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 alpha1-, beta1-, or gamma1-LN domains or with duplicated beta1- or alpha1-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 alpha-, beta-, and gamma-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.
Highlights
In this study we have focused on the contributions of laminin domains to the assembly process
Secreted protein was purified from medium by metal chelating chromatography as described [10]. (c) Type IV collagen was extracted from lathyritic mouse EHS tumor and purified by salt fractionation and DEAE-cellulose chromatography as described [30]. (d) Laminin-111 was extracted with EDTA from lathyritic EHS tumor and purified by gel filtration and DEAE-Sephacel chromatography as described [3]
A correlation was made in this study between binding and self-assembling activities detected in basement membrane components by biochemical means with behavior of the components in a condition-limited cellular model of basement membrane assembly
Summary
LAMB1 genes coding for the common ␥1- and 1-laminin subunits revealed that laminins are essential for the formation of several embryonic basement membranes (16 –18). Lm-111 heterotrimers were generated by recombinant expression in human embryonic kidney 293 cells and evaluated for their ability to polymerize, interact with nidogen-1 and type IV collagen, and form a basement membrane on cultured Schwann cells These cells, involved in laminin-dependent congenital muscular dystrophy, were chosen because it has been found that native laminin-111 assembles a basement membrane-type ECM on the cell surfaces in culture, that laminin attachment to the cells (anchorage) is substantially mediated by sulfatides, and that dystroglycan, not required for assembly, associates with this ECM resulting in the functional readouts of Src activation and utrophin recruitment [25]. The data of this study support the hypothesis that all three LN domains are essential for laminin polymerization and formation of a basement membrane They reveal a requirement of the laminin LG domains for basement membrane assembly consistent with the concept that laminin becomes tethered to the cell surface through its LG domains, whereas type IV collagen and nidogen become tethered primarily to laminin. The data provide cellular evidence for the importance of nidogen serving as a bridge between the laminin and type IV collagen polymers
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