Identification of megalin as the sole rat kidney sialoglycoprotein containing poly alpha2,8 deaminoneuraminic acid.
Recently, poly alpha2,8 deaminoneuraminic acid (poly alpha2,8 KDN) was demonstrated in various embryonic and adult mammalian tissues. This study reports the purification and characterization of the single poly alpha2,8 KDN-bearing glycoprotein from rat kidney. Amino acid sequences of proteolytic fragments shared homology with megalin, a member of the LDL receptor family. Immunochemical analysis supported this finding, since immunoprecipitated poly alpha2,8 KDN-bearing glycoprotein was immunoreactive with anti-megalin antibodies in Western blotting and conversely immunoprecipitated megalin was immunoreactive with the monoclonal anti-poly alpha2,8 KDN antibody. Furthermore, receptor-associated protein affinity-purified megalin reacted with the anti-poly alpha2,8 KDN antibody. By immunoelectron microscopy, labeling for both poly alpha2,8 KDN and megalin coincided in the brush border, endocytic invaginations and vesicles, and apical dense tubules of proximal convoluted tubules. Immunoreactivity for poly alpha2,8 KDN on purified megalin was abolished by beta-elimination reaction but not by N-glycosidase F treatment. These data identified megalin as the sole glycoprotein of rat kidney, which contains poly alpha2,8 KDN present on O-glycosidically linked oligosaccharides. Furthermore, this study shows that megalin carries N-glycosidically linked hybrid and complex-type oligosaccharides terminating with sialic acid. Both poly alpha2,8 KDN and sialic acids on megalin may contribute to the binding of Ca2+ and cationic ligands.
Ziak
M
M
Department of Pathology, University of Zürich, Switzerland.
Kerjaschki
D
D
Farquhar
M G
MG
Roth
J
J
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D026541:Heymann Nephritis Antigenic Complex; D007120:Immunochemistry; D007668:Kidney / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000378:metabolism*; D011233:Precipitin Tests; D051381:Rats / Q000378:metabolism*; D012795:Sialoglycoproteins / Q000378:metabolism*
D026541:Heymann Nephritis Antigenic Complex; D008562:Membrane Glycoproteins; D011134:Polysaccharides; D012795:Sialoglycoproteins; C099023:poly(2,8-deaminoneuraminic acid)