Generation of Chicken Monoclonal Antibodies Against thea1,a2, anda3 Subunit Isoforms of Vacuolar-type Proton ATPase

Sun‐Wada, Ge‐Hong; Tabata, Hiroyuki; Kuhara, Motoki; Kitahara, Iori; Takashima, Yoshiko; Wada, Yoh

doi:10.1089/hyb.2010.0087

Cited by 13 publications

(16 citation statements)

References 29 publications

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“…In order to assess the localization of a3, a chicken monoclonal antibody specific for the mouse a3 was employed. This antibody was previously shown to be specific for the a3 isoform of the mouse V-ATPase and has been used previously to assess a3 expression in mouse melanoma cells [32, 36]. The mouse and human subunit a isoform sequences exhibit a high degree of homology (Supplementary Figure S1A).…”

Section: Resultsmentioning

confidence: 99%

“…Mouse and human subunit a3 protein sequences are 84% identical [37–39]. The a3 antibody was raised against amino acids 658-720 of the mouse a3 sequence, corresponding to amino acids 657-716 in the human sequence (Supplementary Figure S1B) [36]. This region is unique as compared to the a1, a2, and a4 human protein sequences (Supplementary Figure S1C) [36–39].…”

Section: Resultsmentioning

confidence: 99%

“…The a3 antibody was raised against amino acids 658-720 of the mouse a3 sequence, corresponding to amino acids 657-716 in the human sequence (Supplementary Figure S1B) [36]. This region is unique as compared to the a1, a2, and a4 human protein sequences (Supplementary Figure S1C) [36–39]. Moreover, the anti-a3 antibody recognizes a band of the expected molecular mass (∼120-kDa) by Western blot of whole cell lysates from human breast cells (see below), indicating that this antibody recognizes the human a3 protein.…”

Section: Resultsmentioning

confidence: 99%

“…The chemiluminescence substrate for horseradish peroxidase was purchased from General Electric and the signal detected using Kodak BioMax Light film. The chicken monoclonal a3 antibody that recognizes the V-ATPase V 0 a3 subunit was described previously [36]. A mouse monoclonal antibody recognizing α-tubulin was purchased from Genscript.…”

Section: Methodsmentioning

confidence: 99%

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The a3 isoform of subunit a of the vacuolar ATPase localizes to the plasma membrane of invasive breast tumor cells and is overexpressed in human breast cancer

et al. 2016

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The vacuolar (H+)-ATPases (V-ATPases) are a family of ATP-driven proton pumps that acidify intracellular compartments and transport protons across the plasma membrane. Previous work has demonstrated that plasma membrane V-ATPases are important for breast cancer invasion in vitro and that the V-ATPase subunit a isoform a3 is upregulated in and critical for MDA-MB231 and MCF10CA1a breast cancer cell invasion. It has been proposed that subunit a3 is present on the plasma membrane of invasive breast cancer cells and is overexpressed in human breast cancer. To test this, we used an a3-specific antibody to assess localization in breast cancer cells. Subunit a3 localizes to the leading edge of migrating breast cancer cells, but not the plasma membrane of normal breast epithelial cells. Furthermore, invasive breast cancer cells express a3 throughout all intracellular compartments tested, including endosomes, the Golgi, and lysosomes. Moreover, subunit a3 knockdown in MB231 breast cancer cells reduces in vitro migration. This reduction is not enhanced upon addition of a V-ATPase inhibitor, suggesting that a3-containing V-ATPases are critical for breast cancer migration. Finally, we have tested a3 expression in human breast cancer tissue and mRNA prepared from normal and cancerous breast tissue. a3 mRNA was upregulated 2.5-47 fold in all breast tumor cDNA samples tested relative to normal tissue, with expression generally correlated to cancer stage. Furthermore, a3 protein expression was increased in invasive breast cancer tissue relative to noninvasive cancer and normal breast tissue. These studies suggest that subunit a3 plays an important role in invasive human breast cancer.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The a3 isoform of subunit a of the vacuolar ATPase localizes to the plasma membrane of invasive breast tumor cells and is overexpressed in human breast cancer

et al. 2016

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“…JJN3 cells (1×10 3 /96-well) were fixed with 10% neutral-buffered formalin, incubated with 3% BSA-PBS blocking solution, and then with a3V-ATPase antibody (1/200) (39) at 4°C overnight, followed by Alexa Fluor® 594 anti-chicken secondary antibody (1/1,000) and Alexa Fluor® 488 Phalloidin (1/2,000) were then added. Nuclei were counter-stained with DAPI.…”

Section: Methodsmentioning

confidence: 99%

Bone Pain Induced by Multiple Myeloma Is Reduced by Targeting V-ATPase and ASIC3

et al. 2017

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Multiple myeloma (MM) patients experience severe bone pain (MMBP) that is undertreated and poorly understood. In this study, we studied MMBP in an intratibial mouse xenograft model which employs JJN3 human MM cells. In this model, mice develop MMBP associated in bone with increased sprouting of calcitonin gene-related peptide-positive (CGRP+) sensory nerves and in dorsal root ganglia (DRG) with upregulation of phosphorylated ERK1/2 (pERK1/2) and pCREB, two molecular indicators of neuron excitation. We found that JJN3 cells expressed a vacuolar proton pump (V-ATPase) that induced an acidic bone microenvironment. Inhibition of JJN3-colonized bone acidification by a single injection of the selective V-ATPase inhibitor, bafilomycin A1, decreased MMBP, CGRP+ SN sprouting, and pERK1/2 and pCREB expression in DRG. CGRP+ sensory nerves also expressed increased levels of the acid-sensing nociceptor ASIC3. Notably, a single injection of the selective ASIC3 antagonist APETx2 dramatically reduced MMBP in the model. Mechanistic investigations in primary DRG neurons co-cultured with JJN3 cells showed increased neurite outgrowth and excitation inhibited by bafilomycin A1 or APETx2. Further, combining APETx2 with bafilomycin A1 reduced MMBP to a greater extent than either agent alone. Lastly, combining bafilomycin A1 with the osteoclast inhibitor zoledronic acid was sufficient to ameliorate MMBP which was refractory to zoledronic acid. Overall, our results show that osteoclasts and MM cooperate to induce an acidic bone microenvironment that evokes MMBP as a result of the excitation of ASIC3-activated sensory neurons. Further, they present a mechanistic rationale for targeting ASIC3 on neurons along with the MM-induced acidic bone microenvironment as a strategy to relieve MMBP in patients.

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Further characterization of ATP6V0A2-related autosomal recessive cutis laxa

et al. 2012

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Autosomal recessive cutis laxa (ARCL) syndromes are phenotypically overlapping, but genetically heterogeneous disorders. Mutations in the ATP6V0A2 gene were found to underlie both, autosomal recessive cutis laxa type 2 (ARCL2), Debré type, and wrinkly skin syndrome (WSS). The ATP6V0A2 gene encodes the a2 subunit of the V-type H(+)-ATPase, playing a role in proton translocation, and possibly also in membrane fusion. Here, we describe a highly variable phenotype in 13 patients with ARCL2, including the oldest affected individual described so far, who showed strikingly progressive dysmorphic features and heterotopic calcifications. In these individuals we identified 17 ATP6V0A2 mutations, 14 of which are novel. Furthermore, we demonstrate a localization of ATP6V0A2 at the Golgi-apparatus and a loss of the mutated ATP6V0A2 protein in patients' dermal fibroblasts. Investigation of brefeldin A-induced Golgi collapse in dermal fibroblasts as well as in HeLa cells deficient for ATP6V0A2 revealed a delay, which was absent in cells deficient for the ARCL-associated proteins GORAB or PYCR1. Furthermore, fibroblasts from patients with ATP6V0A2 mutations displayed elevated TGF-β signalling and increased TGF-β1 levels in the supernatant. Our current findings expand the genetic and phenotypic spectrum and suggest that, besides the known glycosylation defect, alterations in trafficking and signalling processes are potential key events in the pathogenesis of ATP6V0A2-related ARCL.

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Generation of Chicken Monoclonal Antibodies Against thea1,a2, anda3 Subunit Isoforms of Vacuolar-type Proton ATPase

Cited by 13 publications

References 29 publications

The a3 isoform of subunit a of the vacuolar ATPase localizes to the plasma membrane of invasive breast tumor cells and is overexpressed in human breast cancer

The a3 isoform of subunit a of the vacuolar ATPase localizes to the plasma membrane of invasive breast tumor cells and is overexpressed in human breast cancer

Bone Pain Induced by Multiple Myeloma Is Reduced by Targeting V-ATPase and ASIC3

Further characterization of ATP6V0A2-related autosomal recessive cutis laxa

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