Refinement of the canine CD1 locus topology and investigation of antibody binding to recombinant canine CD1 isoforms
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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.
KeywordsCanine CD1 genes Canine CD1 proteins Antibody recognition of canine CD1 Single nucleotide polymorphisms (SNPs) Antibody epitope identification Canine dendritic cells
We kindly acknowledge Dr. Bruce Roe and his team at Advanced Center for Genome Technology, Oklahoma University for preliminary 454 Sanger sequencing of two of the three BAC sequences. We would like to thank Dr. Christopher Dascher (Havas Life, NY, USA) for input on guinea pig CD1 background, and we thank Dr. Steven Porcelli (Albert Einstein College of Medicine, NY, USA) for generously donating guinea pig hybridomas for use in this study. Furthermore, we thank Biolegend (San Diego, CA, USA) and AbD Serotec (Raleigh, NC, USA) for kindly donating anti-CD1 antibodies. We would like to thank Ken Jackson (UC Davis, CA, USA) for invaluable advice with cloning procedures and culture of BAC clones. This study was partly funded by a PhD scholarship from the Graduate School of Health and Medical Sciences, University of Copenhagen, Denmark. The PacBio analysis and bioinformatics analysis of this study was funded by two pilot grants received from the UC Davis DNA Technologies Core and the UC Davis Bioinformatics Core, respectively.
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Conflict of interest
The authors declare that they have no competing interests.
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