Laminin gamma 3 chain binds to nidogen and is located in murine basement membranes

Abstract

Recently a novel laminin γ 3 chain was identified in mouse and human and shown to have the same modular structure as the laminin γ 1 chain. We expressed two fragments of the γ 3 chain in mammalian cells recombinantly. The first, domain VI/V, consisting of laminin N-terminal (domain VI) and four laminin-type epidermal growth factor-like (domain V) and laminin N-terminal modules, was shown to be essential for self-assembly of laminins. The other was domain III3-5, which consists of three laminin-type epidermal growth factor-like modules and is predicted to bind to nidogens. The γ 3 VI/V fragment was a poor inhibitor for laminin-1 polymerization as was the γ 2 VI/ V fragment. The γ 3 III3-5 fragment bound to nidogen-1 and nidogen-2 with lower affinity than the γ 1 III3-5 fragment. These data suggested that laminins containing the γ 3 chain may assemble networks independent of other laminins. Polyclonal antibodies raised against γ 3 VI/V and γ 3 III3-5 showed no cross-reaction with homologous fragments from the γ 1 and γ 2 chains of laminin and allowed the establishment of γ chain-specific radioimmunoassays and light and electron microscopic immunostaining of tissues. This demonstrated a 20-100-fold lower content of the γ 3 chain compared with the γ 1 chain in various tissue extracts of adult mice. The expression of γ 3 chain was highly tissue-specific. In contrast to earlier assumptions, the antibodies against the γ 3 chain showed light microscopic staining exclusively in basement membrane zones of adult and embryonic tissues, such as the brain, kidney, skin, muscle, and testis. Ultrastructural immunogold staining localized the γ 3 chain to basement membranes of these tissues.