Title

Expression of HLA class I complexes assembled with structural variants of beta2-microglobulin

Date of Completion

January 1999

Keywords

Biology, Molecular|Biology, Cell|Health Sciences, Immunology

Degree

Ph.D.

Abstract

β2-microglobulin (β2m) is an 11.5–12 kD protein constructed of 99 amino acids that fold into two anti-parallel β-pleated sheets connected by a disulfide bond. It is well established that the role of β2m, the only invariant component of the MHC class I complex, is to promote assembly and exocytosis of this ternary complex to the cell surface. This project explored the influence of specific structural elements within β2m on the expression of the MHC class I complex. To dissect out critical elements, genes encoding structural variants of β2m were constructed using molecular biological techniques. Subsequently, the genes were transfected into the β 2m-null human melanoma cell line, FO-1. Expressed products were analyzed to assess the effect of each change on expression of the complex. Studies reported here reveal that it is the four-stranded β-pleated sheet of β 2m that controls cell surface expression of the HLA class I complex. Although individual amino acid residues within this region of β 2m could not be implicated in regulation of the class I complex, these data support the role of heavy chain contact residues in surface expression and stability of the complex. ^ In contrast, the three stranded face of β2m was associated with the major antigenic properties of the molecule. Analysis of chimeric and mutant β2m molecules in anti-human β2m antibody binding studies showed that an immunodominant region of human β 2m maps to the solvent exposed, three stranded β-pleated sheet; more precisely to the linear stretch of amino acids bounded by residues 34 and 51 and including residues 38, 44, and 45. ^ Finally, the structural elements of β2m required to generate the epitope recognized by the mAb W6/32, an α2 HLA class I heavy chain epitope, were defined. Initially, structural variants of β 2m localized critical elements to the NH2-terminus of the molecule. Further work demonstrated recovery of the W6/32 epitope in FO-1 cells transfected with a point mutant of mouse β2m, thus confirming the role of position three in the formation of this antigenic determinant. The studies reported here identify, for the first time, critical structural elements within β2m that affect MHC class I expression. ^