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      Unexpected lack of specificity of a rabbit polyclonal TAP-L (ABCB9) antibody

      research-article
      a , 1 , 2 , 3 ,   1 , 2 , 3
      F1000Research
      F1000Research
      ABCB9, TAP-L transporter, dendritic cell, antigen presentation, MHC, peptide, lysosome

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          Abstract

          In this article, we describe the surprising non-specific reactivity in immunoblots of a rabbit polyclonal antibody (ref. Abcam 86222) expected to recognize the transporter associated with antigen processing like (TAP-L, ABCB9) protein. Although this antibody, according to company documentation, recognizes a band with the expected molecular weight of 84 kDa in HeLa, 293T and mouse NIH3T3 whole-cell lysates, we found that this band is also present in immunoblots of TAP-L deficient bone marrow-derived dendritic cell (BMDC) whole-cell lysates in three independent replicates. We performed extensive verification by multiple PCR tests to confirm the complete absence of the ABCB9 gene in our TAP-L deficient mice. We conclude that the antibody tested cross-reacts with an unidentified protein present in TAP-L knockout cells, which coincidentally runs at the same molecular weight as TAP-L. These findings underline the pitfalls of antibody specificity testing in the absence of cells lacking expression of the target protein.

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          Purification and reconstitution of the antigen transport complex TAP: a prerequisite for determination of peptide stoichiometry and ATP hydrolysis.

          The transporter associated with antigen processing (TAP) is an essential machine of the adaptive immune system that translocates antigenic peptides from the cytosol into the endoplasmic reticulum lumen for loading of major histocompatibility class I molecules. To examine this ABC transport complex in mechanistic detail, we have established, after extensive screening and optimization, the solubilization, purification, and reconstitution for TAP to preserve its function in each step. This allowed us to determine the substrate-binding stoichiometry of the TAP complex by fluorescence cross-correlation spectroscopy. In addition, the TAP complex shows strict coupling between peptide binding and ATP hydrolysis, revealing no basal ATPase activity in the absence of peptides. These results represent an optimal starting point for detailed mechanistic studies of the transport cycle of TAP by single molecule experiments to analyze single steps of peptide translocation and the stoichiometry between peptide transport and ATP hydrolysis.
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            Identification of a lysosomal peptide transport system induced during dendritic cell development.

            The delivery of protein fragments to major histocompatibility complex (MHC)-loading compartments of professional antigen-presenting cells is essential in the adaptive immune response against pathogens. Apart from the crucial role of the transporter associated with antigen processing (TAP) for peptide loading of MHC class I molecules in the endoplasmic reticulum, TAP-independent translocation pathways have been proposed but not identified so far. Based on its overlapping substrate specificity with TAP, we herein investigated the ABC transporter ABCB9, also named TAP-like (TAPL). Remarkably, TAPL expression is strongly induced during differentiation of monocytes to dendritic cells and to macrophages. TAPL does not, however, restore MHC class I surface expression in TAP-deficient cells, demonstrating that TAPL alone or in combination with single TAP subunits does not form a functional transport complex required for peptide loading of MHC I in the endoplasmic reticulum. In fact, by using quantitative immunofluorescence and subcellular fractionation, TAPL was detected in the lysosomal compartment co-localizing with the lysosome-associated membrane protein LAMP-2. By in vitro assays, we demonstrate a TAPL-specific translocation of peptides into isolated lysosomes, which strictly requires ATP hydrolysis. These results suggest a mechanism by which antigenic peptides have access to the lysosomal compartment in professional antigen-presenting cells.
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              Selective and ATP-dependent translocation of peptides by the homodimeric ATP binding cassette transporter TAP-like (ABCB9).

              The transporter associated with antigen processing (TAP)-like (TAPL, ABCB9) belongs to the ATP-binding cassette transporter family, which translocates a vast variety of solutes across membranes. The function of this half-size transporter has not yet been determined. Here, we show that TAPL forms a homodimeric complex, which translocates peptides across the membrane. Peptide transport strictly requires ATP hydrolysis. The transport follows Michaelis-Menten kinetics with low affinity and high capacity. Different nucleotides bind and energize the transport with a slight predilection for purine bases. The peptide specificity is very broad, ranging from 6-mer up to at least 59-mer peptides with a preference for 23-mers. Peptides are recognized via their backbone, including the free N and C termini as well as side chain interactions. Although related to TAP, TAPL is unique as far as its interaction partners, transport properties, and substrate specificities are concerned, thus excluding that TAPL is part of the peptide-loading complex in the classic route of antigen processing via major histocompatibility complex class I molecules.
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                Author and article information

                Journal
                F1000Res
                F1000Res
                F1000Research
                F1000Research
                F1000Research (London, UK )
                2046-1402
                22 May 2015
                2015
                : 4
                : 125
                Affiliations
                [1 ]Institut National de la Santé et de la Recherche Médicale (INSERM) U1151, Paris, 75015, France
                [2 ]Centre national de la recherche scientifique (CNRS), Unité mixte de recherche (UMR) 8253, Paris, 75015, France
                [3 ]Université Paris Descartes, Sorbonne Paris Cité, 75015, France
                [1 ]Department of Microbiology and Immunology, University of Michigan Medical School, MI, USA
                [1 ]Department of Translational Immunology, German Cancer Research Center, Heidelberg, Germany
                [1 ]Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY, USA
                Author notes

                ML designed, performed and interpreted experiments and wrote the manuscript. PvE designed and interpreted experiments and edited the manuscript.

                Competing interests: Both authors confirm that they have no conflict of interest.

                Competing interests: No competing interests were disclosed.

                Competing interests: No competing interests were disclosed.

                Competing interests: No competing interests were disclosed.

                Article
                10.12688/f1000research.6535.1
                4893942
                27335633
                6853ccfa-7513-47ba-9fb7-9f8f4143610f
                Copyright: © 2015 van Endert P and Lawand M

                This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 21 May 2015
                Funding
                Funded by: Fondation pour la Recherche Médicale
                Award ID: DEQ20130326539
                Supported by a grant from the Fondation pour la Recherche Médicale (Equipe FRM DEQ20130326539) to PvE.
                Categories
                Antibody Validation Article
                Articles
                Antigen Processing & Recognition
                Cell Signaling
                Stem Cells & Regeneration

                abcb9,tap-l transporter,dendritic cell,antigen presentation,mhc,peptide,lysosome

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