Disulfide-Dependent Self-Assembly of Adiponectin Octadecamers from Trimers and Presence of Stable Octadecameric Adiponectin Lacking Disulfide Bonds <italic>in Vitro</italic><xref rid="fs1"></xref>

Adiponectin is a circulating insulin-sensitizing hormone that homooligomerizes into trimers, hexamers, and higher molecular weight (HMW) species. Low levels of circulating HMW adiponectin appear to increase the risk for insulin resistance. Currently, assembly of adiponectin oligomers and, consequently, mechanisms responsible for decreased HMWadiponectin in insulin resistance are not well understood. In the work reported here, we analyzed the reassembly of the most abundant HMW adiponectin species, the octadecamer, following its collapse to smaller oligomers in vitro. Purified bovine serum adiponectin octadecamer was treated with reducing agents at pH 5 to obtain trimers. These reduced trimers partially and spontaneously reassembled into octadecamers upon oxidative formation of disulfide bonds. Disulfide bonds appear to occupy a greater role in the process of oligomerization than in the structural stabilization of mature octadecamer. Stable octadecamers lacking virtually all disulfide bonds could be observed in abundance using native gel electrophoresis, dynamic light scattering, and collision-induced dissociation nanoelectrospray ionization mass spectrometry. These findings indicate that while disulfide bonds help to maintain the mature octadecameric adiponectin structure, their more important function is to stabilize intermediates during the assembly of octadecamer. Adiponectin oligomerization must proceed through intermediates that are at least partially reduced. Accordingly, fully oxidized adiponectin hexamers failed to reassemble into octadecamers at a rate comparable to that of reduced trimers. As the findings from the present study are based on in vitro experiments, their in vivo relevance remains unclear. Nevertheless, they describe a redox environment-dependent model of adiponectin oligomerization that can be tested using cell-based approaches. Adiponectin is a peptide hormone secreted from adipocytes with insulin-sensitizing and vascular and cardiac protective functions (1-4). Expression of the adiponectin gene and circulating adiponectin levels are subjected to regulation by a variety of hormones, cytokines, and transcription factors (1). Low levels of circulating adiponectin are associated with insulin resistance, coronary artery disease, and obesity, especially visceral obesity, in humans and animals (5, 6). Mice lacking adiponectin display increased proliferation of vascular smooth muscle cells (7) and hypertrophic cardiomyopathy (8) and are predisposed to develop insulin resistance (9-11). Similarly, hypoadiponectinemia appears to be a risk factor for developing insulin resistance or type 2 diabetes in populations from diverse ethnic backgrounds (6). The primary sequence of adiponectin consists of three domains: an N-terminal region, a collagenous domain, and a C-terminal globular domain (12, 13). Through hydrophobic interactions, three individual globular domains form a globular head that is evidenced by X-ray crystallography (14). The three collagenous domains extending from the globular head presumably adopt a triple-helical structure that appears on electron micrographs as the stick on the lollipop-shaped adiponectin trimer (15, 16). The N-terminal region contains a conserved cysteine at position 22 of mature mouse adiponectin (position 39 including signal peptide) that is required for oligomerization of adiponectin trimers into hexamers and higher molecular weight (HMW) oligomers (15-17). Using different assay methods, different groups have alternatively referred to trimers as low molecular weight (LMW) adiponectin (17) and hexamers as either medium molecular weight (MMW) (17, 18) or LMW (15, 19) adiponectin. Trimers, hexamers, and the largest HMW species, an octadecamer (20), are the three major adiponectin oligomers present in mouse or human serum (15, 17, 21). Although a consensus is yet to be reached, accumulating evidence indicates that levels of circulating HMW adiponectin correlate more closely with insulin action than total This work was supported by a Junior Faculty Award from the American Diabetes Association (1-08-JF-54) and by a grant from the Arizona Biomedical Research Commission to T.-S.T.We also acknowledge financial support from the NSF (DBI Grant CHE 024447) for development of the QToF instrument and the NIH (R01 GM-051387) for support of experiments on fragmentation. C.M.J. is the recipient of a Pfizer Graduate Research Fellowship in Analytical Chemistry. MALDI-TOF mass spectrometric data were acquired by the Arizona Proteomics Consortium with support from the NIEHS (ES06694) to the University of Arizona Southwest Environmental Health Sciences Center, from the NIH/NCI (CA023074) to the Arizona Cancer Center, and from the BIO5 Institute of the University of Arizona. *To whom correspondence should be addressed. Tel: (520) 626-9755. Fax: (520) 626-3644. E-mail: [email protected]. Abbreviations: IAA, iodoacetamide; NEM, N-ethylmaleimide; AMS, 4-acetamido-40-maleimidylstilbene-2,20-disulfonic acid; DTT, dithiothreitol; βME, β-mercaptoethanol; EDTA, ethylenediaminetetraacetic acid; SDS, sodium dodecyl sulfiate; PAGE, polyacrylamide gel electrophoresis; MS, mass spectrometry; CID, collision-induced dissociation; HMW, higher molecular weight; PBS, phosphate-buffered saline; kDa, kilodaltons; ER, endoplasmic reticulum. 12346 Biochemistry, Vol. 48, No. 51, 2009 Briggs et al. adiponectin (22-26). The cause for the selective decrease of HMW adiponectin in insulin resistance is currently not understood. It is critical to study the biogenesis of adiponectin oligomers because defining the assembly pathways of different oligomers may help us understand the cause of the reduced circulating levels of HMW adiponectin in insulin resistance. In addition, numerous studies have demonstrated discrepancies between changes in adipose tissue adiponectin mRNA levels and circulating adiponectin concentrations (27-29). This suggests that factors other than adiponectin gene expression strongly influence circulating adiponectin levels (30-34). The ability of the endoplasmic reticulum (ER) to undergo oxidative protein folding and properly assemble adiponectin oligomers likely represents one such factor. Assembly of adiponectin oligomers is poorly understood. The C22 residues in hexameric and HMW adiponectin are fully oxidized to disulfides (16). In addition, replacement of the C22 residue near the N-terminus of adiponectin with alanine or serine precluded formation of hexamers or HMW species (15-17), indicating the importance of disulfide bonds in adiponectin oligomerization beyond trimers. However, while HMW adiponectin is extremely stable under conditions of high salt or pH, it is readily collapsed to hexamers upon modest reduction in pH to 4 or 5 (15, 17). This indicates strong intermolecular forces other than disulfide bonds are needed to maintain the structure of HMW adiponectin. An ER chaperone, ERp44, has been shown to formmixed disulfides with adiponectin via the cysteine residue near the N-terminus (35). Downregulation of ERp44 in cultured adipocytes led to increased secretion of trimeric adiponectin and decreased secretion of HMW adiponectin (35). Another recently discovered ER chaperone, DsbA-L, has also been shown to promote formation of HMW adiponectin (36). However, the molecular mechanisms by which these chaperones promote HMW adiponectin formation are unclear because a basic understanding of the adiponectin oligomer assembly pathway remains lacking. Studying adiponectin oligomerization in vivo entails labeling nascent adiponectin followed by isolation of labeled adiponectin in intact oligomeric complexes. Due to the technical difficulties surrounding these types of studies, we have developed an in vitro assay to analyze adiponectin oligomerization using purified bovine serum adiponectin. We show that octadecameric adiponectin could be assembled spontaneously from reduced trimers, but not from fully oxidized hexamers, indicating formation of mature hexamers is a process that is distinct from that of HMW adiponectin. We also identified a non-disulfide-bonded octadecameric complex whose oligomerization state was confirmed by multiple analytical methods. This finding underscores the importance of disulfide bond formation in adiponectin assembly because it is not critical for maintaining the octadecameric structure. Indeed, disulfide bond formation accompanied the appearance of octadecameric adiponectin, and alkylation of cysteines blocked the assembly of octadecameric adiponectin. Taken together, these findings suggest that free sulfhydryls must be available on adiponectin intermediates in order for disulfide bonds to form between distinct trimer subunits during the oligomerization process. The present study provides a framework for defining the assembly process of adiponectin oligomers, which may assist in understanding the decreased levels of HMW adiponectin observed in insulinresistant states. EXPERIMENTAL PROCEDURES Purification of Adiponectin Octadecamers. Adiponectin octadecamers were purified to homogeneity from fetal bovine serum (Atlanta Biologicals, Atlanta, GA) or calf serum (Invitrogen, Carlsbad, CA) as described previously (37, 38) with one additional chromatography step. The eluate from the zinc chelation column was added to reactive green 19 resin (Sigma, St. Louis, MO) equilibrated with PBS, pH 7.6. This mixture was allowed to rock overnight at 4 C, and the supernatant was recovered and applied to the next chromatography step. The final preparations contained predominantly octadecamers with small amounts of hexamers. The oligomerization state of purified adiponectin octadecamer was confirmed by established gel filtration chromatography and equilibrium sedimentation techniques (20, 21). Native and Denaturing PAGE Analysis of Adiponectin Oligomers. To analyze oligomerization states of adiponectin under native conditions, samples were diluted with a concentrated native loading buffer to a final composition of 31.25 mM Tris, pH 6.8, 12% glycerol, and 0.05% Orange G. Adiponectin oligomers were fractionated in 7% native Tris-acetate gels that were either purchased (Invitrogen, Carlsbad, CA) or prepared from 30% acrylamide stock solution (37.5:1 acrylamide:bisacrylamide; Bio-Rad Laboratories, Hercules, CA) buffered with 375 mM Tris base titrated to pH 8.5 with glacial acetic acid. The composition of the native running buffer was 25 mM Tris base and 192 mM glycine at pH 8.3. Gels were run at 18 V/cm for 2 h. The oxidation states of the cysteine residue near the N-terminus of adiponectin were determined by nonreducing denaturing PAGE. Samples were denatured by heating at 85 C for 10-20 min in 246 mM Tris, pH 8.5, 10% glycerol, 0.51 mM EDTA, 0.2 mM Serva Blue R, 0.175 mM Phenol Red, and 3% SDS. Monomeric (reduced) and dimeric (oxidized) adiponectin molecules were separated by discontinuous (11% separating, 5% stacking) SDS-PAGE (39) or precast 10%Bis-Tris gels in either MOPSor MES-based SDS running buffer (Invitrogen, Carlsbad, CA). Gels were stained with Krypton IR (Pierce, Rockford, IL) or Coomassie and visualized using the LI-COR Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE). Densitometry was performed using the Odyssey software to quantify the intensity of the bands corresponding to adiponectin