Refinement of the canine CD1 locus topology and investigation of antibody binding to recombinant canine CD1 isoforms

Mette Schjaerff, Stefan M. Keller, Joseph Fass, Lutz Froenicke, Robert A Grahn, Leslie A Lyons, Verena K Affolter, Annemarie T. Kristensen, Peter F Moore

Research output: Contribution to journalArticle

4 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)191-204
Number of pages14
JournalImmunogenetics
Volume68
Issue number3
DOIs
StatePublished - Mar 1 2016

Fingerprint

Canidae
Protein Isoforms
Antibodies
Green Fluorescent Proteins
CD1 Antigens
Immunity
Proteins
Monoclonal Antibodies
Dogs
Lipids
Bacterial Artificial Chromosomes
Antibody Specificity
HEK293 Cells
Antigen Presentation
Dendritic Cells
Genes
Single Nucleotide Polymorphism
Exons
Glycoproteins
Alleles

Keywords

  • Antibody epitope identification
  • Antibody recognition of canine CD1
  • Canine CD1 genes
  • Canine CD1 proteins
  • Canine dendritic cells
  • Single nucleotide polymorphisms (SNPs)

ASJC Scopus subject areas

  • Immunology
  • Medicine(all)
  • Genetics

Cite this

Refinement of the canine CD1 locus topology and investigation of antibody binding to recombinant canine CD1 isoforms. / Schjaerff, Mette; Keller, Stefan M.; Fass, Joseph; Froenicke, Lutz; Grahn, Robert A; Lyons, Leslie A; Affolter, Verena K; Kristensen, Annemarie T.; Moore, Peter F.

In: Immunogenetics, Vol. 68, No. 3, 01.03.2016, p. 191-204.

Research output: Contribution to journalArticle

Schjaerff, Mette ; Keller, Stefan M. ; Fass, Joseph ; Froenicke, Lutz ; Grahn, Robert A ; Lyons, Leslie A ; Affolter, Verena K ; Kristensen, Annemarie T. ; Moore, Peter F. / Refinement of the canine CD1 locus topology and investigation of antibody binding to recombinant canine CD1 isoforms. In: Immunogenetics. 2016 ; Vol. 68, No. 3. pp. 191-204.
@article{8e074351b15a4880b01693c5636894c0,
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.",
keywords = "Antibody epitope identification, Antibody recognition of canine CD1, Canine CD1 genes, Canine CD1 proteins, Canine dendritic cells, Single nucleotide polymorphisms (SNPs)",
author = "Mette Schjaerff and Keller, {Stefan M.} and Joseph Fass and Lutz Froenicke and Grahn, {Robert A} and Lyons, {Leslie A} and Affolter, {Verena K} and Kristensen, {Annemarie T.} and Moore, {Peter F}",
year = "2016",
month = "3",
day = "1",
doi = "10.1007/s00251-015-0889-3",
language = "English (US)",
volume = "68",
pages = "191--204",
journal = "Immunogenetics",
issn = "0093-7711",
publisher = "Springer Verlag",
number = "3",

}

TY - JOUR

T1 - Refinement of the canine CD1 locus topology and investigation of antibody binding to recombinant canine CD1 isoforms

AU - Schjaerff, Mette

AU - Keller, Stefan M.

AU - Fass, Joseph

AU - Froenicke, Lutz

AU - Grahn, Robert A

AU - Lyons, Leslie A

AU - Affolter, Verena K

AU - Kristensen, Annemarie T.

AU - Moore, Peter F

PY - 2016/3/1

Y1 - 2016/3/1

N2 - 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.

AB - 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.

KW - Antibody epitope identification

KW - Antibody recognition of canine CD1

KW - Canine CD1 genes

KW - Canine CD1 proteins

KW - Canine dendritic cells

KW - Single nucleotide polymorphisms (SNPs)

UR - http://www.scopus.com/inward/record.url?scp=84958751668&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84958751668&partnerID=8YFLogxK

U2 - 10.1007/s00251-015-0889-3

DO - 10.1007/s00251-015-0889-3

M3 - Article

C2 - 26687789

AN - SCOPUS:84958751668

VL - 68

SP - 191

EP - 204

JO - Immunogenetics

JF - Immunogenetics

SN - 0093-7711

IS - 3

ER -