Expression of HLA class I molecules assembled with structural variants of human beta2-microglobulin.

Abstract

Mouse and human beta2-microglobulin (beta2m), which differ by 30% in their primary sequence, give rise to disparate levels of HLA class I heavy chain expression in transfectants of the beta2m-null FO-1 human melanoma cell line, i.e., mouse beta2m directs expression of HLA class I heavy chains that is only approximately 20%-30% of that observed for heavy chains assembled with human beta2m. In this report we describe our efforts to better understand the structural basis of this regulatory phenomenon. Initial insight into the importance of the N-terminus of beta2m on HLA expression came from studies with FO-1 cells transfected with chimeric (human X mouse) B2m genes. Chimeric beta2m molecules containing residues 1-69 from human beta2m and residues 70-99 from mouse beta2m (designated HM- beta2m) induced expression of HLA heavy chains to a significantly greater extent ( approximately 80% of level observed with cognate beta2m) than the reverse chimeric construct (designated MH- beta2m) (10%-15% of level observed with cognate beta2m). These data are consistent with the view that the major determinants of HLA class I heavy chain expression map to the portion of the beta2m molecule which forms the four-stranded beta-pleated sheet, comprised of S1, S2, S4, and S5, and one strand of the three-stranded sheet (S3). The mapping of class I regulatory sites to the portion of beta2m containing the four-stranded beta-pleated sheet supports the interpretation that the heavy chain contact residues on beta2m play the major role in regulating major histocompatibility (MHC) class I expression. To further dissect beta2m-mediated regulation of HLA class I expression, site-directed mutants of beta2m were prepared by conversion of human beta2m to the mouse sequence at individual amino acid positions within the four-stranded and three-stranded beta-pleated sheets. Human to mouse amino acid substitutions were made in each divergent residue between positions 1-66, and as controls for COOH-terminal modification, several residues between positions 75 and 94. Cytofluorometry with HLA class I-specific antibodies indicated that cell surface expression of HLA class I heavy chains was largely insensitive to each of the individual substitutions. It is concluded that a combination of divergent residues mapping to positions of heavy chain contact are responsible for the differences observed in MHC class I expression by heterologous forms of beta2m.