Adiponectin multimerization is dependent on conserved lysines in the collagenous domain: evidence for regulation of multimerization by alterations in posttranslational modifications.
Adiponectin is a secreted, multimeric protein with insulin-sensitizing, antiatherogenic, and antiinflammatory properties. Serum adiponectin consists of trimer, hexamer, and larger high-molecular-weight (HMW) multimers, and these HMW multimers appear to be the more bioactive forms. Multimer composition of adiponectin appears to be regulated; however, the molecular mechanisms involved are unknown. We hypothesize that regulation of adiponectin multimerization and secretion occurs via changes in posttranslational modifications (PTMs). Although a structural role for intertrimer disulfide bonds in the formation of hexamers and HMW multimers is established, the role of other PTMs is unknown. PTMs identified in murine and bovine adiponectin include hydroxylation of multiple conserved proline and lysine residues and glycosylation of hydroxylysines. By mass spectrometry, we confirmed the presence of these PTMs in human adiponectin and identified three additional hydroxylations on Pro71, Pro76, and Pro95. We also investigated the role of the five modified lysines in multimer formation and secretion of recombinant human adiponectin expressed in mammalian cell lines. Mutation of modified lysines in the collagenous domain prevented formation of HMW multimers, whereas a pharmacological inhibitor of prolyl- and lysyl-hydroxylases, 2,2'-dipyridyl, inhibited formation of hexamers and HMW multimers. Bacterially expressed human adiponectin displayed a complete lack of differentially modified isoforms and failed to form bona fide trimers and larger multimers. Finally, glucose-induced increases in HMW multimer production from human adipose explants correlated with changes in the two-dimensional electrophoresis profile of adiponectin isoforms. Collectively, these data suggest that adiponectin multimer composition is affected by changes in PTM in response to physiological factors.
Richards
Ayanthi A
AA
Centre for Diabetes and Endocrine Research, University of Queensland, Princess Alexandra Hospital, Brisbane, Queensland 4102, Australia.
Stephens
Tim
T
Charlton
Hayley K
HK
Jones
Alun
A
Macdonald
Graeme A
GA
Prins
Johannes B
JB
Whitehead
Jonathan P
JP
D015082:2,2'-Dipyridyl / Q000494:pharmacology; D052242:Adiponectin / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D003094:Collagen / Q000378:metabolism; D017124:Conserved Sequence; D006224:Cricetinae; D019281:Dimerization; D015870:Gene Expression; D005947:Glucose / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D006900:Hydroxylation / Q000187:drug effects; D008239:Lysine / Q000378:metabolism; D013058:Mass Spectrometry; D008954:Models, Biological; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000378:metabolism*; D011392:Proline / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry
C499918:ADIPOQ protein, human; D052242:Adiponectin; D046912:Multiprotein Complexes; D020033:Protein Isoforms; D011994:Recombinant Proteins; D015082:2,2'-Dipyridyl; D003094:Collagen; D011392:Proline; D005947:Glucose; D008239:Lysine