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 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.

Core 2 GlcNAc transferase and kidney tubular cell-specific expression.

The expression of glycan chains is precisely regulated in a time- and space-dependent manner. We summarize here our recent work on the kidney tubular cell-specific regulation of core 2 beta-1,6-GlcNAc transferase. Gsl5 gene was first identified by genetic analysis on the basis of polymorphic expression of kidney glycolipids among inbred strains of mice and turned out to be a regulatory gene controlling the level of mRNA of kidney-specific core 2 beta-1,6-GlcNAc transferase. This kidney-specific core 2 GlcNAc transferase takes glycolipids having Gal beta 1-3GalNAc at their termini, Gal beta 1-3GalNAc alpha 1- and beta 1-oligosaccharide derivatives, and glycoproteins having core 1 structure, as substrates. Immunohistochemistry with anti-core 2-Le( x ) monoclonal antibody demonstrated that vesicles located just below the microvillous membrane of proximal tubule cells were clearly stained in a Gsl5 -wild type mouse. Western blotting with the monoclonal antibody detected a major glycoprotein with a molecular mass of 500 kDa in the microsomal fraction of the wild type mouse kidney. In situ hybridization with anti-sense cDNA of kidney-specific core 2 GlcNAc transferase confirmed that Gsl5 gene controls the expression of the core 2 beta-1,6-GlcNAc transferase mRNA in a proximal tubular cell-specific manner. The 5' upstream sequences of the kidney-specific core 2 GlcNAc transferase gene in inbred and wild-derived strains of mice were analyzed, and the phylogenetic analysis of these sequences suggests that functional Gsl5 gene might be produced by the time of subspeciation of M. musculus, about one million years ago.