Molecular basis of ligand recognition by the mannose/N-acetylgalactosamine-4-sulfate receptor specificity through multivalency

The multifunctional Mannose/GalNAc-4-SO₄ Receptor (Man/GalNAc-4-SO ₄ receptor) both determines glycoprotein hormone circulating half-life and plays a role in innate immunity. An unusual characteristic of this receptor is that it harbors the capacity to bind unrelated carbohydrate moieties at distinct sites within its polypeptide sequence. In addition, the receptor can take on different forms, differing in ligand specificity and cellular expression. In order to elucidate the mechanism accounting for the different ligand binding characteristics, I investigated the molecular properties of two forms of the Man/GalNAc-4-SO₄ receptor isolated from rat liver and lung. I found that the GalNAc-4-SO₄-specific receptor in liver is dimeric whereas the Man-specific receptor found in lung is monomeric, demonstrating receptor oligomeric structure as a mechanism for determining distinct binding specificities.; Surface plasmon resonance (SPR) and equilibrium dialysis studies were used to characterize both the number of GalNAc-4-SO₄ binding sites per cysteine-rich domain and the affinity and stoichiometry of binding for ligands bearing different numbers and/or configurations of terminal β1,4-linked GalNAc-4-SO₄ moieties. I determined that each cysteine-rich domain has single GalNAc-4-SO₄ binding site and that both the number and spacing of N-linked GalNAc-4-SO₄ moieties on a glycoprotein is critical for recognition.; The specificity for sulfated ligands by the receptor was investigated in several different binding assays using monovalent and multivalent forms of a trisaccharide sulfated at either the C4 or C3 hydroxyl of terminal GalNAc. The affinities displayed by the monovalent and multivalent C3 sulfated ligands were 16.2 and 0.170 μM respectively, while those for the C4 ligands were 25.8 and 0.013 μM, demonstrating that the receptor displays greatly enhanced specificity for polyvalent ligands compared to their monovalent counterparts. By performing slight modifications of the same assay, I found that this 95-fold versus 2000-fold change in affinity for the multivalent ligands was shown to reflect conformational differences.; The binding properties of a soluble monomeric Man/GalNAc-4-SO₄ receptor chimera and various deletion mutants were examined to decipher the structural features that mediate receptor dimerization. My findings indicate that dimerization does not rely on several key residues but rather the presence of multiple contacts throughout the entire extracellular domain of the receptor. These findings have expanded our understanding of the impact of multivalency on carbohydrate-protein interactions and revealed a novel mechanism for modulating ligand specificity.