Immunoreactive Proteins in the Esophageal Gland Cells of Anisakis Simplex Sensu Stricto Detected by MALDI-TOF/TOF Analysis.

Lee Robertson,María Del Carmen Mena,Noelia Carballeda-Sanguiao,Alfonso Navas,Mercedes Careche,Susana C Arcos,Miguel Gonzalez-Muñoz,Sergio Ciordia,Isabel Sánchez-Alonso

doi:10.3390/genes11060683

Abstract

In plant and animal nematode parasites, proteins derived from esophageal gland cells have been shown to be important in the host-nematodes relationship but little is known about the allergenic potential of these proteins in the genus Anisakis. Taking into account the increase of anisakiasis and allergies related to these nematodes, immunoreactive properties of gland cell proteins were investigated. Two hundred ventricles were manually dissected from L3 stage larvae of Aniskakis simplex s.s. to allow direct protein analysis. Denaturing gel electrophoresis followed by monochromatic silver staining which revealed the presence of differential (enriched) proteins when compared to total nematode extracts. Such comparison was performed by means of 1D and 2D electrophoresis. Pooled antisera from Anisakis spp.-allergic patients were used in western blots revealing the presence of 13 immunoreactive bands in the ventricular extracts in 1D, with 82 spots revealed in 2D. The corresponding protein bands and spots were excised from the silver-stained gel and protein assignation was made by MALDI-TOF/TOF. A total of 13 (including proteoforms) were unambiguously identified. The majority of these proteins are known to be secreted by nematodes into the external environment, of which three are described as being major allergens in other organisms with different phylogenetic origin and one is an Anisakis simplex allergen.

Highlights

Humans are incidental hosts of Anisakis simplex and become infected through eating live L3 larvae found in raw or undercooked fish and cephalopod meat, developing the disease known as anisakiosis or anisakiasis
Differences are apparent between the total extracts depending of the applied method (T1 or T2) when same amount of protein is used; the same bands pattern is detected comparing total extracted proteins (TE2 ) and gland cells (GC1 an GC2 ) extracted in phosphate buffer when equal quantities of protein were loaded in each well
The SDS-PAGE analysis of the proteins extracted from total nematodes and those extracted from the gland cells clearly show differences in protein banding

Summary

Introduction

Humans are incidental hosts of Anisakis simplex and become infected through eating live L3 larvae found in raw or undercooked fish and cephalopod meat, developing the disease known as anisakiosis or anisakiasis. To date there are 19 described allergens in Anisakis [8]; a recent proteomic study combining 2D gel analysis and western blotting described 28 immunoreactive proteins present in of the species complex (A. simplex, A. pegreffii, and their hybrid), including intraspecies variations which could be assessed as potential allergens. None of these new potential allergens are considered as members of the protein secretome and were recovered in the whole-body extract of assayed species complex [9]

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