Figure 1.
Three boxes represent 3 exons of ADIPOQ. The coding regions are indicated as filled boxes. The positions of 18 variations identified in this study are located. The variations in upper panel are novel and those in the lower panel were reported. The variations which were indentified in the patients with T2D but not in non-diabetic individuals are indicated with asterisks.
Figure 2.
Adiponectin protein structure and amino acid alignment.
A: Adiponectin protein is divided in to signal sequence, variable region, collageneous domain and globular domain. The upper panel indicates variants identified in this study. Those identified only in T2D patients are indicated with asterisks. The lower panel represents the variants that have been previously reported. B and C: Multiple alignments of adiponectin protein from different species using BioEdit Sequence Alignment Editor. The positions of amino acid substitutions are located. B: Variations located in collageneous domain. C: Variations located in globular domain.
Table 1.
Amino acid substitutions in collagenous and globular domains of adiponectins and their effects.
Figure 3.
Three dimensional structure of modeled human adiponectin globular domain and positions of amino acid variations.
Human adiponectin was constructed by homology modeling using mouse adiponectin [16] as a template. Each monomer of the adiponectin trimer structure is shown in different color. The positions of amino acids being substituted are indicated with arrows.
Figure 4.
Local structural alterations of amino acid substitutions (R112C, R131H, and I164T) in modeled human adiponectin globular domain.
Wild-type and variant adiponectin trimer are superimposed. Three monomers of adiponectin wild-type are indicated in green, cyan, and magenta and labeled with the color letters corresponding to their chain. The variant residues are showed with the gray sticks and labeled with the black letters. The side chains of amino acids within hydrophobic core are shown in line structure with surrounding dots. The O, N, and S atoms are shown with red, blue, and yellow, respectively. A–C: The local structural alterations for R112C, R131H, and I164T variants, respectively.
Figure 5.
Expression, secretion, and oligomerization of adiponectin variants.
Cell lysate (A) and culturing media (B) of HEK293T cells transiently expressed adiponectin wild-type and variants under reducing and heat-denaturing condition. Cell lysate (C) and culturing media (D) of HEK293T cells transiently expressed adiponectin wild-type and variants under non-reducing and non heat-denaturing condition. The total protein of each sample was equally loaded. Results shown are typical of three independent experiments.
Figure 6.
Co-expression of wild-type and variant adiponectins.
Adiponectin variants tagged with FLAG sequence were co-expressed with wild-type tagged with c-Myc sequence. A–D: Immunoblotting of wild-type protein by using anti-Myc antibody. E–H: Immunoblotting of variant proteins by using anti-FLAG antibody. A, B, E and F: Cell lysates (A and E) and culturing media (B and F) of adiponectin variants co-expressed with wild-type under reducing and heat-denaturing condition. C, D, G, and H: Cell lysate (C and G) and culturing media (D and H) of adiponectin variants co-expressed with wild-type under non-reducing and non heat-denaturing condition. The total protein of each sample was equally loaded. Results shown are typical of three independent experiments.
Figure 7.
Sub-cellular localizations of wild-type, R112C and R131H adiponectins.
The wild-type, R112C and R131H adiponectins linked with FLAG epitope transiently expressed in HEK293T cells were detected with mouse anti-FLAG antibody, and with either rabbit anti-Calnexcin (A), rabbit anti-Giantin (B), rabbit anti-TGN46 (C) or rabbit anti-EEA1 (D) antibody, which are antibodies specific to ER, Golgi, TGN and early endosome markers, respectively. Goat anti-mouse IgG-conjugated with Alexa Fluor® 488 (green) and donkey anti-rabbit IgG-conjugated with Cy3 (red) were secondary antibodies for staining adiponectin and the organelle markers, respectively. The nucleus was stained with Hoechst 33342 fluorescent dye (blue).
Figure 8.
Investigation of lysosomal degradation of wild-type, R112C and R131H adiponectins.
At 24 hours after transfection, HEK293T cells were treated with proteasomal/lysosomal or lysosomal inhibitors for 8 hours. The concentrations of reagents are as followed: MG132 10 mM, BFA 5 µg/mL, NH4Cl 25 mM and chloroquine (CHQ) 100 µM. Equal amount of protein from each sample was immunoblotted with anti-FLAG. A–C: Intracellular adiponectin levels of wild-type (A), R112C (B) and R131H (C) after treating with degradation inhibitors. The bar graphs represented relative levels of adiponectin (adiponectin/actin) represented in folds of samples treated with DMSO. Data are mean ± SD of 3 independent experiments. * P value<0.05, analyzed by Mann-Whitney U test. D–F: Extracellular adiponectin levels of wild-type (D), R112C (E) and R131H (F) after treating with degradation inhibitors. Results shown are typical of 3 independent experiments.
Table 2.
Clinical and laboratory characteristics of studies subjects.