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Native adiponectin in serum binds to mammalian cells expressing T-cadherin, but not AdipoRs or calreticulin

  1. Shunbun Kita  Is a corresponding author
  2. Shiro Fukuda
  3. Norikazu Maeda
  4. Iichiro Shimomura
  1. Osaka University, Japan
Short Report
Cite this article as: eLife 2019;8:e48675 doi: 10.7554/eLife.48675
3 figures, 1 table, 1 data set and 1 additional file

Figures

Figure 1 with 2 supplements
Mouse serum adiponectin binds only to the hek293 cells expressing t-cadherin.

(A) Experimental outline. HEK293 cells were transfected with mammalian expression vectors coding mouse T-cadherin (mCdh13), Calreticulin (mCalr), or AdipoR1 (mAdipor1). (B) Native-page analysis of adiponectin preparations. Adiponectin concentrations were measured by ELISA and the equal amount (50 ng) of adiponectin was analyzed. (C) Absolute copy numbers of mRNA levels of mouse mCdh13, mCalr, mAdipor1, and mAdipor2 were quantified. (D) Binding of adiponectin to HEK293 cells expressing none (N), mock (M), T-cadherin (T), or AdipoR1 (R1) (E) Dose-response cell-based binding study. Mouse serum (20 μg adiponectin/mL) was diluted and applied to the cells expressing mock or T-cadherin (left). The bound adiponectin was evaluated by blot intensity (right). Cell lysate following binding was separated by SDS-page and native-page. Essentially same results were obtained from more than three independent experiments.

https://doi.org/10.7554/eLife.48675.002
Figure 1—figure supplement 1
Mouse serum adiponectin binding.

(A) Binding of mouse adiponectin in mouse serum to HEK293 cells. Multimer analysis of bound adiponectin by native page. Essentially same results were obtained from more than three independent experiments.

https://doi.org/10.7554/eLife.48675.003
Figure 1—figure supplement 2
Human serum adiponectin binds only to the hek293 cells expressing t-cadherin.

(A) Experimental outline. HEK293 cells were transfected with mammalian expression vectors coding human T-cadherin (hCDH13), Calreticulin (hCALR), or AdipoR1 (hADIPOR1). (B) Absolute copy numbers of mRNA levels of human T-cadherin (hCDH13), Calreticulin (hCALR), AdipoR1 (hADIPOR1), and AdipoR2 (hADIPOR2) were quantified. (C) Binding of human adiponectin in human serum to HEK293 cells. (D) Multimer analysis of bound adiponectin by native page. Essentially same results were obtained from more than three independent experiments.

https://doi.org/10.7554/eLife.48675.004
Cell surface expression of adiponectin receptors.

(A) Experimental outline of transient expression in HEK293 cells. (B) Surface protein biotinylation analysis. Cell surface biotinylated proteins trapped on Streptavidin beads were eluted and analyzed in SDS-page in two lanes; x1 and x5 concentrations. Note that PA-tag antibody NZ-1 react with human podoplanin (40 KDa) in addition to PA-tagged proteins. GAPDH; control cytosolic protein. (C) Binding of mouse adiponectin in mouse serum (MS) to HEK293 cells. (D) Experimental outline of stable CHO cells expressing adiponectin receptors. (E) stable expressions detected by NZ-1. (F) Binding of mouse adiponectin in mouse serum to CHO cells. Adiponectin-binding from western blots (left) was calculated and expressed in right graph. Data are mean ± SEM. n = 4 ***p<0.01 (unpaired t-test). (G) Binding of NZ-1 antibody to CHO cells. NZ-1-binding from western blots (left) was calculated and expressed in right graph. Data are mean ± SEM. n = 4 ***p<0.001 (unpaired t-test). (H) Confocal immunofluorescence micrographs of CHO cells stained with anti-PA tag antibody NZ-1 (green). Cell nuclei were counterstained with DAPI (blue). Scale bars = 250 nm.

https://doi.org/10.7554/eLife.48675.005
Knockdown of T-cadherin but not Adipors nor calreticulin affected native adiponectin binding to c2c12 myotubes.

(A) Experimental outline. (B) Absolute expression levels of T-cadherin (mCdh13), AdipoR1 (mAdipor1), and AdipoR2 (mAdipor2) in differentiated C2C12 myotubes. (C) Knockdown efficiencies of adiponectin receptors. Data are mean ± SEM. n = 3 ***p<0.001 (unpaired t-test). (D) Binding of native adiponectin in mouse serum to C2C12 cells. Bound adiponectin was shown in representative western blot with a-tubulin as internal control and T-cadherin. (E) Adiponectin-binding. Data are mean ± SEM. n = 4 ***p<0.001 (unpaired t-test). (F) Amount of T-cadherin protein expression. Data are mean ± SEM. n = 4 ***p<0.001 (unpaired t-test). (G) Correlation between bound adiponectin and T-cadherin protein expression. n = 16 a linear regression r2 = 0.9896.

https://doi.org/10.7554/eLife.48675.006

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or referenceIdentifiersAdditional
information
Antibodyanti-mouse adiponectinR and DAF1119goat polyclonal
WB (1:5000)
Antibodyanti-human adiponectinR and DAF1065goat polyclonal
WB (1:5000)
Antibodyanti-T-cadherinR and DAF3264goat polyclonal
WB (1:5000)
Antibodyanti-α-tubulinCell Signaling11H10rabbit polyclonal
WB (1:1000)
Antibodyanti-PA-tag (NZ-1)FUJIFILM012–25863rat monoclonal
WB (1:1000)
Commercial assay, kitCell Surface Biotinylation KitThermo Fisher (Pierce)89881
Biological sample (Mus musculus)SerumCLEA JapanC57BL6J jclFreshly isolated from C57BL6J mice, male
Biological sample (Homo sapiens)SerumFreshly isolated from healthy volunteers, male
Peptide, recombinant proteinFull-length mammalian recombinant mouse adiponectinBioVendorRD272023100
Peptide, recombinant proteinhigh-molecular weight purified mouse adiponectinFukuda et al., 2017
Sequence-based reagentmRplp0_FwGene DesignGGCCAATAAGGTGCCAGCT
Sequence-based reagentmRplp0_RvGene DesignTGATCAGCCCGAAGGAGAAG
Sequence-based reagentAdipor1_FwGene DesignAATGGGGCTCCTTCTGGTAAC
Sequence-based reagentAdipor1_RvGene DesignGGATGACTCTCCAACGTCCCT
Sequence-based reagentAdipor2_FwGene DesignGGAGTGTTCGTGGGCTTAGG
Sequence-based reagentAdipor2_RvGene DesignGCAGCTCCGGTGATATAGAGG
Sequence-based reagentmCdh13_FwGene DesignGCCCTCGTGAGCCTTCTTC
Sequence-based reagentmCdh13_RvGene DesignCACCCTGAGGTCCGTGATGT
Sequence-based reagentmCalr_FwGene DesignAAGATGCCCGATTTTACGCAC
Sequence-based reagentmCalr_RvGene DesignCCCACAGTCGATATTCTGCTC
Sequence-based reagenthRPLP0_FwGene DesignGGCGACCTGGAAGTCCAACT
Sequence-based reagenthRPLP0_RvGene DesignCCATCAGCACCACAGCCTTC
Sequence-based reagenthCDH13_FGene DesignAGTGTTCCATATCAATCAGCCAG
Sequence-based reagenthCDH13_RGene DesignCGAGACCTCATAGCGTAGCTT
Sequence-based reagenthADIPOR1_FGene DesignTCCTGCCAGTAACAGGGAAG
Sequence-based reagenthADIPOR1_RGene DesignGGTTGGCGATTACCCGTTTG
Sequence-based reagenthADIPOR2_FGene DesignCTGGATGGTACACGAAGAGGT
Sequence-based reagenthADIPOR2_RGene DesignTGGGCTTGTAAGAGAGGGGAC
Sequence-based reagenthCALR_FwGene DesignCTCTGTCGGCCAGTTTCGAG
Sequence-based reagenthCALR_RvGene DesignTGTATTCTGAGTCTCCGTGCAT
Cell line
(Homo sapiens)
HEK293 cellsATCCCRL-1573
RRID:CVCL_0045
DMEM+10%FBS
Cell line
(Cricetulus griseus)
CHO cellsATCCCCL-61
RRID:CVCL_0214
Ham's F12+10%FBS
Cell line
(Homo sapiens)
Plat-E cellsCosmobioRV-101
RRID:CVCL_B488
Ecotropic retrovirus packaging
DMEM+10%FBS
Cell line
(Mus musculus)
C2C12 cellsRIKEN cell bankRCB0987
RRID:CVCL_0188
C2C12 sleletal myoblast
DMEM+10%FBS
Recomninant DNA reagentmCdh13This paperMaterials and methods: plasmids
Recomninant DNA reagentmCalrThis paperMaterials and methods: plasmids
Recomninant DNA reagentmAdipor1This paperMaterials and methods: plasmids
Recomninant DNA reagenthCDH13This paperMaterials and methods: plasmids
Recomninant DNA reagenthCALRThis paperMaterials and methods: plasmids
Recomninant DNA reagenthADIPOR1This paperMaterials and methods: plasmids
Recomninant DNA reagentmCat1This paperMaterials and methods: plasmids

Data availability

All data were deposited in Dryad at https://doi.org/10.5061/dryad.82557c0.

The following data sets were generated
  1. 1
    Dryad Digital Repository
    1. S Kita
    2. S Fukuda
    3. N Maeda
    4. I Shimomura
    (2019)
    Data from: Native adiponectin in serum binds to cells expressing T-cadherin, but not AdipoRs or calreticulin.
    https://doi.org/10.5061/dryad.82557c0

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