DIFFERENTIAL EXPRESSION OF 2 BASEMENT-MEMBRANE COLLAGEN GENES, COL4A6AND COL4A5, DEMONSTRATED BY IMMUNOFLUORESCENCE STAINING USING PEPTIDE-SPECIFIC MONOCLONAL-ANTIBODIES

Genes for the human alpha 5(IV) and alpha 6(IV) collagen chains have a unique arrangement in that they are colocalized on chromosome Xq22 in a head-to-head fashion and appear to share a common bidirectional pro moter. In addition we reported a novel observation that the COL4A6 gen e is transcribed from two alternative promoters in a tissue-specific m anner (Sugimoto, M., T, Oohashi, and Y. Ninomiya. 1994, Proc. Natl. Ac ad. Sci. USA. 91:11679-11683). To know whether the translation product s of both genes are colocalized in various tissues, we raised alpha 5( IV) and alpha 6(IV) chain-specific rat monoclonal antibodies against s ynthetic peptides reflecting sequences near the carboxy terminus of ea ch noncollagenous (NC)1 domain. By Western blotting alpha 6(IV) chain- specific antibody recognized 27-kD monomers and associated dimers of t he human type IV collagen NC1 domain, which is the first demonstration of the presence in tissues of the alpha 6(IV) polypeptide as predicte d from its cDNA sequence. Immunofluorescence studies using anti-alpha 6(IV) antibody demonstrated that in human adult kidney the alpha 6(IV) chain was never detected in the glomerular basement membrane, whereas the basement membranes of the Bowman's capsules and distal tubules we re positive. The staining pattern of the glomerular basement membrane was quite different from that obtained with the anti-alpha 5(IV) pepti de antibody. The alpha 5(IV) and alpha 6(IV) chains were colocalized i n the basement membrane in the skin, smooth muscle cells, and adipocyt es; however, little if any reaction was seen in basement membranes of cardiac muscles and hepatic sinusoidal endothelial cells. Thus, both g enes are expressed in a tissue-specific manner, perhaps due to the uni que function of the bidirectional promoter for both genes, which is pr esumably different from that for COL4A1 and COL4A2.