Kunitz-type Serine Protease Inhibitor Research Articles

Characterization of Kunitz-Domain Anticoagulation Peptides Derived from Acinetobacter baumannii Exotoxin Protein F6W77.

Recent studies have revealed that the coagulation system plays a role in mammalian innate defense by entrapping bacteria in clots and generating antibacterial peptides. So, it is very important for the survival of bacteria to defend against the host coagulation system, which suggests that bacterial exotoxins might be a new source of anticoagulants. In this study, we analyzed the genomic sequences of Acinetobacter baumannii and a new bacterial exotoxin protein, F6W77, with five Kunitz-domains, KABP1-5, was identified. Each Kunitz-type domain features a classical six-cysteine framework reticulated by three conserved disulfide bridges, which was obviously similar to animal Kunitz-domain peptides but different from plant Kunitz-domain peptides. Anticoagulation function evaluation showed that towards the intrinsic coagulation pathway, KABP1 and KABP5 had apparently inhibitory activity, KABP4 had weak inhibitory activity, and KBAP2 and KABP3 had no effect even at a high concentration of 20 μg/mL. All five Kunitz-domain peptides, KABP1-5, had no inhibitory activity towards the extrinsic coagulation pathway. Enzyme-inhibitor experiments showed that the high-activity anticoagulant peptide KABP1 had apparently inhibitory activity towards two key coagulation factors, Xa and XIa, which was further confirmed by pull-down experiments that showed that KABP1 can bind to coagulation factors Xa and XIa directly. Structure-function relationship analyses of five Kunitz-type domain peptides showed that the arginine of the P1 site of three new bacterial anticoagulants, KABP1, KABP4 and KABP5, might be the key residue for their anticoagulation activity. In conclusion, with bioinformatics analyses, peptide recombination, and functional evaluation, we firstly found bacterial-exotoxin-derived Kunitz-type serine protease inhibitors with selectively inhibiting activity towards intrinsic coagulation pathways, and highlighted a new interaction between pathogenic bacteria and the human coagulation system.

Supplementation of polyclonal antibodies, developed against epitope-string toxin-specific peptide immunogens, to commercial polyvalent antivenom, shows improved neutralization of Indian Big Four and Naja kaouthia snake venoms

Snakebites profoundly impact the rural population of tropical nations, leading to significant socio-economic repercussions. Polyvalent antivenom (PAV) therapy faces several limitations, including intra-specific variations and poor efficacy against some major toxins and low molecular mass, poorly immunogenic toxins, which contribute to increased mortality and morbidity rates. Innovative strategies for developing novel antivenoms are continuously explored to address these challenges. The present study focuses on designing of 17 epitope-string toxin-specific peptide immunogens from pharmacologically active major and/or poorly immunogenic toxins (snake venom metalloprotease, Kunitz-type serine protease inhibitor, phospholipase A2, three-finger toxin) from the venom of the ‘Big Four’ venomous snakes and Naja kaouthia (NK) in India. These custom peptide antibodies demonstrated robust immuno-reactivity against the venoms ‘Big Four’ and NK. When these antibodies were supplemented with commercial PAV at a defined ratio (formulated polyvalent antivenom or FPAV), it significantly enhanced the neutralization of snake venom enzymes and in vivo neutralization of lethality and pharmacological activities such as haemorrhage, necrosis, pro-coagulant, defibrinogenation, and myotoxicity of ‘Big Four’ and NK venoms compared to PAV in mice. The present study highlights a promising strategy for developing next-generation antivenoms using synthetic peptide-based immunogens, offering a targeted approach to address the limitations of current antivenom therapy.

Assessment of the tissue factor pathway inhibitor (TFPI-rs7586970 variant) polymorphism as a risk factor in myocardial infarction patients

Context The tissue factor pathway inhibitor (TFPI), a Kunitz-type serine protease inhibitor, inhibits the extrinsic pathway of pathways, and might significantly contribute to the pathogenesis of coronary artery diseases. Aim To assess the TFPI-rs7586970 genetic variant as a risk factor in patients with myocardial infarction (MI). Settings and design A descriptive case–control study in the Premises of the Department of Clinical Pathology and Cardiology in Suez Canal University Hospitals included 156 individuals; 78 MI patients and 78 individuals as a control group. Methods and material An interview questionnaire, clinical and laboratory assessment, and molecular genotyping of the TFPI-rs7586970 genetic variant were done using real-time PCR Eva-Green-based high-resolution melting curve (HRM) analysis. Informed consent was taken from all the participants and the study protocol was approved by the Ethics Committee, Faculty of Medicine, Suez Canal University. Statistical analysis used For statistical analysis, t-test, Mann-Whitney U test, chi-squared-test (χ2), one-way ANOVA (analysis of variance), Kruskal-Wallis test, and binary logistic regression model were used. Results The MI group recorded 26 allele copies of the TFPI-rs7586970 genetic variant whereas the control group recorded six copies. The binary logistic regression model for MI occurrence showed that the TFPI-rs7586970 genetic variant polymorphism is a significant predictor for the development of MI. Conclusions TFPI-rs7586970 genetic variant polymorphism is a significant risk factor for the development of MI.

Evaluation of venom diversity and antivenom quality from the venom of long-term captive vs recently wild captured Pseudocerastes persicus snake: An In vitro and In vivo study

Snakebite envenomation is a life-threatening condition and antivenoms are used as the most effective treatment. Venom obtained from snakes in long-term captivity showed some variations in comparison to the venom of the wild snakes. The objective of this study is to compare the venom of the Pseudocerastes persicus under long-term captivity and wild conditions as well as the antivenom obtained from these venoms. We have analyzed venom samples and produced trivalent antivenoms using the venom of long-term captive (LTC) or recently wild-captured (RWC) Pseudocerastes persicus, and RWC Macrovipera lebetina, and Echis carinatus. The HPLC analysis revealed that the RWC snakes' venom had three peaks that were not present in the LTC snake's venom. Further analysis using MALDI-TOF and MS/MS showed that the fraction with a retention time (RT) of 14 min contained a toxin from the Kunitz-type serine protease inhibitor (KUT) class, while the fraction with RT 21 a peptide identified within the snake venom metalloproteinase (SVMP) class. The third peak was identified as a sphingolipid.Interestingly, the in vivo preclinical tests showed no significant differences in the effectiveness of the antivenoms. which could be due to the cross-immunogenicity or cross-reactivity between different toxins in the venom. According to our results, small variations in the venom composition of a species do not lead to a decrease in the efficacy of the polyvalent antivenom.

SPINT2 mutations in the Kunitz domain 2 found in SCSD patients inactivate HAI-2 as prostasin inhibitor via abnormal protein folding and N-glycosylation.

Mutations in the Kunitz-type serine protease inhibitor HAI-2, encoded by SPINT2, are responsible for the pathogenesis of syndromic congenital sodium diarrhea (SCSD), an intractable secretory diarrhea of infancy. Some of the mutations cause defects in the functionally required Kunitz domain 1 and/or subcellular targeting signals. Almost all SCSD patients, however, harbor SPINT2 missense mutations that affect the functionally less important Kunitz domain 2. How theses single amino acid substitutions inactivate HAI-2 was, here, investigated by the doxycycline-inducible expression of three of these mutants in HAI-2-knockout Caco-2 human colorectal adenocarcinoma cells. Examining protein expressed from these HAI-2 mutants reveals that roughly 50% of the protein is synthesized as disulfide-linked oligomers that lose protease inhibitory activity due to the distortion of the Kunitz domains by disarrayed disulfide bonding. Although the remaining protein is synthesized as monomers, its glycosylation status suggests that the HAI-2 monomer remains in the immature, lightly glycosylated form, and is not converted to the heavily glycosylated mature form. Heavily glycosylated HAI-2 possesses full anti-protease activity and appropriate subcellular targeting signals, including the one embedded in the complex-type N-glycan. As predicted, these HAI-2 mutants cannot suppress the excessive prostasin proteolysis caused by HAI-2 deletion. The oligomerization and glycosylation defects have also been observed in a colorectal adenocarcinoma line that harbors one of these SPINT2 missense mutations. Our study reveals that the abnormal protein folding and N-glycosylation can cause widespread HAI-2 inactivation in SCSD patents.

Proteomic Investigation of Cape Cobra (Naja nivea) Venom Reveals First Evidence of Quaternary Protein Structures.

Naja nivea (N. nivea) is classed as a category one snake by the World Health Organization since its envenomation causes high levels of mortality and disability annually. Despite this, there has been little research into the venom composition of N. nivea, with only one full venom proteome published to date. Our current study separated N. nivea venom using size exclusion chromatography before utilizing a traditional bottom-up proteomics approach to unravel the composition of the venom proteome. As expected by its clinical presentation, N. nivea venom was found to consist mainly of neurotoxins, with three-finger toxins (3FTx), making up 76.01% of the total venom proteome. Additionally, cysteine-rich secretory proteins (CRISPs), vespryns (VESPs), cobra venom factors (CVFs), 5'-nucleotidases (5'NUCs), nerve growth factors (NGFs), phospholipase A2s (PLA2), acetylcholinesterases (AChEs), Kunitz-type serine protease inhibitor (KUN), phosphodiesterases (PDEs), L-amino acid oxidases (LAAOs), hydrolases (HYDs), snake venom metalloproteinases (SVMPs), and snake venom serine protease (SVSP) toxins were also identified in decreasing order of abundance. Interestingly, contrary to previous reports, we find PLA2 toxins in N. nivea venom. This highlights the importance of repeatedly profiling the venom of the same species to account for intra-species variation. Additionally, we report the first evidence of covalent protein complexes in N. nivea venom, which likely contribute to the potency of this venom.

The Sensitization Profile for Selected Food Allergens in Polish Children Assessed with the Use of a Precision Allergy Molecular Diagnostic Technique.

Individual populations show a variety of sensitization patterns, which may be associated with the geographic region, climate, dietary habits, or ways of preparing food. The purpose of this study was to comprehensively assess the food allergy sensitization profile in Polish children, particularly to eight food allergens (so-called "the Big 8"): cow milk, eggs, wheat, soybeans, fish, crustacean shellfish, tree nuts, and peanuts. To assess the prevalence and serum levels of specific immunoglobulins E (sIgE), we analyzed the results obtained from selected laboratories located in all regions of Poland that used the multiplex ALEX® test in the period from 2019 to 2022. Results from 3715 children were obtained. The mean age of the study population was 7.0 years. The results were stratified by age: <12 months (3.63%), 1-5 years (39.54%), 6-13 years (46.32%), and 14-18 years (10.0%). The final analysis included the sIgE results obtained with 95 food extracts and 77 food allergen molecules. The highest rates of sIgE to food allergen extracts were found for peanut (29.20%), hazel (28.20%), and apple (23.60%), and those to allergenic molecules were found for the PR-10 family of molecules (Cor a 1.0401 (23.77%), Mal d 1 (22.37%), Ara h 8 (16.93%), and globulin 7/8S (Ara h 1; 15.59%)). The lowest rates of sIgE reactivity to extracts were found for strawberry (0.40%), oregano (0.30%), and thornback ray (0.16%), and those to allergenic molecules were found for Mal d 2 (0.27%) (thaumatin-like protein, TLP), Ani s 1 (0.30%) (Kunitz-type serine protease inhibitor), and Che a 1 (0.43%) (Ole e 1 family). The rates of sensitization to storage proteins of the analyzed "the Big 8" molecules decreased significantly (p < 0.05) with age. Conversely, the rates of sensitization to PR-10 family proteins increased significantly with age. The three most common allergens in Poland, regardless of whether IgE was assayed against extracts or molecules of food allergens, were peanut, hazel, and apple (in different order depending on the ranking). A detailed analysis of sensitization to the extracts and molecules of main food allergens based on the results of a multiplex ALEX® test demonstrated the sensitization profile in Polish children (including molecular sensitization, particularly the "the Big 8" food allergen molecules), which shows considerable differences in comparison with those in other countries. Serum sIgE analysis of children from all regions of Poland revealed a food allergen molecular sensitization profile that changes with age.

A Novel Kunitz-Type Serine Protease Inhibitor (HcKuSPI) is Involved in Antibacterial Defense in Innate Immunity and Participates in Shell Formation of Hyriopsis cumingii.

Serine protease inhibitors (SPIs) are abundantly reported for its inhibition against specific proteases involved in the immune responses, but SPI data related to calcareous shells are scarce. Previously, our research group has reported the proteome analysis of non-nucleated pearl powder, and a candidate matrix protein containing two Kunitz domains in the acid soluble fraction caught our attention. In the present study, the full-length cDNA sequence of HcKuSPI was obtained from Hyriopsis cumingii. HcKuSPI was specifically expressed in the mantle, with hybridization signals mainly concentrated to dorsal epithelial cells at the mantle edge and weak signals at the mantle pallium, suggesting HcKuSPI was involved in shell formation. HcKuSPI expression in the mantle was upregulated after Aeromonas hydrophila and Staphylococcus aureus challenge to extrapallial fluids (EPFs). A glutathione S transferase (GST)-HcKuSPI recombinant protein showed strong inhibitory activity against the proteases, trypsin and chymotrypsin. Moreover, HcKuSPI expression in an experimental group was significantly higher when compared with a control group during pellicle growth and crystal deposition in shell regeneration processes, while the organic shell framework of newborn prisms and nacre tablets was completely destroyed after HcKuSPI RNA interference (RNAi). Therefore, HcKuSPI secreted by the mantle may effectively neutralize excess proteases and bacterial proteases in the EPF during bacterial infection and could prevent matrix protein extracellular degradation by suppressing protease proteolytic activity, thereby ensuring a smooth shell biomineralization. In addition, GST-HcKuSPI was also crucial for crystal morphology regulation. These results have important implications for our understanding of the potential roles of SPIs during shell biomineralization.

Biological activities of Vipegrin, an anti-adhesive Kunitz-type serine proteinase inhibitor purified from Russell's viper venom

Vipegrin is a 6.8 kDa Kunitz-type serine proteinase inhibitor purified from Russell's viper (Vipera russelii russelii) venom. Kunitz-type serine proteinase inhibitors are non-enzymatic proteins and are ubiquitous constituents of viper venoms. Vipegrin could significantly inhibit the catalytic activity of trypsin. It also posseses disintegrin-like properties and could inhibit collagen and ADP-induced platelet aggregation in a dose-dependent manner. Vipegrin is cytotoxic to MCF7 human breast cancer cells and restricts its invasive property. Confocal microscopic analysis revealed that Vipegrin could induce apoptosis in MCF7 cells. Vipegrin disrupts cell to cell adhesion of MCF7 cells through its disintegrin-like activity. It also causes disruption of attachment of MCF7 cells to synthetic (poly L-lysine) and natural (fibronectin, laminin) matrices. Vipegrin did not cause cytotoxicity on non-cancerous HaCaT, human keratinocytes. The observed properties indicate that Vipegrin may help the development of a potent anti-cancer drug in future.

Exploring Toxin Genes of Myanmar Russell's Viper, Daboia siamensis, through De Novo Venom Gland Transcriptomics.

The Russell's viper (Daboia siamensis) is a medically important venomous snake in Myanmar. Next-generation sequencing (NGS) shows potential to investigate the venom complexity, giving deeper insights into snakebite pathogenesis and possible drug discoveries. mRNA from venom gland tissue was extracted and sequenced on the Illumina HiSeq platform and de novo assembled by Trinity. The candidate toxin genes were identified via the Venomix pipeline. Protein sequences of identified toxin candidates were compared with the previously described venom proteins using Clustal Omega to assess the positional homology among candidates. Candidate venom transcripts were classified into 23 toxin gene families including 53 unique full-length transcripts. C-type lectins (CTLs) were the most highly expressed, followed by Kunitz-type serine protease inhibitors, disintegrins and Bradykinin potentiating peptide/C-type natriuretic peptide (BPP-CNP) precursors. Phospholipase A2, snake venom serine proteases, metalloproteinases, vascular endothelial growth factors, L-amino acid oxidases and cysteine-rich secretory proteins were under-represented within the transcriptomes. Several isoforms of transcripts which had not been previously reported in this species were discovered and described. Myanmar Russell's viper venom glands displayed unique sex-specific transcriptome profiles which were correlated with clinical manifestation of envenoming. Our results show that NGS is a useful tool to comprehensively examine understudied venomous snakes.

HAI-1 is required for the novel role of FGFBP1 in maintenance of cell morphology and F-actin rearrangement in human keratinocytes.

Formation and maintenance of skin barrier function require tightly controlled membrane-associated proteolysis, in which the integral membrane Kunitz-type serine protease inhibitor, HAI-1, functions as the primary inhibitor of the membrane-associated serine proteases, matriptase and prostasin. Previously, HAI-1 loss in HaCaT human keratinocytes resulted in an expected increase in prostasin proteolysis but a paradoxical decrease in matriptase proteolysis. The paradoxical decrease in shed active matriptase is further investigated in this study with an unexpected discovery of novel functions of fibroblast growth factor-binding protein 1 (FGFBP1), which acts as an extracellular ligand that can rapidly elicit F-actin rearrangement and subsequently affect the morphology of human keratinocytes. This novel growth factor-like function is in stark contrast to the canonical activity of this protein through interactions with FGFs for its pathophysiological functions. This discovery began with the observation that HAI-1 KO HaCaT cells lose the characteristic cobblestone morphology of the parental cells and exhibit aberrant F-actin formation along with altered subcellular targeting of matriptase and HAI-2. The alterations in cell morphology and F-actin status caused by targeted HAI-1 deletion can be restored by treatment with conditioned medium from parental HaCaT cells, in which FGFBP1 was identified by tandem mass spectrometry. Recombinant FGFBP1 down to 1ng/ml was able to revert the changes caused by HAI-1 loss. Our study reveals a novel function of FGFBP1 in the maintenance of keratinocyte morphology, which depends on HAI-1.

PmKuSPI is regulated by pmo-miR-bantam and contributes to hemocyte homeostasis and viral propagation in shrimp

The Kunitz-type serine protease inhibitor (KuSPI) is a low molecular weight protein that plays a role in modulating a range of biological processes. In Penaeus monodon, the PmKuSPI gene has been found to be highly expressed in the white spot syndrome virus (WSSV)-infected shrimp and is predicted to be regulated by a conserved microRNA, pmo-miR-bantam. We reported that, despite being upregulated at the transcriptional level, the PmKuSPI protein was also upregulated after WSSV infection. Silencing the PmKuSPI gene in healthy shrimp had no effect on phenoloxidase activity or apoptosis but resulted in a delay in the mortality of WSSV-infected shrimp as well as a reduction in the total hemocyte number and WSSV copies. According to an in vitro luciferase reporter assay, the pmo-miR-bantam bound to the 3′UTR of the PmKuSPI gene as predicted. In accordance with the loss of function studies using dsRNA-mediated RNA interference, the administration of the pmo-miR-bantam mimic into WSSV-infected shrimp lowered the expression of the PmKuSPI transcript and the PmKuSPI protein, as well as the WSSV copy number. According to these results, the protease inhibitor PmKuSPI is posttranscriptionally controlled by pmo-miR-bantam and plays a role in hemocyte homeostasis, which in turn affects shrimp susceptibility to WSSV infection.

Abstract 2584: Overcoming cetuximab resistance in colorectal cancer through inhibition of HGF processing

Abstract Cetuximab is a monoclonal antibody against epidermal growth factor receptor (EGFR) and can prolong survival in metastatic colorectal cancer (CRC) patients. However, therapeutic resistance can limit its efficiency and use. Our lab has previously identified that the activation of receptor tyrosine kinases (RTKs) MET and RON contributed to cetuximab resistance. In the absence of activating mutations of MET and RON, the activity of these RTKs depends on the availability of their ligands, hepatocyte growth factor (HGF) and hepatocyte growth factor-like (HGFL), respectively. To become biologically active, the inactive precursors of HGF and HGFL must be processed by serine proteases (HGFA, matriptase, and hepsin). A survey of TCGA datasets indicated that MET and HGF were overexpressed in several CRC CMS subtypes. However, we did not observe any significant overexpression of the serine proteases. Hepatocyte growth factor activator inhibitor type 1 and 2 (HAI-1/2) are two Kunitz-type serine protease inhibitors that displayed reduced expression in TCGA datasets. The addition of recombinant human HGF conferred cetuximab resistance to cetuximab-sensitive cells and cetuximab-sensitive cells engineered to overexpress HGF also exhibited cetuximab-resistant phenotype. Cetuximab resistance induced by HGF addition or overexpression could be overcome by downstream inhibition with crizotinib, a multi-targeted RTK inhibitor. ZFH7116 is a small molecule inhibitor of the serine proteases that mimics HAI-1 activity. ZFH7116 suppressed HGF cleavage and maturation in our cetuximab-resistant SC cells. We observed cooperative effect of ZFH7116 with cetuximab to reduce SC cell growth in 3D collagen cultures. We are currently testing if upstream inhibition with recombinant human HAI-1 addition can suppress HGF cleavage and overcome cetuximab resistance induced by HGF overexpression. Collectively, our findings suggest that inhibiting HGF cleavage and processing may be a unique strategy for overcoming EGFR-targeted therapy resistance in colorectal cancer. Citation Format: Vivian Truong Jones, Ramona Graves-Deal, Galina Bogatcheva, Zheng Cao, James Higginbotham, Sarah Harmych, Gregory Ayers, Marisol Ramirez, Qi Liu, James Janetka, Bhuminder Singh. Overcoming cetuximab resistance in colorectal cancer through inhibition of HGF processing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2584.

Decomplexation of Venom Proteome of Pakistani Cobra (Naja naja naja)

The venom proteome of Naja naja from Sindh, Pakistan was decomplexed utilizing reverse phase HPLC and SDS PAGE. The results were compared with already reported Naja naja species of the region. The banding pattern represents all the major families of proteins including three-finger toxins, phospholipase A2, snake venom metalloproteases, L-amino acid oxidases, phosphodiesterase, nucleotidases, cysteine-rich secretory proteins, serine proteases, nerve growth factor, cobra venom factor, acetylcholinesterases, Kunitz-type serine protease inhibitors and C-type lectin proteins. The decomplexation of the venom showed the best possible separation through RP-HPLC elution of venom components containing small peptides, small and large proteins based on hydrophobicity. The SDS PAGE under reducing and non-reducing conditions of HPLC fractions highlighted the presence of several proteins.

Possible role of combined therapy targeting MET and pro-HGF activation for renal cell carcinoma: analysis by human HGF-producing SCID mice

MET is a high-affinity receptor tyrosine kinase of HGF (hepatocyte growth factor). HGF is secreted as an inactive single-chain precursor (pro-HGF), which requires proteolytic activation for conversion to an active form. HGF activator inhibitor (HAI)-2 is a transmembrane Kunitz-type serine protease inhibitor, which inhibits all pro-HGF-activating enzymes. In RCC, increased expression of MET and decreased expression of HAI-2 were reported to be poor prognostic factors. In the current study, we tried to inhibit the growth of RCC cells by dual inhibition of both MET phosphorylation and pro-HGF-activation using MET inhibitor and HAI-2 overexpression. A transgenic mouse model which expressed human HGF (HGF mouse) was used for in vivo analysis to evaluate the HGF/MET signaling axis accurately. Initially, doxycycline-induced HAI-2 overexpression RCC cells (786-O-HAI2) were prepared. The cells were cultured with pro-HGF, and inhibitory effect of MET inhibitor (SCC244) and HAI-2 was evaluated by phosphorylation of MET and cell proliferation. Next, the cells were subcutaneously implanted to HGF mice and the growth inhibition was determined by SCC244 and HAI-2. Single use of each inhibitor showed significant inhibition in MET phosphorylation, migration and proliferation of 786-O-HAI2 cells; however, the strongest effect was observed by combined use of both inhibitors. Although in vivo analysis also showed apparent downregulation of MET phosphorylation and growth inhibition in combined treatment, statistical significance was not observed compared with single use of MET inhibitor. Combined treatment with MET-TKI and HAI-2 suggested to consider as a candidate for new strong therapy for RCC.

N-glycosylation on Asn-57 is required for the correct HAI-2 protein folding and protease inhibitory activity.

Hepatocyte growth factor activator inhibitor (HAI)-2 is an integral membrane Kunitz-type serine protease inhibitor that regulates the proteolysis of matriptase and prostasin in a cell-type selective manner. The cell-type selective nature of HAI-2 function depends largely on whether the inhibitor and potential target enzymes are targeted to locations in close proximity. The N-glycan moiety of HAI-2 can function as a subcellular targeting signal. HAI-2 is synthesized with 1 of 2 different N-glycan modifications: one of oligomannose-type, which largely remains in the endoplasmic reticulum/GA, and another of complex-type, which is targeted toward the apical surface in vesicle-like structures, and could function as an inhibitor of matriptase and prostasin. HAI-2 contains 2 putative N-glycosylation sites, Asn-57 and Asn-94, point mutations of which were generated and characterized in this study. The protein expression profile of the HAI-2 mutants indicates that Asn-57, and not Asn-94, is responsible for the N-glycosylation of both HAI-2 species, suggesting that the form with oligomannose-type N-glycan is the precursor of the form with complex-type N-glycan. Unexpectedly, the vast majority of non-glycosylated HAI-2 is synthesized into multiple disulfide-linked oligomers, which lack protease inhibitory function, likely due to distorted conformations caused by the disarrayed disulfide linkages. Although forced expression of HAI-2 in HAI-2 knockout cells artificially enhances HAI-2 oligomerization, disulfide-linked HAI-2 oligomers can also be observed in unmodified cells. These results suggest that N-glycosylation on Asn-57 is required for folding into a functional HAI-2 with full protease suppressive activity and correct subcellular targeting signal.

Gene cloning and functional study of PmKSPI from Pinctada fucata martensii

Kunitz-type serine protease inhibitors (KSPI) are a family of serine protease inhibitors (SPIs) and are extensively found in animals, plants, and microbes. SPI can inhibit proteases that may be harmful or unwanted to its cells. Here, a four-domain Kunitz-type SPI, PmKSPI, was cloned by RACE in the pearl oyster Pinctada fucata martensii. The full-length cDNA sequence of PmKSPI was 1318 bp, including the 5′ UTR (25 bp), the 3′ UTR (96 bp) and ORF (1197 bp). Homology analysis indicated that PmKSPI had the highest resemblance (30.14%) with its homolog in Crassostrea gigas. Phylogenetic analysis revealed that PmKSPI clustered with homologs in other molluscs. We found that PmKSPI mRNA expression in P. f. martensii was distributed in all six tissues, with the highest level in the mantle, and almost no expression in other tissues. After PAMPs challenge, expression of PmKSPI mRNA in the mantle was significantly up-regulated. The recombinant protein rPmKSPI significantly inhibited the growth of 5 kinds of Gram-negative bacteria but had little effect on Gram-positive bacterial activity. Transmission electron microscopy showed that plasmolysis occurred in two Gram-negative bacteria species when treated with rPmKSPI. rPmKSPI may thus have a bactericidal effect by destroying the bacterial cell membrane or cell walls and releasing its contents. Therefore, our results suggest that PmKSPI is tightly associated with the immunological defence of P. f. martensii.

Comparative transcriptomic analysis of the larval and adult stages of Dibothriocephalus dendriticus (Cestoda: Diphyllobothriidea).

Tapeworms of the genus Dibothriocephalus are widely distributed throughout the world, some of which are agents of human diphyllobothriasis, one of the most important fish-borne zoonoses caused by a cestode parasite. Genomic and transcriptomic data can be used to develop future diagnostic tools and epidemiological studies. The present work focuses on a comparative analysis of the transcriptomes of adult and plerocercoid D. dendriticus and the identification of their differentially expressed genes (DEGs). Transcriptome assembly and analysis yielded and annotated 35,129 unigenes, noting that 16,568 (47%) unigenes were not annotated in known databases, which may indicate a unique set of expressed transcripts for D. dendriticus. A total of 8022 differentially expressed transcripts were identified, including 3225 upregulated and 4797 downregulated differentially expressed transcripts from the plerocercoid and adult animals. The analysis of DEGs has shown that among the most differentially expressed genes, there are important genes characteristic of each stage. Thus, several genes are characteristic of D. dendriticus plerocercoids, including fatty acid-binding protein and ferritin. Among the most highly expressed DEGs of the adult stage of D. dendriticus is the Kunitz-type serine protease inhibitor, in two putative isoforms. The analyses of GO and KEGG metabolic pathways revealed that a large number of the DEGs of D. dendriticus are associated with the biosynthesis of various substances such as arginine and folate, as well as with various metabolic pathways such as galactose metabolism, selenocompound metabolism, and phosphonate and phosphinate metabolism. This will contribute to further research aimed at identifying targets for new generation drugs and the development of specific vaccines.

Evolution of nacre- and prisms-related shell matrix proteins in the pen shell, Atrina pectinata

The molluscan shell is a good model for understanding the mechanisms underlying biomineralization. It is composed of calcium carbonate crystals and many types of organic molecules, such as the matrix proteins, polysaccharides, and lipids. The pen shell Atrina pectinata (Pterioida, Pinnidae) has two shell microstructures: an outer prismatic layer and an inner nacreous layer. Similar microstructures are well known in pearl oysters (Pteriidae), such as Pinctada fucata, and many kinds of shell matrix proteins (SMPs) have been identified from their shells. However, the members of SMPs that consist of the nacreous and prismatic layers of Pinnidae bivalves remain unclear. In this study, we identified 114 SMPs in the nacreous and prismatic layers of A. pectinata, of which only seven were found in both microstructures. 54 of them were found to bind calcium carbonate. Comparative analysis of nine molluscan shell proteomes showed that 69 of 114 SMPs of A. pectinata were found to have sequential similarity with at least one or more SMPs of other molluscan species. For instance, nacrein, tyrosinase, Pif/BMSP-like, chitinase (CN), chitin-binding proteins, CD109, and Kunitz-type serine proteinase inhibitors are widely shared among bivalves and gastropods. Our results provide new insights for understanding the complex evolution of SMPs related to nacreous and prismatic layer formation in the pteriomorph bivalves.

Structural and functional properties of rBmTI-A: A Kunitz-BPTI serine protease inhibitor with therapeutical potential

Serine proteases are an important group of enzymes present in several organisms such as viruses, bacteria and eukaryotes involved in several physiological and pathological processes such as cancer, neurodegeneration, tissue inflammation and infections. Kunitz-type serine protease inhibitors have been studied as therapeutical targets with positive results in many of these diseases. rBmTI-A (recombinant B. microplus Trypsin Inhibitor A) is a Kunitz-BPTI type inhibitor based on the native protein BmTI-A. BmTI-A was extracted from tick larvae and presented inhibitory activity against trypsin, human plasma kallikrein (HuPK), human neutrophil elastase (HNE) and human plasmin. rBmTI-A presented the same inhibitory activities of the BmTI-A and its thermostability has already been demonstrated. In emphysema induced by porcine pancreatic elastase (PPE) and by cigarette smoke animal models, the treatment using rBmTI-A showed a protective effect against the development of pulmonary emphysema and prevented the increase of inflammatory cells. In chronic allergic animal model, rBmTI-A treatment resulted in attenuated bronchial hyperresponsiveness, inflammation, remodeling. These are important physiological results in emphysema and lung inflammatory animal models with rBmTI-A treatment confirming its therapeutical potential.