Identification of novel three allergens from Anisakis simplex by chemiluminescent immunoscreening of an expression cDNA library

Anisakis simplex is a representative nematode parasitizing marine organisms, such as fish and squids, and causes not only anisakiasis but also IgE-mediated allergy. Although 10 kinds of proteins have so far been identified as A. simplex allergens, many unknown allergens are considered to still exist. In this study, a chemiluminescent immunoscreening method with higher sensitivity than the conventional method was developed and used to isolate IgE-positive clones from an expression cDNA library of A. simplex. As a result, three kinds of proteins, Ani s 11 (307 amino acid residues), Ani s 11-like protein (160 residues) and Ani s 12 (295 residues), together with three known allergens (Ani s 5, 6 and 9), were found to be IgE reactive. Furthermore, ELISA data showed that both recombinant Ani s 11 and 12 expressed in Escherichia coli are recognized by about half of Anisakis-allergic patients. Ani s 11 and Ani s 11-like protein are characterized by having six and five types of short repetitive sequences (5–16 amino acid residues), respectively. Both proteins share as high as 78% sequence identity with each other and also about 45% identity with Ani s 10, which includes two types of short repetitive sequences. On the other hand, Ani s 12 is also structurally unique in that it has five tandem repeats of a CX13–25CX9CX7,8CX6 sequence, similar to Ani s 7 having 19 repeats of a CX17–25CX9–22CX8CX6 sequence. The repetitive structures are assumed to be involved in the IgE-binding of the three new allergens.

Graphical AbstractThree proteins (Ani s 11, Ani s 11-like protein and Ani s 12) were suggested to be new allergens by chemiluminescent immunoscreening of an expression cDNA library constructed from Anisakis simplex. Both recombinant Ani s 11 and 12 were confirmed to be IgE-reactive by ELISA using patient sera.Figure optionsDownload as PowerPoint slideResearch Highlights
► A chemiluminescent immunoscreening method is useful to identify new allergens.
► Ani s 11, Ani s 11-like protein and Ani s 12 are new Anisakis allergens.
► Ani s 11 and Ani s 11-like protein have several short repetitive sequences.
► Ani s 12 has five tandem repeats of a novel sequence.