Core 2 GlcNAc modification and megalin ligand-binding activity

Megalin, a receptor-like transporter glycoprotein, is expressed on kidney proximal tubular cells and reabsorbs small-molecular-weight proteins from the glomerular filtrate. Here, we report that mouse megalins differently modified with core 2 β6GlcNAc transferase had different kinetic properties to a fluorescence-labeled ligand, retinol-binding protein (RBP). BALB/c mice, a wild-type strain in terms of the expression of kidney-specific core 2 β6GlcNAc transferase, express megalin carrying the core 2 extended Lex epitope, while DBA/2 mice, a mutant-strain of the core 2 β6GlcNAc transferase, express megalin lacking the epitope. We purified these two types of megalin using lentil lectin chromatography and measured the ligand-binding activities of the megalins using Cy5-labeled RBP by applying gel permeation chromatography (GPC) and fluorescence correlation spectroscopy (FCS). The analysis by GPC indicated that the apparent Vmax of the interaction between Cy5-labeled RBP and the megalins of BALB/c and DBA/2 mice was 60 μM and 30 μM, respectively, and the apparent Km was 11 μM and 17 μM, respectively. Scatchard analysis demonstrated the presence of two binding sites. Linear regression analysis resulted in a two-binding-site model characterized by a high-affinity site (KdBALB = 12.0 μM; KdDBA = 20.9 μM) and a low-affinity site (KdBALB = 36.2 μM; KdDBA = 58.8 μM). FCS analysis exhibited quite different Km and Vmax values from those obtained by GPC, but similar Km values for the two types of megalin, and a lower Vmax value for DBA/2 megalin than BALB/c megalin. These results suggest that the core 2 GlcNAc extended glycan chains on megalin can change the ligand-binding affinity and capacity.