Molecular cloning and expression of two new allergens from Anisakis simplex

Abstract

The nematode Anisakis simplex is a marine parasite that causes allergy as well as anisakiasis. Although five Anisakis allergens have already been identified, immunoblotting studies suggested that unidentified allergens still exist. In this study, an expression cDNA library constructed from A. simplex was subjected to immunoscreening using an Anisakis-allergic patient serum, and two positive clones coding for allergens (named Ani s 5 and 6) were obtained. Ani s 5 (152 amino acid residues) is homologous with nematode proteins belonging to the SXP/RAL-2 protein family and Ani s 6 (84 amino acid residues) with serine protease inhibitors from various animals. Of the 28 patient sera examined, seven and five reacted to recombinant Ani s 5 and 6 expressed in Escherichia coli, respectively. By inhibition immunoblotting experiments using the recombinant allergens as inhibitors, natural Ani s 5 could be identified as a 15-kDa protein in the crude extract of A. simplex but natural Ani s 6 could not be identified probably due to its low expression. In conclusion, Ani s 5 and 6 are new allergens of A. simplex that are specific to some Anisakis-allergic patients.

Anisakis simplex: from Obscure Infectious Worm to Inducer of Immune Hypersensitivity

“…Four major protease inhibitor classes have been identified (Table 3) according to the catalytic type of protease inhibited, namely, (i) serine (serpins and smapins), (ii) cysteine (stefins, cystatins, and kininogens), (iii) aspartic (aspins), and (iv) metalloproteases (reviewed by Knox [156]). Very recently, Ani s 4 and Ani s 6 (Table 4) have been found to be the first protease inhibitors of cystatin and serine classes, respectively, of the class Nematoda that cause allergy in humans (158,229). Until now, only two protease inhibitors (belonging to the aspin and cystatin classes) had been identified in nematodes as major allergens for sheep (245) and rodents (111), respectively.…”

“…Ani s 4 (9 kDa) and Ani s 6 are ES allergens and are inhibitors of cysteine and serine protease, respectively, and they were the first nematode protease inhibitors found to be allergens to humans (158,229). Ani s 4 belongs to the cystatin family of cysteine protease inhibitors, both by its sequence similarity to other cystatins and by its experimentally demonstrable protease activity (against papain) (229).…”

“…It is located both in the excretory gland and below the cuticle, and it is heat stable (boiling for 30 min) and resistant to pepsin digestion (48). Ani s 6 is not a major allergen, and it is similar in sequence to serine protease inhibitors of other animals and inhibits ␣-chymotrypsin, but not trypsin, in a dose-dependent manner (158).…”

“…Ani s 5 (15 kDa) and Ani s 8 (15 kDa) are heat-stable ES allergens that show antigenic cross-reactivity and share amino acid sequence similarity with several members of the nematode SXP/RAL-2 protein family (158,159). Ani s 5 is not a major allergen of A. simplex (158).…”

“…Ani s 5 is not a major allergen of A. simplex (158). Ani s 7 is a glycoprotein present in ES and is the target of the UA3 MAb, and it seems to be recognized by 100% of allergic patients (228).…”