An Antibody to the Lutheran Glycoprotein (Lu) Recognizing the LU4 Blood Type Variant Inhibits Cell Adhesion to Laminin α5

Abstract

BackgroundThe 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 FindingsIn 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 Arg175, 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/SignificanceThis 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.

An Anti-Human Lutheran Glycoprotein Phage Antibody Inhibits Cell Migration on Laminin-511: Epitope Mapping of the Antibody

“…The Lu-Fc binding assay was performed as described previously [19]. Briefly, Lu-Fc was diluted with PBS(-) containing 10 μg/ml of antibody or 5–12 × 10 11 pfu/ml of phage antibodies and was then placed on adult mouse kidney sections.…”

“…As described in our previous study [19], cDNAs encoding the mutated Lu proteins were generated by PCR using the primer sets listed in S1 Table and were subcloned into the human IgG 1 Fc expression vector. Transfectants were selected in DMEM containing 10% FBS and 100 μg/ml Zeocin (ThermoFisher Scientific).…”

“…Our recent report showed that Lu and B-CAM promote the migration of lung carcinoma cells on laminin-511 (LM-511), which is composed of α5, β1, and γ1 chains [18]. Of the commercially available antibodies, we also discovered that one monoclonal antibody against Lu can inhibit its binding to laminin α5 [19]. Furthermore, the function-blocking antibody against Lu effectively inhibits the migration of lung carcinoma cells on LM-511.…”

The Lutheran/Basal Cell Adhesion Molecule Promotes Tumor Cell Migration by Modulating Integrin-mediated Cell Attachment to Laminin-511 Protein

“…Sol-Lu treated with trypsin or CDB was used for SDS-PAGE and binding assays. Mouse kidney sections were prepared as described in our previous study (27). Animal studies were permitted by the Animal Research Committee of Tokyo University of Pharmacy and Life Sciences.…”

“…Lu-Fc bound to endogenous laminin ␣5 was detected with anti-human IgG antibody conjugated with Alexa Fluor 488. Quantitation of the recombinant proteins bound to laminin ␣5 was performed as described in our previous study (27).…”