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.

Organization and expression of basement membrane collagen IV genes and their roles in human disorders.

Six distinct genes have been identified as belonging to the type IV collagen gene family. They can be organized into three sets, i.e., COL4A1/COL4A2, COL4A3/COL4A4, and COL4A5/COL4A6, which are localized on three different chromosomes in humans, 13, 2, and X, respectively. Within each set the genes are aligned head-to-head and their expression is regulated by bidirectional promoters between the genes. Transcriptional regulation of the COL4A1/COL4A2 set has been well characterized. The transcription of COL4A6 seems to be controlled by two alternative promoters. While collagen IV molecules composed of alpha1 and alpha2 chains are broadly distributed, molecules comprising combinations of the other four chains, alpha3-alpha6, are important components of specialized basement membranes. The precise chain composition of triple-helical molecules assembled from the alpha3-alpha6 chains is not entirely clear, but it is hypothesized that alpha3-alpha5 chains and alpha5 and alpha6 chains form heterotrimeric molecules. Several pieces of evidence indicate that alpha3/alpha4/alpha5 molecules and alpha5/alpha6 molecules are components of the basement membrane network. This helps explain the observation that the kidney and skin basement membranes from patients with Alport syndrome caused by mutations in the alpha5 coding gene, COL4A5, are defective in the alpha3, alpha4, and alpha6 chains together with the alpha5 chain. Large deletions involving the COL4A5 and COL4A6 genes have been found in rare cases of diffuse leiomyomatosis associated with Alport syndrome.

Two genes, COL4A3 and COL4A4 coding for the human alpha3(IV) and alpha4(IV) collagen chains are arranged head-to-head on chromosome 2q36.

We first isolated and characterized genomic DNA fragments that cover the 5' flanking sequences of COL4A3 and COL4A4 encoding the human basement membrane alpha3(IV) and alpha4(IV) collagen chains, respectively. Nucleotide sequence analysis indicated that the two genes are arranged head-to-head. To determine transcription start site for COL4A4 gene, we performed RACE and RNase protection assays, indicating that there are two alternative transcripts presumably derived from two different promoters. Interestingly, one transcription start site (from exon 1') of COL4A4 is only 5 bp away from the reported transcription start site of COL4A3, whereas the other transcript (from exon 1) starts 373 nucleotides downstream from the first one, generating the two kinds of transcripts that differ in the 5' UTR regions. Expression of these two transcripts appears tissue-specific; exon 1 transcript was expressed predominantly in epithelial cells, while exon 1' transcript showed rather ubiquitous and low expression. The nucleotide sequence of the promoter region is composed of dense CpG dinucleotides, GC boxes, CTC boxes and a CCAAT box but no TATA box. These results provide information to delineate the promoter activity for the tissue-specific expression of the six type IV collagen genes and basement membrane assembly in different tissues and organs.