Lutheran blood group glycoprotein and its newly characterized mouse homologue specifically bind α5 chain-containing human laminin with high affinity

Abstract

Lutheran blood group glycoproteins (Lu gps) are receptors for the extracellular matrix protein, laminin. Studies suggest that Lu gps may contribute to vasoocclusion in sickle cell disease and it has recently been shown that sickle cells adhere to laminin isoforms containing the ␣5 chain (laminin 10/11). Laminin ␣5 is present in the subendothelium and is also a constituent of bone marrow sinusoids, suggesting a role for the Lu/ laminin interaction in erythropoiesis. The objectives of the current study were to define more precisely the molecular interactions of the extracellular and intracellular regions of human Lu and to clone and characterize a mouse homologue. To this end, complementary DNA and genomic clones for the mouse homologue were sequenced and the mouse Lu gene mapped to a region on chromosome 7 with conserved synteny with human 19q13.2. Mouse and human Lu gps are highly conserved (72% identity) at the amino acid sequence level and both mouse and human Lu gps specifically bind laminin 10/11 with high affinity. Furthermore, the first 3, N-terminal, immunoglobulin superfamily domains of human Lu are critical for this interaction. The results indicated that the cytoplasmic domain of BRIC 221-labeled human Lu gp is linked with the spectrin-based skeleton, affording the speculation that this interaction may be critical for signal transduction. These results further support a role for Lu gps in sickle cell disease and indicate the utility of mouse models to explore the function of Lu gp-laminin 10/11 interaction in normal erythropoiesis and in sickle cell disease. IntroductionThe Lutheran blood group is composed of a complex set of antigens expressed on 2 integral membrane glycoprotein isoforms of 85 and 78 kd. 1,2 The complementary DNA (cDNA) encoding the 85-kd isoform has been cloned, 3 and the predicted structure is that of a type 1 membrane protein. There are 5 disulfide-bonded, extracellular, immunoglobulin superfamily (IgSF) domains, a single hydrophobic membrane span, and a cytoplasmic tail of 59 residues. 3 The composition of the extracellular IgSF domains puts Lutheran blood group glycoproteins (Lu gps) in the subset of adhesion molecules that includes the human tumor marker MUC18/ MCAM4 and the chicken neural adhesion molecule Gicerin. [4][5][6] Chicken gicerin binds neurite outgrowth factor, a variant of the extracellular matrix (ECM) protein laminin 7,8 and, interestingly, recent studies suggest that Lu gp also functions as a laminin receptor. [9][10][11] The Lu gp cytoplasmic tail contains an SH3 binding motif and 5 potential phosphorylation sites, consistent with receptor signaling function. Of note, differences in the structure of the cytoplasmic tail distinguish the 2 isoforms. The 78-kd isoform (also termed B-CAM 12 or Lu[v13] 13 ) is generated by alternative splicing of intron 13 and differs from the larger form by having a truncated cytoplasmic tail lacking the SH3 binding motif as well as the potential phosphorylation sites. A recent study in epithelial cells 14 suggests that the cytop...

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

“…On the other hand, sol-Lu did not bind to laminin-1 (␣1␤1␥1). This specificity for laminins containing the ␣5 chain is consistent with published results (20,27). We therefore conclude that sol-Lu has binding properties similar to native cell surface Lu and is an appropriate tool to investigate and identify Lu-binding sites on the laminin ␣5 chain.…”

“…Udai et al (25) demonstrated that the major laminin receptor present on sickle cells is the Lu/B-CAM protein. Furthermore, K562 cells transfected with human Lu adhere to laminin-10/11 but not to laminins lacking the ␣5 chain (27). In our previous studies we made an antibody specific for mouse Lu, and we determined its expression pattern (28).…”

“…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.…”

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

“…At least some studies have shown that B-CAM/LU is associated with the cytoskeleton in red blood cells [17], making our hypothesis a reasonable one to explore. Therefore, to determine if the cytoskeleton is involved in regulation of B-CAM/LUmediated cell adhesion to laminin, we performed adhesion studies under both static and continuousflow conditions using MEL cells expressing B-CAM with a cytoplasmic tail truncated to five amino acids (B-CAM-ST5/MEL).…”

“…The red cell laminin receptor B-CAM/LU [15] is the protein that bears Lutheran blood group antigens and binds specifically to laminins containing the a5 chain (laminins 10 and 11) [16,17]. B-CAM/LU comprises two protein spliceoforms derived from a single gene; B-CAM lacks the last 40 cytosolic amino acids of the LU protein [18,19].…”