Identification and Cloning of Prs a 1, a 32-kDa Endochitinase and Major Allergen of Avocado, and Its Expression in the Yeast Pichia pastoris

Abstract

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.

Dimethyl isotope labeling assisted de novo peptide sequencing

“…The gap in the sequence because of the missing c 3 -ion was compensated by the corresponding z-ion being present. The result from the ETD spectrum was confirmed by the corresponding CID spectrum (data not shown) and the peptide sequence matches to a 32 kDa avocado endochitinase [38]. The presented examples clearly demonstrate that the introduced mass signatures facilitate annotation and consequently sequence determination.…”

“…Besides the seven peptides from ascorbate peroxidase, we de novo sequenced peptides from proteins in the 26 kDa band that have high sequence homology either to chitinases or to a proteasome subunit alpha. The abundant peptide KVADR(I/L)GFY from the 31 kDa band ( Figure 6) had sequence homology with a peptide of a known and already described 32 kDa endochitinase from avocado [38].…”

Cataract surgery for natural rubber latex allergic patients

“…37 The use of recombinant NRL proteins has shed much light on the possible role of crossreaction of plant allergens and may provide a standard extract for in vivo testing in the future. 38,39 The disadvantages of in vivo testing include the risk for severe anaphylaxis in the test subject, so full resuscitation facilities must be at hand. Unfortunately, skin prick testing also suffers from reliability and specificity issues.…”

Features and Mode of Action of Cross-reactive Plant Allergens Relevant to Latex-fruit Syndrome

“…Patients sensitized to hevein (Hev b 6.02) or the N-terminal region of prohevein are therefore predicted to show cross-reactivity to class I endochitinases in various fruits and vegetables. Class I endochitinases in avocado, banana, and chestnut were indeed confirmed as cross-reactive allergens for hevein-sensitized patients Diaz-Perales et al, 1998;Mikkola et al, 1998;Sowka et al, 1998a;Blanco et al, 1999;Diaz-Perales et al, 1999;Posch et al, 1999;. The hevein-like domains of class I endochitinases are supposed to have a binding capacity to chitin, which is a major component of the external walls of pathogenic fungi (Raikhel et al, 1993;Beintema, 1994).…”

“…Likewise, antifungal endochitinases are going to be intentionally expressed in crops to enhance their resistance to infections fungi (Graham & Sticklen, 1994;Yun et al, 1997). Again, we should remember that class I endochitinases in vegetable foods as well as hevein have already been registered as cross-reactive allergens (Sowka et al, 1998a). Synergistic effects of endochitinases and ␤-1,3-glucanases against fungal infection have also been reported (Zhu et al, 1994;Jongedijk et al, 1995).…”