Expression of distinct conformations of free HLA-Cw4 heavy chains in transfected neuroblastoma cells.
Epitope mapping of HLA-Cw4 indicates that the two monoclonal antibodies (mAbs) L31 and M38, specific for beta 2-microglobulin (beta2m)-free HLA-C heavy chains, react preferentially with the KYK motif, located in the binding groove (alpha1 domain). Transfection of HLA-Cw4 cDNA into a neuroblastoma cell line, which normally expresses negligible HLA class I, resulted in the constitutive surface expression of molecules displaying different reactivities with the two mAbs. This cellular system was used to determine whether L31 and M38 recognize distinct conformations of beta2m-free HLA-C proteins, and to investigate their mechanism of expression. Interferon-gamma greatly enhanced the expression of L31-reactive free chains, while abolishing that of M38-reactive molecules. The cytokine-induced expression of L31-reactive molecules was inhibited by anti-sense oligonucleotides specific for beta2m mRNA, while constitutive expression of L31-reactive molecules was only partially affected. Exogenous beta2m resulted in a reduction of constitutive L31 reactivity, and in a concomitant increase of M38 reactivity. These results indicate that: 1) at the cell surface, L31 and M38 react with two distinct conformations of HLA-Cw4 beta2m-free heavy chains, of which the L31-reactive conformation is the least folded; 2) the expression of both conformers can be modulated by endogenous or exogenous beta2m; and (3) L31-reactive molecules exposed at the cell surface are likely to derive from the dissociation of empty HLA-Cw4/beta2m complexes.
Marozzi
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A
Dipartimento di Biologia e Genetica per le Science Mediche, Università di Milano, Via Viotti 3/5, 20133 Milano, Italy.
Meneveri
R
R
De Santis
C
C
Robbioni
P
P
Molteni
E
E
Beretta
A
A
Siccardi
A G
AG
Ginelli
E
E
D000911:Antibodies, Monoclonal; D001483:Base Sequence; D018604:Epitope Mapping; D005434:Flow Cytometry; D015236:HLA-C Antigens / Q000737:chemistry*; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000276:immunology; D011487:Protein Conformation; D011994:Recombinant Proteins; D014162:Transfection; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D015236:HLA-C Antigens; C039966:HLA-C*04 antigen; D011994:Recombinant Proteins