Characterisation Of The Laminin 10/11 Binding Site On The Lutheran Glycoprotein Suggests A Novel Type Of Protein-Protein Interaction.

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.