Human basement membrane collagen (type IV). The amino acid sequence of the alpha 2(IV) chain and its comparison with the alpha 1(IV) chain reveals deletions in the alpha 1(IV) chain

The cDNA and protein sequences of the N-terminal 60% of the alpha 2(IV) chain of human basement membrane collagen have been determined. By repeated primer extension with synthetic oligodeoxynucleotides and mRNA from either HT1080 cells or human placenta overlapping clones were obtained which cover 3414 bp. The derived protein sequence allows for the first time a comparison and alignment of both alpha chains of type IV collagen from the N terminus. This alignment reveals an additional 43 amino acid residues in the alpha 2(IV) chain as compared to the alpha 1(IV) chain. 21 of these additional residues form a disulfide-bridged loop within the triple helix which is unique among all known collagens.     

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)     

Deletion analysis of the mouse alpha 1(III) collagen promoter.

A chimeric gene was constructed by fusing the DNA sequences containing the 5' flanking region of the mouse alpha 1(III) collagen gene to the coding sequence of the bacterial chloramphenicol acetyltransferase (CAT) gene. Transient transfection experiments indicated that the alpha 1(III) promoter is active in NIH 3T3 fibroblasts and BC3H1 smooth muscle cells. The activity of the alpha 1(III) collagen promoter-CAT plasmid is stimulated approximately ten fold by the presence of the SV40 enhancer element. Removing sequences upstream of -200 stimulates the activity of the chimeric gene eight fold. Further deletion analysis identified sequences located between -350 and -300 that were instrumental in repressing the activity of the promoter. This 50 bp region contains a direct repeat sequence that may be involved in the regulation of the mouse alpha 1(III) collagen gene. Truncating the alpha 1(III) promoter to -80 further stimulated expression. We propose that the positive regulatory elements of this gene appear to be located within the first 80 bp of the promoter, whereas elements located further upstream exert a negative effect on the expression of the gene. Regulation of the alpha 1(III) gene contrasts with that of the alpha 2(I) collagen gene, which appears to be regulated by several positive elements located in various regions of the promoter.     

Extensive homology between the carboxyl-terminal peptides of mouse alpha 1(IV) and alpha 2(IV) collagen

We have determined the complete primary structure for the carboxyl-terminal peptides of mouse alpha 1(IV) and alpha 2(IV) collagen; which have 229 and 227 amino acids, respectively. The amino acid sequences are 63% identical and conservatively substituted in 28 positions. A striking feature of these peptides is that the first half of each sequence is homologous with the second half, 37% in alpha 1(IV) and 36% in alpha 2(IV). These results suggest that the carboxyl-terminal peptides of type IV collagen are closely related in their structure and evolution. Presumably, they were first derived by internal duplication of a common ancestral DNA sequence which later, by gene duplication, gave rise to the two different but homologous carboxyl-terminal peptides of type IV collagen.     

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.     

The complete primary structure of a nematode alpha 2(IV) collagen and the partial structural organization of its gene.

We have isolated and characterized cDNA and genomic DNA clones which encode an alpha 2(IV) collagen chain from the parasitic nematode Ascaris suum. In addition we have determined, by nucleic acid sequence analysis, the structural organization of approximately two-thirds of the gene. This analysis has shown that the gene contains at least 15 introns, and those that have been characterized range in size from 141 to 854 base pairs. The derived protein sequence contains 1763 amino acids and includes a putative 26-amino acid signal sequence. The collagenous triple-helical region contains 17 interruptions, many of which occur in the same positions as those in the human alpha 1(IV) and alpha 2(IV) chains. Comparison of the genomic DNA sequence with the cDNA sequence has revealed the presence of a sequence within the gene which appears to be an intact and normal exon that is not represented in our cDNA sequence. The presence of this putative exon raises the possibility that the A. suum alpha 2(IV) collagen gene may undergo alternative splicing.     

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.     

Head-to-head arrangement of murine type IV collagen genes

The genes for the two dissimilar subunits of type IV collagen are organized in a head-to-head manner with their translation initiation codons within 874 base pairs. Murine genomic clones which contain portions of both genes have been isolated and characterized. These clones contain the first exon of the alpha 1(IV) chain and the first 3 exons for the alpha 2(IV) chain within a 1.7-kilobase HindIII fragment. The intergenic region appears not to resemble previously described bidirectional promoters. The HindIII fragment is present as a single copy in the mouse genome ruling out the presence of one of these gene fragments as a pseudogene. These findings agree with linkage studies of these two genes and differ from the known organization of human fibrillar collagen genes which have been found to be dispersed within the human genome.     

Tissue-specific expression of the mouse alpha 2(I) collagen promoter. Studies in transgenic mice and in tissue culture cells.

We sought to determine the cis-acting elements responsible for the pattern of tissue specific expression of the mouse alpha 2(I) collagen gene. Using an RNase protection assay we first verified that expression of the alpha 2(I) collagen gene is mainly confined to tendons, bone, and skin in mice. Both transgenic mice and DNA transfection of tissue culture cells were used as experimental approaches. Transgenic mice lines were generated harboring chloramphenicol acetyltransferase (CAT) chimeric genes that contained either (a) 2000 base pairs (bp) of 5'-flanking sequences of the mouse alpha 2(I) collagen gene plus additional sequences between +418 and +1524 of the first intron of this gene or (b) the same promoter sequences without intron sequences or (c) the 350-bp proximal promoter sequences. Transgenic mice containing both types of 2000-bp promoters showed a pattern of CAT expression that was tissue specific. The presence of sequences of the first intron in the transgene did not increase the level of promoter activity. Transgenic mice harboring the 350-bp alpha 2(I) collagen promoter also showed a pattern that was tissue-specific except that high level expression also occurred in the brain. This suggests that negative regulation is an important component of tissue-specific expression. In order to analyze the first 350 bases in detail, we performed transient expression experiments, using promoter fragments attached to the luciferase reporter gene. Fibroblasts, which show a high level expression of the endogenous alpha 2(I) collagen gene, and B cells, in which the gene is silent, were transfected with a series of deletions and substitution mutations within the proximal 350-bp promoter. These experiments were unable to define unique cell-specific cis-acting elements. However, when the sequence between -315 and -284 was tandemly repeated upstream of a minimal alpha 2(I) collagen promoter (-41 to +54), the activity of this construction was considerably higher in fibroblasts than in B cells when compared with the minimal promoter itself. In gel retardation assays, the levels of complexes that bind to this sequence were higher in fibroblast nuclear extracts than in myeloma nuclear extracts. Our results are consistent with the hypothesis that the -315 to -284 DNA sequence participates in the cell-specific control of the alpha 2(I) collagen gene in fibroblasts.     

Differential binding of a CCAAT DNA binding factor to the promoters of the mouse alpha 2(I) and alpha 1(III) collagen genes

An exonuclease III assay (Wu, C. (1985) Nature 317, 84-87) was used to identify in nuclear extracts of NIH 3T3 cells a factor which binds to the CCAAT segment of the alpha 2(I) collagen promoter between -80 and -84. This sequence is located on the coding strand in the alpha 2(I) collagen promoter. Binding is specific since only promoter fragments which contain the CCAAT box sequences on one or the other DNA strand inhibit binding to the alpha 2(I) collagen CCAAT box. The CCAAT binding factor protects approximately 26 base pairs of the alpha 2(I) collagen promoter from exonuclease III digestion. Binding to the alpha 2(I) collagen promoter CCAAT box is not inhibited by a fragment of the alpha 1(III) collagen promoter (from -396 to +16), which does not contain a CCAAT sequence on either one or the other strand. Our data suggest that two genes such as the alpha 2(I) and alpha 1(III) collagen genes, which are coordinately expressed in many tissues, are not necessarily regulated by the same trans-acting DNA binding proteins.     

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.     

The binding of heparin to type IV collagen: domain specificity with identification of peptide sequences from the alpha 1(IV) and alpha 2(IV) which preferentially bind heparin

Three distinctive heparin-binding sites were observed in type IV collagen by the use of rotary shadowing: in the NC1 domain and at distances 100 and 300 nm from the NC1 domain. Scatchard analysis indicated different affinities for these sites. Electron microscopic analysis of heparin-type IV collagen interaction with increasing salt concentrations showed the different affinities to be NC1 greater than 100 nm greater than 300 nm. The NC1 domain bound specifically to chondroitin/dermatan sulfate side chains as well. This binding was observed at the electron microscope and in solid-phase binding assays (where chondroitin sulfate could compete for the binding of [3H]heparin to NC1-coated substrata). The triple helix-rich, rod-like domain of type IV collagen did not bind to chondroitin/dermatan sulfate side chains. In solid-phase binding assays only heparin could compete for the binding of [3H]heparin to this domain. In order to more precisely map potential heparin-binding sites in type IV collagen, we chemically synthesized 17 arginine- and lysine-containing peptides from the alpha 1(IV) and alpha 2(IV) chains. Three peptides from the known sequence of the alpha 1(IV) and alpha 2(IV) chains were shown to specifically bind heparin: peptide Hep-I (TAGSCLRKFSTM), from the alpha 1(NC1) chain, peptide Hep-II (LAGSCLARFSTM), a peptide corresponding to the same sequence in peptide Hep-I from the alpha 2 (NC1) chain, and peptide Hep-III (GEFYFDLRLKGDK) which contained an interruption of the triple helical sequence of the alpha 1(IV) chain at about 300 nm from the NC1 domain, were demonstrated to bind heparin in solid-phase binding assays and compete for the binding of [3H]heparin to type IV collagen-coated substrata. Therefore, each of these peptides may represent a potential heparin-binding site in type IV collagen. The mapping of the binding of heparin or related structures, such as heparan sulfate proteoglycan, to specific sequences of type IV collagen could help the understanding of several structural and functional properties of this basement membrane protein as well as interactions with other basement membrane and/or cell surface-associated macromolecules.