Complement C9 binding site and the anti-microbial activity of caprine vitronectin are localized in close proximity in the N-terminal region of the protein.

Vitronectin (Vn) is a ligand for complement C9 and modulates its activity that favors bacterial growth and survival. At the same time, the anti-microbial activity of the heparin-binding region of human Vn has been documented. To understand these diverse and opposite functions of the protein, we have analyzed the interaction of caprine Vn with C9 in the homologous system. In a previous study, the C9 binding activity was mapped to the N-fragment of the caprine Vn (N-Vn), representing the first 200 amino acids. Interestingly, this fragment also inhibited bacterial growth. In this study, we have generated four sub-fragments of N-Vn and analyzed C9 binding by ELISA, blot overlay, surface plasmon resonance and circular dichroism spectroscopy. These sub-fragments were also tested for antimicrobial activity against E. coli and S. aureus by drop plate method and analyzing cell death by flow cytometry. Results of these analyses together with previous data suggest that in addition to the second RGD motif (106-108 amino acids), the first 47 residues are also required for C9 binding. The anti-microbial tests employed indicate that the growth inhibitory property is contributed by 101-150 residues of Vn. These results provide an initial insight into two diverse Vn functions.

Staphylococcus aureus autolysins interact with caprine vitronectin without involving the heparin binding domain and the second arginine-glycine-aspartic acid motif of the host protein.

Many bacteria exploit host proteins for their colonization. Vitronectin (Vn), present in the blood and extracellular matrix, is one such protein that acts as a bridge between the bacteria and the host tissues leading to infection. In this study, Vn binding protein of Staphylococcus aureus (COL strain) (SaVnBP) has been characterized as autolysin(s) based on mass spectrometry data and the ability of these proteins to degrade S. aureus substratum. Deletion of the heparin-binding domain (residues 341-380) from the Vn did not affect its ability to interact with SaVnBP. Similarly, change of R to A or D to A in the second arginine-glycine-aspartic (RGD2) motif of Vn had no negative effect on protein-protein interaction. These results imply that the primary heparin-binding site and the second RGD motif of caprine Vn may not be involved in the initial step of S. aureus colonization.

Defining the complement C3 binding site and the antigenic region of Haemonchus contortus GAPDH.

Haemonchus contortus is an economically important parasite that survives the host defense system by modulating the immune response. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is secreted by the parasite and the host responds by producing anti-enzyme antibodies. The enzyme inhibits complement cascade, an arm of the innate immunity, by binding to complement C3. In this study, the C3 binding site and the antigenic region of the enzyme were identified by generating short recombinant fragments and deleting a defined region of the enzyme. Using these proteins in ligand overlay and plate binding assay, the C3 binding region of GAPDH was localized within the 38 residues represented by 77-114 amino acids whereas one of the antigenic regions was identified in between 77 and 171 amino acids. In addition, deletion of amino acids 77 to 171 from GAPDH (fragment AB) also showed weak immunogenicity but lacked C3 binding activity. Fragment D comprising 95 residues (77-171), had both the C3 binding activity as well as immunogenicity like the parent enzyme, also stimulated host peripheral blood mononuclear cells in vitro. This truncated GAPDH moiety was stable at refrigerated temperature for at least 12 weeks and appears as a promising new therapeutic tool considering its longer shelf life as compared to the parent protein.

Functional Diversity of the Excretory/Secretory Proteins of Nematode Parasites.

INTRODUCTION: Parasites release a wide array of protein as excretory and secretory products (ESPs). Irrespective of their mode of propagation, ESPs are found to be secreted or excreted by both naturally occurring and laboratory-cultivated parasites. Mass spectrometry-based approaches have been extensively used to identify and characterize the ESP constituents. ESPs are involved in various cellular activities such as immune modulation, proteolysis, inhibition of proteases and protection of cells against oxidants. Specifically, their role in host immune evasion by down-regulation of pro-inflammatory cytokines and up-regulation of anti-inflammatory cytokines attracts scientific attention. A thorough investigation of functional diversity of ESPs may be helpful in planning control strategies against many parasites. METHODS: This review focuses on diversity of ES proteins, various approaches to identify them and discusses about the biochemical and functional aspects of such proteins. RESULTS: The diverse array of proteins secreted or excreted (a, GST-1, acetylcholinesterase, GAPDH) by the parasites are also described emphasizing their role in cellular physiology. CONCLUSION: Finally, it concludes by citing some of these proteins as potential therapeutic agents against helminth challenge.

Enhancing the Stability of Haemonchus contortus Glyceraldehyde-3-Phosphate Dehydrogenase and Binding of Host Albumin to the Parasite Enzyme.

INTRODUCTION: Haemonchus contortus is an economically important parasite of domestic animals. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) excreted in the ES product of H. contortus can be a promising vaccine candidate for controlling the parasite infection. Unfortunately, the parasite enzyme breaks down rapidly. The current study focusses on stabilizing the recombinant GAPDH (rGAPDH) of H. contortus. METHODS: The rGAPDH was purified and stored in two different buffers (sodium phosphate + EDTA and bicarbonate-sodium chloride) to check the stability. The affinity of the parasite enzyme towards host serum (Goat) components was evaluated by affinity chromatography. The interacting component was identified by mass spectrometry. RESULTS: Here, we report that the enzyme can be stabilized for at least 3 months if stored in bicarbonate-sodium chloride. This should facilitate testing of the enzyme in challenge trials. Additionally, we show that the parasite enzyme has affinity for host albumin; this interaction may have significance in host-parasite relationship. CONCLUSION: The present study reports a combination of sodium bicarbonate (0.1 M) with 0.5 M sodium chloride as a suitable buffer to enhance the stability of H. contortus GAPDH.

Role of low ouabain-sensitive isoform of Na+-K+-ATPase in the regulation of basal tone and agonist-induced contractility in ovine pulmonary artery.

Although Na-K-ATPase plays an important role in vascular smooth muscle function, it is unknown which isoforms of the enzyme are present in the pulmonary vasculature and whether they possess different affinities for ouabain. Unlike rodents, Na-K-ATPase in sheep and humans displays greater affinity for ouabain. Thus, the present study examined the presence of various isoforms of the enzyme by Western blot analysis and their sensitivity to inhibition by ouabain (biochemical estimation of enzyme activity/K-relaxations) in the ovine pulmonary artery. Further, we studied the effect of ouabain on the basal tone and agonist-induced contractions in this vessel. Our results show the presence of both alpha1 and alpha2 isoforms of Na-K-ATPase in this vessel. The biphasic shape of the ouabain inhibition curve indicates that the alpha1 and alpha2 isoforms of the enzyme may possess low and high affinity, respectively, for the cardiac glycoside. Concentrations of ouabain <1 microM had no significant effect on the basal tone of the vessel. At 1 microM concentration, however, there was a significant increase in the basal tension (55% of 5-HT 1 microM contraction). Ouabain (0.1 microM) selectively increased the vasoconstrictor potency of 5-HT (pD2 6.81 +/- 0.10 versus control pD2 5.95 +/- 0.07), but not that of phenylephrine (pD2 5.80 +/- 0.07 versus control pD2 6.05 +/- 0.05). Neither endothelium removal nor treatment with PKG inhibitor KT 5823 (2 microM) had any effect on the sodium pump function. These results indicate that the low, but not the high, ouabain-sensitive isoform of Na-K-ATPase regulates basal tone and agonist-induced contractility in the ovine pulmonary artery.

Goat vitronectin: characterization and binding to Staphylococcus aureus.

Vitronectin (Vn) is a multifunctional protein present in plasma and in the extracellular matrix. Previous studies have demonstrated binding of many bacteria to human Vn. In this study, we have characterized goat Vn and studied its interaction with S. aureus considering the importance of this bacterium in animal husbandry. Goat Vn possesses two RGD motifs, at positions 45 and 106, and two multimerization sites that were identified from the recombinant fragments of the protein. The first site was localized at the N-terminus of the protein and the second at the C-terminus that did not require a full heparin-binding region, as partial deletion of this site did not affect multimerization. The 40 kDa N-terminal fragment, Vn1-200, supported S. aureus binding. Similarly, two fragments representing the C-terminus of the protein (35 kDa Vn183-444 and the 22 kDa Vn323-444) with complete heparin-binding site also supported S. aureus binding whereas the 14 kDa fragment, Vn363-444, with truncated heparin-binding site did not. Thus, a complete heparin-binding site at the C-terminus of Vn is essential for S. aureus binding. Maximum S. aureus binding was observed with Vn isolated by immunoaffinity chromatography, which predominantly consisted of multimers. This observation is significant considering the fact that the multimeric Vn is a component of the matrix surrounding the cells and may play an important part in initial bacterial adhesion and subsequent colonization.

Characterization of Haemonchus contortus calreticulin suggests its role in feeding and immune evasion by the parasite.

Haemonchus contortus, a gastrointestinal parasite of sheep and goat feeds on the blood of its host and causes bleeding at the biting site. In this report, we demonstrate that the Ca2+ binding protein, calreticulin (CalR), is present in excretory/secretory products of adult worms. The secreted CalR enhanced plasma coagulation time. Using recombinant fragments, this property has been mapped to C-terminal part of the molecule which has binding sites for Ca2+ as well as clotting factors. Complement protein C1q bound to immobilized CalR and C1q dependent lysis of sensitized sheep erythrocytes was inhibited by CalR, a function mapped to N-domain of the protein. Factor X and a 24 kDa polypeptide derived from prothrombin but not prothrombin bound to immobilized CalR. The binding site for 24 kDa polypeptide in the CalR molecule has been localized in the P-domain. Our results suggest at least two functions for secreted CalR: first, to prevent blood clotting by binding to Ca2+ and clotting factors thus enabling parasite to feed on the host blood and second to modulate the host immune response by binding to complement C1q thereby facilitating parasite's survival within the host.

Characterization of goat plasma vitronectin.

Vitronectin (VN) was isolated and characterized from goat plasma in native and denatured state. Native VN consisted of 160 and >250 kDa polypeptides, whereas denatured VN showed bands of 81 and >250 kDa on SDS-gel. Storage of 81 kDa polypeptide for 3 days at 4 degrees C resulted in formation of 160 and >250 kDa proteins. Hence high molecular weight forms of VN may be dimer and multimeric forms of 81 kDa monomer. Both native as well as denatured VN showed cell adhesive activity. Cells bound to native VN were round, whereas cells adhered to denatured VN were fully spread, a characteristic also observed with 81 kDa polypeptide. The 81 kDa VN bound to Heparin, whereas the 160 kDa preparation did not bind to Heparin in presence of urea. Absence of EDTA resulted in the degradation of goat VN. Similarly, addition of excess Ca(2+) caused total degradation of VN polypeptides in buffers with EDTA, suggesting metalloprotease activity inthe protein.

Sero-surveillance for surra in cattle using native surface glycoprotein antigen from Trypanosoma evansi.

Surra, caused by Trypanosoma evansi affects a wide range of domestic and wild animals in the tropics, taking a huge toll on the already impoverished economy here. In bovines surra normally develops into a chronic infection that is often associated with severe production losses, yet with no distinct clinical signs making its adequate diagnosis vital. Though direct microscopic observation of T. evansi in circulation may be the diagnostic gold standard for surra, it is insensitive and impractical for population prevalence studies, making sero-diagnosis the preferred choice for the latter. In this study, we standardize an ELISA with Concanavalin-A (Con-A) affinity purified T. evansi surface glycoprotein antigen and compare its sensitivity and specificity to direct microscopy of stained thin smears and molecular (PCR) diagnostics. The ELISA was then put on field trial for sero-surveillance of cattle for surra in three geographically distinct populations in the Indian subcontinent, to yield an overall sensitivity and specificity of 100% and 89.15% compared to standard stained thin smear examinations and 95.23% and 90.84% compared to blood PCR examinations.

Interferon stimulated gene 15 (ISG15): molecular characterization and expression profile in endometrium of buffalo (Bubalus bubalis).

Interferon stimulated gene 15 (ISG15), one of the several proteins induced by conceptus derived Type I and/or a Type II interferon (IFN), is implicated as an important factor in determining the uterine receptivity and conceptus development. However, presence as well as specific role of the ISG15 in buffalo (Bubalus bubalis) reproduction is yet to be elucidated. In the present study, both genomic and cDNA sequences of bubaline (bu) ISG15 were cloned and investigated for its expression in different tissues of female reproductive tract of buffalo. Sequence analysis revealed 100% identity among the genomic sequences (1014 bp) of buISG15 from three different breeds of buffalo (viz., Murrah: Acc. No. DQ118137, Mehsana: Acc. No. DQ118138, and Nagpuri: Acc. No. DQ118136) and cDNAs (Acc. Nos. HM543268-HM543270). As in cattle, the buISG15 was comprised of two exons of 57 bp and 520 bp encoding a peptide of 154 amino acids. Moreover, the buISG15 cDNA sequence exhibited 98.3% and 98.5% identity with that of taurine and indicine cattle, respectively. Subsequent reverse transcription PCR analysis revealed expression of the buISG15 in the uterine endometrium, corpus luteum (CL), corpus hemorrhagicum and oviduct. Quantitative Real Time PCR (RTqPCR) analysis also confirmed the constitutive expression of the buISG15 in the uterine endometrium during different stages (i.e. estrus, diestrus and proestrus) of estrous cycle and also during early (∼d 30-40) pregnancy. Western blot analysis of the endometrial extract from both estrous cyclic as well as pregnant buffalo demonstrated the presence of only conjugated ISG15 which was >40 kDa. ISG15 mRNA and immune-reactive proteins were localized in the stromal as well as glandular epithelial cells of the uterine endometrium of estrous cyclic as well as pregnant buffalo. However, there was no significant difference in amount of ISG15 mRNA across the different reproductive phases. To conclude, this study will be helpful for the further understanding of the roles of the ISG15 in pregnancy of buffalo cows.

Protein acyltransferase function of purified calreticulin: the exclusive role of P-domain in mediating protein acylation utilizing acyloxycoumarins and acetyl CoA as the acyl group donors.

The distinct biochemical function of endoplasmic reticulum (ER) protein Calreticulin (CR) catalyzing the transfer of acyl group from acyloxycoumarin to a receptor protein was termed calreticulin transacylase (CRTAase). The present study, unlike the previous reports of others utilizing CR-deficient cells alone, dealt with the recombinant CR domains of Heamonchus contortus (rhCRTAase) in order to examine their CRTAase activity. P-domain of rhCR unlike N- and C-domains was found to be endowed with CRTAase function. We have also observed for the first time acetyl CoA, as a substrate for rhCRTAase/P-domain mediated acetylation of recombinant Schistosoma japonicum glutathione S-transferase (rGST). rhCRTAase/P-domain were also found to undergo autoacylation by acyloxycoumarins. Also, the isolated autoacylated rhCRTAase/P-domain in non-denatured form alone exhibited the ability to transfer acyl group to rGST indicating the stable intermediate nature of acylated CR. P-domain catalyzed acetylation of rGST by 7,8-Diacetoxy-4-methylcoumarin or acetyl CoA resulted in the modification of several lysine residues in common was evidenced by LC-MS/MS analysis. The putative site of the binding of acyloxycoumarins with CR was predicted by computational blind docking studies. The results showed the involvement of two lysine residues Lys-173 and Lys-174 present in P-domain for binding acyloxycoumarins and acetyl CoA thus highlighting that the active site for the CRTAase activity would reside in the P-domain of CR. Certain ER proteins are known to undergo acetylation under the physiological conditions involving acetyl CoA. These results demonstrating CRTAase mediated protein acetylation by acetyl CoA may hint at CR as the possible protein acetyltransferase of the ER lumen.

Molecular characterization and expression profile of uterine serpin (SERPINA14) during different reproductive phases in water buffalo (Bubalus bubalis).

Uterine serpins (SERPINA14) play important roles during pregnancy in the farm animals. In this study, we have cloned and characterized cDNA sequence encoding the bubaline SERPINA14. We also studied its spatio-temporal expression in the uterine endometrium. The bubaline SERPINA14 has an open reading frame of 1299bp. Itshares ∼90% identity with the SERPINA14 of other ruminant livestock species. Phylogenetically, bubaline SERPINA14 has been placed in the same clade that contained other mammalian homologues with a maximum identity to bovine SERPINA14. Using an anti-ovine monoclonal antibody, Western blot analysis of the uterine fluid of buffalo during the early stage of pregnancy confirmed the presence of SERPINA14 of about 48,000Da. The results of quantitative real time PCR (RT-qPCR) as well as in situ hybridization demonstrated a stage and tissue specific expression of bubaline SERPINA14. The level of SERPINA14 mRNA was low during stage I (Days 3-5), which increased (P<0.05) during stage II (Days 6-15) and then subsequently declined during stage III (Days 16-21) of the estrus cycle. During early pregnancy (Days ∼30 of gestation) the level of SERPINA14 mRNA was as high as that during stage II of the estrus cycle. The SERPINA14 mRNA was localized in the glandular epithelium. The differential spatio-temporal expression of SERPINA14 in the uterine endometrium of buffalo suggests its plausible important roles in reproduction.

Protein acyltransferase function of purified calreticulin. Part 1: characterization of propionylation of protein utilizing propoxycoumarin as the propionyl group donor.

We have earlier reported that an endoplasmic reticulum luminal protein calreticulin (CR) mediated the acetylation of certain receptor proteins such as glutathione S-transferase (GST) by polyphenolic acetates, leading to irreversible inhibition. This function of calreticulin was termed calreticulin transacetylase. In this communication, we have demonstrated for the first time the ability of the purified recombinant calreticulin of a parasitic nematode Haemonchus contortus to transfer propionyl group from 7,8-Dipropoxy-4-methylcoumarin (DPMC) to recombinant Schistosoma japonicum glutathione S-transferase (rGST). Calreticulin transacetylase exhibited hyperbolic kinetics and yielded K(m) (140 microM) and V(max) (105 units) when the concentration of DPMC was varied keeping the concentration of rGST constant. rGST thus propionylated was found to positively interact with anti-acetyl lysine antibody. Also, the nanoscale LC-MS/MS analysis identified the propionylation sites on three lysine residues: Lys-11, -180 and -181 of rGST. These results highlight the transacylase function of calreticulin (CRTAase).