The antibody response was followed weekly during 68 weeks in 17 Balb/c mice intraperitoneally (i.p.) infected with 2000 Echinococcus granulosus protoscoleces (PSC) and in three mice i.p. immunized with 2000 dead PSC. Antibodies against hydatid cyst fluid (HCFA) and its peptidic (periodate-resistant) and carbohydrate (periodate-sensitive) epitopes were titrated by ELISA. Avidity and the antigen recognition pattern of antibodies were also analysed during infection and immunization by ELISA and immunoblot, respectively. The antibody response of infected mice showed quantitative and qualitative variations during infection, since both titre as well as recognition of peptide and carbohydrate epitopes in HCFA depended on time post infection. No avidity maturation was evident during the course of infection. Sera from infected mice recognized the 38 kDa subunit of Ag5 but did not react with the 8 kDa subunit of AgB. On the contrary, the antibodies response of immunized mice showed only one peak of antibodies that recognized both peptidic and carbohydrate epitopes of HCFA. In addition, sera from these mice recognized mainly 60 and 110 kDa bands. Our results suggest that: a) avidity and antigen recognition patterns of antibodies in mice treated with live PSC are different from those treated with dead PSC; b) antibodies against HCFA glucidic or peptidic epitopes appear at different times post infection.
We have compiled MultitaskProtDB, available online at http://wallace.uab.es/multitask, to provide a repository where the many multitasking proteins found in the literature can be stored. Multitasking or moonlighting is the capability of some proteins to execute two or more biological functions. Usually, multitasking proteins are experimentally revealed by serendipity. This ability of proteins to perform multitasking functions helps us to understand one of the ways used by cells to perform many complex functions with a limited number of genes. Even so, the study of this phenomenon is complex because, among other things, there is no database of moonlighting proteins. The existence of such a tool facilitates the collection and dissemination of these important data. This work reports the database, MultitaskProtDB, which is designed as a friendly user web page containing >288 multitasking proteins with their NCBI and UniProt accession numbers, canonical and additional biological functions, monomeric/oligomeric states, PDB codes when available and bibliographic references. This database also serves to gain insight into some characteristics of multitasking proteins such as frequencies of the different pairs of functions, phylogenetic conservation and so forth.
The antibody response was followed during 68 weeks in 17 Balb/c mice intraperitoneally (i.p.) infected with Echinococcus granulosus protoscoleces (PSC) and in three mice i.p. immunized with dead PSC. Titres of antibodies recognizing peptidic and glucidic PSC epitopes, as well as their isotypic and avidity profiles were followed by ELISA. In addition, antigen recognition patterns were analysed by immunoblot. The response against carbohydrate epitopes was dominant in infected and immunized mice but stronger in the first group. Infected mice showed similar profiles of specific IgG and IgM with maximum titres from week 38 to 53. Although IgG1 and IgG3 were the predominant antibody subclasses, the ratio of IgG1/IgG3 antibody titres as well as antibody avidity decreased during the experiment, encompassing a decrease in recognition of peptidic epitopes. Immunized mice did not show significant levels of specific IgM and, after week 15, showed IgG titres lower than the infected mice. IgG1 was the predominant IgG subclass during all the experiment with background levels of IgG3. The mean Ab avidity was high and showed no significant changes during immunization. Different patterns of response were thus produced by dead and developing live parasites. Although high avidity IgG1 antibodies were early found in both cases, lower avidity IgG3 antibodies were increasingly produced afterwards only in infected animals. The isotype switch and avidity decrease observed only during infection are consistent with a possible parasitic mechanism to evade host immunity.
Multitasking, or moonlighting, is the capability of some proteins to execute two or more biological functions. MultitaskProtDB-II is a database of multifunctional proteins that has been updated. In the previous version, the information contained was: NCBI and UniProt accession numbers, canonical and additional biological functions, organism, monomeric/oligomeric states, PDB codes and bibliographic references. In the present update, the number of entries has been increased from 288 to 694 moonlighting proteins. MultitaskProtDB-II is continually being curated and updated. The new database also contains the following information: GO descriptors for the canonical and moonlighting functions, three-dimensional structure (for those proteins lacking PDB structure, a model was made using Itasser and Phyre), the involvement of the proteins in human diseases (78% of human moonlighting proteins) and whether the protein is a target of a current drug (48% of human moonlighting proteins). These numbers highlight the importance of these proteins for the analysis and explanation of human diseases and target-directed drug design. Moreover, 25% of the proteins of the database are involved in virulence of pathogenic microorganisms, largely in the mechanism of adhesion to the host. This highlights their importance for the mechanism of microorganism infection and vaccine design. MultitaskProtDB-II is available at http://wallace.uab.es/multitaskII.
Multitasking or moonlighting is the capability of some proteins to execute two or more biochemical functions. Usually, moonlighting proteins are experimentally revealed by serendipity. For this reason, it would be helpful that Bioinformatics could predict this multifunctionality, especially because of the large amounts of sequences from genome projects. In the present work, we analyze and describe several approaches that use sequences, structures, interactomics, and current bioinformatics algorithms and programs to try to overcome this problem. Among these approaches are (a) remote homology searches using Psi-Blast, (b) detection of functional motifs and domains, (c) analysis of data from protein–protein interaction databases (PPIs), (d) match the query protein sequence to 3D databases (i.e., algorithms as PISITE), and (e) mutation correlation analysis between amino acids by algorithms as MISTIC. Programs designed to identify functional motif/domains detect mainly the canonical function but usually fail in the detection of the moonlighting one, Pfam and ProDom being the best methods. Remote homology search by Psi-Blast combined with data from interactomics databases (PPIs) has the best performance. Structural information and mutation correlation analysis can help us to map the functional sites. Mutation correlation analysis can only be used in very specific situations – it requires the existence of multialigned family protein sequences – but can suggest how the evolutionary process of second function acquisition took place. The multitasking protein database MultitaskProtDB (), previously published by our group, has been used as a benchmark for the all of the analyses.
The aim of this work was to assess the influence in the diagnostic value for human hydatid disease of the composition of bovine hydatid cyst fluid (BHCF) obtained from fertile (FC) and non-fertile cysts (NFC). Eight batches from FC and 5 from NFC were prepared and analysed with respect to chemical composition: total protein, host-derived protein, carbohydrate and lipid contents. No differences were observed in the first two parameters but carbohydrate and lipid contents were shown to be higher in batches from FC than in those from NFC. Bands of 38 and 116 kD in SDS-PAGE profiles were observed to be present in BHCF from FC only. Two pools were prepared from BHCF batches obtained from FC (PFC) and NFC (PNFC), respectively. Antigen recognition patterns were analysed by immunoblot. Physicochemical conditions for adsorption of antigens to the polystyrene surface (ELISA plates) were optimized. The diagnostic value of both types of BHCF as well as the diagnostic relevance of oxidation of their carbohydrate moieties with periodate were assessed by ELISA using 42 serum samples from hydatid patients, 41 from patients with other disorders, and 15 from healthy donors. Reactivity of all sera against native antigen were tested with and without free phosphorylcholine. The best diagnostic efficiency was observed using BHCF from periodate-treated PFC using glycine buffer with strong ionic strength to coat ELISA plates.
We analysed specific antibody (Ab) and circulating antigen (CAg) profiles along experimental mouse infection using as control a group of mice immunized with intact but dead parasites. Results from this experiment showed an early major CAg peak followed by a larger Ab peak which partially overlaps with other minor CAg peaks. These results suggest that CAg may be a marker of early mouse infection. In order to study the relevance of these findings in humans we similarly analysed by ELISA 148 sera provided by retrospective post-surgical follow-up of 19 patients. Available records showed that 14 patients developed new cysts one to ten years after surgery while no new disease was observed in the-other five. Some of the former patients showed CAg, as early as two months after surgery while no CAg was observed in the other five patients at any time. In addition, a collection of 38 sera obtained before surgery were similarly tested and five of them showed only CAg, while 18 showed only Ab and 12 sera showed Ab&CAg. These results in humans are consistent with the findings in the mouse experimental model and suggest that CAg may be an early marker of hydatid infection, thus being relevant for post-surgical follow-up.
Protein multitasking or moonlighting is the capability of certain proteins to execute two or more unique biological functions. This ability to perform moonlighting functions helps us to understand one of the ways used by cells to perform many complex functions with a limited number of genes. Usually, moonlighting proteins are revealed experimentally by serendipity, and the proteins described probably represent just the tip of the iceberg. It would be helpful if bioinformatics could predict protein multifunctionality, especially because of the large amounts of sequences coming from genome projects. In the present article, we describe several approaches that use sequences, structures, interactomics and current bioinformatics algorithms and programs to try to overcome this problem. The sequence analysis has been performed: (i) by remote homology searches using PSI-BLAST, (ii) by the detection of functional motifs, and (iii) by the co-evolutionary relationship between amino acids. Programs designed to identify functional motifs/domains are basically oriented to detect the main function, but usually fail in the detection of secondary ones. Remote homology searches such as PSI-BLAST seem to be more versatile in this task, and it is a good complement for the information obtained from protein-protein interaction (PPI) databases. Structural information and mutation correlation analysis can help us to map the functional sites. Mutation correlation analysis can be used only in very restricted situations, but can suggest how the evolutionary process of the acquisition of the second function took place.
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