title: Core 2 GlcNAc modification and megalin ligand-binding activity.
abstract: 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 beta6GlcNAc 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 beta6GlcNAc transferase, express megalin carrying the core 2 extended Le(x) epitope, while DBA/2 mice, a mutant-strain of the core 2 beta6GlcNAc 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 V(max) of the interaction between Cy5-labeled RBP and the megalins of BALB/c and DBA/2 mice was 60 microM and 30 microM, respectively, and the apparent K(m) was 11 microM and 17 microM, 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 (K(dBALB)=12.0 microM; K(dDBA)=20.9 microM) and a low-affinity site (K(dBALB)=36.2 microM; K(dDBA)=58.8 microM). FCS analysis exhibited quite different K(m) and V(max) values from those obtained by GPC, but similar K(m) values for the two types of megalin, and a lower V(max) 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.
authors.affiliation: Sphingolipid Expression Laboratory, RIKEN Frontier Research System, Wako, Saitama, Japan.
authors.forename: Heng
authors.initials: H
authors.lastname: Zhang
authors.forename: Shigemi
authors.initials: S
authors.lastname: Yoshioka
authors.forename: Masao
authors.initials: M
authors.lastname: Miyazaki
authors.forename: Reiji
authors.initials: R
authors.lastname: Kannagi
authors.forename: Akemi
authors.initials: A
authors.lastname: Suzuki
mesh_terms: D000818:Animals; D002232:Carbocyanines; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008024:Ligands; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008811:Mice, Inbred DBA; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D011485:Protein Binding; D012177:Retinol-Binding Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D002232:Carbocyanines; D008024:Ligands; D026561:Low Density Lipoprotein Receptor-Related Protein-2; D012177:Retinol-Binding Proteins; C085321:cyanine dye 5; D017351:N-Acetylglucosaminyltransferases; C053024:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-acetylglucosaminyl transferase