RhoA vesicle trafficking–mediated transglutaminase 2 membrane translocation promotes IgA1 mesangial deposition in IgA nephropathy
Zhong Zhong, Zhijian Li, Yanjie Li, Lanping Jiang, Qingyu Kong, Wei Chen, Shaozhen Feng
Zhong Zhong, Zhijian Li, Yanjie Li, Lanping Jiang, Qingyu Kong, Wei Chen, Shaozhen Feng
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Research Article Nephrology

RhoA vesicle trafficking–mediated transglutaminase 2 membrane translocation promotes IgA1 mesangial deposition in IgA nephropathy

Abstract

Transglutaminase 2 (TGase2) has been shown to contribute to the mesangial IgA1 deposition in a humanized mouse model of IgA nephropathy (IgAN), but the mechanism is not fully understood. In this study, we found that inhibition of TGase2 activity could dramatically decrease the amount of polymeric IgA1 (pIgA1) isolated from patients with IgAN that interacts with human mesangial cells (HMC). TGase2 was expressed both in the cytosol and on the membrane of HMC. Upon treatment with pIgA1, there were more TGase2 recruited to the membrane. Using a cell model of mesangial deposition of pIgA1, we identified 253 potential TGase2-associated proteins in the cytosolic fraction and observed a higher concentration of cellular vesicles and increased expression of Ras homolog family member A (RhoA) in HMC after pIgA1 stimulation. Both the amount of pIgA1 deposited on HMC and membrane TGase2 level were decreased by inhibition of the vesicle trafficking pathway. Mechanistically, TGase2 was found to be coprecipitated with RhoA in the cellular vesicles. Membrane TGase2 expression was greatly increased by overexpression of RhoA, while it was reduced by knockdown of RhoA. Our in vitro approach demonstrated that TGase2 was transported from the cytosol to the membrane through a RhoA-mediated vesicle-trafficking pathway that can facilitate pIgA1 interaction with mesangium in IgAN.

Authors

Zhong Zhong, Zhijian Li, Yanjie Li, Lanping Jiang, Qingyu Kong, Wei Chen, Shaozhen Feng

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Figure 3

TGase2 interactome in the cytosol of human mesangial cells treated with pIgA1.

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TGase2 interactome in the cytosol of human mesangial cells treated with ...
(A) HMC were treated with or without pIgA1 (10 μg/mL) for 48 hours. Cytosolic (C) and membrane (M) fractions were separated and validated for enrichment of β-tubulin and Na+/K+ ATPase by Western blot. TGase2-associated proteins in the cytosol were immunoprecipitated using anti-TGase2 antibody (n = 2). The isotype control antibody cross-link to protein A/G magnetic beads was used as a control (n = 2). (B) The TGase2-associated proteins after IP were analyzed via MS. The number of proteins was identified as specifically associated with TGase2 after exclusion of nuclear, mitochondrial, and ribosomal proteins. (C and D) Functional distribution of TGase2-associated proteins from cytosolic fractions without (C) or with pIgA1 deposition (D). Proteins were clustered based on their functions in Homo sapiens, which were determined by protein identification search in the UniProtKB database (https://www.uniprot.org/). Column charts display the distribution of different functions of TGase2-associated proteins with numbers and the percentage of proteins falling in the various functional categories. (E) The protein-interaction network was built from TGase2-associated proteins from cytosolic fraction without pIgA1 treatment (208 proteins) or with pIgA1 treatment (253 proteins). The protein-interaction network was mapped against the Homo sapiens reference database using the STRING tool. Candidates were selected using both known and predicted protein interactions with a threshold confidence level of 0.5. Networks were imported into Cytoscape software, and the direct partners of TGase2 were shown. The size and color shade of circles are proportional to the number of protein interactions, while the thickness and color shade of the lines are proportional to the confidence of the interactions. Here, only 1 interactome of cytosolic TGase2 is shown because it remained the same in HMC without or with pIgA1 treatment.