cDNA cloning and sequencing reveal the major horse allergen Equ c1 to be a glycoprotein member of the lipocalin superfamily.
Abstract: The gene encoding the major horse allergen, designated Equus caballus allergen 1 (Equ c1), was cloned from total cDNA of sublingual salivary glands by reverse transcription-polymerase chain reaction using synthetic degenerate oligonucleotides deduced from N-terminal and internal peptide sequences of the glycosylated hair dandruff protein. A recombinant form of the protein, with a polyhistidine tail, was expressed in Escherichia coli and purified by immobilized metal affinity chromatography. The recombinant protein is able to induce a passive cutaneous anaphylaxis reaction in rat, and it behaves similarly to the native Equ c1 in several immunological tests with allergic patients' IgE antibodies, mouse monoclonal antibodies, or rabbit polyclonal IgG antibodies. Amino acid sequence identity of 49-51% with rodent urinary proteins from mice and rats suggests that Equ c1 is a new member of the lipocalin superfamily of hydrophobic ligand-binding proteins that includes several other major allergens. An RNA blot analysis demonstrates the expression of mRNA Equ c1 in liver and in sublingual and submaxillary salivary glands.
Publication Date: 1996-12-20 PubMed ID: 8955138DOI: 10.1074/jbc.271.51.32951Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
The researchers have identified and cloned the gene that produces the major horse allergen, Equ c1. They found that this allergen is a member of the lipocalin superfamily, a group of proteins that binds to small molecules.
Gene Isolation and Cloning
- The gene of the allergen Equ c1 was extracted from the sublingual salivary glands’ total cDNA. This was achieved by using the Reverse Transcription-Polymerase Chain Reaction (RT-PCR) method, which amplifies a specific DNA segment from a DNA template.
- The researchers utilized synthetic degenerate oligonucleotides deduced from the N-terminal and internal peptide sequences of a specific glycosylated protein found in horse dandruff. This served as primers for the PCR.
Recombinant Protein Expression and Testing
- Equ c1 protein was then created in a laboratory setting with a polyhistidine tail, a process referred to as recombinant protein expression. Escherichia coli cells were used to facilitate this process.
- Using Immobilized Metal Affinity Chromatography (IMAC), the scientists were able to purify the recombinant protein. IMAC is a technique utilized to separate proteins based on their interaction with metal ions.
- Tests showed that the recombinant protein can induce allergic reactions similarly to the native Equ c1 protein. Allergy tests were conducted on rats, causing a passive cutaneous anaphylaxis reaction, a serious and widespread allergic reaction.
- Furthermore, the recombinant protein showed similar behavior as the native Equ c1 in various immunological tests with allergic patients’ IgE antibodies, as well as with mouse monoclonal and rabbit polyclonal IgG antibodies.
Lipocalin Superfamily Affiliation and mRNA Expression
- The amino acid sequence of Equ c1 displayed 49-51% identity with rodent urinary proteins from mice and rats. This similarity indicated that Equ c1 is a member of the lipocalin superfamily, a group of proteins that binds to small, usually hydrophobic, molecules.
- The research team performed RNA blot analysis (also known as Northern blotting) to confirm the expression of the Equ c1 mRNA. They found this mRNA is expressed in the liver as well as in sublingual and submaxillary salivary glands.
Cite This Article
APA
Gregoire C, Rosinski-Chupin I, Rabillon J, Alzari PM, David B, Dandeu JP.
(1996).
cDNA cloning and sequencing reveal the major horse allergen Equ c1 to be a glycoprotein member of the lipocalin superfamily.
J Biol Chem, 271(51), 32951-32959.
https://doi.org/10.1074/jbc.271.51.32951 Publication
Researcher Affiliations
- Unité d'Immuno-Allergie, Département de Physiopathologie, Institut Pasteur, 28 rue du Dr Roux, 75024 Paris Cedex 15, France. cgreg@pasteur.fr
MeSH Terms
- Allergens / genetics
- Allergens / immunology
- Amino Acid Sequence
- Animals
- Base Sequence
- Cloning, Molecular
- DNA, Complementary / genetics
- Glycoproteins / genetics
- Glycoproteins / immunology
- Horses / genetics
- Horses / immunology
- Humans
- Lipocalins
- Lipoproteins / genetics
- Lipoproteins / immunology
- Models, Molecular
- Molecular Sequence Data
- Multigene Family
- Recombinant Proteins
- Salivary Proteins and Peptides / genetics
- Salivary Proteins and Peptides / immunology
- Sequence Homology, Amino Acid
- Solubility
- Tissue Distribution
Citations
This article has been cited 23 times.- Victor S, Lampa E, Rask Andersen A, Gafvelin G, Grönlund H, Elfman L. Measurement of Horse Allergens Equ c 1 and Equ c 2: A Comparison among Breeds.. Int Arch Allergy Immunol 2022;183(11):1166-1177.
- Virtanen T. Inhalant Mammal-Derived Lipocalin Allergens and the Innate Immunity.. Front Allergy 2021;2:824736.
- Baran Ketencioğlu B, Yilmaz I, Tutar N, Gülmez I, Oymak FS. Horse allergen sensitivity and respiratory symptoms among horse farm workers.. Turk J Med Sci 2020 Jun 23;50(4):910-916.
- Caraballo L, Valenta R, Puerta L, Pomés A, Zakzuk J, Fernandez-Caldas E, Acevedo N, Sanchez-Borges M, Ansotegui I, Zhang L, van Hage M, Abel-Fernández E, Karla Arruda L, Vrtala S, Curin M, Gronlund H, Karsonova A, Kilimajer J, Riabova K, Trifonova D, Karaulov A. The allergenic activity and clinical impact of individual IgE-antibody binding molecules from indoor allergen sources.. World Allergy Organ J 2020 May;13(5):100118.
- Haka J, Niemi MH, Mattila P, Jänis J, Takkinen K, Rouvinen J. Development of hypoallergenic variants of the major horse allergen Equ c 1 for immunotherapy by rational structure based engineering.. Sci Rep 2019 Dec 27;9(1):20148.
- Victor S, Binnmyr J, Lampa E, Rask-Andersen A, Elfman L. Levels of horse allergen Equ c 4 in dander and saliva from ten horse breeds.. Clin Exp Allergy 2019 May;49(5):701-711.
- Zahradnik E, Janssen-Weets B, Sander I, Kendzia B, Mitlehner W, May C, Raulf M. Lower allergen levels in hypoallergenic Curly Horses? A comparison among breeds by measurements of horse allergens in hair and air samples.. PLoS One 2018;13(12):e0207871.
- Curin M, Hilger C. Allergy to pets and new allergies to uncommon pets.. Allergol Select 2017;1(2):214-221.
- Hentges F, Léonard C, Arumugam K, Hilger C. Immune responses to inhalant Mammalian allergens.. Front Immunol 2014;5:234.
- Liukko AL, Kinnunen TT, Rytkönen-Nissinen MA, Kailaanmäki AH, Randell JT, Maillère B, Virtanen TI. Human CD4+ T cell responses to the dog major allergen Can f 1 and its human homologue tear lipocalin resemble each other.. PLoS One 2014;9(5):e98461.
- Zahradnik E, Raulf M. Animal allergens and their presence in the environment.. Front Immunol 2014;5:76.
- Gawlik R, Pitsch T, Dubuske L. Anaphylaxis as a manifestation of horse allergy.. World Allergy Organ J 2009 Aug;2(8):185-9.
- Hilger C, Kuehn A, Hentges F. Animal lipocalin allergens.. Curr Allergy Asthma Rep 2012 Oct;12(5):438-47.
- Liccardi G, Emenius G, Merritt AS, Salzillo A, D'Amato M, D'Amato G. Direct and indirect exposure to horse: risk for sensitization and asthma.. Curr Allergy Asthma Rep 2012 Oct;12(5):429-37.
- McDonald RE, Fleming RI, Beeley JG, Bovell DL, Lu JR, Zhao X, Cooper A, Kennedy MW. Latherin: a surfactant protein of horse sweat and saliva.. PLoS One 2009 May 29;4(5):e5726.
- Logan DW, Marton TF, Stowers L. Species specificity in major urinary proteins by parallel evolution.. PLoS One 2008 Sep 25;3(9):e3280.
- Greene LH, Chrysina ED, Irons LI, Papageorgiou AC, Acharya KR, Brew K. Role of conserved residues in structure and stability: tryptophans of human serum retinol-binding protein, a model for the lipocalin superfamily.. Protein Sci 2001 Nov;10(11):2301-16.
- Smith AM, Pomes A, Chapman MD. Molecular biology of indoor allergens.. Clin Rev Allergy Immunol 2000 Jun;18(3):265-83.
- Loebel D, Scaloni A, Paolini S, Fini C, Ferrara L, Breer H, Pelosi P. Cloning, post-translational modifications, heterologous expression and ligand-binding of boar salivary lipocalin.. Biochem J 2000 Sep 1;350 Pt 2(Pt 2):369-79.
- Rabjohn P, Helm EM, Stanley JS, West CM, Sampson HA, Burks AW, Bannon GA. Molecular cloning and epitope analysis of the peanut allergen Ara h 3.. J Clin Invest 1999 Feb;103(4):535-42.
- Santa H, Saarela JT, Laatikainen R, Rautianen J, Virtanen T, Rytkönen M, Mäntyjärvi R. A bovine dander allergen, comparative modeling, and similarities and differences in folding with related proteins.. J Protein Chem 1998 Oct;17(7):657-62.
- Musu T, Grégoire C, David B, Dandeu JP. The relationships between the biochemical properties of allergens and their immunogenicity.. Clin Rev Allergy Immunol 1997 Winter;15(4):485-98.
- Musu T, Rabillon J, Pelletier C, David B, Dandeu JP. Simultaneous quantitation of specific IgE against 20 purified allergens in allergic patients sera by checkerboard immunoblotting (CBIB).. J Clin Lab Anal 1997;11(6):357-62.
Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
