Makoto Hirano1, Kiichiro Totani2, Tomohiko Fukuda3, Jianguo Gu3, Akemi Suzuki4. 1. Institute of Glycoscience, Tokai University, 4-1-1 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan; Department of Materials and Life Science, Faculty of Science and Technology, Seikei University, 3-3-1 Kichijoji-kita, Musashino, Tokyo 180-8633, Japan. Electronic address: mhirano@st.seikei.ac.jp. 2. Department of Materials and Life Science, Faculty of Science and Technology, Seikei University, 3-3-1 Kichijoji-kita, Musashino, Tokyo 180-8633, Japan. 3. Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsusima, Aoba, Sendai, Miyagi 981-8558, Japan. 4. Institute of Glycoscience, Tokai University, 4-1-1 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan; Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsusima, Aoba, Sendai, Miyagi 981-8558, Japan.
Abstract
BACKGROUND: Megalin is a 600-kDa single-spanning transmembrane glycoprotein and functions as an endocytic receptor, distributed not only in the kidney but also in other tissues. Structurally and functionally distinct ligands for megalin have been identified. Megalin has 30 potential N-glycosylation sites in its extracellular domain. We found that megalin interacts with its ligands in a glycoform-dependent manner. METHODS: Distribution of megalin and glycans was histochemically analyzed in mouse kidneys. Kidney absorption of Cy5-labeled ligands was examined in vivo. Megalin-ligand interactions were analyzed using ligand blotting and ELISA. RESULTS: Megalins expressed on renal proximal convoluted tubules (PCTs) and proximal straight tubules (PSTs) have different N-glycans. PCT megalin stained with Lens culinaris agglutinin (LCA), which recognizes core-fucosyl N-glycans catalyzed by α1,6-fucosyltransferase (Fut8). In contrast, PST megalin stained with wheat germ agglutinin (WGA), which recognizes hybrid-type N-glycans. Retinol-binding protein-Cy5 (RBP-Cy5) was endocytosed by megalin on PCTs but minimally endocytosed by PSTs. BSA-Cy5 was endocytosed nearly equally by both tubules. The purified LCA-positive glycoform megalin had higher binding activity for RBP and vitamin D-binding protein than did WGA-positive glycoform megalin. Both glycoforms had nearly the same BSA- and kanamycin-binding activities. RBP-binding analysis of megalin lacking core fucose, in Fut8-/- mouse kidneys, had significantly decreased binding activity. CONCLUSIONS: N-Glycosylation of megalin can modulate its ligand-binding activity. Core fucosylation, in particular, is a modification crucial for megalin-RBP interactions. GENERAL SIGNIFICANCE: Cell type-specific glycoforms of megalin exist in the proximal tubular cells and modulate ligand absorption capacity. Copyright Â
BACKGROUND:Megalin is a 600-kDa single-spanning transmembrane glycoprotein and functions as an endocytic receptor, distributed not only in the kidney but also in other tissues. Structurally and functionally distinct ligands for megalin have been identified. Megalin has 30 potential N-glycosylation sites in its extracellular domain. We found that megalin interacts with its ligands in a glycoform-dependent manner. METHODS: Distribution of megalin and glycans was histochemically analyzed in mouse kidneys. Kidney absorption of Cy5-labeled ligands was examined in vivo. Megalin-ligand interactions were analyzed using ligand blotting and ELISA. RESULTS:Megalins expressed on renal proximal convoluted tubules (PCTs) and proximal straight tubules (PSTs) have different N-glycans. PCT megalin stained with Lens culinaris agglutinin (LCA), which recognizes core-fucosyl N-glycans catalyzed by α1,6-fucosyltransferase (Fut8). In contrast, PST megalin stained with wheat germ agglutinin (WGA), which recognizes hybrid-type N-glycans. Retinol-binding protein-Cy5 (RBP-Cy5) was endocytosed by megalin on PCTs but minimally endocytosed by PSTs. BSA-Cy5 was endocytosed nearly equally by both tubules. The purified LCA-positive glycoformmegalin had higher binding activity for RBP and vitamin D-binding protein than did WGA-positive glycoformmegalin. Both glycoforms had nearly the same BSA- and kanamycin-binding activities. RBP-binding analysis of megalin lacking core fucose, in Fut8-/- mouse kidneys, had significantly decreased binding activity. CONCLUSIONS: N-Glycosylation of megalin can modulate its ligand-binding activity. Core fucosylation, in particular, is a modification crucial for megalin-RBP interactions. GENERAL SIGNIFICANCE: Cell type-specific glycoforms of megalin exist in the proximal tubular cells and modulate ligand absorption capacity. Copyright Â
Authors: Rodrigo Pacheco Silva-Aguiar; Nathália C F Bezerra; Miguel C Lucena; Gabriela M Sirtoli; Roberto T Sudo; Gisele Zapata-Sudo; Christina M Takiya; Ana Acacia S Pinheiro; Wagner Barbosa Dias; Celso Caruso-Neves Journal: J Biol Chem Date: 2018-06-28 Impact factor: 5.157