Role of Laminin Terminal Globular Domains in Basement Membrane Assembly

Abstract

Laminins contribute to basement membrane assembly through interactions of their N-and C-terminal globular domains. To further analyze this process, recombinant laminin-111 heterotrimers with deletions and point mutations were generated by recombinant expression and evaluated for their ability to self-assemble, interact with nidogen-1 and type IV collagen, and form extracellular matrices on cultured Schwann cells by immunofluorescence and electron microscopy. Wild-type laminin and laminin without LG domains polymerized in contrast to laminins with deleted ␣1-, ␤1-, or ␥1-LN domains or with duplicated ␤1-or ␣1-LN domains. Laminins with a full complement of LN and LG domains accumulated on cell surfaces substantially above those lacking either LN or LG domains and formed a lamina densa. Accumulation of type IV collagen onto the cell surface was found to require laminin with separate contributions arising from the presence of laminin LN domains, nidogen-1, and the nidogen-binding site in laminin. Collectively, the data support the hypothesis that basement membrane assembly depends on laminin self-assembly through formation of ␣-, ␤-, and ␥-LN domain complexes and LG-mediated cell surface anchorage. Furthermore, type IV collagen recruitment into the laminin extracellular matrices appears to be mediated through a nidogen bridge with a lesser contribution arising from a direct interaction with laminin. The laminins (Lm)2 are a major family of basement membrane glycoproteins each consisting of ␣-, ␤-, and ␥-subunits joined together through a coiled-coil (1). The ␣1-, ␣2-, ␣3B-, and ␣5-laminins possesses globular LN domains at the N terminus of each of the three subunits. In contrast, ␣4-laminins possess only two short arms because of truncation of the ␣-subunit short arm. All laminins terminate in a group of LG domains distal to the coiled-coil. Previous studies have shown that laminin-111 (Lm-111, ␣1␤1␥1-subunit composition) self-assembles into a polymer (2-6). This polymerization is entropy-driven, thermally reversible, cooperative with a critical concentration of 0.1 M, and calcium-dependent. The three-dimensional ultrastructure of the polymer was seen as a dense array of interconnecting struts in freeze-dried Pt/C replicas (4). Analysis of interactions among laminin proteolytic fragments supported a model of self-assembly in which the essential repeating complex of polymerization consists of the three LN domains joined together with each domain derived from a different laminin molecule (3). A subsequent study of different members of the laminin family led to a hypothesis that those truncated laminins lacking one or more short arms would not polymerize. More recently, evaluation of binding interactions between recombinant N-terminal laminin protein fragments by plasmon resonance spectroscopy and chemical cross-linking identified not only the proposed binding between ␣1-␤1-, ␣1-␥1-, and ␤1-␥1-LN-LEa pairs, but also selfbinding between fragments containing the ␣1-LN domain (7). This type of homologous inter-domain bind...

Increased aldehyde-modification of collagen type IV in symptomatic plaques – A possible cause of endothelial dysfunction

“…14,15 In addition, collagen type IV has been shown to be present in the intima of fatty streaks and in advanced lesions. 16 Collagen type IV interaction to endothelial cells is important for maintaining endothelial cell function.…”

Basement Membranes: Cell Scaffoldings and Signaling Platforms

“…The LN domains appear to require their adjacent LE repeats to fold properly because of an unusual interdomain pattern of disulfide-binding (Kalkhof et al 2008). Studies with laminin-111 have revealed that selfassembly is calcium-dependent and mediated by cooperative binding among a, b, and g subunit LN domains to form ternary complexes among adjacent laminins (Yurchenco and Cheng 1993;McKee et al 2007) (see Fig. 3A).…”

“…The intercomponent binding interactions and polymerizations of laminins and type IV collagen that create a relevant scaffold matrix were first identified and characterized by a study of purified components in vitro. Assembly on cells, however, has been found to be facilitated by anchorage to select cell surfaces that serves to increase the local surface concentration of components, promoting the interactions (Kalb and Engel 1991;Li et al 2005b;McKee et al 2007). This initial anchoring interaction appears to occur primarily through the laminin LG domains (Figs.…”

“…However, one also needs to consider modifying interactions arising from sites distal to the LEa domain as well as spatial constraints that may affect the rules of self-assembly for intact heterotrimers. Recent studies with recombinant laminin-111 heterotrimers bearing domain modifications favored a strict ab-g LN ternary interaction as a requirement for self-assembly and basement membrane formation (McKee et al 2007;McKee et al 2009). These studies also revealed that deletion of the aLN domain (preventing polymerization) resulted in assembly of a laminin-poor, collagenrich ECM and suggested that laminins lacking an a-short arm such as laminin-411 would assemble in this alternative manner (Fig.…”

“…2) through a multistep process that is substantially mass action-driven (selfassembly) and that is initiated by the binding of laminins to competent (i.e., laminin-binding) cell surfaces (Smyth et al 1998;Li et al 2002;Li et al 2005b;McKee et al 2007;McKee et al 2009). The intercomponent binding interactions and polymerizations of laminins and type IV collagen that create a relevant scaffold matrix were first identified and characterized by a study of purified components in vitro.…”