The interaction of beta 2‐microglobulin (β2m) with mouse class I major histocompatibility antigens and its ability to support peptide binding. A comparison of human and mouse β₂m

Abstract

The function of major histocompatibility complex (MHC) class I molecules is to sample peptides derived from intracellular proteins and to present these peptides to CD8+ cytotoxic T lymphocytes. In this paper, biochemical assays addressing MHC class I binding of both peptide and beta 2-microglobulin (beta 2m) have been used to examine the assembly of the trimolecular MHC class I/beta 2m/peptide complex. Recombinant human beta 2m and mouse beta 2ma have been generated to compare the binding of the two beta 2m to mouse class I. It is frequently assumed that human beta 2m binds to mouse class I heavy chain with a much higher affinity than mouse beta 2m itself. We find that human beta 2m only binds to mouse class I heavy chain with slightly (about 3-fold) higher affinity than mouse beta 2m. In addition, we compared the effect of the two beta 2m upon peptide binding to mouse class I. The ability of human beta 2m to support peptide binding correlated well with its ability to saturate mouse class I heavy chains. Surprisingly, mouse beta 2m only facilitated peptide binding when mouse beta 2m was used in excess (about 20-fold) of what was needed to saturate the class I heavy chains. The inefficiency of mouse beta 2m to support peptide binding could not be attributed to a reduced affinity of mouse beta 2m/MHC class I complexes for peptides or to a reduction in the fraction of mouse beta 2m/MHC class I molecules participating in peptide binding. We have previously shown that only a minor fraction of class I molecules are involved in peptide binding, whereas most of class I molecules are involved in beta 2m binding. We propose that mouse beta 2m interacts with the minor peptide binding (i.e. the "empty") fraction with a lower affinity than human beta 2m does, whereas mouse and human beta 2m interact with the major peptide-occupied fraction with almost similar affinities. This would explain why mouse beta 2m is less efficient than human beta 2m in generating the peptide binding moiety, and identifies the empty MHC class I heavy chain as the molecule that binds human beta 2m preferentially.

Asialoerythropoietin is a nonerythropoietic cytokine with broad neuroprotective activity in vivo

“…For binding assays, asialoEPO and rhEPO were radiolabeled according to a standard protocol by using chloramine T as described (29). The radioligand activity corresponded to Ϸ50…”

Specific killing of P53 mutated tumor cell lines by a cross-reactive human HLA-A2-restricted P53-specific CTL line

“…Wiley and Garboczi, was transformed into E. coli strain XA90 and protein production and purification was performed as described elsewhere. 20 Binding of peptide to recombinant MHC class I heavy chains was performed essentially as described previously 21 using purified denatured heavy-chain solution diluted 100-fold in binding buffer (20 mM tris pH 6.7 150 mM NaCL, 1 mM EDTA, 1 M ␤ 2 m and 1-2 nM tracer peptide) (Pedersen et al, unpublished). The p53 peptides showed comparable affinity for the HLA-A2 molecule with IC50 values ranging from 70 to 200 nM (Table III).…”

The Effect of Human β2-Microglobulin on Major Histocompatibility Complex I Peptide Loading and the Engineering of a High Affinity Variant

“…For the development of murine vaccination models, the availability of a naturally occurring higher affinity ␤ 2 m (h␤ 2 m) provides this essential component (31,59). In contrast, there are no known naturally occurring ␤ 2 ms available with higher affinity for human MHC I heavy chains than h␤ 2 m. The availability of three-dimensional structural data for a number of MHC I molecules as well as our previous studies examining chimeric m␤ 2 m/h␤ 2 m molecules (26) led to the identification of a specific region and, ultimately, a single residue of ␤ 2 m (serine 55) to mutate in order to increase its affinity for human MHC I heavy chains.…”