Occurrence of secretory glycoprotein-specific GalNAc beta 1-->4GlcNAc sequence in N-glycans in MDCK cells.
Many reports show that N-glycans of glycoproteins play important roles in vectorial transport in MDCK cells. To assess whether structural differences in N-glycans exist between secretory glycoproteins and membrane glycoproteins, we studied the N-glycan structures of the glycoproteins isolated from MDCK cells. Polarized MDCK cells were metabolically labeled with [3H]glucosamine, and (3)H-labeled N-glycans of four glycoprotein fractions, secretory glycoproteins in apical and basolateral media, and apical and basolateral membrane glycoproteins, were released by glycopeptidase F. The structures of the free N-glycans were comparatively analyzed using various lectin column chromatographies and sequential glycosidase digestion. The four samples commonly contained high-mannose-type glycans and bi- and tri-antennary glycans with a bisected or non-bisected trimannosyl core. However, secretory glycoproteins in both media predominantly contained (sialyl)LacdiNAc sequences, +/-Sia alpha 2-->6GalNAc beta 1-->4GlcNAc beta 1-->R, which linked only to a non-bisected trimannosyl core. beta1-->4N-acetylgalactosaminyltransferase (beta 4GalNAc-T) activity in MDCK cells preferred non-bisected glycans to bisected ones in accordance with the proposed N-glycan structures. This secretory glycoprotein-predominant LacdiNAc sequence was also found in the case of human embryonic kidney 293 cells. These results suggest that the secretory glycoprotein-specific (sialyl)LacdiNAc sequence and the corresponding beta 4GalNAc-T are involved in transport of secretory glycoproteins.
Ohkura
Takashi
T
Department of Biochemistry, Sasaki Institute and Core Research for Evolutional Science and Technology of the Japan Science and Technology Corporation, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan.
Seko
Akira
A
Hara-Kuge
Sayuri
S
Yamashita
Katsuko
K
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002458:Cell Fractionation; D002460:Cell Line; D004187:Disaccharides / Q000737:chemistry* / Q000378:metabolism; D004285:Dogs; D005944:Glucosamine / Q000737:chemistry / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology; D007785:Lactose / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D017350:N-Acetylgalactosaminyltransferases / Q000378:metabolism*; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism; D037121:Plant Lectins; D014316:Tritium / Q000378:metabolism
D004187:Disaccharides; D006023:Glycoproteins; D009844:Oligosaccharides; D037121:Plant Lectins; D014316:Tritium; C093701:N-acetylgalactosaminyl-1-4-N-acetylglucosamine; D017350:N-Acetylgalactosaminyltransferases; D007785:Lactose; D005944:Glucosamine