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.
Highlights
The Lutheran glycoprotein (Lu) carries the antigen of the Lutheran blood group system
Here we used Lu-Fc as a probe on adult mouse tissue sections because it could be detected by anti-human IgG antibody conjugated to Alexa488
The binding of Lu-Fc to laminin a5 in tissue sections allowed us to examine whether anti-Lu/basal cell adhesion molecule (B-CAM) monoclonal antibodies could block binding (Fig. 1C)
Summary
The Lutheran glycoprotein (Lu) carries the antigen of the Lutheran blood group system. A splice variant of Lu known as basal cell adhesion molecule (B-CAM) [4] has the same extracellular and transmembrane domains as Lu, but it lacks the COOH-terminal 40 amino acids of the cytoplasmic tail. Recent studies have shown that adrenergic stimuli increase Lu/B-CAM binding to laminin via both a PKAdependent pathway and exchange proteins activated by the cAMP-dependent Rap pathway [9,10]. It is unclear if there is synergistic cross-talk between these pathways. The Lutheran blood group glycoprotein (Lu), an Ig superfamily (IgSF) transmembrane receptor, is known as basal cell adhesion molecule (B-CAM). 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 a5 via Lu/B-CAM
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