Randall K. Ribaudo

The Lectin-Like NK Cell Receptor Ly-49A Recognizes a Carbohydrate-Independent Epitope on Its MHC Class I Ligand

The mouse NK inhibitory Ly-49A receptor specifically interacts with a peptide-induced conformational determinant on its MHC class I ligand, H-2Dd. In addition, it binds the polysaccharide fucoidan, consistent with its C-type lectin homology and the hypothesis that Ly-49A interacts with carbohydrates on Dd. Herein, however, we demonstrate that Ly-49A recognizes Dd mutants lacking N-glycosylation. Fucoidan competes for binding with anti-Ly-49A antibodies that inhibit Ly-49A-Dd interaction, and blocks apparent Ly-49A binding to unglycosylated Dd. We confirm that Ly-49A recognizes the alpha1 and amino-terminal alpha2 domains of Dd by analysis of recombinant H-2Kd-H-2Dd molecules. These studies indicate that Ly-49A recognizes carbohydrate-independent epitope(s) on Dd and suggest that Ly-49A has two distinct ligands, carbohydrate and MHC class I.

Functional comparison of bovine, murine, and human β2-microglobulin: interactions with murine MHC i molecules

Fetal calf serum is a well known source of bovine beta2-microglobulin (beta2m) which can exchange with endogenous beta2m from, as well as promote peptide binding to, class I major histocompatibility (MHC I) molecules on cells cultured in vitro. Recombinant bovine beta2m was expressed and purified for direct functional comparison to human and murine beta2m for interactions with murine MHC I molecules H-2Kb, Db, Kd, Ld, and Dd. Bovine and human beta2m were equivalent in stabilizing MHC I heavy chains and facilitating peptide loading, suggesting similar affinities for murine MHC I heavy chains. The activity of murine beta2m was significantly weaker, consistent with previous work that demonstrated the lower affinity of murine human beta2m for murine heavy chains compared to human beta2m. Analysis of bovine beta2m in fetal calf serum revealed ten-fold higher concentrations than in adult bovine serum, levels shown to significantly affect MHC I stability and peptide loading. The ramifications for the study of MHC I molecules from cells in culture and the evolutionary implications of the higher affinity interactions of human and bovine beta2m are discussed.

Differential association of β2-microglobulin mutants with MHC class I heavy chains and structural analysis demonstrate allele-specific interactions

Dynamic interactions between major histocompatibility complex (MHC) class I heavy chains and beta2-microglobulin (beta2m) play a critical role in their stability on the cell surface, and their ability to present peptide antigens to CD8+ T-cells. A cursory review of protein sequence homologies and three-dimensional crystal structures of MHC complexes might indicate very similar modes of interaction between the heavy and light chains. In this report, a panel of human beta2m mutants was screened to probe the interactions of beta2m with the murine MHC molecules H-2Kb, -Db, -Kd, -Ld, and -Dd. Binding experiments coupled with analyses of existing three-dimensional crystal structures demonstrate allelic differences in their interaction with beta2m. A comprehensive analysis of the existing murine MHC structures indicates a conformational flexibility on the part of murine beta2m that is not present in beta2m of the human structures. This flexibility is in a region directly interacting with the heavy chain and may relate to its lower affinity for murine heavy chains relative to human beta2m. This defined panel of beta2m mutants of differing affinity may also be useful for subsequent studies of thymic selection, T-cell recognition, and more refined algorithms for protein structure prediction.

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

The ability to directly load cell surface major histocompatibility complex (MHC) class I molecules with peptides provides a potentially powerful approach toward the development of vaccines to generate cell-mediated immunity. We demonstrate that exogenous β2-microglobulin (β2m) stabilizes human cell surface MHC I molecules and facilitates their loading with exogenous peptides. Additionally, using three-dimensional crystal structures and known interaction sites between MHC I heavy chains and β2m, we engineered variants of human β2m (hβ2m) with a single serine substitution at residue 55. This alteration was predicted to promote hydrophobic interactions at the MHC I heavy chain/β2m interface and displace an ordered water molecule. Compared with hβ2m, the serine to valine substitution at residue 55 had improved ability to bind to cell surface HLA-A1, HLA-A2, and HLA-A3 molecules, facilitate exogenous peptide loading, and promote recognition by peptide-specific T cells. The inclusion of hβ2m or higher affinity variants when pulsing cells with MHC-restricted peptides increases the efficiency of peptide loading 50–80-fold. Therefore, the inclusion of hβ2m in peptide-based vaccines may increase cell surface antigen densities above thresholds that allow recognition of peptide antigens by the immune system, particularly for cryptic, subdominant, or marginally antigenic peptides.

Preparation of RNA from tissues and cells.

Most procedures for isolating RNA from eukaryotic cells involve lysing and denaturing cells to liberate total nucleic acids. Additional steps are then required to remove DNA.

Isolation and Purification of Large DNA Restriction Fragments from Agarose Gels

This unit describes methods for recovering and purifying DNA restriction fragments from agarose gels. The first protocol describes electroelution of the fragment of interest from standard agarose gels using buffer‐filled dialysis bags, followed by concentration and purification using an Elutip column. This approach can be used effectively for fragments of all sizes from 50 to 20,000 bp. Electrophoresis directly onto NA‐45 paper ( second protocol ) provides relatively high yields for fragments <2000 bp. Fragments >1000 bp can also be separated on low gelling/melting agarose gels and purified by phenol extraction ( third protocol ), b‐agarase digestion of the gel (first alternate protocol ), or via glass beads extraction (second alternate protocol ). Removing linkers from a fragment using a column rather than a gel is described, followed by a method for estimating DNA concentrations in solution.

Class I MHC/Peptide/ β 2-Microglobulin Interactions:The Basis of Cytotoxic T-Cell Recognition

The past decade has witnessed a major revolution in our thinking and understanding of the molecular basis of T-cell recognition. In this brief review we outline the historical development of this knowledge and how it has drawn upon advances in cellular immunology, virology, genetics, molecular biology, and structural biology. The current status of our view of the molecular details is summarized, and an outline of critical molecular and cellular questions for the future is presented.