N-Glycoform-dependent interactions of megalin with its ligands

• Published in: Biochimica et biophysica acta. General subjects Vol. 1861; no. 1; pp. 3106 - 3118
• Main Authors: Hirano, Makoto, Totani, Kiichiro, Fukuda, Tomohiko, Gu, Jianguo, Suzuki, Akemi
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2017
• Subjects: absorption; agglutinins; Animals; Carbocyanines - metabolism; Chromatography, Affinity; Endocytic receptor; Female; fucose; Fucosyltransferases - deficiency; Fucosyltransferases - metabolism; Glycoform; glycoproteins; Glycosylation; Kidney - metabolism; Kidney Tubules, Proximal - cytology; Kidney Tubules, Proximal - metabolism; Lens culinaris; life cycle assessment; Ligands; Ligand–receptor interaction; Low Density Lipoprotein Receptor-Related Protein-2 - metabolism; mice; Mice, Inbred C57BL; Mice, Knockout; Organ Specificity; Plant Lectins - metabolism; polysaccharides; Polysaccharides - metabolism; Protein Binding; proximal tubules; Retinol-Binding Proteins - metabolism; tissues; vitamin D-binding protein; wheat germ; Wheat Germ Agglutinins - metabolism; RAP; OCT; RBP; Glycoform; PSTs; PNGase F; TBST; Endocytic receptor; RBP-Cy5; PFA; LCA; LRP; JCGGDB; SGLT-2; WGA; Fut8; DBP; NP-40; PCTs; Kan; MAS; Ligand–receptor interaction
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2016.10.015

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. 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. 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. N-Glycosylation of megalin can modulate its ligand-binding activity. Core fucosylation, in particular, is a modification crucial for megalin–RBP interactions. Cell type-specific glycoforms of megalin exist in the proximal tubular cells and modulate ligand absorption capacity. •Segment-specific N-glycoforms of megalin exist in a nephron.•Megalin glycoforms exhibit different ligand-binding activities.•Lack of core-fucose reduces the binding activity to retinol-binding protein.•N-Glycosylation of megalin modulates the ligand-binding capacity.