The Laminin 511/521–binding site on the Lutheran blood group glycoprotein is located at the flexible junction of Ig domains 2 and 3

Abstract

The Lutheran blood group glycoprotein, first discovered on erythrocytes, is widely expressed in human tissues. It is a ligand for the ␣5 subunit of Laminin 511/521, an extracellular matrix protein. This interaction may contribute to vaso-occlusive events that are an important cause of morbidity in sickle cell disease. Using x-ray crystallography, small-angle x-ray scattering, and site-directed mutagenesis, we show that the extracellular region of Lutheran forms an extended structure with a distinctive bend between the second and third immunoglobulin-like domains. The linker between domains 2 and 3 appears to be flexible and is a critical determinant in maintaining an overall conformation for Lutheran that is capable of binding to Laminin. Mutagenesis studies indicate that Asp312 of Lutheran and the surrounding cluster of negatively charged residues in this linker region form the Laminin-binding site. Unusually, receptor binding is therefore not a function of the domains expected to be furthermost from the plasma membrane. These studies imply that structural flexibility of Lutheran may be essential for its interaction with Laminin and present a novel opportunity for the development of therapeutics for sickle cell disease. IntroductionThe Lutheran glycoprotein (Lu gp) cellular adhesion molecule is widely expressed in human tissues 1 and is known for carrying antigens of the Lutheran blood group system. On erythrocytes, Lu gp is expressed as 2 isoforms of 78 and 85 kDa. 2 Both share a common extracellular portion that has previously been predicted to comprise 5 immunoglobulin superfamily (IgSF) domains. 1 The 78-kDa isoform (also known as BCAM 3 or Lu[v13] 4 ) results from alternative splicing and lacks 40 C-terminal amino acids within the cytoplasmic domain that contain an SH3-binding motif, a dileucine motif responsible for basolateral targeting, 5 and 5 potential phosphorylation sites. 1 Lu gp binds specifically and with high affinity to the extracellular matrix (ECM) protein Laminin (Ln) containing the ␣5 subunit 6-9 (Laminin 511 and Laminin 521 (Ln511/521) (numbering as in Aumailley et al 10 ). This interaction plays a direct role in the pathophysiology of sickle cell disease by mediating adhesion of sickle cells, via Lu gp, to exposed Ln511/521 of inflamed or damaged vascular endothelium. 9,11,12 Recent studies have shown that higher than normal intracellular levels of cAMP in sickle erythrocytes influence a protein kinase A-mediated or Rap1-mediated signaling pathway resulting in increased adhesion of sickle cells to the basement membrane glycoproteins Ln511/ 521. 13,14 Furthermore, phosphorylation of the 85-kDa Lu gp at S621 in epinephrine-stimulated K562 cells alters adhesion to Ln511/521. 15 The Ln-binding activity of Lu gp is a property of the amino terminal region of Lu gp, and has been localized to a region predicted to form the first 3 IgSF domains (D1D2D3). 6,16 The complementary binding site on Ln is located within a large carboxyl-terminal globular domain of the ␣5 subunit, known to comprise 5 ...

Glycophorin-C sialylation regulates Lu/BCAM adhesive capacity during erythrocyte aging

“…[36][37][38][39][40] An initial study by Zen et al, using murine erythroleukemia cells expressing various Lu/BCAM domains, determined that the membrane proximal domain, domain 5, contributes to adhesion of these cells to laminin-a5. 38 Later, this finding was seemingly contradicted by 2 groups that used recombinant Lu/BCAM protein and BIACORE technology to address the same question.…”

Adhesive activity of Lu glycoproteins is regulated by interaction with spectrin

“…This linker between the second and third domains appears flexible and is crucial for creating the Lu conformation required for laminin binding. 23 Our current data now detail an important interaction of the Lu cytoplasmic tail linking it to the cytoskeleton.Another major finding of the current study was that specifically disrupting the Lu-spectrin interaction by exogenously incorporated ␣R4 led to an increased adhesivity of resealed ghosts to laminin. The basis of the increased adhesion to the laminin matrix when Lu is released from the membrane cytoskeleton is uncertain, but a plausible mechanism requires the freely floating transmembrane Lu molecules to cluster and thereby generate a large adhesive force.…”

“…This linker between the second and third domains appears flexible and is crucial for creating the Lu conformation required for laminin binding. 23 Our current data now detail an important interaction of the Lu cytoplasmic tail linking it to the cytoskeleton.…”

“…1,5,21,22 We have also determined that the laminin binding site is present in the 3 membrane distal IgSF domains 22 and is located at the flexible junction of Ig domains 2 and 3. 23 Interactions of receptor molecule cytoplasmic domains with the cytoskeleton can play critical roles in regulating receptor function. Earlier we determined that Lu has a high degree of connectivity to the erythrocyte membrane cytoskeleton.…”

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

“…5b). To determine the putative involvement of the Lu/BCAM-laminin 511/521 interaction in the stress fibers formation, MDCK cells expressing the D343R Lu/BCAM mutant, which is unable to bind to laminin 511/521 (the mutated residue is the same as residue 312 in reference [21]), were analyzed. These cells did not form stress fibers neither on laminin 511/521 nor on fibronectin (Fig.…”