N-Linked Glycosylation of Mouse Adiponectin

Abstract

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.

Key words

adiponectin - modification - adipocyte

• References

• 1 Berg AH, Combs TP, Scherer PE. ACRP30/adiponectin: an adipokine regulating glucose and lipid metabolism. Trends Endocrinol Metab 2002; 13: 84-89
• 2 Hotamisligil GS, Spiegelman BM. Tumor necrosis factor alpha: a key component of the obesity-diabetes link. Diabetes 1994; 43: 1271-1278
• 3 Matsuzawa Y, Funahashi T, Nakamura T. Molecular mechanism of metabolic syndrome X: contribution of adipocytokines adipocyte-derived bioactive substances. Ann N Y Acad Sci 1999; 892: 146-154
• 4 Shimomura I, Funahashi T, Takahashi M, Maeda K, Kotani K, Nakamura T, Yamashita S, Miura M, Fukuda Y, Takemura K, Tokunaga K, Matsuzawa Y. Enhanced expression of PAI-1 in visceral fat: possible contributor to vascular disease in obesity. Nat Med 1996; 2: 800-803
• 5 Hotamisligil GS, Shargill NS, Spiegelman BM. Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science 1993; 259: 87-91
• 6 Kahn BB, Flier JS. Obesity and insulin resistance. J Clin Invest 2000; 106: 473-481
• 7 Maeda N, Shimomura I, Kishida K, Nishizawa H, Matsuda M, Nagaretani H, Furuyama N, Kondo H, Takahashi M, Arita Y, Komuro R, Ouchi N, Kihara S, Tochino Y, Okutomi K, Horie M, Takeda S, Aoyama T, Funahashi T, Matsuzawa Y. Diet-induced insulin resistance in mice lacking adiponectin/ACRP30. Nat Med 2002; 8: 731-737
• 8 Trayhurn P. Endocrine and signalling role of adipose tissue: new perspectives on fat. Acta Physiol Scand 2005; 184: 285-293
• 9 Yamauchi T, Kamon J, Waki H, Terauchi Y, Kubota N, Hara K, Mori Y, Ide T, Murakami K, Tsuboyama-Kasaoka N, Ezaki O, Akanuma Y, Gavrilova O, Vinson C, Reitman ML, Kagechika H, Shudo K, Yoda M, Nakano Y, Tobe K, Nagai R, Kimura S, Tomita M, Froguel P, Kadowaki T. The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nat Med 2001; 7: 941-946
• 10 Maeda K, Okubo K, Shimomura I, Funahashi T, Matsuzawa Y, Matsubara K. cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1 (AdiPose Most abundant Gene transcript 1). Biochem Biophys Res Commun 1996; 221: 286-289
• 11 Arita Y, Kihara S, Ouchi N, Takahashi M, Maeda K, Miyagawa J, Hotta K, Shimomura I, Nakamura T, Miyaoka K, Kuriyama H, Nishida M, Yamashita S, Okubo K, Matsubara K, Muraguchi M, Ohmoto Y, Funahashi T, Matsuzawa Y. Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity. Biochem Biophys Res Commun 1999; 257: 79-83
• 12 Hotta K, Funahashi T, Arita Y, Takahashi M, Matsuda M, Okamoto Y, Iwahashi H, Kuriyama H, Ouchi N, Maeda K, Nishida M, Kihara S, Sakai N, Nakajima T, Hasegawa K, Muraguchi M, Ohmoto Y, Nakamura T, Yamashita S, Hanafusa T, Matsuzawa Y. Plasma concentrations of a novel, adipose-specific protein, adiponectin, in type 2 diabetic patients. Arterioscler Thromb Vasc Biol 2000; 20: 1595-1599
• 13 Araki H, Nishihara T, Matsuda M, Fukuhara A, Kihara S, Funahashi T, Kataoka TR, Kamada Y, Kiyohara T, Tamura S, Hayashi N, Shimomura I. Adiponectin plays a protective role in caerulein-induced acute pancreatitis in mice fed a high-fat diet. Gut 2008; 57: 1431-1440
• 14 Ebina K, Oshima K, Matsuda M, Fukuhara A, Maeda K, Kihara S, Hashimoto J, Ochi T, Banda NK, Yoshikawa H, Shimomura I. Adenovirus-mediated gene transfer of adiponectin reduces the severity of collagen-induced arthritis in mice. Biochem Biophys Res Commun 2009; 378: 186-191
• 15 Kumada M, Kihara S, Ouchi N, Kobayashi H, Okamoto Y, Ohashi K, Maeda K, Nagaretani H, Kishida K, Maeda N, Nagasawa A, Funahashi T, Matsuzawa Y. Adiponectin specifically increased tissue inhibitor of metalloproteinase-1 through interleukin-10 expression in human macrophages. Circulation 2004; 109: 2046-2049
• 16 Okamoto Y, Kihara S, Ouchi N, Nishida M, Arita Y, Kumada M, Ohashi K, Sakai N, Shimomura I, Kobayashi H, Terasaka N, Inaba T, Funahashi T, Matsuzawa Y. Adiponectin reduces atherosclerosis in apolipoprotein E-deficient mice. Circulation 2002; 106: 2767-2770
• 17 Tsubakio-Yamamoto K, Matsuura F, Koseki M, Oku H, Sandoval JC, Inagaki M, Nakatani K, Nakaoka H, Kawase R, Yuasa-Kawase M, Masuda D, Ohama T, Maeda N, Nakagawa-Toyama Y, Ishigami M, Nishida M, Kihara S, Shimomura I, Yamashita S. Adiponectin prevents atherosclerosis by increasing cholesterol efflux from macrophages. Biochem Biophys Res Commun 2008; 375: 390-394
• 18 Yokota T, Oritani K, Takahashi I, Ishikawa J, Matsuyama A, Ouchi N, Kihara S, Funahashi T, Tenner AJ, Tomiyama Y, Matsuzawa Y. Adiponectin, a new member of the family of soluble defense collagens, negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages. Blood 2000; 96: 1723-1732
• 19 Richards AA, Stephens T, Charlton HK, Jones A, Macdonald GA, Prins JB, Whitehead JP. Adiponectin multimerization is dependent on conserved lysines in the collagenous domain: evidence for regulation of multimerization by alterations in posttranslational modifications. Mol Endocrinol 2006; 20: 1673-1687
• 20 Wang Y, Xu A, Knight C, Xu LY, Cooper GJ. Hydroxylation and glycosylation of the four conserved lysine residues in the collagenous domain of adiponectin. Potential role in the modulation of its insulin-sensitizing activity. J Biol Chem 2002; 277: 19521-19529
• 21 Frizzell N, Rajesh M, Jepson MJ, Nagai R, Carson JA, Thorpe SR, Baynes JW. Succination of thiol groups in adipose tissue proteins in diabetes: succination inhibits polymerization and secretion of adiponectin. J Biol Chem 2009; 284: 25772-25781
• 22 Sato C, Yasukawa Z, Honda N, Matsuda T, Kitajima K. Identification and adipocyte differentiation-dependent expression of the unique disialic acid residue in an adipose tissue-specific glycoprotein, adipo Q. J Biol Chem 2001; 276: 28849-28856
• 23 Richards AA, Colgrave ML, Zhang J, Webster J, Simpson F, Preston E, Wilks D, Hoehn KL, Stephenson M, Macdonald GA, Prins JB, Cooney GJ, Xu A, Whitehead JP. Sialic acid modification of adiponectin is not required for multimerization or secretion but determines half-life in circulation. Mol Endocrinol 2010; 24: 229-239
• 24 Wang Y, Lam KS, Chan L, Chan KW, Lam JB, Lam MC, Hoo RC, Mak WW, Cooper GJ, Xu A. Post-translational modifications of the four conserved lysine residues within the collagenous domain of adiponectin are required for the formation of its high molecular weight oligomeric complex. J Biol Chem 2006; 281: 16391-16400
• 25 Scherer PE, Williams S, Fogliano M, Baldini G, Lodish HF. A novel serum protein similar to C1q, produced exclusively in adipocytes. J Biol Chem 1995; 270: 26746-26749
• 26 Zielinska DF, Gnad F, Wisniewski JR, Mann M. Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints. Cell 2010; 141: 897-907
• 27 Maeda N, Takahashi M, Funahashi T, Kihara S, Nishizawa H, Kishida K, Nagaretani H, Matsuda M, Komuro R, Ouchi N, Kuriyama H, Hotta K, Nakamura T, Shimomura I, Matsuzawa Y. PPARgamma ligands increase expression and plasma concentrations of adiponectin, an adipose-derived protein. Diabetes 2001; 50: 2094-2099
• 28 Iwaki M, Matsuda M, Maeda N, Funahashi T, Matsuzawa Y, Makishima M, Shimomura I. Induction of adiponectin, a fat-derived antidiabetic and antiatherogenic factor, by nuclear receptors. Diabetes 2003; 52: 1655-1663
• 29 Morita S, Kojima T, Kitamura T. Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther 2000; 7: 1063-1066
• 30 Tarentino AL, Quinones G, Trumble A, Changchien LM, Duceman B, Maley F, Plummer Jr TH. Molecular cloning and amino acid sequence of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase from flavobacterium meningosepticum. J Biol Chem 1990; 265: 6961-6966