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.

Circulating Anti-Coatomer Protein Complex Subunit Epsilon (COPE) Autoantibodies as a Potential Biomarker for Cardiovascular and Cerebrovascular Events in Patients with Obstructive Sleep Apnea

“…COPI, which is among the best-characterized coat complexes, is a cytosolic complex that coats Golgi-derived vesicles and is involved in protein transport from the Golgi apparatus to the endoplasmic reticulum via recruitment by Arf1. [21][22][23] Although the precise cellular mechanisms of COPI, and circulating COPE-Ab, in patients with OSA were not revealed by the current study, lipid inflammation might be a key element associated with elevated COPE-Ab levels in this patient population. Previous studies have reported that the Arf1-COPI vesicular transport machinery regulates droplet morphology and lipid storage/utilization within the vesicle-trafficking pathway.…”

Characterization of the N-glycans of female Angiostrongylus cantonensis worms

“…In a recent proteomic study of the 31-kDa antigen from A. cantonensis, recognition of this antigen by human antibodies was found to be dependent on the carbohydrate moieties present (Morassutti et al, 2012). Correspondingly, attempts to produce recombinant antigens of Angiostrongylus in prokaryotic systems have failed (Morassutti et al, 2013), potentially due to a lack of proper glycosylation .…”

“…Previously, by used carbohydrate oxidation, our group demonstrated that glycans are essential for an immunodiagnosis of angiostrongyliasis (Morassutti et al, 2012). In the current study, we further demonstrate the importance of glycans for immunoglobulin recognition, and also show that this recognition is specifically due to N-glycans, since total abrogation of sera recognition was The MALDI-TOF spectra of the N-glycans present in the TE prepared from female A. cantonensis worms (Table 1), that were enriched using five different lectin columns, trapped a very similar set of N-glycans in each fraction.…”

“…For example, when an antigen prepared from the reproductive tubes of female A. costaricensis worms, a species which can cause abdominal angiostrongyliasis, high antigenicity was observed with human sera (Bender et al, 2003). In addition, the main diagnostic antigen for CA is a glycoprotein that is obtained from female A. cantonensis worms (Morassutti et al, 2012).…”

Molecular and pathological studies on Angiostrongylus species in southeast Queensland

“…However these signs are not specific enough to relate the symptoms to neural angiostrongyliasis. 102,103 , excretory products in a coagglutination assay 104 and AW-3C2 antigen 105 have been utilized in many recent studies to improve the sensitivity and specificity of the ELISA test.…”