Hydrophobic interaction chromatography for isolation and purification of Equ.cl, the horse major allergen

Dandeu, J P; Rabillon, J.; Divanovic, A.; Carmi–Leroy, Annick; David, B.

doi:10.1016/0378-4347(93)80072-c

Cited by 35 publications

(34 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…fied from salivary glands and dander extracts by a combination of size exclusion chromatography in fast protein liquid chromatography (FPLC) and hydrophobic interaction chromatography as described previously (17).…”

Section: Methodsmentioning

confidence: 99%

“…In contrast, the N-terminal end of the protein initially purified from hair dandruff (SDVAI) would result from a cleavage between Asn 20 and Ser 21 , which is not predicted by Von Heijne's rules. Equ c1 was purified from saliva, and the microsequencing of its N-terminal peptide revealed a mixture of three sequences, one of them beginning at the predicted Gln 16 , but the others at Glu 18 and Ser 21 , respectively (Fig. 3B).…”

Section: Molecularmentioning

confidence: 99%

“…The major horse allergen, Equ c1, is a potent allergen responsible for about 80% of anti-horse IgE antibody response in patients who are chronically exposed to horse allergens. Although much work has been carried out on the isolation and identification of the horse allergenic agents responsible for human hypersensitivity response (12)(13)(14)(15)(16), the major horse allergen was only recently purified from hair and dandruff (17). A previous study by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing-PAGE showed that Equ c1 appears as a single polypeptide with a relative molecular mass of 21,500 daltons and a pI of 3.9.…”

mentioning

confidence: 99%

“…A previous study by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing-PAGE showed that Equ c1 appears as a single polypeptide with a relative molecular mass of 21,500 daltons and a pI of 3.9. The purification of Equ c1 allowed the sequencing of the 27 N-terminal amino acids and of internal peptides (18).…”

mentioning

confidence: 99%

See 3 more Smart Citations

cDNA Cloning and Sequencing Reveal the Major Horse Allergen Equ c1 to Be a Glycoprotein Member of the Lipocalin Superfamily

Grégoire¹,

Rosinski‐Chupin

Rabillon³

et al. 1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

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.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Molecularmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

cDNA Cloning and Sequencing Reveal the Major Horse Allergen Equ c1 to Be a Glycoprotein Member of the Lipocalin Superfamily

Grégoire¹,

Rosinski‐Chupin

Rabillon³

et al. 1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The animals responsible for allergy are obviously familiar domestic cats and dogs, but also the horse, which is often incriminated. Five main horse allergens have been isolated and purified (1,2). Among these, the Equ c 1 protein (molecular mass 21.5 kDa, 187 amino acids, pI ϭ 4.5) has been defined as a major allergen, because it induces an IgE-mediated type I allergic reaction in a majority of the patients allergic to horses.…”

mentioning

confidence: 99%

Crystal Structure of the Allergen Equ c 1

Lascombe¹,

Grégoire²,

Poncet³

et al. 2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

The three-dimensional structure of the major horse allergen Equ c 1 has been determined at 2.3 Å resolution by x-ray crystallography. Equ c 1 displays the typical fold of lipocalins, a ␤-barrel flanked by a C-terminal ␣-helix. The space between the two ␤-sheets of the barrel defines an internal cavity that could serve, as in other lipocalins, for the binding and transport of small hydrophobic ligands. Equ c 1 crystallizes in a novel dimeric form, which is distinct from that observed in other lipocalin dimers and corresponds to the functional form of the allergen. Binding studies of point mutants of the allergen with specific monoclonal antibodies raised in mouse and IgE serum from horse allergic patients allowed to identify putative B cell antigenic determinants. In addition, total inhibition of IgE serum recognition by a single specific monoclonal antibody revealed the restricted nature of the IgE binding target on the molecular surface of Equ c 1.The incidence of allergic diseases is increasing in developed countries resulting from growing exposure to allergens, altered stimulation of the immune system during development, and probably facilitating an adjuvant effect of the environment. Allergy to animals is distinguishable by its intensity and the possibility of sensitization by a limited contact with danders or hair. The animals responsible for allergy are obviously familiar domestic cats and dogs, but also the horse, which is often incriminated. Five main horse allergens have been isolated and purified (1, 2). Among these, the Equ c 1 protein (molecular mass 21.5 kDa, 187 amino acids, pI ϭ 4.5) has been defined as a major allergen, because it induces an IgE-mediated type I allergic reaction in a majority of the patients allergic to horses.Equ c 1 belongs to the lipocalin family (3, 4). Although members of this family display low sequence similarity (often lower than 20% of amino acid identities), all share a conserved folding pattern, an 8-stranded ␤-barrel flanked by an ␣-helix at the C-terminal end of the polypeptide chain. The lipocalin ␤-barrel often defines a central apolar cavity that serves for the binding and transport of small hydrophobic molecules such as retinol (retinol-binding protein (5, 6)), odorant molecules (bovine odorant-binding protein (7, 8)), or pheromones (mouse major urinary protein, mMUP 1 (9), and rat urinary ␣2-globulin (10)). Several lipocalins have been described as allergenic proteins, among which the mouse major urinary protein mMUP (11), rat urinary ␣2-globulin rat urinary ␣2-globulin (12), bovine ␤-lactoglobulin (13), cockroach allergen Bla g 4 (14), dog allergens Can f 1 and Can f 2 (15), and bovine allergen Bos d 2 (16).Whether a protein exhibits special structural characteristics that are responsible for its allergenic properties is an issue that remains poorly understood. Although the three-dimensional structures of some allergenic lipocalins are known (mMUP (9), rat urinary ␣2-globulin (10), bovine ␤-lactoglobulin (17, 18), and Bos d 2 (19)), little information is availa...

show abstract

Simultaneous quantitation of specific IgE against 20 purified allergens in allergic patients sera by checkerboard immunoblotting (CBIB)

Musu

Rabillon

Pelletier

et al. 1997

J. Clin. Lab. Anal.

View full text Add to dashboard Cite

A simple technique, checkerboard immunoblotting (CBIB), is described for simultaneous quantitation of specific IgE antibodies against several allergens in human sera. Using as little as 50 μl of each of the 20 sera examined against 20 different allergens, it was possible in a single run to achieve 400 tests. To guarantee high specificity and sensitivity of the assay, this new application of CBIB employs purified allergens, cyanogen bromide‐activated nitrocellulose membrane, and Phosphorimager technology. Results are expressed both qualitatively (five classes) and quantitatively in kilo units per liter equilibrated against the World Health Organization (WHO) standard for IgE. There was excellent agreement between the results of CBIB and the results of Pharmacia Cap System, an alternative method widely used for measuring serum‐specific IgE. The CBIB method certainly could be useful in any laboratory interested in allergy clinical research for easy screening and relative quantitation of allergen‐specific human IgE. J. Clin. Lab. Anal. 11:357–362, 1997.© 1997 Wiley‐Liss, Inc.

show abstract

Hydrophobic interaction chromatography for isolation and purification of Equ.cl, the horse major allergen

Cited by 35 publications

References 16 publications

cDNA Cloning and Sequencing Reveal the Major Horse Allergen Equ c1 to Be a Glycoprotein Member of the Lipocalin Superfamily

cDNA Cloning and Sequencing Reveal the Major Horse Allergen Equ c1 to Be a Glycoprotein Member of the Lipocalin Superfamily

Crystal Structure of the Allergen Equ c 1

Simultaneous quantitation of specific IgE against 20 purified allergens in allergic patients sera by checkerboard immunoblotting (CBIB)

Contact Info

Product

Resources

About