Molecular and functional defects in kidneys of mice lacking collagen alpha 3(IV): implications for Alport syndrome

Collagen IV is a major structural component of all basal laminae (BLs). Six collagen IV alpha chains are present in mammals; alpha 1 and alpha 2(IV) are broadly expressed in embryos and adults, whereas alpha 3- 6(IV) are restricted to a defined subset of BLs. In the glomerular BL of the kidney, the alpha 1 and alpha 2(IV) chains are replaced by the alpha 3-5(IV) chains as development proceeds. In humans, mutation of the collagen alpha 3, alpha 4, or alpha 5(IV) chain genes results in a delayed onset renal disease called Alport syndrome. We show here that mice lacking collagen alpha 3(IV) display a renal phenotype strikingly similar to Alport syndrome: decreased glomerular filtration (leading to uremia), compromised glomerular integrity (leading to proteinuria), structural changes in glomerular BL, and glomerulonephritis. Interestingly, numerous changes in the molecular composition of glomerular BL precede the onset of renal dysfunction; these include loss of collagens alpha 4 and alpha 5(IV), retention of collagen alpha 1/2(IV), appearance of fibronectin and collagen VI, and increased levels of perlecan. We suggest that these alterations contribute, along with loss of collagen IV isoforms per se, to renal pathology.     

Major rearrangements in the alpha 5(IV) collagen gene in three patients with Alport syndrome.

The gene coding for the alpha 5 chian of type IV collagen (alpha 5(IV) collagen), which maps to Xq22, is a candidate gene for the X-linked dominant disease Alport syndrome (AS). Using three cDNA clones, covering the 3' end of the alpha 5(IV) collagen gene, 3 of 38 patients have been identified with mutations in this gene. Each of these patients shows a gross rearrangement of DNA: a deletion of at least 35 kb, an insertion/deletion event involving approximately 25 kb, and a duplication of at least 35 kb of DNA.     

DNA rearrangements in the alpha 5(IV) collagen gene (COL4A5) of individuals with Alport syndrome: further refinement using pulsed-field gel electrophoresis.

Alport syndrome (AS), an X-linked kidney disorder, has been shown to be caused by mutations in the gene for the alpha 5-chain of type IV collagen (COL4A5), which maps to Xq22. On the basis of the results of conventional Southern blot analysis of AS patient DNAs, we employed pulsed-field gel electrophoresis to characterize further three gene rearrangements at the 3'-end of alpha 5(IV). We were able to construct long-range restriction maps for all three of these patients and deduce the extent and nature of each rearrangement. One of these mutations is a 450-kb simple deletion that includes 12 kb of the alpha 5(IV) gene. A second mutation has been shown to be a direct duplication of 35 kb of alpha 5(IV) genomic DNA, and a third mutation involves a complex insertion/deletion event resulting in an overall loss of 25 kb.     

The alloantigenic sites of alpha3alpha4alpha5(IV) collagen: pathogenic X-linked alport alloantibodies target two accessible conformational epitopes in the alpha5NC1 domain

Anti-glomerular basement membrane (GBM) antibody nephritis is caused by an autoimmune or alloimmune reaction to the NC1 domains of alpha3alpha4alpha5(IV) collagen. Some patients with X-linked Alport syndrome (XLAS) develop post-transplant nephritis mediated by pathogenic anti-GBM alloantibodies to collagen IV chains present in the renal allograft but absent from the tissues of the patient. In this work, the epitopes targeted by alloantibodies from these patients were identified and characterized. All XLAS alloantibodies recognized conformational epitopes in the NC1 domain of alpha5(IV) collagen, which were mapped using chimeric alpha1/alpha5 NC1 domains expressed in mammalian cells. Allograft-eluted alloantibodies mainly targeted two conformational alloepitopes mapping to alpha5NC1 residues 1-45 and 114-168. These regions also encompassed the major epitopes of circulating XLAS alloantibodies, which in some patients additionally targeted alpha5NC1 residues 169-229. Both kidney-eluted and circulating alloantibodies to alpha5NC1 distinctively targeted epitopes accessible in the alpha3alpha4alpha5NC1 hexamers of human GBM, unlike anti-GBM autoantibodies, which targeted sequestered alpha3NC1 epitopes. The results identify two immunodominant alpha5NC1 epitopes as major alloantigenic sites of alpha3alpha4alpha5(IV) collagen specifically implicated in the pathogenesis of post-transplant nephritis in XLAS patients. The contrast between the accessibility of these alloepitopes and the crypticity of autoepitopes indicates that distinct molecular forms of antigen may initiate the immunopathogenic processes in the two forms of anti-GBM disease.     

Single base mutation in alpha 5(IV) collagen chain gene converting a conserved cysteine to serine in Alport syndrome.

We have identified a point mutation in the type IV collagen alpha 5 chain gene (COL4A5) in Alport syndrome. Variant PstI (Barker et al., 1990, Science 248, 1224-1227), and BglII restriction sites with complete linkage with the Alport phenotype have been found in the 3' end of the COL4A5 gene in the large Utah Kindred P. The approximate location of the variant sites was determined by restriction enzyme mapping, after which this region of the gene (1028 bp) was amplified with the polymerase chain reaction (PCR) from DNA of normal and affected individuals for sequencing analysis. The PCR products showed the absence or presence of the variant PstI and BglII sites in DNA from normal and affected individuals, respectively. DNA sequencing revealed a single base change in exon 3 (from the 3' end) in DNA from affected individuals, changing the TGT codon of cysteine to the TCT codon for serine. This single base mutation also generated new restriction sites for PstI and BglII. The mutation involves a cysteine residue that has remained conserved in the carboxyl-end noncollagenous domain (NC domain) of all known type IV collagen alpha chains from Drosophila to man. It is presumably crucial for maintaining the right conformation of the NC domain, which is important for both triple-helix formation and the formation of intermolecular cross-links of type IV collagen molecules.     

The alpha 4(IV) chain of basement membrane collagen. Isolation of cDNAs encoding bovine alpha 4(IV) and comparison with other type IV collagens.

Renal basement membranes are believed to contain five distinct type IV collagens. An understanding of the specific roles of these collagens and the specificities of their interactions will be aided by knowledge of their comparative structures. Genes for alpha 1(IV), alpha 2(IV), alpha 3(IV), and alpha 5(IV) have been cloned and the deduced peptide sequences compared. A fifth chain, alpha 4(IV), has been identified in glomerular and other basement membranes. Using a polymerase chain reaction-based strategy and short known peptide sequences from the noncollagenous domain (NC1), we have cloned and characterized partial bovine cDNAs of alpha 4(IV). Sequence analysis shows that this molecule has characteristic features of type IV collagens including an NH2-terminal Gly-X-Y domain which is interrupted at several points and a COOH-terminal NC1 domain with 12 cysteine residues in positions identical to those of other type IV collagens. Within the NC1 domain bovine alpha 4(IV) has 70, 59, 58, and 53% amino acid identity with human alpha 2(IV), alpha 1(IV), alpha 5(IV), and alpha 3(IV), respectively. Alignment of the peptides also shows that alpha 4(IV) is most closely related to alpha 2(IV). Nevertheless, in the extreme COOH-terminal region of the NC1 domain there are structural features that are unique to alpha 4(IV). Cloning of the region of alpha 4(IV) that encodes the NC1 domain allows comparison of all five type IV collagens and highlights certain regions that are likely to be important in the specificities of NC1-NC1 interactions and in other discriminant functions of these molecules.     

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.     

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.     

Construction of a yeast artificial chromosome contig encompassing the human alpha 5(IV) collagen gene (COL4A5).

A PCR-based screening approach was used to isolate six yeast artificial chromosome (YAC) clones containing segments of the human alpha 5(IV) collagen gene (COL4A5). This gene is located at Xq22 and is known to be involved in the kidney disorder known as Alport syndrome (AS). By analyzing sequence-tagged sites, cDNA content, and rare-cutting restriction site patterns in these YAC clones, a contig that spans the entirety of the alpha 5(IV) gene was constructed. This contig may contain as much as 690 kb of DNA from the alpha 5(IV) locus. On the basis of the information obtained from these YAC clones, the genomic map and gene structure of the alpha 5(IV) gene have been refined. This study has also provided a valuable resource for subsequent studies of the alpha 5(IV) gene and its flanking DNA sequences.     

Integrin alpha3beta1, a novel receptor for alpha3(IV) noncollagenous domain and a trans-dominant Inhibitor for integrin alphavbeta3

Exogenous soluble human alpha3 noncollagenous (NC1) domain of collagen IV inhibits angiogenesis and tumor growth. These biological functions are attributed to the binding of alpha3NC1 to integrin alphavbeta3. However, in some tumor cells that express integrin alphavbeta3, the alpha3NC1 domain does not inhibit proliferation, suggesting that integrin alphavbeta3 expression is not sufficient to mediate the anti-tumorigenic activity of this domain. Therefore, in the present study, we searched for novel binding receptors for the soluble alpha3NC1 domain in cells lacking alphavbeta3 integrin. In these cells, soluble alpha3NC1 bound integrin alpha3beta1; however, unlike alphavbeta3, alpha3beta1 integrin did not mediate cell adhesion to immobilized alpha3NC1 domain. Interestingly, in cells lacking integrin alpha3beta1, adhesion to the alpha3NC1 domain was enhanced due to activation of integrin alphavbeta3. These findings indicate that integrin alpha3beta1 is a receptor for the alpha3NC1 domain and transdominantly inhibits integrin alphavbeta3 activation. Thus integrin alpha3beta1, in conjunction with integrin alphavbeta3, modulates cellular responses to the alpha3NC1 domain, which may be pivotal in the mechanism underpinning its anti-angiogenic and anti-tumorigenic activities.     

Differential expression of mouse alpha5(IV) and alpha6(IV) collagen genes in epithelial basement membranes

We first completed the primary structure of the mouse alpha5(IV) and alpha6(IV) chains, from which synthetic peptides were produced and a chain-specific monoclonal antibodies were raised. Expression of collagen IV genes in various basement membranes underlying specific organ epithelia was analyzed by immunohistochemical staining using these monoclonal antibodies and other antibodies from human and bovine sequences. It was possible to predict the presence of the three collagen IV molecules: [alpha1(IV)](2) alpha2(IV), alpha3(IV)alpha4(IV)alpha5(IV), and [alpha5(IV)](2)alpha6(IV). In skin basement membrane two of the three forms, [alpha1(IV)](2)alpha2(IV) and [alpha5(IV)](2)alpha6(IV), were detected. The alpha3(IV)alpha4(IV)alpha5(IV) molecule was observed as the major form in glomerulus, alveolus, and choroid plexus, where basement membranes function as filtering units. The molecular form [alpha5(IV)](2)alpha6(IV) was present in basement membranes in tubular organs such as the epididymis, where the tubes need to expand in diameter. Thus, the distribution of the basement membranes with different molecular composition is consistent with tissue-specific function.     

Identification of S-hydroxylysyl-methionine as the covalent cross-link of the noncollagenous (NC1) hexamer of the alpha1alpha1alpha2 collagen IV network: a role for the post-translational modification of lysine 211 to hydroxylysine 211 in hexamer assembly

Collagen IV networks are present in all metazoans as components of basement membranes that underlie epithelia. They are assembled by the oligomerization of triple-helical protomers, composed of three alpha-chains. The trimeric noncollagenous domains (NC1) of each protomer interact forming a hexamer structure. Upon exposure to acidic pH or denaturants, the hexamer dissociates into monomer and dimer subunits, the latter reflect distinct interactions that reinforce/cross-link the quaternary structure of hexamer. Recently, the cross-link site of the alpha1alpha1alpha2 network was identified, on the basis of x-ray crystal structures at 1.9-A resolution, in which the side chains of Met93 and Lys211 were proposed to be connected by a novel thioether bond (Than, M. E., Henrich, S., Huber, R., Ries, A., Mann, K., Kuhn, K., Timpl, R., Bourenkov, G. P., Bartunik, H. D., and Bode, W. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 6607-6612); however, at the higher resolution of 1.5 A, we found no evidence for this cross-link (Vanacore, R. M., Shanmugasundararaj, S., Friedman, D. B., Bondar, O., Hudson, B. G., and Sundaramoorthy, M. (2004) J. Biol. Chem. 279, 44723-44730). Given this discrepancy in crystallographic findings, we sought chemical evidence for the location and nature of the reinforcement/cross-link site. Trypsin digestion of monomer and dimer subunits excised a approximately 5,000-Da complex that distinguished dimers from monomers; the complex was characterized by mass spectrometry, Edman degradation, and amino acid composition analyses. The tryptic complex, composed of two peptides of 44 residues derived from two alpha1 NC1 monomers, contained Met93 and Lys211 post-translationally modified to hydroxylysine (Hyl211). Truncation of the tryptic complex with post-proline endopeptidase reduced its size to 14 residues to facilitate characterization by tandem mass spectrometry, which revealed a covalent linkage between Met93 and Hyl211. The novel cross-link, termed S-hydroxylysyl-methionine, reflects at least two post-translational events in its formation: the hydroxylation of Lys211 to Hyl211 within the NC1 domain during the biosynthesis of alpha-chains and the connection of Hyl211 to Met93 between the trimeric NC1 domains of two adjoining triple-helical protomers, reinforcing the stability of collagen IV networks.     

Colocalization of the genes for the alpha 3(IV) and alpha 4(IV) chains of type IV collagen to chromosome 2 bands q35-q37.

Each type of basement membrane in man contains between two and five genetically distinct type IV collagens: alpha 1(IV)-alpha 5(IV). Genes for alpha 1(IV), alpha 2(IV), alpha 3(IV), and alpha 5(IV) have been isolated. We have recently isolated partial cDNAs for the fifth member of the family, designated alpha 4(IV). On the basis of comparison of the deduced peptide sequences of all five chains, the type IV collagens can be divided into two families: alpha 1-like, comprising alpha 1(IV), alpha 3(IV), and alpha 5(IV); and alpha 2-like, comprising alpha 2(IV) and alpha 4(IV). Genes encoding the alpha 1(IV) and alpha 2(IV) chains (COL4A1 and COL4A2) both map to human chromosome 13q34 and have been shown to be transcribed from opposite DNA strands using a common bidirectional promoter that allows coordinate regulation of the two chains. Indeed, these two chains are commonly found together in basement membrane and form [alpha 1]2.[alpha 2] heterotrimers. Whereas alpha 1(IV) and alpha 2(IV) have been found in all basement membranes studied hitherto, it has been shown that alpha 3(IV) and alpha 4(IV) are found in only a subset of basement membranes. In basement membranes where either of these molecules is present, however, they are found together. In view of this relationship and the structural similarities between alpha 1(IV) and alpha 3(IV) and between alpha 2(IV) and alpha 4(IV), we hypothesized that COL4A3 and COL4A4, the genes encoding alpha 3(IV) and alpha 4(IV), respectively, have a genomic organization similar to that of COL4A1 and COL4A2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quaternary organization of the goodpasture autoantigen,the alpha 3(IV) collagen chain. Sequestration of two cryptic autoepitopes by intrapromoter interactions with the alpha4 and alpha5 NC1 domains

Goodpasture's (GP) disease is caused by autoantibodies that target the alpha3(IV) collagen chain in the glomerular basement membrane (GBM). Goodpasture autoantibodies bind two conformational epitopes (E(A) and E(B)) located within the non-collagenous (NC1) domain of this chain, which are sequestered within the NC1 hexamer of the type IV collagen network containing the alpha3(IV), alpha4(IV), and alpha5(IV) chains. In this study, the quaternary organization of these chains and the molecular basis for the sequestration of the epitopes were investigated. This was accomplished by physicochemical and immunochemical characterization of the NC1 hexamers using chain-specific antibodies. The hexamers were found to have a molecular composition of (alpha3)(2)(alpha4)(2)(alpha5)(2) and to contain cross-linked alpha3-alpha5 heterodimers and alpha4-alpha4 homodimers. Together with association studies of individual NC1 domains, these findings indicate that the alpha3, alpha4, and alpha5 chains occur together in the same triple-helical protomer. In the GBM, this protomer dimerizes through NC1-NC1 domain interactions such that the alpha3, alpha4, and alpha5 chains of one protomer connect with the alpha5, alpha4, and alpha3 chains of the opposite protomer, respectively. The immunodominant Goodpasture autoepitope, located within the E(A) region, is sequestered within the alpha3alpha4alpha5 protomer near the triple-helical junction, at the interface between the alpha3NC1 and alpha5NC1 domains, whereas the E(B) epitope is sequestered at the interface between the alpha3NC1 and alpha4NC1 domains. The results also reveal the network distribution of the six chains of collagen IV in the renal glomerulus and provide a molecular explanation for the absence of the alpha3, alpha4, alpha5, and alpha6 chains in Alport syndrome.     

Alpha(v)beta3 and alpha(v)beta5 integrins bind both the proximal RGD site and non-RGD motifs within noncollagenous (NC1) domain of the alpha3 chain of type IV collagen: implication for the mechanism of endothelia cell adhesion

The NC1 domains of human type IV collagen, in particular alpha3NC1, are inhibitors of angiogenesis and tumor growth (Petitclerc, E., Boutaud, A., Prestayko, A., Xu, J., Sado, Y., Ninomiya, Y., Sarras, M. P., Jr., Hudson, B. G., and Brooks, P. C. (2000) J. Biol. Chem. 275, 8051-8061). The recombinant alpha3NC1 domain contained a RGD site as part of a short collagenous sequence at the N terminus, designated herein as RGD-alpha3NC1. Others, using synthetic peptides, have concluded that this RGD site is nonfunctional in cell adhesion, and therefore, the anti-angiogenic activity is attributed exclusively to alpha(v)beta(3) integrin interactions with non-RGD motifs of the RGD-alpha3NC1 domain (Maeshima, Y., Colorado, P. C., and Kalluri, R. (2000) J. Biol. Chem. 275, 23745-23750). This nonfunctionality is surprising given that RGD is a binding site for alpha(v)beta(3) integrin in several proteins. In the present study, we used the alpha3NC1 domain with or without the RGD site, expressed in HEK 293 cells for native conformation, as an alternative approach to synthetic peptides to assess the functionality of the RGD site and non-RGD motifs. Our results demonstrate a predominant role of the RGD site for endothelial adhesion and for binding of alpha(v)beta(3) and alpha(v)beta(5) integrins. Moreover, we demonstrate that the two non-RGD peptides, previously identified as the alpha(v)beta(3) integrin-binding sites of the alpha3NC1 domain, are 10-fold less potent in competing for integrin binding than the native protein, indicating the importance of additional structural and/or conformational features of the alpha3NC1 domain for integrin binding. Therefore, the RGD site, in addition to non-RGD motifs, may contribute to the mechanisms of endothelial cell adhesion in the human vasculature and the anti-angiogenic activity of the RGD-alpha3NC1 domain.     

Effect of glycine substitutions on alpha5(IV) chain structure and structure-phenotype correlations in Alport syndrome

The phenotype variety caused by glycine substitutions in alpha5(IV) chain in X-linked Alport syndrome (XLAS) prompted the complexity of structure changes of alpha5(IV) chain that was little to know now. In this study, we expressed a domain of alpha5(IV) chain containing different glycine substitutions (G1015V and G1030S, respectively) which were revealed in two XLAS pedigrees with different phenotype severities and the corresponding domain of a control in Escherichia coli. The recombinant proteins were characterized by immunoblot and mass spectrometry and analyzed the secondary structure by using circular dichroism (CD) spectroscopy. CD analysis showed that the recombinant protein containing G1015V mutation identified in the pedigree of juvenile-onset XLAS exhibited 12.9% alpha-helix that was not found in the control recombinant protein. The spectrum of the recombinant protein containing G1030S mutation identified in the pedigree of adult-onset XLAS was slightly different from that of the control, that is, mostly with the random coil and the beta-sheet, while without alpha-helix. These results demonstrated that two kinds of glycine substitutions, although in the same domain of alpha5(IV) chain, displayed the distinctly different secondary structures. The changes of the secondary structure could explain the phenotypic diversities of XLAS, which would be hardly understood solely by analyzing genomic DNA or mRNA of alpha5(IV) chain.     

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.     

Glomerular basement membrane: evidence for collagenous domain of the alpha 3 and alpha 4 chains of collagen IV

A collagenous component(s) of Mr = 60K was extracted from glomerular basement membrane with urea and was purified. Upon digestion, it yielded a collagenase-resistant fragment(s) of Mr = 23.5K. Both component and fragment showed immunochemical identity with the noncollagenous domains of the new alpha 3 & alpha 4 chains of collagen IV. The component is characterized by a collagenous domain of about 280 residues and a noncollagenous domain of about 250 residues. These findings further establish these new chains as distinct entities of collagen IV.     

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.