Differential expression of two basement membrane collagen genes, COL4A6 and COL4A5, demonstrated by immunofluorescence staining using peptide-specific monoclonal antibodies.

Abstract

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 promoter. In addition we reported a novel observation that the COL4A6 gene is transcribed from two alternative promoters in a tissue-specific manner (Sugimoto, M., T. Oohashi, and Y. Ninomiya. 1994. Proc. Natl. Acad. Sci. USA. 91:11679-11683). To know whether the translation products of both genes are colocalized in various tissues, we raised alpha 5(IV) and alpha 6(IV) chain-specific rat monoclonal antibodies against synthetic peptides reflecting sequences near the carboxy terminus of each noncollagenous (NC)1 domain. By Western blotting alpha 6(IV) chain-specific antibody recognized 27-kD monomers and associated dimers of the human type IV collagen NC1 domain, which is the first demonstration of the presence in tissues of the alpha 6(IV) polypeptide as predicted 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 were positive. The staining pattern of the glomerular basement membrane was quite different from that obtained with the anti-alpha 5(IV) peptide antibody. The alpha 5(IV) and alpha 6(IV) chains were colocalized in the basement membrane in the skin, smooth muscle cells, and adipocytes; however, little if any reaction was seen in basement membranes of cardiac muscles and hepatic sinusoidal endothelial cells. Thus, both genes are expressed in a tissue-specific manner, perhaps due to the unique function of the bidirectional promoter for both genes, which is presumably different from that for COL4A1 and COL4A2.

Glomerular Expression of Type IV Collagen Chains in Normal and X-Linked Alport Syndrome Kidneys

“…Monoclonal antibodies against the NC1 domain of the ␣4 chain of type IV collagen (mAb 85) (22) were a gift from MM Kleppel, (22) and monoclonal antibodies against the NC1 domain of the ␣6(IV) chain (H63) were from Y Sado. 23 Affinity-purified fluorescein isothiocyanate-conjugated sheep immunoglobulin G anti-rabbit and anti-mouse immunoglobulins were from Silenius (Victoria, Australia). Fluorescein-conjugated affinity-purified donkey antisheep immunoglobulin G (HϩL) antibodies were from Jackson Laboratories (West Grove, PA).…”

“…In the human and rodent kidney, immunohistochemical studies have shown a low-level expression of ␣1(IV) to ␣2(IV) in mature GBM whereas the ␣3(IV) to ␣5(IV) chains are highly expressed. [22][23][24][25][26][27] Little is known about the different isoforms of triple-helical type IV collagen molecules, 5,28 and their supramolecular organization in the different basement membranes. However, different subpopulations of NC1 hexamers, which reflect the association of two triple-helical molecules within the type IV collagen network, have been described recently in GBM as well as in other basement membranes.…”

Epitope mapping of anti-glomerular basement membrane (GBM) antibodies with synthetic peptides

“…It has been shown that 3(IV)-and 4(IV)-chains co-localize and are only present in specialized basement membranes, for example in the kidney, eye, cochlea, lung and brain, whereas the 1(IV)-and 2(IV)-chains are present in all basement membranes [6]. Results show that the 5(IV)-and 6(IV)-chains also have a limited distribution in different basement membranes [5,7,8].…”

The Inner Ear of Dogs with X-Linked Nephritis Provides Clues to the Pathogenesis of Hearing Loss in X-Linked Alport Syndrome

“…In most patients with Alport syndrome, the GBM contains the ␣1 and ␣2 chains and lacks the ␣3, ␣4, and ␣5 chains. 8,9,14,15 The altered ␣-chain composition seems to reflect an arrest of an early developmental switch, wherein an immature ␣1/␣2 network persists and is not replaced by the ␣3/␣4/␣5 network. 10,16 The existence of an ␣3/␣4/␣5 network provides a plausible explanation for the absence of these chains in Alport syndrome in the setting of COL4A3, COL4A4, or COL4A5 mutations, in that all three chains are required for the assembly of this network.…”

“…Type IV collagen is assembled from a family of distinct ␣-chains designated ␣1 to ␣6, which are encoded by six different genes, COL4A1 to COL4A6, respectively. 7 The glomerular basement membrane (GBM) is composed of the ␣1 to ␣5 chains, 8,9 which are assembled in triple helices that in turn selfassemble into supramolecular networks. Two distinct ␣1/ ␣2-containing and ␣3/␣4/␣5-containing networks have been identified at the biochemical level.…”