Identification of active sequences in the L4a domain of laminin α5 promoting neurite elongation.

Laminin α5 is an extracellular matrix protein containing multiple domains implicated in various biological processes, such as embryogenesis and renal function. In this study, we used recombinant proteins and synthetic peptides to identify amino acid residues within the short arm region of α5 that were critical for neurite outgrowth activity. The short arm of α5 contains three globular domains (LN, L4a, and L4b) and three rodlike elements (LEa, LEb, and LEc). Recombinant proteins comprised of the α5 short arm fused with a Fc tag produced in 293 cells were assayed for PC12 (pheochromocytoma) cell adhesion and neurite outgrowth activities. Although it did not have cell attachment activity, neurite outgrowth was promoted by the recombinant protein. To narrow the region involved in neurite outgrowth activity, two truncated recombinant proteins were produced in 293 cells. A recombinant protein lacking L4a and LEb lost activity. Furthermore, we synthesized 78 partially overlapping peptides representing most of the amino acid sequences of L4a and LEb. Of the peptides, A5-76 [mouse laminin α5 928-939 (TSPDLFRLVFRY) in L4a] exhibited neurite outgrowth activity. Mutagenesis studies showed that Phe(933) and Arg(934) were involved in neurite outgrowth activity. Moreover, inhibition assays using anti-integrin monoclonal antibodies showed that neurite outgrowth on the α5 short arm was partially mediated by integrin α1ß1. However, the antibodies to integrin α1 and ß1 did not inhibit neurite elongation on the A5-76 peptide. These results suggest that in addition to cellular interactions with the active site in the L4a domain, the binding of integrin α1ß1 seems to modulate neurite elongation on the short arm of α5.

An antibody to the lutheran glycoprotein (Lu) recognizing the LU4 blood type variant inhibits cell adhesion to laminin α5.

BACKGROUND: The Lutheran blood group glycoprotein (Lu), an Ig superfamily (IgSF) transmembrane receptor, is also known as basal cell adhesion molecule (B-CAM). Lu/B-CAM is a specific receptor for laminin α5, a major component of basement membranes in various tissues. Previous reports have shown that Lu/B-CAM binding to laminin α5 contributes to sickle cell vaso-occlusion. However, as there are no useful tools such as function-blocking antibodies or drugs, it is unclear how epithelial and sickled red blood cells adhere to laminin α5 via Lu/B-CAM. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we discovered a function-blocking antibody that inhibits Lu binding to laminin α5 using a unique binding assay on tissue sections. To characterize the function-blocking antibody, we identified the site on Lu/B-CAM recognized by this antibody. The extracellular domain of Lu/B-CAM contains five IgSF domains, D1-D2-D3-D4-D5. The antibody epitope was localized to D2, but not to the D3 domain containing the major part of the laminin α5 binding site. Furthermore, mutagenesis studies showed that Arg(175), the LU4 blood group antigenic site, was crucial for forming the epitope and the antibody bound sufficiently close to sterically hinder the interaction with α5. Cell adhesion assay using the antibody also showed that Lu/B-CAM serves as a secondary receptor for the adhesion of carcinoma cells to laminin α5. CONCLUSION/SIGNIFICANCE: This function-blocking antibody against Lu/B-CAM should be useful for not only investigating cell adhesion to laminin α5 but also for developing drugs to inhibit sickle cell vaso-occlusion.