Characterization of naturally processed and presented peptides associated with bovine major histocompatibility complex (BoLA) class II DR molecules S. Sharif, B. A. Mallard and B. N. Wilkie Department of Pathobiology, University of Guelph, Guelph, Ontario, Canada Summary Differential regulation of genetic resistance to infectious disease may partially be explained by variation in the binding affinity and the repertoire of pathogen-derived antigenic peptides associated with major histocompatibility complex (MHC) molecules. In this study, we investigated characteristics of peptides that bind to the bovine MHC allele BoLA- DRB3* 2703, which is associated with occurrence of clinical mastitis in Holstein dairy cattle, and assigned a putative peptide-binding motif to this allele. This was achieved by in vitro expression of allele * 2703 as well as a control allele, BoLA-DRB3* 1201 which is present at high frequency in Holsteins. Transfected cell lines alone (for allele * 1201) or in combination with blood mononuclear cells from an animal homozygous for allele * 2703 were used as the source of naturally processed and presented peptides. Subsequent to elution of peptides from BoLA-DR + cells, their sequences were determined by electrospray ionization mass spectrometry. Eluted peptides were between 13 and 20 amino acids long and the majority were in sets of overlapping sequences. These peptides were derived from intra- and extracellular proteins, as well as foreign proteins present in the culture medium. Some peptides had originated from molecular chaperones present in the endoplasmic reti- culum, such as ER-60 and GRP78, pointing to some degree of overlap and cross-sampling between MHC class I and class II antigen presentation pathways. Consistent with reports of human and mouse MHC class II-associated peptides, putative peptide-binding motifs could be assigned to alleles * 2703 and * 1201, comprising a hydrophobic or an aromatic residue at relative position 1, a hydrophobic residue at position 4 and a small residue at position 6 of the eluted peptides. These findings provide the foundation for future studies of molecular mechanisms of MHC-disease associations of cattle. Keywords antigen-binding groove, antigen presentation, BoLA class II alleles, disease association, mass spectrometry, peptide-binding motifs, transfectants. Introduction Genetic regulation of host resistance to disease and immune responsiveness is polygenic. Among the many loci involved in induction and regulation of immune response, the major histocompatibility complex (MHC) genes explain a sub- stantial portion of genetic variation in immune respon- siveness (Biozzi et al. 1974; Mallard et al. 1989). Cell surface expression of two bovine MHC (BoLA) class II genes, DR and DQ, has been documented (Lewin 1997). The BoLA-DR molecules are heterodimers, formed by a mono- morphic DRA and a highly polymorphic DRB3 chain. More than 60 alleles have been identified in the DRB3 locus and much of this polymorphism is concentrated in the second exon of this gene, which encodes the antigen-binding groove of the molecule ( 2 Davies et al. 1997). Several groups have reported strong statistical associa- tions between allelic variants of the BoLA-DRB3 gene and immune responsiveness (Dietz et al. 1997), as well as with occurrence of disease (Xu et al. 1993; Maillard et al. 1996; Starkenburg et al. 1997). More recently, a significant association was detected between BoLA-DRB3.2* 23 allele, which corresponds to various subtypes of BoLA-DRB3* 27, Address for correspondence Dr Shayan Sharif, Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON 1G 2W1, Canada. E-mail: shayan@uoguelph.ca Accepted for publication 13 October 2002 Ó 2003 International Society for Animal Genetics, Animal Genetics, 34, 116–123 116