title: Refinement of the canine CD1 locus topology and investigation of antibody binding to recombinant canine CD1 isoforms.
abstract: CD1 molecules are antigen-presenting glycoproteins primarily found on dendritic cells (DCs) responsible for lipid antigen presentation to CD1-restricted T cells. Despite their pivotal role in immunity, little is known about CD1 protein expression in dogs, notably due to lack of isoform-specific antibodies. The canine (Canis familiaris) CD1 locus was previously found to contain three functional CD1A genes: canCD1A2, canCD1A6, and canCD1A8, where two variants of canCD1A8, canCD1A8.1 and canCD1A8.2, were assumed to be allelic variants. However, we hypothesized that these rather represented two separate genes. Sequencing of three overlapping bacterial artificial chromosomes (BACs) spanning the entire canine CD1 locus revealed canCD1A8.2 and canCD1A8.1 to be located in tandem between canCD1A7 and canCD1C, and canCD1A8.1 was consequently renamed canCD1A9. Green fluorescent protein (GFP)-fused canine CD1 transcripts were recombinantly expressed in 293T cells. All proteins showed a highly positive GFP expression except for canine CD1d and a splice variant of canine CD1a8 lacking exon 3. Probing with a panel of anti-CD1 monoclonal antibodies (mAbs) showed that Ca13.9H11 and Ca9.AG5 only recognized canine CD1a8 and CD1a9 isoforms, and Fe1.5F4 mAb solely recognized canine CD1a6. Anti-CD1b mAbs recognized the canine CD1b protein, but also bound CD1a2, CD1a8, and CD1a9. Interestingly, Ca9.AG5 showed allele specificity based on a single nucleotide polymorphism (SNP) located at position 321. Our findings have refined the structure of the canine CD1 locus and available antibody specificity against canine CD1 proteins. These are important fundamentals for future investigation of the role of canine CD1 in lipid immunity.
authors.affiliation: Department of Veterinary Clinical and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Dyrlaegevej 16, 1870, Frederiksberg C, Denmark.|Department of Veterinary Pathology, Microbiology and Immunology, University of California, One Shields Avenue, Davis, CA, 95616, USA.
authors.forename: Mette
authors.initials: M
authors.lastname: Schjaerff
authors.affiliation: Department of Veterinary Pathology, Microbiology and Immunology, University of California, One Shields Avenue, Davis, CA, 95616, USA.
authors.forename: Stefan M
authors.initials: SM
authors.lastname: Keller
authors.affiliation: UC Davis Genome Center - Bioinformatics Core, University of California, 451 Health Science Drive, Davis, CA, 95616, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Fass
authors.affiliation: UC Davis Genome Center - DNA Technologies and Expression Analysis Cores, University of California, 451 Health Science Drive, Davis, CA, 95616, USA.
authors.forename: Lutz
authors.initials: L
authors.lastname: Froenicke
authors.affiliation: Veterinary Genetics Laboratory, University of California, 980 Old Davis Road, Davis, CA, 95616, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Grahn
authors.affiliation: Lyons Feline and Comparative Genetics, Department of Veterinary Medicine and Surgery, University of Missouri, 1600 E Rollins Street, Colombia, MO, 65211, USA.
authors.forename: Leslie
authors.initials: L
authors.lastname: Lyons
authors.affiliation: Department of Veterinary Pathology, Microbiology and Immunology, University of California, One Shields Avenue, Davis, CA, 95616, USA.
authors.forename: Verena K
authors.initials: VK
authors.lastname: Affolter
authors.affiliation: Department of Veterinary Clinical and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Dyrlaegevej 16, 1870, Frederiksberg C, Denmark.
authors.forename: Annemarie T
authors.initials: AT
authors.lastname: Kristensen
authors.affiliation: Department of Veterinary Pathology, Microbiology and Immunology, University of California, One Shields Avenue, Davis, CA, 95616, USA. pfmoore@ucdavis.edu.
authors.forename: Peter F
authors.initials: PF
authors.lastname: Moore
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D000918:Antibody Specificity; D018949:Antigens, CD1 / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D019295:Computational Biology; D004285:Dogs; D056426:Genetic Loci; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide; D011485:Protein Binding; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
chemical_list: D000911:Antibodies, Monoclonal; D018949:Antigens, CD1; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins
keywords: Antibody epitope identification; Antibody recognition of canine CD1; Canine CD1 genes; Canine CD1 proteins; Canine dendritic cells; Single nucleotide polymorphisms (SNPs)