Characterization of the Laminin Binding Domains of the Lutheran Blood Group Glycoprotein

Abstract

Lutheran (Lu) blood group antigens and the basal cell adhesion molecule antigen reside on two glycoproteins that belong to the Ig superfamily (IgSF) and carry five Ig-like extracellular domains. These glycoproteins act as widely expressed adhesion molecules and represent the unique receptors for laminin-10/11 in erythroid cells. Here, we report the mapping of IgSF domains responsible for binding to laminin. In plasmonic resonance surface experiments, only recombinant Lu proteins containing the N-terminal IgSF domains 1-3 were able to bind laminin-10/11 and to inhibit binding of laminin to Lu-expressing K562 cells. Mutant recombinant proteins containing only IgSF domain 1, domains 1 ؉ 2, domains 1 ؉ 3, domains 2 ؉ 3, domain 3, domain 4, domain 5, and domains 4 ؉ 5 failed to bind laminin as well as a construct containing all of the extracellular domains except domain 3. Altogether, these results indicate that IgSF domains 1-3 are involved in laminin binding and that a specific spatial arrangement of these three first domains is most probably necessary for interaction. Neither the RGD nor the N-glycosylation motifs present in IgSF domain 3 were involved in laminin binding. Lutheran (Lu)1 blood group antigens and the basal cell adhesion molecule antigen (a marker of epithelial ovarian cancers) are carried by two glycoprotein (gp) isoforms of 85 kDa (Lu) and 78 kDa (Lu(v13)) that are translated from spliceoforms of a single pre-mRNA (1, 2). The Lu gps have a wide tissue distribution with a predominant expression in the basal layer of the epithelium and the endothelium of blood vessel walls (1). Lu and Lu(v13) are type I membrane proteins of the immunoglobulin superfamily (IgSF) that share a similar extracellular domain composed of five Ig-like domains (two V-set and three C2-set) and a single transmembrane domain but differ by the presence or absence of a cytoplasmic C-terminal domain of 40 amino acids (59 versus 19, respectively) (2). The extracellular domain of Lu gps contains a potential integrin binding site in the third IgSF domain as well as five N-glycosylation consensus motifs, one in the third domain and the others in the fourth domain. The 40 C-terminal residues of the Lu gp cytoplasmic tail carry three characteristic motifs involved in protein-protein interaction, polarization to plasma membrane, and intracellular signaling pathways (3), such as (i) a proline-rich motif for potential binding of Src homology 3 domains, (ii) a dileucine motif responsible for the basolateral expression of Lu gp in polarized epithelial cells (4), and (iii) potential phosphorylation motifs. These motifs are absent in the Lu(v13) gp isoform which exhibits a nonpolarized expression in epithelial cells (4). Recent studies have shown that the C-terminal intracellular domain of Lu gps interacts with the membrane skeleton (5, 6).Interestingly, the Lu gps share a significant sequence similarity with the CD146 (MUC18) and CD166 (activated leukocyte cell adhesion molecule) adhesion molecules found in human tissues (7,8), and Lu and Lu(v...

B‐CAM/LU expression and the role of B‐CAM/LU activation in binding of low‐ and high‐density red cells to laminin in sickle cell disease

“…El Nemer et al and our work showed that the ability of RBC to bind soluble laminin increases with increased expression of B-CAM/LU [20][21][22]. However, data from Lee and colleagues have suggested that HD SS RBC account for the SS RBC most adherent to immobilized laminin during continuous flow exerting the physiological shear stress of 1 dyne/cm 2 [16].…”

“…Both spliceoforms contain five identical extracellular immunoglobulin (Ig) superfamily (SF) domains, and both B-CAM and LU recombinant proteins expressed on the cell surface bind well to soluble and immobilized laminin [20,21]. Our previous studies of SS RBC showed about 50% more B-CAM/LU molecules on lowdensity (LD) SS RBC enriched in reticulocytes than on high-density (HD) SS RBC [21,22] and SS reticulocytes themselves express about four times as much B-CAM/LU than do mature SS RBC [20].…”

Identification of the Binding Site for the Lutheran Blood Group Glycoprotein on Laminin α5 through Expression of Chimeric Laminin Chains in Vivo

“…The extracellular domain of Lu contains N-glycosylation consensus motifs. Recently it was reported that Ig-like domains 1-3 are involved in binding to laminin ␣5 (27,29), but the N-glycosylation motifs were not found to be involved in laminin binding. A bacterial Lu recombinant protein that we initially prepared did not bind to laminin-10/11 (data not shown), suggesting that proper glycosylation is required to ensure proper folding.…”

“…In transgenic mouse hearts that overexpress laminin ␣5, Lu levels are elevated, suggesting that the increased ␣5 in cardiomyocyte basement membranes recruits additional Lu to the cell surface through a direct interaction. Although the laminin-binding site on Lu has been mapped to a first approximation (20,27,29), there is little insight into the structural basis for Ig superfamily members binding to laminins. To understand better the interaction between Lu and the laminin ␣5 chain, it is important to determine the site of Lu binding on ␣5.…”

Global Profiling of the Cell Surface Proteome of Cancer Cells Uncovers an Abundance of Proteins with Chaperone Function

“…2). The Lutheran glycoprotein, a membrane protein belonging to the immunoglobulin superfamily, has been shown to act as a specific receptor for laminin in cell-matrix interaction (17). Furthermore, the 4F2 heavy chain antigen (also known as CD98) was identified in SH-SY5Y, A549, LoVo, and the Sup-B15 leukemia cell lines.…”