The 31-kDa antigen of Angiostrongylus cantonensis comprises distinct antigenic glycoproteins

Abstract

Human angiostrongyliasis results from accidental infection with Angiostrongylus, an intra-arterial nematode. Angiostrongylus cantonensis infections result in eosinophilic meningitis, and A. costaricensis infections cause eosinophilic enteritis. Immunological methodologies are critical to the diagnosis of both infections, since these parasites cannot be isolated from fecal matter and are rarely found in cerebrospinal fluid samples. A. costaricensis and A. cantonensis share common antigenic epitopes which elicit antibodies that recognize proteins present in either species. Detection of antibodies to a 31-kDa A. cantonensis protein present in crude adult worm extracts is a sensitive and specific method for immunodiagnosis of cerebral angiostrongyliasis. The objective of the present work was to isolate and characterize the 31-kDa proteins using soluble protein extracts derived from adult female worms using both one- (1DE) and two-dimensional (2DE) gel electrophoresis. Separated proteins were blotted onto nitrocellulose and probed using sera from infected and non-infected controls. The 31-kDa band present in 1DE gels and the 4 spots identified in 2DE gels were excised and analyzed by electrospray ionization mass spectrometry. Using the highest scores obtained following Mascot analysis, amino acid sequences were obtained that matched four unique proteins: tropomyosin, the 14-3-3 phosphoserine-binding protein, a protein containing a nascent polypeptide-associated complex domain, and the putative epsilon subunit of coatomer protein complex isoform 2. Oxidative cleavage of diols using sodium m-periodate demonstrated that carbohydrate moieties are essential for the antigenicity of all four spots of the 31-kDa antigen. In this article we describe the identification of the 31-kDa antigen, and provide DNA sequencing of the targets. In conclusion, these data suggest that reactivity to the 31-kDa proteins may represent antibody recognition of more than one protein, and recombinant protein-based assays for cerebral angiostrongyliasis diagnosis may require eukaryotic expression systems to maintain antigenicity.

FIG. 1.

Identification of 31-kDa molecules on 1DE. Female worm total protein extract (TE) was resolved in 1DE gel and probed on Western Blot with: lane 1, pool of positive controls for abdominal angiostrongyliasis; lane 2, pool of normal human sera. The square represents the band excised from the gel for MS analyses. (Color image available at www.liebertpub.com/vbz).

FIG. 2.

Identification of the 31-kDa protein complex on 2DE. (a) TE pH range 3–11, silver staining. (b) TE pH range 3–11 Western blot using sera derived from pooled A. costaricensis and A. cantonensis infection. (c) TE pH range 3–6 sera derived from A. costaricensis infection. (d) TE pH range 3–6 sera derived from A. cantonensis infection. (e) Normal human sera. (f) Carbohydrate oxidation. (g) Coomassie blue staining. The four spots of the 31-kDa proteins are indicated on the pH 3–11 strip. Circles represent the spots excised for MS analyses. (Color image available at www.liebertpub.com/vbz).