Identification and cloning of prs a 1, a 32-kDa endochitinase and major allergen of avocado, and its expression in the yeast Pichia pastoris.
Avocado, the fruit of the tropical tree Persea americana, is a source of allergens that can elicit diverse IgE-mediated reactions including anaphylaxis in sensitized individuals. We characterized a 32-kDa major avocado allergen, Prs a 1, which is recognized by 15 out of 20 avocado- and/or latex-allergic patients. Natural Prs a 1 was purified, and its N-terminal and two tryptic peptide sequences were determined. We isolated the Prs a 1 encoding cDNA by PCR using degenerate primers and 5'-rapid amplification of cDNA ends. The Prs a 1 cDNA coded for an endochitinase of 326 amino acids with a leader peptide of 25 amino acids. We expressed Prs a 1 in the yeast Pichia pastoris at 50 mg/liter of culture medium. The recombinant Prs a 1 showed endochitinase activity, inhibited growth and branching of Fusarium oxysporum hyphae, and possessed IgE binding capacity. IgE cross-reactivity with latex proteins including a 20-kDa allergen, most likely prohevein, was demonstrated, providing an explanation for the commonly observed cross-sensitization between avocado and latex proteins. Sequence comparison showed that Prs a 1 and prohevein had 70% similarity in their chitin-binding domains. Characterization of chitinases as allergens has implications for engineering transgenic crops with increased levels of chitinases.
Department of General and Experimental Pathology, University of Vienna, AKH-EBO-3Q, Waehringer Guertel 18-20, A-1090 Vienna, Austria.
S
S
Sowka
L S
LS
Hsieh
M
M
Krebitz
A
A
Akasawa
B M
BM
Martin
D
D
Starrett
C K
CK
Peterbauer
O
O
Scheiner
H
H
Breiteneder
D000485:Allergens / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D001483:Base Sequence; D002688:Chitinases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D003429:Cross Reactions; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D006967:Hypersensitivity; D007073:Immunoglobulin E / Q000378:metabolism; D020315:Latex Hypersensitivity; D019770:Lauraceae / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D010940:Plant Proteins / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000485:Allergens; D052179:Antigens, Plant; D018076:DNA, Complementary; D010940:Plant Proteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D002688:Chitinases; C115191:Prs a 1 allergen