The NC1 Domain of Collagen IV Encodes a Novel Network Composed of the α1, α2, α5, and α6 Chains in Smooth Muscle Basement Membranes

Abstract

Type IV collagen, the major component of basement membranes (BMs), is a family of six homologous chains (␣1-␣6) that have a tissue-specific distribution. The chains assemble into supramolecular networks that differ in the chain composition. In this study, a novel network was identified and characterized in the smooth muscle BMs of aorta and bladder. The noncollagenous (NC1) hexamers solubilized by collagenase digestion were fractionated by affinity chromatography using monoclonal antibodies against the ␣5 and ␣6 NC1 domains and then characterized by two-dimensional gel electrophoresis and Western blotting. Both BMs were found to contain a novel ␣1⅐␣2⅐␣5⅐␣6 network besides the classical ␣1⅐␣2 network. The ␣1⅐␣2⅐␣5⅐␣6 network represents a new arrangement in which a protomer (triplehelical isoform) containing the ␣5 and ␣6 chains is linked through NC1-NC1 interactions to an adjoining protomer composed of the ␣1 and ␣2 chains. Re-association studies revealed that the NC1 domains contain recognition sequences sufficient to encode the assembly of both networks. These findings, together with previous ones, indicate that the six chains of type IV collagen are distributed in three major networks (␣1⅐␣2, ␣3⅐␣4⅐␣5, and ␣1⅐␣2⅐␣5⅐␣6) whose chain composition is encoded by the NC1 domains. The existence of the ␣1⅐␣2⅐␣5⅐␣6 network provides a molecular explanation for the concomitant loss of ␣5 and ␣6 chains from the BMs of patients with X-linked Alport's syndrome. The basement membrane (BM),1 a continuous sheet of extracellular matrix, separates epithelial cells from the underlying stroma and plays important roles in normal biological functions (such as cell adhesion, growth, and differentiation; tissue repair; and molecular ultrafiltration) as well as in pathological events (such as cancer cell invasion and metastasis). Moreover, degradation and de novo synthesis of vascular BMs are critical events in the angiogenesis processes. BMs function is impaired in hereditary and acquired diseases in which type IV collagen is affected, including Alport's syndrome, a hereditary form of progressive renal disease; diffuse leiomyomatosis, a benign proliferation of smooth muscle cells; and Goodpasture syndrome, an anti-type IV collagen autoimmune disease (1).Type IV collagen is the major structural component of the BM, and it consists of a family of six homologous ␣(IV) chains, designated ␣1-␣6 (1). Each chain is characterized by a long collagenous domain of ϳ1400 residues of Gly-X-Y repeats, interrupted by ϳ20 short noncollagenous sequences, and by a noncollagenous (NC1) domain of ϳ230 residues at the carboxyl terminus. Three ␣(IV) chains assemble into triple-helical molecules (protomers) that further associate to form supramolecular networks by dimerization at the carboxyl terminus through NC1 domains and by formation of tetramers at the amino terminus (2). The chain composition, and thus the properties of the type IV collagen networks are influenced by two factors. First, the chain composition of networks is limited by chain availability...

Breakpoint determination of X;autosome balanced translocations in four patients with premature ovarian failure

“…11 The a5-and a6-chains are found in the basement membrane of skin, smooth muscle and kidney. 12 Two transcripts of COL4A6 are known, isoforms A and B (Figure 3b). The protein structure of collagen type IV contains an amino-terminal collagenous domain (also called 7S domain), a triple-helical region (Gly-X-Y) and a carboxyl-terminal (14) 14 Breakpoint determination of X;autosome balanced translocations A Nishimura-Tadaki et al non-collagenous (NC1) domain (Figure 3b).…”

Quaternary Organization of the Goodpasture Autoantigen, the α3(IV) Collagen Chain

“…However, whereas mAb H43 recognized cryptic epitopes and required pretreatment of tissue sections with acid urea, the newly produced mAbs stained native sections, indicating that they bind to epitopes exposed in the NC1 hexamer. For further use in the affinity fractionation of NC1 hexamers, RH45 IgG was purified on protein G-Sepharose (Amersham Biosciences) and immobilized to Affi-Gel-10 (Bio-Rad), as described for mAbs B51 and B66 (17).…”

“…2 Several NC1 hexamer-binding mAbs were used for the immunoaffinity fractionation of native NC1 hexamers from human GBM. Mab1 and Mab3 were used for immunoprecipitation of ␣1-and ␣3-containing native NC1 hexamers, and immobilized rat mAbs B51 (to ␣5) and B66 (to ␣6) were used for affinity chromatography of hexamers containing the ␣5 and ␣6 chains, respectively, as described (17). In addition, several novel hexamer-binding mAbs specific for the ␣4 NC1 domain were produced by the rat lymph node method (18) in Wistar-Kyoto rats immunized with either human r-␣4 NC1 domain (mAbs RH42 and RH45) or with total human GBM hexamers (mAb N42).…”

“…Whether this hexamer population consisted of ␣3/␣4 or ␣3/␣5 or ␣3/␣4/␣5 heterohexamers could not be determined because only an ␣3-precipitating antibody was available. Recently, we developed precipitating mAbs for ␣5 and ␣6 NC1 domains, designated B51 and B66, respectively (17). Here, we introduce a novel precipitating mAb specific for the ␣4 NC1 domain, RH45.…”

“…Because of the special position of NC1 hexamers, connecting two adjoining protomers, the identity of monomer and dimer subunits of the hexamers reflects the chain composition of protomers, and the dimer subunits identify which chains are connected by covalent interactions between protomers. A strategy perfected for the analysis of NC1 hexamers from smooth muscle basement mem-branes that led to the discovery of a novel ␣1⅐␣2⅐␣5⅐␣6(IV) network (17) was used here to analyze the collagen IV network containing the ␣3, ␣4, and ␣5 chains.…”