Salivary Lipocalin Research Articles

Assessment of Salivary Neutrophil Gelatinase-Associated Lipocalin Levels in Diseased and Healthy Periodontium

Background: periodontal disease is one of the most frequent human diseases. Depending on the severity of the inflammation, the destructive process may affect both the gingiva (gingivitis) and/or the periodontal ligament and alveolar bone that surround and support the teeth (periodontitis). Objective: The study aimed to determine and compare the level of salivary Neutrophil gelatinase-associated lipocalin in periodontal health and disease (gingivitis and periodontitis stage I-III). Subjects and Methods: A total of 90 individuals participated in the research separated into five groups, clinically healthy (n=10), gingivitis (n=20), stage I periodontitis (n=20), stage II periodontitis (n=20) and stage III periodontitis (n=20), periodontal parameters were recorded, and the level of salivary Neutrophil gelatinase-associated lipocalin was assessed by using the enzyme-linked immunosorbent assay. Results: Neutrophil gelatinase-associated lipocalin salivary level was significantly higher (p<0.05) in periodontal disease groups compared to healthy controls. A statistically significant difference was also found between gingivitis and advanced periodontitis (stageII-III) (p<0.05). No significant difference (p>0.05) was found between different stages of periodontitis. Conclusions: Neutrophil gelatinase-associated lipocalin is associated with periodontal diseases and my play a role in its pathogenesis

PSXIV-17 Examining Mammary Gland Transcriptomic Profiles Using Rna-Seq in Slick and Wild-Type Haired Holstein Cattle Under Tropical Conditions

Abstract In lactating dairy cows, heat stress reduces feed intake, alters the metabolism of the animal, and compromises milk production. Holstein cattle carrying the prolactin receptor gene mutation (SLICK) have been shown to have superior performance under tropical conditions by exhibiting increased milk yield compared with wild-type haired Holstein cattle (WT). Recent research indicated that SLICK cattle have greater mammary gland blood flow and lower vaginal temperatures, while maintaining prolonged voluntary exposure to direct solar radiation than their WT counterparts. Nevertheless, differences in molecular mechanisms between genotypes at the mammary gland level have been unexplored. The objective of the current study was to investigate possible differences in the mammary gland transcriptome from SLICK (n = 7) and WT (n = 6) Puerto Rican Holstein cattle. At 160 ± 3 days in milk (DIM), ultrasound guided mammary biopsies were collected from the left rear quarter of each animal and samples were snap frozen. Unstranded RNA-seq libraries were created from the frozen mammary tissue and sequenced using Illumina Novaseq 2x150bp. Quantification of expression was done by salmon [v1.9.0] using the Bos taurus (ARS UCD1.3) transcriptome database. Testing for significant differential expression (DE) was done using a pipeline consisting of the R packages tximport [v1.26.1] and edgeR [v3.40.2]. The fry method from edgeR was used to do gene set enrichment analysis (GSEA) for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) terms. A single gene (GeneID:513329, major allergen Equ c 1 isoform X2) showed significant (B.H. adjusted P-value ≤ 0.05) differential expression. Aligning the protein sequence for this gene using the BLASTp against the non-redundant (NR) database revealed high similarity to two pheromones, salivary lipocalin 2 and salivary lipocalin-like. Both the KEGG pathway and GO term GSEA showed no statistically significant enrichment at B.H. adjusted P-value ≤ 0.05; however, using a less stringent criteria with a normal P-value ≤ 0.05, multiple relevant pathways were identified. For example, the KEGG pathway analysis revealed that the arachidonic acid metabolism pathway (bta00590) was enriched, and the tight junction function pathway (bta4530) was depleted in the SLICK Holstein samples. Among the GO analysis, genes were found to be downregulated in response to cold (GO:0009409), hair follicle development (GO:0001942), and vasoconstriction (GO:0042310), whereas positive regulation of oxytocin production (GO:0140668), and defense response to Gram-positive bacterium (GO:0050830) were upregulated. In conclusion, differences in gene expression were limited, but after enrichment interesting pathways were revealed and deserve further exploration to aid in elucidating different molecular mechanisms between SLICK and WT cattle.

Pheromones, binding proteins, and olfactory systems in the pig (Sus scrofa): An updated review.

Pigs utilize multimodal communication for reproductive and other behaviors, and chemical communication is one of the key components. The success of reproduction relies on chemical communication favored by the steroid pheromones from boar saliva. These steroids were proven to be involved in advancing puberty in gilts (the boar effect) and in promoting estrus behaviors in gilts/sows, thereby helping to detect estrus and facilitating the timing of artificial insemination. The steroid pheromones bound with carrier proteins are evidenced in the mandibular (submandibular) salivary secretions of the boar. These salivary steroids bind with carrier proteins in the nasal mucus and vomeronasal organ (VNO) of the sows, eventually triggering a cascade of activities at the olfactory and endocrine levels. Besides steroid pheromones, pig appeasing pheromones (from mammary skin secretions of sows) have also been demonstrated to bind with carrier proteins in the nasal mucus and VNO of the piglets. Thus far, four different proteins have been identified and confirmed in the nasal mucus and VNO of pigs, including odorant binding proteins (OBPs), salivary lipocalin (SAL), pheromaxein, and Von Ebner's Gland Protein (VEGP). The critical roles of the chemosensory systems, main olfactory systems and VNO, have been comprehensively reported for pigs. This review summarizes the current knowledge on pheromones, their receptor proteins, and the olfactory systems of porcine species.

Increase in serum and salivary neutrophil gelatinase-associated lipocalin levels with increased periodontal inflammation.

Objective:This study aimed to determine serum and salivary levels of neutrophil gelatinase-associated lipocalin (NGAL) and evaluate NGAL correlation with key anti-interleukin 10 (IL-10) and pro-inflammatory (IL-1β) cytokines in different severities of periodontal diseases. We also calculated the systemic inflammation using the periodontal inflamed surface area (PISA) to evaluate its correlation with NGAL in the study groups.Methodology:Eighty systemically healthy and non-smoking individuals were separated into four groups of 20: clinically healthy (Group 1), gingivitis (Group 2), stage I generalized periodontitis (Group 3, Grade A), and stage III generalized periodontitis (Group 4, Grade A). Sociodemographic characteristics and periodontal parameters were recorded, and PISA was calculated. The serum and salivary levels of interleukin (IL)-1β, IL-10, and NGAL were determined using the enzyme-linked immunosorbent assay (ELISA).Results:We observed a significant increase in serum and salivary NGAL levels from healthy to periodontitis groups (p=0.000). Group 2 presented significantly higher serum and salivary IL-10 levels and salivary IL-1β levels than Group 3 (p=0.000). Serum and salivary parameters (IL-1β, IL-10, and NGAL levels) were strongly positively correlated to periodontal parameters and PISA values (p=0.000). Groups 2 and 3 showed overlapping PISA values.Conclusion:The overlapping PISA values found in Groups 2 and 3 suggest that gingivitis might progress to a systemic inflammatory burden somewhat comparable to stage I periodontitis. This finding is supported by the higher serum and salivary cytokines/mediators levels in the gingivitis group than in stage I periodontitis group. Serum and salivary NGAL levels increased proportionally to disease severity and PISA. NGAL seems to play a role in the pathogenesis of periodontal disease, within the limitation of our study.

On the characterisation of the porcine gland-specific salivary proteome

To expand the knowledge on the porcine salivary proteome, secretions from the three major salivary glands were collected from anaesthetised piglets. Pilocarpine and isoproterenol were simultaneously injected intraperitoneally to increase the volume and protein concentration of the saliva, respectively. The protein composition and relative protein-specific abundance of saliva secreted by the parotid gland and by the mandibular and monostomatic sublingual gland, were determined using iTRAQ. When combining two detection methods, MALDI-TOF/TOF MS and Q-Exactive orbitrap MS/MS, a total of 122 porcine salivary proteins and 6 mammalian salivary proteins with a predicted porcine homolog were identified. Only a quantitative and not a qualitative difference was observed between both ductal secretions. The 128 proteins were detected in both secretions, however, at different levels. Twenty-four proteins (20 porcine and 4 mammalian with a predicted porcine homolog) were predominantly secreted by the parotid gland, such as carbonic anhydrase VI and alpha-amylase. Twenty-nine proteins (all porcine) were predominantly secreted by the mandibular and sublingual glands, for example salivary lipocalin and submaxillary apomucin protein. Data are available via ProteomeXchange with identifier PXD008853. SignificanceIn humans, more than 3000 salivary proteins have been identified. To our knowledge, previous studies on porcine saliva only identified a total of 34 proteins. This research increased the total number of identified proteins in porcine saliva to 143. This insight into the porcine salivary proteome will facilitate the search for potential biomarkers that may help in the early detection of pathologies and follow-up of animal welfare. Moreover, it can also endorse the value of a porcine animal model and contribute to a better understanding of the animal's physiology. Additionally, this was the first study to collect and analyse gland specific saliva of pigs. The obtained relative-quantitative knowledge of the identified proteins is valuable when comparing data of stimulated (chewing on a device) vs. unstimulated (passive) saliva collection in the future, since salivary stimulation changes the relative contribution of the major salivary glands to the whole saliva in the oral cavity. For example, carbonic anhydrase VI, which is present in higher concentrations in parotid saliva, has a higher concentration in stimulated whole saliva because of the larger contribution of the parotid gland after stimulation by chewing.

Periconceptional undernutrition affects the levels of DNA methylation in the peri-implantation pig endometrium and in embryos

Maternal undernutrition during the periconceptional period alters the transcriptomic profile of pig endometrium and embryos. Herein, we tested the hypothesis that restricted maternal consumption by females during the periconceptional period impairs the pattern of DNA methylation in both the endometrium and embryos during the peri-implantation period (Day 15–16 of gestation). Affected genes in restricted-diet-fed pig endometrium and embryos were identified using quantitative methylation-specific PCR and comprised those genes which are known to be important in reproductive, metabolic and epigenetic function, thereby exhibiting altered transcriptomic expression in endometrium and embryos of restricted-diet-fed gilts. Specifically, levels of DNA methylation of selected genes with altered expression in the endometrium included acid phosphatase type 2C (PPAP2C), salivary lipocalin (SAL1), endothelin receptor type B (EDNRB), regulator of G-protein signalling 12 (RGS12), type 4 17β-hydroxysteroid dehydrogenase (HSD17B4), toll-like receptor 3 (TLR3), and adiponectin receptor 1 (ADIPOR1). In embryos, adiponectin receptor 2 (ADIPOR2), prostaglandin-endoperoxide synthase 2 (PTGS2), arachidonate 12-lipoxygenase (ALOX12), progestin and adipoQ receptor family member 7 (PAQR7), progesterone receptor membrane component 2 (PGRMC2), steroidogenic acute regulatory protein (STAR), and serpin family A member 1 (SERPINA1) were altered. Finally, 5 acid phosphatase tartrate resistant (ACP5), high mobility group box 2 (HMGB2), and DNA (cytosine-5)-methyltransferase 1 (DNMT1) were altered in both the endometrium and in embryos. In the endometrium, the methylation levels of ACP5 (regulation of endometrial-conceptus iron transport), RGS12 (protein-coupled receptor signalling), and TLR3 (immune response) were increased, while that of EDNRB (corpus luteum maintenance) was decreased. In embryos, the methylation levels of ADIPOR2 (metabolic homeostasis) and DNMT1 (DNA methylation maintenance) were increased. The levels of methylation in other studied endometrial and embryonic genes were unchanged. DNA methylation levels in both the peri-implantation pig endometrium and embryos may be altered in response to female nutritional restriction.

Salivary Neutrophil Gelatinase-Associated Lipocalin Sampling Feasibility in Acute Renal Colic.

Neutrophil gelatinase-associated lipocalin (NGAL) levels in the serum and urine are predictive biomarkers of acute kidney injury with correlation to complication and survival in major surgery. Salivary levels of NGAL during acute renal colic may reflect the degree of renal injury as it appears in different compartments encompassing body response in time perspective. Our aim is to evaluate and examine the feasibility and correlation of salivary NGAL with serum and urine levels in acute renal colic event. A prospective controlled study of all patients presenting to the emergency room with acute renal colic event diagnosed with single ureteral stone obstruction by noncontrast CT. Saliva, urine, and blood samples were collected in patients and a control group during the first morning of admission. The study groups consisted of 44 patients and 13 controls, mean age 47 ± 15 years, body mass index 29 ± 6, mean stone size 6 ± 4 mm, mean creatinine levels 1.3 ± 0.7 mg/dL, mean white blood count 10,900 ± 3100 counts per field, and C-reactive protein 29 ± 55. Serum (190 ± 120 ng/mL vs 81 ± 24; p < 0.001) and predominantly salivary (474 ± 185 vs 328 ± 134 ng/mL; p < 0.05) NGAL levels were significantly elevated in patients compared with controls. Salivary NGAL sampling is feasible during the acute phase of renal colic. High levels of salivary NGAL are observed in a single sampling during acute ureteral stone obstruction and may advance clinical decision-making.

Gender influence on the salivary protein profile of finishing pigs

A study on gender differences in the normal range of biomarkers in porcine saliva samples has the scope for further attention. In the present study, the salivary protein profiles of age-matched healthy male and female finishing pigs were compared. The levels of salivary adenosine deaminase (ADA) activity, haptoglobin (Hp) and C-reactive protein (CRP) have been quantified in 32 male and 32 female pigs to ensure the presence of gender effect on the median levels of salivary biomarkers. Moreover, the total salivary protein content was quantified and compared. The overall salivary protein distribution was compared with SDS-PAGE in 14 male and 14 female pigs and the possible gender influence in the salivary protein profile was analysed by 2DE in 6 male and 6 female pigs. Statistically significant differences were observed in the median values of Hp, CRP, and ADA between male and female pigs (p<0.005). Although the total salivary protein content was not different between the sexes, the salivary protein distribution and profile showed specific gender differences in three proteins of the lipocalin family: the odorant-binding protein, salivary lipocalin and lipocalin 1. These proteins have been related to animal immune status and should be further explored as possible porcine salivary biomarkers. SignificanceThe biological relevance of the reported research is based on the possible gender influence on the discovery of salivary biomarkers in porcine production. As differences have been reported in the salivary protein distribution in male pigs in comparison to that of female pigs, the normal-range values, according to gender, of the newly discovered biomarkers should be explored and defined prior to its application in the porcine production system. A hormonal sexual influence is highly hypothesized.

Influence of different sample preparation strategies on the proteomic identification of stress biomarkers in porcine saliva

BackgroundThe influence of two different sample treatments comprising the enrichment of glycoproteins by boronic acid and dynamic range compression by hexapeptide libraries, on the detection of stress markers in saliva of pigs was evaluated in this study. For this purpose, saliva samples collected before and after the application of an acute stress model consisting of nasal restraining in pigs were processed without any treatment and with the two different treatments mentioned above. Protein separation by two-dimensional gel electrophoresis (2-DE) followed by identification of proteins using MALDI-TOF/TOF mass spectrometry (MS) was used as proteomic technique.ResultsThe application of each of the two different sample treatment protocols allowed the identification of unique proteins that could be potential salivary acute stress markers in pigs: lipocalin 1, protein S100-A8 and immunoglobulin M by enrichment of glycoproteins; protein S100-A9, double headed protease inhibitor submandibular gland, and haemoglobin by dynamic range compression; and protein S100-A12 by both protocols. Salivary lipocalin, prolactin inducible protein, light chain of immunoglobulins, adenosine deaminase and carbonic anhydrase VI were identified as potential markers in untreated saliva as well as one of the other treatments.ConclusionThe use of different procedures allowed the detection of different potential stress markers. Although from a practical point of view, the use of saliva without further treatment as well as the enrichment of glycoproteins are less expensive and easy to do procedures.

TSGP4 from Ornithodoros moubata: molecular cloning, phylogenetic analysis and vaccine efficacy of a new member of the lipocalin clade of cysteinyl leukotriene scavengers

Recently obtained evidence indicated that an orthologue of the O. savignyi TSGP4 salivary lipocalin was present in the saliva of O. moubata. TSGP4 is known to act as a cysteinyl leukotrienes scavenger helping in the prevention of inflammation and oedema at the tick bite site. Since this function seems to be crucial for successful tick feeding, the novel O. moubata TSGP4 turned into a potential vaccine target. The purposes of the current work were: (i) to clone and characterize the O. moubata TSGP4 and, (ii) to produce it as recombinant to evaluate its protective efficacy as vaccine antigen. The results of these experiments indicated that the O. moubata TSGP4 shows high sequence and structural identity with the O. savignyi orthologue suggesting identical function in the physiology of the tick-host relationship. The mature native TSGP4 is not immunogenic when it is inoculated to host with tick saliva during feeding, but host vaccination with the recombinant protein TSGP4 in Freund’s adjuvants induced strong humoral immune responses that recognized both the recombinant and native TSGP4 and protected the host with a 14.1% efficacy. So, the O. moubata TSGP4 can be considered a silent salivary antigen; however, in the light of the current results, its inclusion in the current repertory of protective antigens to be targeted by anti-tick vaccines could be controversial.

Antihistamine response: a dynamically refined function at the host-tick interface.

BackgroundTicks counteract host inflammatory responses by secreting proteins from their saliva that compete for histamine binding. Among these tick salivary proteins are lipocalins, antiparallel beta-barrel proteins that sequester small molecules. A tick salivary lipocalin has been structurally resolved and experimentally shown to efficiently compete for histamine with its native receptor (e.g., H1 histamine receptor). To date, molecular dynamics simulations focus on protein-protein and protein-ligand interactions, but there are currently no studies for simultaneous ligand exploration between two competing proteins.MethodsAided by state-of-the-art, high-throughput computational methods, the current study simulated and analyzed the dynamics of competitive histamine binding at the tick-host interface using the available crystal structures of both the tick salivary lipocalin histamine-binding protein from Rhipicephalus appendiculatus and the human histamine receptor 1.ResultsThe attraction towards the tick salivary lipocalin seems to depend on the protonated (adding a hydrogen ion) state of histamine since the current study shows that as histamine becomes more protonated it increases its exploration for the tick salivary lipocalin. This implies that during tick feeding, histamine may need to be protonated for the tick salivary lipocalin to efficiently sequester it in order to counteract inflammation. Additionally, the beta-hairpin loops (at both ends of the tick salivary lipocalin barrel) were reported to have a functional role in sequestering histamine and the results in the current study concur and provide evidence for this hypothesis. These beta-hairpin loops of the tick salivary lipocalin possess more acidic residues than a structurally similar but functionally unrelated lipocalin from the butterfly, Pieris brassicae; comparative results indicate these acidic residues may be responsible for the ability of the tick lipocalin to out-compete the native (H1) receptor for histamine.ConclusionsThree explanatory types of data can be obtained from the current study: (i) the dynamics of multiple binding sites, (ii) competition between two proteins for a ligand, and (iii) the intrinsic molecular components involved in the competition. These data can provide further insight at the atomic level of the host-tick interface that cannot be experimentally determined. Additionally, the methods used in this study can be applied in rationally designing drugs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-014-0491-9) contains supplementary material, which is available to authorized users.

Detection of potential markers for systemic disease in saliva of pigs by proteomics: A pilot study

Animals with different health status have been studied in order to extend the knowledge about protein composition of porcine saliva samples and to discover potential salivary markers for systemic disease in porcine production. Clinical examination of animals was performed at farm level where 10 healthy pigs and 10 animals with evident clinical signs of disease were randomly selected. Saliva and blood samples were obtained and afterwards animals were humanely sacrificed to perform a complete necropsy. Levels of two acute phase proteins, haptoglobin and C-reactive protein, were used to identify possible active infections of the animals. Moreover, serological analysis, to the main porcine infectious diseases in the area, was performed. Salivary proteins were separated by two-dimensional gel electrophoresis followed by mass spectrometry for the identification of specific proteins. A total of 58 spots out of 75 were successfully identified by MS, which correspond to 20 unique proteins. Two different approaches were used to perform a statistical comparison of saliva protein patterns from healthy and diseased animals using the relative spot volume (% spot volume/total volume of all spot in the gel, approach “A”) or taking also into account the total protein content of each saliva sample (μg of spot/mL of saliva, approach “B”). Both analyses showed three proteins in common that are differentially regulated between states. However, approach B was selected for biomarker searching since it gave an estimation of protein concentration and showed differential expression of proteins between both health states in a total of 10 proteins, which were up-regulated in disease. Mass spectrometric analysis identified those proteins as salivary lipocalin, lipocalin 1, double headed protease inhibitor protein, adenosine deaminase, haptoglobin, albumin fragments, S100-A8, S100-A9, S100-A12 and pancreatic alpha amylase. These proteins could be considered as potential salivary markers of disease.

The evolutionary history of the SAL1 gene family in eutherian mammals

BackgroundSAL1 (salivary lipocalin) is a member of the OBP (Odorant Binding Protein) family and is involved in chemical sexual communication in pig. SAL1 and its relatives may be involved in pheromone and olfactory receptor binding and in pre-mating behaviour. The evolutionary history and the selective pressures acting on SAL1 and its orthologous genes have not yet been exhaustively described. The aim of the present work was to study the evolution of these genes, to elucidate the role of selective pressures in their evolution and the consequences for their functions.ResultsHere, we present the evolutionary history of SAL1 gene and its orthologous genes in mammals. We found that (1) SAL1 and its related genes arose in eutherian mammals with lineage-specific duplications in rodents, horse and cow and are lost in human, mouse lemur, bushbaby and orangutan, (2) the evolution of duplicated genes of horse, rat, mouse and guinea pig is driven by concerted evolution with extensive gene conversion events in mouse and guinea pig and by positive selection mainly acting on paralogous genes in horse and guinea pig, (3) positive selection was detected for amino acids involved in pheromone binding and amino acids putatively involved in olfactory receptor binding, (4) positive selection was also found for lineage, indicating a species-specific strategy for amino acid selection.ConclusionsThis work provides new insights into the evolutionary history of SAL1 and its orthologs. On one hand, some genes are subject to concerted evolution and to an increase in dosage, suggesting the need for homogeneity of sequence and function in certain species. On the other hand, positive selection plays a role in the diversification of the functions of the family and in lineage, suggesting adaptive evolution, with possible consequences for speciation and for the reinforcement of prezygotic barriers.

Cloning, characterization and diagnostic performance of the salivary lipocalin protein TSGP1 from Ornithodoros moubata

The argasid tick Ornithodoros moubata is distributed throughout South and East Africa and Madagascar, where it colonizes wild and domestic habitats and feeds on warthogs, domestic swine, and humans. This argasid transmits the spirochete Borrelia duttonii, causing East African tick-borne relapsing fever in humans, and the African swine fever virus, which causes a highly lethal haemorrhagic disease in pigs. Tick surveillance and the elimination of O. moubata from synanthropic environments (human dwellings and pigsties) would facilitate the control and prevention of these two diseases. Since direct surveillance methods are impractical in this context, the development of an indirect method for the detection of specific antibodies against tick salivary proteins in samples taken from animal or human hosts living in the area under study would provide a more convenient surveillance and diagnostic tool. Previous work has indicated that the 20A1 salivary antigen of O. moubata could be an optimal candidate for the development of a specific serological test and identified it as an orthologue of the Ornithodoros savignyi TSGP1 lipocalin. The objectives of the present work were to clone, sequence, and molecularly characterize the O. moubata TSGP1, as well as its production as a recombinant protein in order to assess its usefulness as a diagnostic antigen in an ELISA test for tick surveillance. Our results show that O. moubata TSGP1 (OmTSGP1) conserves the tertiary structure of lipocalins and contains the biogenic amine-binding motif. We also show that OmTSGP1 shares 65% sequence identity with the O. savignyi TSGP1, demonstrating that they represent orthologous proteins and suggesting they share identical function as biogenic amine scavengers. A recombinant form of OmTSGP1 was produced, showing 100% sensitivity and 99.4% specificity in an ELISA test for the detection of anti-O. moubata antibodies in pig sera. This recombinant antigen represents a promising epidemiological tool for O. moubata surveillance that may help to implement control measures against O. moubata-borne diseases.

Dipetalodipin, a Novel Multifunctional Salivary Lipocalin That Inhibits Platelet Aggregation, Vasoconstriction, and Angiogenesis through Unique Binding Specificity for TXA2, PGF2α, and 15(S)-HETE

Dipetalodipin (DPTL) is an 18 kDa protein cloned from salivary glands of the triatomine Dipetalogaster maxima. DPTL belongs to the lipocalin superfamily and has strong sequence similarity to pallidipin, a salivary inhibitor of collagen-induced platelet aggregation. DPTL expressed in Escherichia coli was found to inhibit platelet aggregation by collagen, U-46619, or arachidonic acid without affecting aggregation induced by ADP, convulxin, PMA, and ristocetin. An assay based on incubation of DPTL with small molecules (e.g. prostanoids, leukotrienes, lipids, biogenic amines) followed by chromatography, mass spectrometry, and isothermal titration calorimetry showed that DPTL binds with high affinity to carbocyclic TXA(2), TXA(2) mimetic (U-46619), TXB(2), PGH(2) mimetic (U-51605), PGD(2,) PGJ(2), and PGF(2α). It also interacts with 15(S)-HETE, being the first lipocalin described to date to bind to a derivative of 15-lipoxygenase. Binding was not observed to other prostaglandins (e.g. PGE(1), PGE(2), 8-iso-PGF(2α), prostacyclin), leukotrienes (e.g. LTB(4), LTC(4), LTD(4), LTE(4)), HETEs (e.g. 5(S)-HETE, 12(S)-HETE, 20-HETE), lipids (e.g. arachidonic acid, PAF), and biogenic amines (e.g. ADP, serotonin, epinephrine, norepinephrine, histamine). Consistent with its binding specificity, DPTL prevents contraction of rat uterus stimulated by PGF(2α) and induces relaxation of aorta previously contracted with U-46619. Moreover, it inhibits angiogenesis mediated by 15(S)-HETE and did not enhance inhibition of collagen-induced platelet aggregation by SQ29548 (TXA(2) antagonist) and indomethacin. A 3-D model for DPTL and pallidipin is presented that indicates the presence of a conserved Arg(39) and Gln(135) in the binding pocket of both lipocalins. Results suggest that DPTL blocks platelet aggregation, vasoconstriction, and angiogenesis through binding to distinct eicosanoids involved in inflammation.

Salivary Lipocalin Is Uniquely Expressed in the Uterine Endometrial Glands at the Time of Conceptus Implantation and Induced by Interleukin 1Beta in Pigs

Uterine secretions are essential for the development of the conceptus during pregnancy. In pigs, various molecules, including transport proteins, growth factors, enzymes, and extracellular matrix proteins, are secreted into the uterine lumen. Our previous work identified salivary lipocalin (SAL1), a steroidal pheromone-binding protein, as present in the porcine uterus. To initiate studies on the role of SAL1 in the porcine uterus, we evaluated 1) the spatial and temporal expression of SAL1 in the uterine endometrium during the estrous cycle and pregnancy, and in the conceptus during early pregnancy; 2) secretion of SAL1 into the uterine lumen on Day (D) 12 of the estrous cycle and pregnancy; and 3) the effects of steroid hormones and cytokines on SAL1 mRNA levels. SAL1 was localized to glandular epithelial cells (GE) in the endometrium during the estrous cycle and pregnancy, with the highest level of SAL1 expression on D12 of pregnancy. In addition, SAL1 protein secretion into the uterine lumen was detected in uterine flushings on D12 of the estrous cycle and pregnancy, with higher levels on D12 of pregnancy. SAL1 protein, but not SAL1 transcript, was also detected in the conceptuses on D12 and D15. In explant culture experiments, SAL1 mRNA levels in the endometrium were increased by interleukin 1beta. The results of a GE- and implantation stage-specific expression and uterine secretion of SAL1 in the porcine uterus suggest that SAL1 present at the maternal-fetal interface may act as a histotroph and play an important role in the establishment of pregnancy.

Innovation in immunotherapy

Innovation in immunotherapy

Ir-LBP, an Ixodes ricinus Tick Salivary LTB4-Binding Lipocalin, Interferes with Host Neutrophil Function

BackgroundDuring their blood meal, ticks secrete a wide variety of proteins that can interfere with their host's defense mechanisms. Among these proteins, lipocalins play a major role in the modulation of the inflammatory response.Methodology/Principal FindingsWe previously identified 14 new lipocalin genes in the tick Ixodes ricinus. One of them codes for a protein that specifically binds leukotriene B4 with a very high affinity (Kd: ±1 nM), similar to that of the neutrophil transmembrane receptor BLT1. By in silico approaches, we modeled the 3D structure of the protein and the binding of LTB4 into the ligand pocket. This protein, called Ir-LBP, inhibits neutrophil chemotaxis in vitro and delays LTB4-induced apoptosis. Ir-LBP also inhibits the host inflammatory response in vivo by decreasing the number and activation of neutrophils located at the tick bite site. Thus, Ir-LBP participates in the tick's ability to interfere with proper neutrophil function in inflammation.Conclusions/SignificanceThese elements suggest that Ir-LBP is a “scavenger” of LTB4, which, in combination with other factors, such as histamine-binding proteins or proteins inhibiting the classical or alternative complement pathways, permits the tick to properly manage its blood meal. Moreover, with regard to its properties, Ir-LBP could possibly be used as a therapeutic tool for illnesses associated with an increased LTB4 production.

Docking and Virtual Screening Studies for New Leads of Boar Salivary Lipocalin

We have performed FlexX docking experiments to predict the best docking poses of 5-androst-16-en-3-ol or 5-androst-16-en-3-one to Boar salivary lipocalin (SAL). Since no steroids were found inside of the binding pocket of the X-ray structure of 1GM6, we tried to find docking structures after opening the pocket using the random tweak option implemented in SYBYL. This operation allowed the ligand to enter the pocket. The best poses generated from FlexX were different from the structures reported earlier, which calculated docking poses by manual docking followed by minimization. Analysis of docking poses allowed us to identify pharmacophores. From this information, virtual screening experiments using UNITY were performed. Among six candidates, 3-(3,7-dimethyloct-6-enylamino)propane-1,2-diol (Leadquest code name: 5755) was chosen for further development. Future work will involve synthesis of some derivatives of 5755 and biological experiments if any derivatives can control the biostimulation and improve reproductive efficiency in pigs.

Structural Determinants of Factor IX(a) Binding in Nitrophorin 2, a Lipocalin Inhibitor of the Intrinsic Coagulation Pathway

Nitrophorin 2 (NP2) is a salivary lipocalin from Rhodnius prolixus that binds with coagulation factors IX (fIX) and IXa (fIXa). Binding of NP2 with fIXa results in potent inhibition of the intrinsic factor Xase complex. A panel of site-directed surface mutants of NP2 was generated to locate determinants of high affinity fIX(a) binding. The locations of the mutations were based on comparisons with the related, but less potent, inhibitor nitrophorin 3 (NP3). Three point mutants (K21A, K92A, and V94A) were found that clearly reduced the inhibitory potency as measured by the activity of a reconstituted factor Xase system. Binding of NP2 with fIXa and fIX as measured by surface plasmon resonance and isothermal titration calorimetry was reduced in a similar manner. Of the three mutants, two (K92A and V94A) were located on the loop connecting beta-strands E and F of the lipocalin beta-barrel. The largest changes were seen with the K92A mutation, which lies at the apex of the loop, with a smaller effect being seen with mutation of Val(94). Combination of four E-F loop mutations (K92A, A93K, V94A, E97A) in a single mutant reduced the inhibitory potency and binding to levels similar to those seen with NP3 without affecting heme or histamine binding.