Tapasin Enhances Assembly of Transporters Associated with Antigen Processing-dependent and -independent Peptides with HLA-A2 and HLA-B27 Expressed in Insect Cells

Lauvau, Grégoire; Gubler, Brigitte; Cohen, H.; Daniel, Soizic; Caillat‐Zucman, Sophie; Endert, Peter Van

doi:10.1074/jbc.274.44.31349

Cited by 36 publications

(38 citation statements)

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“…In the present studies, we investigated functional interactions between purified calreticulin and a peptide-deficient form of the human MHC class I molecule, HLA-A2. Previous analyses of functional interactions between calreticulin and MHC class I proteins have been carried out in Drosophila and Sf9 insect cells, and in these systems, calreticulin has been shown to enhance expression of MHC class I heavy chains and heterodimers (9,10). Our studies indicate direct binding between calreticulin and HLA-A2 heavy chains at elevated temperatures.…”

mentioning

confidence: 48%

“…HLA-A2 is an unusual MHC class I molecule in that it binds to signal peptides within the ER lumen (14). Previous experiments have shown the presence of HLA-A2-specific peptides within the ER of Sf9 insect cells (10), suggesting that the soluble A2 expressed in insect cells may be at least partially peptide-occupied. To assess the peptide occupancy of the soluble HLA-A2, we developed a fluorescencebased peptide binding assay.…”

Section: Hla-a2 Purified From Insect Cells Rapidly Associates With Flmentioning

confidence: 99%

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Calreticulin recognizes misfolded HLA-A2 heavy chains

Mancino

Rizvi

Lapinski

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Our studies investigated functional interactions between calreticulin, an endoplasmic reticulum chaperone, and major histocompatibility complex (MHC) class I molecules. Using in vitro thermal aggregation assays, we established that calreticulin can inhibit heat-induced aggregation of soluble, peptide-deficient HLA-A2 purified from supernatants of insect cells. The presence of HLA-A2-specific peptides also inhibits heat-induced aggregation. Inhibition of heat-induced aggregation of peptide-deficient HLA-A2 by calreticulin correlates with a rescue of the HLA-A2 heavy chain from precipitation, by forming high-molecular-weight complexes with calreticulin. Complex formation between HLA-A2 heavy chains and calreticulin occurs at 50°C but not 37°C, suggesting polypeptide-based interactions between the HLA-A2 heavy chain and calreticulin. Once complexes are formed, the addition of peptide is not sufficient to trigger efficient assembly of heavy chain͞␤2m͞peptide complexes. Using a fluorescent peptidebased binding assay, we show that calreticulin does not enhance peptide binding by HLA-A2 at 37°C. We also show that calreticulin itself is converted to oligomeric species on exposure to 37°C or higher temperatures, and that oligomeric forms of calreticulin are active in inhibiting thermal aggregation of peptide-deficient HLA-A2. Taken together, these results suggest that calreticulin functions in the recognition of misfolded MHC class I heavy chains in the endoplasmic reticulum. However, in the absence of other endoplasmic reticulum components, calreticulin by itself does not enhance the assembly of misfolded MHC class I heavy chains with ␤2m and peptides.

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confidence: 48%

Section: Hla-a2 Purified From Insect Cells Rapidly Associates With Flmentioning

confidence: 99%

Calreticulin recognizes misfolded HLA-A2 heavy chains

Mancino

Rizvi

Lapinski

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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“…One of these evaluates accumulation of a glycosylated radiolabeled peptide in microsomes (17), whereas the other uses appearance of HLA-B27 molecules on the surface of infected insect cells as a readout for TAP function. We have previously shown that insect cell-expressed B27 molecules are loaded with TAP-supplied peptides in the absence of other human accessory molecules, notably tapasin, and that cell surface expression in the Sf9 insect cell system reflects intracellular peptide loading of HLA-B27 (22). In this study, we analyzed peptide loading of HLA-B27 as an additional and physiologically relevant readout for TAP function.…”

Section: Peptide Translocation and Supply To Hla-b27 Molecules Bymentioning

confidence: 99%

“…Immunoprecipitation, Western Blot, and Flow Cytometric AnalysisThese experiments were also performed as described previously by us (18,22). For immunoprecipitation, 30-l (for vesicles prepared up to three months prior to the experiment) or 60-l microsomes (for older vesicles) were lysed in a total volume of 600 l using Nonidet P-40, and TAP complexes were immunoprecipitated with monoclonal antibody (mAb) 148.3 specific for human TAP1 (23).…”

Section: For Details)mentioning

confidence: 99%

Distinct Functions of the ATP Binding Cassettes of Transporters Associated with Antigen Processing

Saveanu

Daniel

Endert

2001

Journal of Biological Chemistry

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The transporters associated with antigen processing (TAP1/TAP2) provide peptides to MHC class I molecules in the endoplasmic reticulum. Like other ATP-binding cassette proteins, TAP uses ATP hydrolysis to power transport. We have studied peptide binding to as well as translocation by TAP proteins with mutations in the Walker A and B sequences that are known to mediate ATP binding and hydrolysis. We show that a mutation in the TAP1 Walker B sequence reported to abrogate class I expression by a lung tumor does not affect ATP binding affinity, suggesting a defect restricted to ATP hydrolysis. This mutation reduces peptide transport by only 50%, suggesting that TAP function can be highly limiting for antigen presentation in non-lymphoid cells. Single substitutions in Walker A sequences (TAP1K544A, TAP2K509A), or their complete replacements, abrogate nucleotide binding to each subunit. Although all of these mutations abrogate peptide transport, they reveal distinct roles for nucleotide binding to the two transporter subunits in TAP folding and in regulation of peptide substrate affinity, respectively. Alteration of the TAP1 Walker A motif can have strong effects on TAP1 and thereby TAP complex folding. However, TAP1 Walker A mutations compatible with correct folding do not affect peptide binding. In contrast, abrogation of the TAP2 nucleotide binding capacity has little or no effect on TAP folding but eliminates peptide binding to TAP at 37°C in the presence of nucleotides. Thus, nucleotide binding to TAP2 but not to TAP1 is a prerequisite for peptide binding to TAP. Based on these results, we propose a model in which nucleotide and peptide release from TAP are coupled and followed by ATP binding to TAP2, which induces high peptide affinity and initiates the transport cycle.The transporters associated with antigen processing (TAP) 1 belong to the family of ATP binding cassette (ABC) transporters, a large group of proteins that use energy provided by nucleotide triphosphates to translocate a vast variety of substrates across intracellular or cell surface membranes (1). All ABC transporters possess two transmembrane domains, each generally composed of six membrane-spanning segments, and two nucleotide binding domains (NBDs) with primary sequence homology across the protein family. Whereas substrate interaction is generally thought to involve the transmembrane domains, the NBDs bind and hydrolyze ATP. Both of the latter events have been shown to lead to conformational changes that upon transmission to substrate binding domains in an undefined fashion result in substrate translocation (2).Given the important role of ABC transporters in diseases such as mucoviscidosis or cancer, the mechanism of substrate transport has been subject to intense scrutiny. Mutagenesis studies and crystallographic analysis of HisP and MalK, the NBDs of bacterial histidine and maltose transporters, respectively, have elucidated the role of several conserved sequence motifs contained in ABC transporter NBDs (3, 4). Thus, the Walker A consensus s...

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“…These authors concluded that tapasin increases the expression of fully assembled class I molecules by retaining empty class I molecules until they bind peptide. Furthermore, using another insect expression system (Lepidoptera), Lauvau et al (23) concluded that tapasin facilitates assembly of peptide with class I independently of mediating their retention in the ER. Finally, other recent studies have suggested that tapasin may be involved in peptide editing of class I in a manner analogous to the role of DM with MHC class II molecules (24 -26).…”

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confidence: 99%

Kb, Kd, and Ld Molecules Share Common Tapasin Dependencies as Determined Using a Novel Epitope Tag

Myers

Harris

Connolly

et al. 2000

The Journal of Immunology

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The endoplasmic reticulum protein tapasin is considered to be a class I-dedicated chaperone because it facilitates peptide loading by proposed mechanisms such as peptide editing, endoplasmic reticulum retention of nonpeptide-bound molecules, and/or localizing class I near the peptide source. Nonetheless, the primary functions of tapasin remain controversial as do the relative dependencies of different class I molecules on tapasin for optimal peptide loading and surface expression. Tapasin dependencies have been addressed in previous studies by transfecting different class I alleles into tapasin-deficient LCL721.220 cells and then monitoring surface expression and Ag presentation to T cells. Indeed, by these criteria, class I alleles have disparate tapasin-dependencies. In this study, we report a novel and more direct method of comparing tapasin dependency by monitoring the ratio of folded vs open forms of the different mouse class I heavy chains, Ld, Kd, and Kb. Furthermore, we determine the amount of de novo heavy chain synthesis required to attain comparable expression in the presence vs absence of tapasin. Our findings show that tapasin dramatically improves peptide loading of all three of these mouse molecules.

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Tapasin Enhances Assembly of Transporters Associated with Antigen Processing-dependent and -independent Peptides with HLA-A2 and HLA-B27 Expressed in Insect Cells

Cited by 36 publications

References 72 publications

Calreticulin recognizes misfolded HLA-A2 heavy chains

Calreticulin recognizes misfolded HLA-A2 heavy chains

Distinct Functions of the ATP Binding Cassettes of Transporters Associated with Antigen Processing

Kb, Kd, and Ld Molecules Share Common Tapasin Dependencies as Determined Using a Novel Epitope Tag

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