Detailed Characterization of Structure-Activity Relationships in the Human Adiponectin Molecule

Abstract

Adiponectin is an abundant circulating adipose tissue-derived hormone, or adipokine, that elicits distinct biological functions through its different oligomeric forms. Posttranslational modifications within its non-globular region have been demonstrated to be crucial for its formation of multimers (multimerization), secretion, stability, and function. Therefore, information concerning these structural modifications and their functional implications is expected to lead to important new insights concerning its structure and function, and possible roles as a candidate molecule for the experimental treatment of adiponectin-deficiency states. In this thesis, expression constructs for human adiponectin were stably transfected into CHO-S cells, and resulting monoclonal lines selected to optimize production of recombinant protein in chemically-defined media. Purified recombinant-human adiponectin was present in three main oligomeric forms, corresponding to trimeric, hexameric and high-molecular-weight (HMW) isoforms or complexes. Analysis by electron microscopy revealed that recombinant-human adiponectin formed bouquetlike HMW structures, consistent with its physiological assembly. Functional assays in skeletal muscle showed the recombinant adiponectin to be bioactive, and therefore appropriate for more detailed analysis of structure-activity relationships. Several sialyl- and O-linked glycosyl-modifications were identified in recombinant human adiponectin by an approach based on two-dimensional gel electrophoresis (2- DGE) combined with targeted endo-proteolytic digestion and liquid-chromatographymass spectrometry (LC-MS). Three sets of O-linked carbohydrate chains (HexNAc-Hex) and up to five sialic acid (Sia) residues were thus identified in enzyme-digested peptides derived from the NH2-terminal region: these were similar to the O-linked HexNAc-Hex-Sia chains that are typically present in mucin-like glycoproteins. These Olinked modification sites were localized to three adjacent NH2-terminal Thr residues (designated as Thr20, Thr21 and Thr22 when numbered as for human pro-adiponectin), which correspond to residues two to four of the mature protein. Hereinafter, the primary structure of adiponectin has been numbered as for pro-adiponectin to make this work consistent with the majority of publications in the field. Mutation of any one of these three threonine residues by replacement with an alanine residue by sitedirected mutagenesis, or alternatively replacement of all three threonine residues thus, demonstrated the loss of pI isoforms as shown by 2DGE: this effect was greatest when all three residues were replaced in the triple-mutant form. Functional analysis clearly showed that these structural modifications are required for full activity. The structural pattern of the threonine O-glycosylation in the NH2-terminal region was further characterized by quantitative protein-level analysis of the distinctive glycosylation patterns of the peptide adiponectin-(19-39) isolated from different sitedirected mutant forms. These studies provide robust evidence for a dominant role of the third residue, Thr22, in determining the degree and extent of NH2-terminal glycosylation of all three of these residues. The structural role of NH2-terminal glycosylation was further investigated in wild-type (WT) and triple-mutant adiponectin (Thr20-22Ala) in vitro by quantitative comparisons between the different oligomeric forms. Quantitative analysis of WT-adiponectin showed that differences in the NH2-terminal glycosylation pattern did not cause significant differences between the different multimeric species, consistent with the view that these threonine modifications do not play direct roles in multimerization or secretion of adiponectin. However, analysis of the triple mutant showed that this modification caused increased susceptibility to chemical reduction or heat-denaturation, resulting in loss of stability and truncation of the amino-terminal residues in most triple-mutant molecules. Thus the NH2-terminal glycosylation plays a major role in protecting the integrity of the NH2-terminal region of the hormone by maintaining its structural integrity. Interestingly, although structural differences were not apparent between the wild-type (WT) and triple mutant proteins by electron microscopy, the AMPK activation assay in C2C12 skeletal myotubes showed that triple-mutant adiponectin was not bioactive compared with the WT-protein. Therefore, the NH2- terminal glycosylation pattern plays a critical role in this key function of adiponectin. In WT-adiponectin, quantitative analysis of residues within the collagenous domain of monomers isolated from individual multimeric states showed similar patterns of modification of hydroxylation of lysyl and prolyl residues. WT-adiponectin trimers exhibit significantly higher levels of lysyl and prolyl modification on residues furthest removed from the globular domain. By contrast, the reverse pattern is present in monomers isolated from HMW complexes: that is, higher levels of modification are present on residues closest to the globular domain. Furthermore, the pattern in hexamers is intermediate between those of the trimeric and HMW forms. These findings are consistent with the existence of different patterns of hydroxylation of lysyl and prolyl residues that could stabilize the different oligomeric forms, consistent with their distinct structural and functional properties. During these studies of the posttranslational modification of lysyl residues (by glycosylation and hydroxylation), a previously-unknown modification in human adiponectin was observed and mapped (by manual interpretation of LC-MS/MS data), to a conserved lysyl residue in the variable region. In addition, the molecular mechanism for 2D-spot laddering within one pI isoform was investigated: the distinct subspots in the vertical-laddering pattern were found to reflect the successive step-wise addition of two-hexose moieties to the four conserved lysyl residues in the collagenous domain. The data presented in this thesis represent a comprehensive analysis of the detailed structural variation caused by numerous posttranslational modifications in recombinant human adiponectin, and provide a strong platform for further analysis of the relationship between function and different post-translational modifications (PTMs) of the adiponectin molecule. Adiponectin is thus revealed to comprise a highlyheterogeneous group of related molecules that are distinguished from each other by their patterns of posttranslational modification, and by linked functional properties. These studies are expected to provide significant insights into the functional roles played by defects in adiponectin synthesis that occur in adiponectin deficiency states such as type-2 diabetes and the metabolic syndrome. They also provide a wellgrounded approach to the structure-activity relationships in the different isoforms of human adiponectin.