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 ...
The Lutheran glycoprotein is a five domain member of the immunoglobulin superfamily (IgSF) with a wide tissue distribution. It is a ligand for Laminin isoforms containing the alpha5 chain (Laminins 10 and 11). Lutheran glycoprotein on erythrocytes is thought to play a role in vasocclusive events that are a serious cause of morbidity in sickle cell anaemia. We have investigated the molecular basis of the Lutheran:Laminin 10/11 interaction. Lutheran binding to Laminin 10/11 is pH and salt dependent suggesting the interaction is influenced by surface charge. Since Laminins are known to contain areas of positive charge that are of importance in binding to other ligands (heparin, alpha-dystroglycan), a molecular model of Lutheran glycoprotein was constructed to identify surface exposed areas of negatively charged aspartic and glutamic acid residues. Selected residues were mutated to alanine and the mutant proteins examined for binding to Laminin 10/11 using ELISA and Surface Plasmon Resonance. Mutations E309A and D310A greatly reduced binding to Laminin 10/11 while D312A completely abolished binding. The Lutheran model predicts a rod-like structure with a flexible hinge region of 6–8 residues between the 2nd and 3rd IgSF domains. Residues E309, D310 and D312 are located on domain 3 proximal to the hinge region. Mutations (H235P, and delta 233–235) within the hinge region also abolished Laminin binding showing the hinge region to be essential for ligand interaction. Electron tomography on recombinant Lutheran-Fc chimeric protein bound to Laminin 10/11 suggested Lutheran glycoprotein bends at the hinge region to expose the critical negatively charged residues on domain 3 and thereby allow Laminin binding. These data suggest Lutheran-Laminin 10/11 interaction is a novel type of protein:protein interaction and provide a foundation for further investigation of its biological significance.
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