N-linked glycosylation of mouse adiponectin

• Published in: Hormone and metabolic research Vol. 43; no. 8; p. 545
• Main Authors: Tanaka, M, Fukuhara, A, Shimomura, I
• Format: Journal Article
• Language: English
• Published: Germany 01-07-2011
• Subjects: 3T3-L1 Cells; Adipocytes - drug effects; Adipocytes - metabolism; Adiponectin - blood; Adiponectin - chemistry; Adiponectin - metabolism; Amino Acid Sequence; Animals; Culture Media, Conditioned - pharmacology; Glycosylation - drug effects; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Tunicamycin - pharmacology
• ISSN: 1439-4286
• DOI: 10.1055/s-0031-1280782

Adiponectin is an insulin-sensitizing adipokine with antidiabetic, anti-atherogenic, anti-inflammatory, and cardioprotective properties. Previously, some types of posttranslational modification on adiponectin have been reported. In this study, we demonstrate that mouse adiponectin protein migrated as 2 bands on SDS-PAGE gel. Slower migrating band of adiponectin was reduced by PNGase treatment. PNGase is known as N-glycosidase, and is able to change the mobility of N-glycosylated protein on SDS-PAGE gel. This result indicates the possibility that slower band shifted and overlapped with faster band by cleavage of N-glycan. To further clarify the N-glycosylation of adiponectin, we investigated the effect of N-glycosylation inhibitor tunicamycin on 3T3-L1 adipocytes. Tunicamycin significantly reduced the ratio of slower band to faster band in culture medium from 3T3-L1 adipocytes. This result also indicates the possibility that slower band of adiponectin is N-glycosylated. Lastly, to identify glycosylated asparagine residues, we established 3T3-L1 cell lines stably expressing wild type and mutant adiponectin in N-glycosylation sites. Wild-type adiponectin protein migrated as double bands, and mutant adiponectin in either asparagine at position 53 or threonine at 55 lacked slower band. These results suggest that a part of mouse adiponectin is modified by N-linked glycosylation at asparagine 53.