The epitope recognized by pan-HLA class I-reactive monoclonal antibody W6/32 and its relationship to unusual stability of the HLA-B27/β 2 -microglobulin complex

Tran, Tri M.; Iványi, P; Hilgert, I; Brdička, Tomáš; Pla, Marika; Breur, B. S.; Flieger, Miroslav; Ivăsková, E; Hořejšı́, Václav

doi:10.1007/s002510100353

Cited by 29 publications

(33 citation statements)

References 17 publications

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“…Thus, although the two H chains have distinct peptide receptivity and affinity for ␤ 2 m, they form a somewhat common reservoir of unstable folding intermediates existing at equilibrium, and this is a likely source of free, unfolded H chains in HLA-E preparations used for immunization. Consistent with this idea, W6/32 has been shown to react with isolated and partially denatured class I H chains (46), and to prevent melting of HLA-E 107R H chains when added to soluble cell extracts immediately after cell lysis (1). Also consistent with the idea of a common pool of unfolded (or partially folded) HLA-E molecules, the MEM Abs (particularly MEM-E/06) detected some ␤ 2 m-associated H chains, although this can be clearly demonstrated only in the case of H chains encoded by the classical class I loci (Figs.…”

Section: Abs To Hla-e Are Biased To Recognize Unfolded H Chainsmentioning

confidence: 48%

HLA-E: Strong Association with β2-Microglobulin and Surface Expression in the Absence of HLA Class I Signal Sequence-Derived Peptides

Monaco¹,

Sibilio²,

Melucci³

et al. 2008

The Journal of Immunology

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), all of the MEM Abs unexpectedly reacted with ␤ 2 -microglobulin (␤ 2 m)-free and denatured (but not ␤ 2 m-associated and folded) HLA-E H chains. Remarkably, other HLA-E-restricted Abs were also reactive with free H chains. Immunodepletion, in vitro assembly, flow cytometry, and three distinct surface-labeling methods, including a modified (conformation-independent) biotin-labeling assay, revealed the coexistence of HLA-E conformers with unusual and drastically antithetic features. MEM-reactive conformers were thermally unstable and poorly surface expressed, as expected, whereas ␤ 2 m-associated conformers were either unstable and weakly reactive with the prototypic conformational Ab W6/32, or exceptionally stable and strongly reactive with Abs to ␤ 2 m even in cells lacking permissive alleles (721.221), TAP (T2), or tapasin (721.220). Noncanonical, immature (endoglycosidase H-sensitive) HLA-E glycoforms were surface expressed in these cells, whereas mature glycoforms were exclusively expressed (and at much lower levels) in cells carrying permissive alleles. Thus, HLA-E is a good, and not a poor, ␤ 2 m assembler, and TAP/tapasin-assisted ligand donation is only one, and possibly not even the major, pathway leading to its stabilization and surface expression.

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Section: Abs To Hla-e Are Biased To Recognize Unfolded H Chainsmentioning

confidence: 48%

HLA-E: Strong Association with β2-Microglobulin and Surface Expression in the Absence of HLA Class I Signal Sequence-Derived Peptides

Monaco¹,

Sibilio²,

Melucci³

et al. 2008

The Journal of Immunology

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“…These effects are primarily mediated by altering peptide binding (42)(43)(44), but additional effects on HC/␤ 2 m interactions, which are particularly strong in HLA-B27 (45,46), cannot be ruled out. That most variants with the lowest thermostability showed a strong interaction with Tpn is in agreement with the role of this chaperone in retaining in the ER those MHC-I molecules that fail to optimize their peptide cargo (47).…”

Section: Discussionmentioning

confidence: 99%

Mutational Analysis Reveals a Complex Interplay of Peptide Binding and Multiple Biological Features of HLA-B27

Galocha

Castro

2010

Journal of Biological Chemistry

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Molecular polymorphism influences the strong association of HLA-B27 with ankylosing spondylitis through an unknown mechanism. Natural subtypes and site-directed mutants were used to analyze the effect of altering the peptide-binding site of this molecule on its stability, interaction with tapasin, folding, and export. The disease-associated subtypes B*2705, B*2702, and B*2704 showed higher thermostability at 50°C than all other subtypes and mutants, except some mimicking B*2702 polymorphism. The lowest values were found among pocket B mutants, most of which interacted strongly with tapasin, but otherwise there was no correlation between thermostability and tapasin interaction. Mutants resulting in increased hydrophobicity frequently acquired their maximal thermostability faster than those with increased polarity, suggesting that this process is largely driven by the thermodynamics of peptide binding. Folding, export, and tendency to misfold were influenced by polymorphism all along the peptide-binding site and were not specifically dependent on any particular region or structural feature. Frequent uncoupling of thermostability, folding/misfolding, and export can be explained by the distinct effect of mutations on the acquisition of a folded conformation, the optimization rate of B27-peptide complexes, and their quality control in the endoplasmic reticulum, all of which largely depend on the ways in which mutations alter peptide binding, without excluding additional effects on interactions with tapasin or other proteins involved in folding and export. The similarity of the generally disease-associated B*2707 to nondisease-associated subtypes in all the features analyzed suggests that molecular properties other than antigen presentation may not currently explain the relationship between HLA-B27 polymorphism and ankylosing spondylitis.HLA-B27 is the major susceptibility factor for a group of chronic inflammatory diseases known as spondyloarthropathies, whose prototype is ankylosing spondylitis (AS).2 This is a disorder characterized by inflammation of the entheses and joints of the axial skeleton, followed by pathological new bone formation, ultimately leading to ankylosis (1). HLA-B27 is involved in triggering the inflammatory process, but the mechanism is unknown. Four main hypotheses, each based on a particular property of HLA-B27, are currently being investigated. The arthritogenic peptide hypothesis (2) is based on the antigen-presenting specificity of HLA-B27 and assumes that molecular mimicry between microbial and self-derived B27 ligands would trigger autoimmune T cell cross-reaction and tissue injury. The misfolding hypothesis (3) is based on the slow folding and tendency to misfold of HLA-B*2705 (4) and assumes that accumulation of misfolded B27 heavy chain (HC) could trigger inflammation by eliciting the unfolded protein response and activation of NFB. The surface homodimer hypothesis (5, 6) is based on the expression of HC homodimers at the cell surface, following dissociation of HLA-B27-peptide complex...

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“…Additional control stainings were done with the W6/32 Mab. This mouse Mab is a broadly used pan-HLA class I-reactive monoclonal antibody that recognises a conformational epitope dependent on association between heavy chains and b 2 -m (Barnstable et al, 1978;Tran et al, 2001). MAb binding was detected using biotinylated rabbit anti-rat IgG (1 : 100; Dako, Copenhagen, Denmark) or anti-mouse IgG (1 : 150; Dako) and streptavidin conjugated to horseradish peroxidase (1 : 500; Zymed, San Francisco, CA, USA).…”

Section: Immunohistochemistrymentioning

confidence: 99%

Increased expression of beta 2-microglobulin in multidrug-resistant tumour cells

et al. 2002

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The rat monoclonal antibody LMR-12 was shown earlier to react with a plasma membrane protein, upregulated in multidrugresistant cell lines. In this study, we observed distinct LMR-12 staining in 36 out of 55 non-drug-selected tumour cell lines, including melanomas, renal cell-, colon-and lung carcinomas, whereas in other tumour types, such as leukaemia and ovarian cancer, LMR-12 staining was generally low or absent. The cDNA encoding the LMR-12 antigen was isolated from a library of the multidrug-resistant human fibrosarcoma cell line HT1080/DR4 by expression cloning in MOP8 cells. Sequence analysis showed that the LMR-12 antigen is identical to the major histocompatibility complex class I molecule beta 2-microglobulin (b 2 -m). The LMR-12/ b 2 -m staining results were confirmed by mRNA microarray data from an independent National Cancer Institute study, as well as by newly obtained reverse transcriptase polymerase chain reaction data. Further analysis of the microarray data showed that b 2 -m levels closely reflected levels of major histocompatibility complex class I heavy chains and the transporter associated with antigen processing. Since the ABC transporter associated with antigen processing was previously shown to contribute to multidrug-resistance, it may very well be that the observed LMR-12/ b 2 -m levels are secondary to (elevated) levels of the transporter associated with antigen processing. A perspective arising from the present study is that drug resistant tumour cells may, by having elevated levels of major histocompatibility complex related molecules, be particular good candidates for alternative therapeutic therapies, such as cytotoxic T cell mediated immune-therapies.

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The epitope recognized by pan-HLA class I-reactive monoclonal antibody W6/32 and its relationship to unusual stability of the HLA-B27/β 2 -microglobulin complex

Cited by 29 publications

References 17 publications

HLA-E: Strong Association with β2-Microglobulin and Surface Expression in the Absence of HLA Class I Signal Sequence-Derived Peptides

HLA-E: Strong Association with β2-Microglobulin and Surface Expression in the Absence of HLA Class I Signal Sequence-Derived Peptides

Mutational Analysis Reveals a Complex Interplay of Peptide Binding and Multiple Biological Features of HLA-B27

Increased expression of beta 2-microglobulin in multidrug-resistant tumour cells

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