The identified peptides present in the 1DE band did not correspond to the peptides identified in the 2DE gel spots. This is likely a result of the better separation obtained during 2DE, which allowed for improved isolation of the antigenic protein. A comparison of the 2DE immunoblot and the total protein stain of the 2DE gel demonstrates that most of the proteins in the 31-kDa range are not immunogenic (). Thus, mass spectrometry analysis following 2DE gel separation allowed for more specific identification of those proteins with antigenic activity. One of the peptides obtained by 1DE showed the highest Mascot score match to the 33-kDa tropomyosin from Heligmosomoides polygyrus, a rodent nematode.
Tropomyosin is a highly conserved muscle protein with potent allergenic potential. This protein is known to induce IgE production in parasitic nematode infections such as anisakiasis and onchocerciasis (Sereda et al. 2008
), but due to similarities between invertebrate tropomyosins, IgE antibodies cross-react with tropomyosins from other species, and therefore tropomyosins are not useful for diagnostic purposes (Sereda et al. 2008
). However, specificity may be further tested by epitope mapping of this protein.
The 14-3-3 proteins are dimeric phosphoserine-binding proteins, which are members of a family of acidic regulatory molecules that participate in signal transduction, transport, and regulation, of several eukaryotic biochemical processes (Obsilova et al. 2008
; Mrowiec and Schwappach 2006
). In some parasites, such as Echinococcus multilocularis
and Schistosoma mansoni
, 14-3-3 proteins have been described to be immunogenic, and therefore have been promoted as potential vaccine targets (Schechtman et al. 2001
; Siles-Lucas et al. 2008
; Wang et al. 2009
). In addition, the 14-3-3 protein has been identified as a prominent product in the S. mansoni
female worm reproductive system (Schechtman et al. 2001
). This may explain previous findings showing the female reproductive system as the main source of antigenic targets useful for the diagnosis of abdominal angiostrongyliasis caused by A. costarecensis
). Moreover, these proteins might directly interact with immune system components, since these interactions have been modulated by 14-3-3 proteins secreted by Toxoplasma gondii
and E. granulosus
(Assossou et al. 2004
; Siles-Lucas et al. 2008
Coatomer proteins (COP) form a coat protein complex that mediates protein transport between the Golgi compartment (COPI), endoplasmic reticulum (COPII), and the plasma membrane (clathrin/adaptin; Lee and Goldberg, 2010
). COPI from rat liver peroxisomes contains stoichiometric amounts of seven subunits, including alpha-COP (160
kDa), beta-COP (107
kDa), beta-prime-COP (102
kDa), delta-COP (57
kDa), epsilon-COP (36
kDa), gamma-COP (97
kDa), and zeta-COP (20
kDa; Lay et al. 2006
). To date, there is no evidence that these proteins can induce immune responses. However, a crystallographic analysis showed that the epsilon-COP and alpha-COP complex were exposed on COPI vesicles, thereby facilitating their extracellular targeting (Hsia and Hoelz, 2010
), suggesting that the complex might be attached to the Golgi membrane while transporting proteins that are eventually exposed to the host's immune system.
The nascent polypeptide-associated complex (NAC) is associated with ribosomes and involved in nascent polypeptide chain folding (Hayashi et al. 2011
). NAC is also implicated in the targeting of ribosomes to the ER membrane (Wiedmann and Prehn, 1999
31-kDa antigen was first described as a glycoprotein, and its antigenicity was considered independent of carbohydrate moieties (Eamsobhana et al. 1998
). In the present study m
-periodate treatment eliminated the recognition by sera from infected individuals (), demonstrating that carbohydrate moieties are essential for antibody recognition of the 31-kDa protein. The better separation of the proteins by 2DE allowed us to distinguish the specific antigenic spots corresponding to the previously described 31-kDa antigen, which was first detectable as a single band in 1DE preparations. Another explanation for this conflicting result is the amount of antigen loaded in the previous characterization of the 31-kDa protein, for which 10
μg were applied in the gel, while here only 3
μg in 2DE gels were loaded. The largest amount of antigen in 1DE gels could contain unspecific sugars being recognized by infected sera. This finding has strong implications for the choice of appropriate vectors to express such recombinant targets for the development of diagnostic tests.
In order to achieve the complete DNA sequence of each identified protein for further recombinant protein studies, we sequenced the DNA in a random way using the parallel sequencing approach (Morassutti et al. in preparation). The NAC domain was revealed to be composed of 185 amino acids, while 14-3-3 protein had 249 amino acids. The sequences were published in Genebank under the numbers GI: 341864443 for NAC domain-containing protein, and GI: 341864441 for 14-3-3.
Analysis of the data presented in this report raises the question of whether the reactivity observed with the native parasite 31-kDa molecules is due to reactivity with one or more of the putative proteins identified by MS/MS. Interestingly, the NAC domain-containing protein, epsilon-COPI, and 14-3-3 protein, all play putative biological roles in protein translocation. Therefore we hypothesize that they may form a cell membrane complex, which may have led to co-isolation of these proteins in the original TE preparation, and may ultimately explain how all three could be antigenic.
In conclusion, the set of proteins with an estimated molecular weight of 31
kDa identified by 2DE consisted of several potential antigens. Cloning of the corresponding cDNAs and expression of these proteins is the next critical step to further define their roles as diagnostic targets, and may represent a tool to better understand host-Angiostrongylus