[Show abstract][Hide abstract] 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.
No preview · Article · Apr 2008 · Biochimica et Biophysica Acta
[Show abstract][Hide abstract] ABSTRACT: 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 β-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 β-1,6-GlcNAc transferase. This kidney-specific core 2 GlcNAc transferase takes glycolipids having Galβ1-3GalNAc at their termini, Galβ1-3GalNAcα1- and β1-oligosaccharide derivatives, and glycoproteins having core 1 structure, as substrates. Immunohistochemistry with anti-core 2-Lex
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 β-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. Published in 2004.
No preview · Article · Feb 2004 · Glycoconjugate Journal
[Show abstract][Hide abstract] ABSTRACT: The expression of glyco-chains is precisely regulated in a time-and-space dependent manner. We summarize two types of regulation: kidney tubular cell-specific regulation of core 2 β1-6GlcNAc transferase by the Gsl5 gene, and the suppression of N-glycolylneuraminic acid (NeuGc) by the regulation of CMP-NeuAc hydroxylase activity.The Gsl5 gene identified by genetic analysis on the basis of the polymorphic expression of kidney glycolipids among inbred strains of mice regulates β1,6-GlcNAc transferase activity. The GlcNAc transferase transfers GlcNAc on Galβ1-3GalNAcβ-and also synthesizes core 2 structure, GlcNAcβ1-6(Galβ1-3)GalNAcα-. Immunohistochemistry with anti-core 2-Lex monoclonal antibody demonstrated that the lysosome-like vesicles of the proximal tubule cells were clearly stained in a Gsl5 wild-type mouse. Western blotting confirmed that the positive staining was due to the microsomal glycoproteins. These results, together with those of in situ hybridization, confirmed that the Gsl5 gene controls the expression of β-1,6-GlcNAc transferase mRNA in a proximal tubular cell-specific manner.The expression of NeuGc is controlled by the activity of CMP-NeuAc hydroxylation, which requires three enzyme proteins, cytochrome b5, cytochrome b5 reductase, and a terminal hydroxylase, in the presence of NADH. We cloned mouse and human hydroxylase cDNAs. Mouse brain contains a very high amount of NeuAc, but the hydroxylase mRNA is not detectable by Northern blotting. Surprisingly, the same phenotype is conserved in humans, even though humans have lost the ability to produce intact hydroxylase enzyme, due to the deletion of 92 bp in the genome.
No preview · Article · Dec 2001 · International Congress Series