The Lutheran Blood Group Glycoproteins, the Erythroid Receptors for Laminin, Are Adhesion Molecules

Abstract

The Lutheran antigens are recently characterized glycoproteins in which the extracellular region contains five immunoglobulin like domains, suggesting some recognition function. A recent abstract suggests that the Lutheran glycoproteins (Lu gps) act as erythrocyte receptors for soluble laminin (Udani, M., Jefferson, S., Daymont, C., Zen, Q., and Telen, M. J. (1996) Blood 88, Suppl. 1, 6 (abstr.)). In the present report, we provided the definitive proof of the laminin receptor function of the Lu gps by demonstrating that stably transfected cells (murine L929 and human K562 cell lines) expressing the Lu gps bound laminin in solution and acquired adhesive properties to laminin-coated plastic dishes but not to fibronectin, vitronectin, transferrin, fibrinogen, or fibrin. Furthermore, expression of either the long-tail (85 kDa) or the short-tail (78 kDa) Lu gps, which differ by the presence or the absence of the last 40 amino acids of the cytoplasmic domain, respectively, conferred to transfected cells the same laminin binding capacity. We also confirmed by flow cytometry analysis that the level of laminin binding to red cells is correlated with the level of Lu antigen expression. Indeed, Lunull cells did not bind to laminin, whereas sickle cells from most patients homozygous for hemoglobin S overexpressed Lu antigens and exhibited an increased binding to laminin, as compared with normal red cells. Laminin binding to normal and sickle red cells as well as to Lu transfected cells was totally inhibited by a soluble Lu-Fc chimeric fragment containing the extracellular domain of the Lu gps. During in vitro erythropoiesis performed by two-phase liquid cultures of human peripheral blood, the appearance of Lu antigens in late erythroid differentiation was concomitant with the laminin binding capacity of the cultured erythroblasts. Altogether, our results demonstrated that long-tail and short-tail Lu gps are adhesion molecules that bind equally well laminin and strongly suggested that these glycoproteins are the unique receptors for laminin in normal and sickle mature red cells as well as in erythroid progenitors.

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

“…We have demonstrated previously that the ability to bind soluble laminin, as detected by flow cytometry, increases proportionally with B-CAM/LU expression levels of both AA and SS RBC [15]. El Nemer et al [20] further showed that recombinant soluble LutheranFc fragment inhibited 90-100% of soluble laminin binding to AA and SS RBC. Our present study of density-separated fractions of SS RBC also confirms that the ability to bind soluble laminin is correlated with B-CAM/LU expression levels in the corresponding cell fractions.…”

“…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].…”

“…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]. 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].…”

Novel role for the Lu/BCAM–spectrin interaction in actin cytoskeleton reorganization

“…At day 7, polarized cells were fixed 20 min with 4% paraformaldehyde, treated with 50 mM NH 4 Cl in PBS, permeabilized 10 min with 0.5% Triton (PBS) and incubated with F241 anti-Lu MoAb (1:10) and the polyclonal anti-DII-spectrin SH3 domain A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.…”

“…10 4 mocktransfected MDCK cells or transfected cells expressing wt-Lu, mt-Lu, D343R Lu mutant or Lu(v13) were added to the wells (in 400 Pl). Cells were incubated in in serum-free medium for 90 min at 37°C in presence or absence of 1 Pg/ml per well of the cell-penetrating form of the Clostridium botulinum C3 toxin, an ADP ribosyltransferase that selectively ribosylates Rho proteins, rendering them inactive After this incubation time, cells were fixed 20 min with 4% paraformaldehyde, treated with 50 mM NH 4 Cl in PBS, permeabilized 10 min with 0.5% Triton (PBS), saturated 30 min in Image-iTFX signal enhancer solution. Cells were washed with PBS-0.5% BSA and incubated with Phalloidin AlexaFluor® 488 (1:50) for 1 h at RT.…”