohjalainen, E.-R., Kadri, A.S., Goddard, A.D., Sheer, D., Solomon, E., and Pihlajaniemi, T. (1990) Am. J. Hum. Genet. 46, 1024-1033). Consequently, the newly discovered alpha 5(IV) collagen chain may have a critical role in inherited diseases of connective tissue.     

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.     

The complete primary structure for the alpha 1-chain of mouse collagen IV. Differential evolution of collagen IV domains

We report here the complete nucleotide and amino acid sequences for the alpha 1-chain of mouse collagen IV which is 1669 amino acids in length, including a putative 27-residue signal peptide. In comparison with the amino acid sequence for the alpha 2-chain (Saus, J., Quinones, S., MacKrell, A. J., Blumberg, B., Muthkumaran, G., Pihlajaniemi, J., and Kurkinen, M. (1989) J. Biol. Chem. 264, 6318-6324), the two chains of collagen IV are 43% identical. Most of the interruptions of the Gly-X-Y repeat are homologously placed but strikingly show no sequence similarity between the two chains. Availability of the amino acid sequences for human collagen IV allows a detailed comparison of the primary structure of collagen IV and reveals evolutionarily conserved domains of the protein. Between the two species, the alpha 1 (IV) chains are 90.6% and the alpha 2 (IV) chains are 83.5% identical in sequence. We discuss these data with respect to differential evolution between and within the collagen IV chain types.     

Isolation and partial characterization of a novel basement membrane collagen

A guanidine-HCl extraction of lens capsule basement membrane dissolves collagenous material. This material was fractionated on an Agarose A-5M column. Fractions 1, 2 and 3 were further purified and partially characterized immunochemically and by amino acid analysis. Fraction 3 has a molecular weight of 55,000 when compared with collagen type I standard. The CNBr peptide pattern and composition of fraction 3 are different from those of alpha 1 (IV) 95K and alpha 2 (IV) 95K chains. The results described suggest the presence of a new chain in lens capsule basement membrane.     

Glomerular basement membrane. Identification of dimeric subunits of the noncollagenous domain (hexamer) of collagen IV and the Goodpasture antigen

The noncollagenous (NC1) domain hexamer of glomerular basement membrane (GBM) collagen is composed of a multiplicity of monomeric and dimeric subunits, and specific subunits are the targets for anti-GBM autoantibodies of patients with Goodpasture (GP) syndrome. The identity of GBM monomers has been established and the alpha 3(IV)NC1 monomer identified as the one that binds GP antibodies (Gunwar, S., Saus, J., Noelken, M. E., and Hudson, B. G. (1990) J. Biol. Chem. 265, 5466-5469). In the present study, the chain origin of 25 dimeric components and the identity of those that bound the anti-GBM antibodies from two GP patients were determined. This was accomplished by NH2-terminal sequence analysis and immunoblotting analysis of dimeric components that were resolved by two-dimensional electrophoresis in combination with high pressure liquid chromatography. The results revealed that (a) the components are mainly homodimers of the NC1 domains of alpha 1, alpha 2, alpha 3, alpha 4, and probably alpha 5 chains of collagen IV, reflecting a specificity of promoter-promoter association and (b) each homodimer had several size and charge isoforms. The GP antibodies bound exclusively to both alpha 3(IV)NC1 monomers and dimers and not to other basement membrane constituents. These findings provided new insights about the structure of GBM collagen and together with our previous findings firmly established the alpha 3(IV) chain as the target for the anti-GBM antibodies that mediate glomerulonephritis and pulmonary hemorrhage in patients with Goodpasture syndrome.     

Properties of the collagenous domain of the alpha 3(IV) chain, the Goodpasture antigen, of lens basement membrane collagen. Selective cleavage of alpha (IV) chains with retention of their triple helical structure and noncollagenous domain

A third chain, alpha 3(IV), of basement membrane collagen was recently discovered and was identified as the primary target for the autoantibodies of patients with Goodpasture syndrome (Saus, J., Wieslander, J., Langeveld, J. P. M., Quinones, S., and Hudson, B. G. (1988) J. Biol. Chem. 263, 13374-13380). In the present study, this chain was excised in the form of a truncated promoter by cleavage of basement membrane with Pseudomonas aeruginosa elastase and characterized. The triple helical structure and NC1 domain were retained. Elastase selectively cleaved at a site within the triple helical domain of the alpha 3 chain that is distinct from the cleavage site of the alpha 1 and alpha 2 chains. The truncated alpha 3 chain was found to contain 1460 residues, of which 1225 comprise the collagenous domain, and is cross-linked within this domain by disulfide bonds, forming a high Mr complex (greater than 300,000). Truncated protomers with a length of 340 nm corresponding to the theoretical length for the truncated alpha 3 chain were observed by electron microscopy as suprastructures in which the triple helical domains of three protomers were interwined. These protomers were also connected to each other and to the 140-nm protomers that appear to be comprised of the alpha 1 and alpha 2 chains. These results extended the known length of the alpha 3 chain by about 1000 residues and suggested that protomers of this chain self-associate through interactions between their triple helical domains and between their NC1 domains.     

Distinct antitumor properties of a type IV collagen domain derived from basement membrane

Vascular basement membrane is an important structural component of blood vessels. During angiogenesis this membrane undergoes many alterations and these changes are speculated to influence the formation of new capillaries. Type IV collagen is a major component of vascular basement membrane, and recently we identified a fragment of type IV collagen alpha2 chain with specific anti-angiogenic properties (Kamphaus, G. D., Colorado, P. C., Panka, D. J., Hopfer, H., Ramchandran, R., Torre, A., Maeshima, Y., Mier, J. W., Sukhatme, V. P., and Kalluri, R. (2000) J. Biol. Chem. 275, 1209-1215). In the present study we characterize two different antitumor activities associated with the noncollagenous 1 (NC1) domain of the alpha3 chain of type IV collagen. This domain was previously discovered to possess a C-terminal peptide sequence (amino acids 185-203) that inhibits melanoma cell proliferation (Han, J., Ohno, N., Pasco, S., Monboisse, J. C., Borel, J. P., and Kefalides, N. A. (1997) J. Biol. Chem. 272, 20395-20401). In the present study, we identify the anti-angiogenic capacity of this domain using several in vitro and in vivo assays. The alpha3(IV)NC1 inhibited in vivo neovascularization in matrigel plug assays and suppressed tumor growth of human renal cell carcinoma (786-O) and prostate carcinoma (PC-3) in mouse xenograft models associated with in vivo endothelial cell-specific apoptosis. The anti-angiogenic activity was localized to amino acids 54-132 using deletion mutagenesis. This anti-angiogenic region is separate from the 185-203 amino acid region responsible for the antitumor cell activity. Additionally, our experiments indicate that the antitumor cell activity is not realized until the peptide region is exposed by truncation of the alpha3(IV)NC1 domain, a requirement not essential for the anti-angiogenic activity of this domain. Collectively, these results effectively highlight the distinct and unique antitumor properties of the alpha3(IV)NC1 domain and the potential use of this molecule for inhibition of tumor growth.     

The structural organization of type IV collagen. Identification of three NC1 populations in the glomerular basement membrane.

Type IV collagen, which has long been assumed to contain two alpha 1(IV) and one alpha 2(IV) chains, also contains alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains. Stoichiometry of collagenous alpha(IV) chains differs among tissues, suggesting the existence of subclasses of type IV collagen, each with a unique chain composition. This study seeks to define, by characterization of subunit compositions of NC1 domain populations, the structural organization of type IV collagen from bovine glomerular basement membrane. NC1 hexamers from type IV collagen were separated on two affinity chromatography columns, one containing monoclonal antibodies to the alpha 3 chain, and another, to the alpha 1 chain. SDS-polyacrylamide gel electrophoresis, immunoblotting, reversed phase high-performance liquid chromatography, and enzyme-linked immunosorbent assay identified three NC1 hexamer populations: 1) a hexamer composed of (alpha 1)2 and (alpha 2)2 homodimers; 2) a hexamer composed of (alpha 3)2 and (alpha 4)2 homodimers; 3) a hexamer containing all four alpha chains connected in heterodimers, alpha 1-alpha 3 and alpha 2-alpha 4. Results suggest that there are two distinct type IV collagen molecules, one composed of alpha 1(IV) and alpha 2(IV) chains and another composed of alpha 3(IV) and alpha 4(IV) chains. Furthermore, polymerization occurs between molecules with the same chain composition and between molecules with different chain composition. Moreover, crosslinking between different alpha chains is restricted, thus limiting the number of possible macromolecular structures.     

Properties of the globular domain of type IV collagen and its relationship to the Goodpasture antigen

The globular domain of type IV collagen from bovine glomerular basement membrane was solubilized by collagenase digestion. Components of this domain include several monomer-size and structurally related dimer-size polypeptides. The monomer-size polypeptides were resolved into three fractions (M1, M2, and M3) with slightly different mobilities upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (nonreduced Mr = 24,500-28,300). Chemical and immunochemical studies indicate that each is a distinct component. M2 is reactive with antibodies from patients with Goodpasture syndrome. The molecular weight by sedimentation equilibrium was 32,000 for M2 and 28,000 for M1. The dimers were characterized as two classes, D1 and D2. D1 consists of two sets of nonreactive components (D1a-d and D1a,c) whereas D2 contains one set of four components (D2a-d), each of which is reactive with Goodpasture sera. Chemical and immunochemical studies indicate that a monomer-dimer relationship exists between M1 and D1 and between M2 and D2. The origin of M3 remains undetermined. Rabbit antibodies to type IV collagen alpha chains react with M1 and M2, and antibodies to M1 and M2 react with type IV collagen alpha chains, which provides additional evidence for the localization of the Goodpasture antigen to one of the chains of type IV collagen.     

Evidence for separate networks of classical and novel basement membrane collagen. Characterization of alpha 3(IV)-alport antigen heterodimer.

The COOH-terminal non-collagenous domains (NC1) of type IV collagen from glomerular basement membranes (GBM), lens capsule basement membranes, and Descemet's membrane varied in the distribution of their NC1 subunits. All of these basement membranes (BMs) contained both classical (alpha 1(IV) and alpha 2(IV)) and novel collagen chains (alpha 3(IV), alpha 4(IV) and the Alport antigen). Whereas GBM had a predominance of disulfide-bonded subunits, the lens capsule and Descemet's membrane were primarily monomeric, differences that are likely related to the functional and structural diversity of collagen in various tissues. A heterodimer formed from monomeric subunits of alpha 3(IV) and the Alport antigen exists in human and bovine GBM. This dimer represents an important cross-link of the NC1 domain of novel collagen. Additionally, immunoaffinity methodology showed that the novel BM collagen hexamers segregate into populations containing only novel BM subunits without the participation of the classical subunits (alpha 1(IV) and alpha 2(IV)). These data provided evidence for the presence of two separate networks of BM collagen: one containing alpha 1(IV) and alpha 2(IV), and the other consisting of the novel collagen chains.     

Physical and immunochemical studies of the globular domain of type IV collagen. Cryptic properties of the Goodpasture antigen

The globular domain of type IV collagen from bovine glomerular basement membrane was isolated under nondenaturing conditions. It was shown to exist in a hexameric form comprising monomeric and dimeric subunits, with the Goodpasture antigen residing in monomer M2 and dimer D2 as previously described (Butkowski, R. J., Wieslander, J., Wisdom, B. J., Barr, J. F., Noelken, M. E., and Hudson, B. G. (1985) J. Biol. Chem. 260, 3739-3747). The epitope, however, is sequestered inside the hexamer, but becomes exposed and binds with the Goodpasture antibody upon dissociation of the hexamer into its subunits after treatment with concentrated guanidine HC1 or dilute acetic acid (pH less than 3.0). The process is completely reversible even from the denatured state. Circular dichroism studies show that the conformation of each subunit is unusually resistant to change in 6 M guanidine HC1 at 25 degrees C. This suggests that exposure of the epitope by dissociation requires minimal or no unfolding of subunits. The results provide additional evidence for localization of the Goodpasture antigen to the globular domain of type IV collagen. Moreover, these studies extend the conclusion (Weber, H., Engel, J., Wiedemann, H., Glanville, R., and Timpl, R. (1984) Eur. J. Biochem. 139, 401-410) about a tumor basement membrane, to an authentic physiological membrane, that the globular domain is a major cross-linking site in the type IV collagen matrix.     

Molecular cloning of alpha 5(IV) collagen and assignment of the gene to the region of the X chromosome containing the Alport syndrome locus.

Type IV collagen is a major structural component of basement membranes. Four constituent polypeptides have been described and characterized to different degrees. Whereas the primary structure of the alpha 1(IV) and alpha 2(IV) chains has been completely established, only short protein sequences have been reported for the recently recognized alpha 3(IV) and alpha 4(IV) subunits. We have isolated overlapping human cDNA clones whose derived amino acid sequence is highly homologous to the alpha 1(IV) and alpha 2(IV) chains. However, these clones code for neither alpha 3(IV) nor alpha 4(IV), and thus this new polypeptide has been designated the alpha 5 chain of type IV collagen. To determine whether the gene encoding the alpha 5(IV) chain is syntenic with the contiguously arranged alpha 1(IV) and alpha 2(IV) genes at 13q34, the alpha 5(IV) cloned DNA was hybridized to genomic DNA from somatic cell hybrids and to metaphase chromosomes. The results demonstrated that the alpha 5(IV) collagen gene is located on the long arm of the X chromosome. Since 14 collagen genes have previously been assigned to nine autosomes, these data represent the first mapping of a collagen gene to the X chromosome. Most important, the alpha 5(IV) gene has been sublocalized to bands Xq22----q23, which are in the same region known to contain the locus for the X-linked form of Alport syndrome. It is therefore possible that this severe dominantly inherited nephritis, manifested by splitting of the glomerular basement membrane, could be caused by mutations in the alpha 5(IV) collagen gene.     

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.     

Ontogenesis of glomerular basement membrane: structural and functional properties

Protein A-gold immunocytochemistry was applied in combination with morphometrical approaches to reveal the alpha 1(IV), alpha 2(IV), and alpha 3(IV) chains of type IV collagen as well as entactin on renal basement membranes, particularly on the glomerular one, during maturation. The results have indicated that a heterogeneity between renal basement membranes appears during the maturation process. In the glomerulus at the capillary loop stage, both the epithelial and endothelial cell basement membranes were labeled for the alpha 1(IV) and alpha 2(IV) chains of type IV collagen and entactin. After fusion, both proteins were present on the entire thickness of the typical glomerular basement membrane. At later stages, the labeling for alpha 1(IV) and alpha 2(IV) chains of type IV collagen decreased and drifted towards the endothelial side, whereas the labeling for the alpha 3(IV) chain increased and remained centrally located. Entactin remained on the entire thickness of the basement membrane during maturation and in adult stage. The distribution of endogenous serum albumin in the glomerular wall was studied during maturation, as a reference for the functional properties of the glomerular basement membrane. This distribution, dispersed through the entire thickness of the basement membrane at early stages, shifted towards the endothelial side of the lamina densa with maturation, demonstrating a progressive acquisition of the permselectivity. These results demonstrate that modifications in the content and organization of the different constituents of basement membranes occur with maturation and are required for the establishment of the filtration properties of the glomerular basement membrane.     

Exon/intron structure of the human alpha 3(IV) gene encompassing the Goodpasture antigen (alpha 3(IV)NC1). Identification of a potentially antigenic region at the triple helix/NC1 domain junction.

The Goodpasture antigen has been identified as the non-collagenous (NC1) domain of alpha 3(IV), a novel collagen IV chain (Saus, J., Wieslander, J., Langeveld, J., Quinones, S., and Hudson, B.G. (1988) J. Biol. Chem. 263, 13374-13380). In the present study, the exon/intron structure and sequence for 285 amino acids of human alpha 3(IV), comprising 53 amino acids of the triple-helical domain and the complete NC1 domain (232 amino acids), were determined. Based on the comparison of the amino acid sequences of the alpha 1(IV), alpha 2(IV), alpha 3(IV), and alpha 5(IV) NC1 domains, a phylogenetic tree was constructed which indicates that alpha 2(IV) was the first chain to evolve, followed by alpha 3(IV), and then by alpha 1(IV) and alpha 5(IV). The exon/intron structure of these domains is consistent with this evolution model. In addition, it appears that alpha 3(IV) changed most after diverging from the parental gene. Analysis of its primary structure reveals that, at the junction between the triple-helical and NC1 domains, there exists a previously unrecognized, highly hydrophilic region (GLKGKRGDSGSPATWTTR) which is unique to the human alpha 3(IV) chain, containing a cell adhesion motif (RGD) as an integral part of a sequence (KRGDSGSP) conforming to a number of protein kinase recognition sites. Based on primary structure data, we outline new aspects to be explored concerning the molecular basis of collagen IV function and Goodpasture syndrome.     

Structure of mouse type IV collagen. Amino-acid sequence of the C-terminal 511-residue-long triple-helical segment of the alpha 2(IV) chain and its comparison with the alpha 1(IV) chain

The sequence of 511 residues from the C-terminal portion of the triple helix of mouse alpha 2(IV) chain was determined by using the pepsin fragment P2 of collagen IV and two cDNA clones selected from an Engelbreth-Holm-Swarm (EHS) tumor library. The sequence contains nine interruptions of the triplet repeat Gly-Xaa-Yaa ranging in size from single insertions or deletions up to stretches of eleven amino acid residues. Five of these interruptions match those present in the homologous segment of the alpha 1(IV) chain but are otherwise different in length and/or sequence. A low homology was found for the triplet regions of the alpha 1(IV) and alpha 2(IV) chain which constitute more than 90% of the sequence. The data indicate a remote evolutionary relationship of the triple-helical sequences of the two constituent chains of basement membrane collagen.     

Assembly of type IV collagen. Insights from alpha3(IV) collagen-deficient mice

Type IV collagen includes six genetically distinct polypeptides named alpha1(IV) through alpha6(IV). These isoforms are speculated to organize themselves into unique networks providing mammalian basement membranes specificity and inequality. Recent studies using bovine and human glomerular and testis basement membranes have shown that unique networks of collagen comprising either alpha1 and alpha2 chains or alpha3, alpha4, and alpha5 chains can be identified. These studies have suggested that assembly of alpha5 chain into type IV collagen network is dependent on alpha3 expression where both chains are normally present in the tissue. In the present study, we show that in the lens and inner ear of normal mice, expression of alpha1, alpha2, alpha3, alpha4, and alpha5 chains of type IV collagen can be detected using alpha chain-specific antibodies. In the alpha3(IV) collagen-deficient mice, only the expression of alpha1, alpha2, and alpha5 chains of type IV collagen was detectable. The non-collagenous 1 domain of alpha5 chain was associated with alpha1 in the non-collagenous 1 domain hexamer structure, suggesting that network incorporation of alpha5 is possible in the absence of the alpha3 chain in these tissues. The present study proves that expression of alpha5 is not dependent on the expression of alpha3 chain in these tissues and that alpha5 chain can assemble into basement membranes in the absence of alpha3 chain. These findings support the notion that type IV collagen assembly may be regulated by tissue-specific factors.     

Structural heterogeneity of the noncollagenous domain of basement membrane collagen

The noncollagenous domain of collagen from three different basement membranes of bovine origin (glomerular, lens capsule, and placental) was excised with bacterial collagenase, purified under nondenaturing conditions, and characterized. In each case the domain existed as a hexamer comprised of four distinct subunits (alpha 1 (IV) NC1, alpha 2 (IV) NC1, M2*, and M3). Each subunit exists in both monomeric and dimeric (disulfide-cross-linked) forms. Certain dimers also exist which contain nonreducible cross-links. The hexamers from the three membranes differ with respect to stoichiometry of subunits and subunit isoforms and to the degree of cross-linking of monomers into dimers. The minor subunits, M2* and M3, vary in quantity over a 20-fold range relative to the major ones among the three hexamers. The results indicate that: 1) at least two populations of triple-helical collagen molecules, differing in chain composition, exist in each membrane and that their relative proportions are tissue-specific; and 2) the chemical nature of the noncollagenous domain of these populations is tissue-specific with regard to subunit isoforms and relative proportion of reducible and nonreducible cross-links in dimers. A novel structural feature of the noncollagenous domain of basement membrane collagen was also evinced from these studies. Namely, that each of the four monomeric subunits exists in charge isoforms.