Disulfide-dependent self-assembly of adiponectin octadecamers from trimers and presence of stable octadecameric adiponectin lacking disulfide bonds in vitro

Abstract

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 HMW adiponectin 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.

Figure 1. (A) Native PAGE analysis of adiponectin oligomeric state following reduction and collapse of octadecamers and subsequent removal of reductant by dialysis

Purified bovine serum octadecamers were collapsed to trimers in 100 mM DTT or 200 mM βME and subsequently dialyzed against PBS over a 3 hr period to remove DTT or βME. Following fractionation in PAGE, the proteins were detected by fluorescence from an infrared stain as described in Experimental Procedures. Two samples, one in DTT and the other in βME, were not dialyzed against PBS to assess if re-oligomerization to octadecamers depended upon removal of reducing agents. (B) Densitometric analysis of adiponectin octadecamer (top panel), hexamer (middle panel), and trimer (bottom panel) levels during removal of reductant by dialysis following collapse of purified octadecamers. The integrated intensity of each band corresponding to a specific oligomer at a particular time point was normalized to the sum of the integrated intensities of all three major oligomers at the same time point to obtain a value for the percentage of total adiponectin. Average percentage values and standard error of the mean from four and five independent experiments whose initial reductant was, respectively, DTT (open circle) and βME (closed square) were plotted. (C) Oligomerization state of adiponectin following titration with different concentrations of DTT. Purified octadecamers were treated with 1, 10, and 100 mM DTT and resulting products’ oligomerization states were analyzed using native PAGE as described above and in Experimental Procedures. (D) Effect of pH and βME concentration on adiponectin oligomerization state. Adiponectin octadecamers were treated with 2, 20, 50 or 200 mM βME at pH 5 or 7 as described in Experimental Procedures. Native PAGE was used to analyze oligomerization states following the treatments.

Figure 2. Effect of oxidizing agent hydrogen peroxide on (A) re-oligomerization of octadecameric adiponectin and (B) oxidation of cysteines near N-terminus to form disulfide bonds

Purified adiponectin octadecamers were first reduced by DTT followed by lowering the pH to 5 as described in Experimental Procedures to produce trimers. The samples were subsequently dialyzed against PBS at pH 7.6 to remove DTT in the absence or presence of 5 mM hydrogen peroxide over a period for 3 hrs as indicated. Samples were removed at different time points during the dialysis and fractionated in native gels to monitor oligomerization states and in non-reducing denaturing gels to distinguish the oxidized (dimer) and reduced (monomer) forms of adiponectin. Oxidation states were fixed by NEM treatment.

Figure 3. Effect of sulfhydryl alkylating agent iodoacetamide (IAA) on (A) re-oligomerization and (B) oxidation of adiponectin

Purified adiponectin octadecamers were first reduced by βME followed by lowering of pH to 5 as described in Experimental Procedures to produce trimers. Samples were neutralized in the absence or presence of IAA as indicated, and dialyzed against PBS containing 25 mM βME to quench IAA and remove hydroiodic acid, a product of the alkylation reaction. Samples were then dialyzed against PBS to remove βME and allow re-oligomerization to occur. Native PAGE analysis was performed to distinguish between the different oligomeric states of adiponectin and assess the extent to which oligomerization was inhibited by lack of disulfide formation following alkylation of reduced cysteines. Oxidation states were fixed by NEM treatment and determined using non-reducing SDS-PAGE. (C–E) Densitometric analysis of (C) octadecameric, (D) hexameric, and (E) trimeric adiponectin during removal of βME following alkylation of cysteines by IAA. Levels of each oligomer as percentages of total adiponectin were calculated as described in legend to Fig. 1B. Average percentage values and standard error of the mean from three independent experiments in which collapsed octadecamers were treated with (open circle) or without (closed square) IAA were plotted. (F) Densitometric analysis of disulfide bond formation in adiponectin complexes. The integrated intensities of protein bands corresponding to adiponectin dimers in non-reducing denaturing gels were normalized to the integrated intensities of bands corresponding to monomers in order to obtain a dimer to monomer ratio. Average ratios and standard error of the mean from three independent experiments in which collapsed octadecamers were treated with (open circle) or without (closed square) IAA were plotted.

Figure 4. Effect of low concentrations of DTT on (A) oligomerization and (B) oxidation states of adiponectin octadecamers using gel electrophoresis and confirmation of oligomerization state in solution by dynamic light scattering under (C) oxidized and (D) reduced conditions

Purified adiponectin octadecamers in PBS were treated with or without 4 mM DTT at 4°C overnight. Oligomerization state was assessed using native and oxidation state using non-reducing denaturing PAGE. AMS was used to quench DTT and alkylate reduced cysteines to prevent their oxidation during sample denaturation. Samples identically treated as those loaded onto native gels were analyzed using dynamic light scattering as described in Experimental Procedures.

Figure 5. (A, C) Native and (B, D) collision-induced dissociation (CID) mass spectra of adiponectin octadecamers in the (A, B) absence or (C, D) presence of 1 mM DTT

Purified bovine serum octadecamers in 200 mM ammonium acetate at pH 7 were treated with or without 1 mM DTT at 4°C overnight. Native and reduced adiponectin samples were ionized via nano-electrospray and their mass-to-charge ratios determined in a quadrupole-time-of-flight mass spectrometer as described in Experimental Procedures. To assess the oxidation states of octadecameric adiponectin detected in the mass spectrometer, a single charged octadecamer species was selected by the quadrupole mass analyzer and fragmented by collision with argon gas. The mass-to-charge ratios of the fragments were determined in TOF and the charge state distributions were calculated using the MassLynx software system. The peaks corresponding to the monomers in the CID spectrum in panel D contain a predominant species with a molecular mass of 26206.7. As dimers consist of monomers with different posttranslational modifications, the peaks corresponding to the dimers in the CID spectrum in panel B are broader than those in panel D. The median molecular mass calculated from the range of dimer peaks is 52412.3.

Figure 6. (A) Comparison of re-oligomerization rates between oxidized hexamers and reduced trimers using native PAGE

Samples of adiponectin octadecamers were converted to mostly trimers by βME treatment at pH 5 while a decrease of pH to 5 alone resulted in mostly hexamers. Samples were dialyzed against PBS at pH 7.6 as described in Experimental Procedures to allow re-oligomerization to occur over a 1 hr period. (B) Native and (C) non-reducing denaturing PAGE analysis of the low pH-derived hexamers’ potential to re-oligomerize to octadecamers. Samples of hexamers generated originally by glycine treatment were dialyzed against PBS for 2 hrs as described in Experimental Procedures. After 2 hrs of dialysis, the samples were treated with βME to convert oxidized hexamers to reduced trimers, and the abilities of these trimers to re-oligomerize to octadecamers and to re-form disulfide bonds over a period of 2 hrs were monitored in native and non-reducing denaturing gels.