Immunoassay Detects Salivary Anti-SSA/Ro-52 Autoantibodies in Seronegative Patients with Primary Sjögren's Syndrome.

The diagnostic work-up for Sjögren's syndrome is challenging and complex, including testing for serum autoantibodies to SSA/Ro and a labial salivary gland biopsy. Furthermore, the diagnosis is often delayed. In this study, we tested the hypothesis that anti-SSA/Ro autoantibodies are detectable in the saliva of patients with primary Sjögren's syndrome (pSS) because the disease affects the salivary glands, and these autoantibodies display greater discriminatory performance in saliva than in serum. SSA/Ro-52 Ags were used to develop what is, to our knowledge, a novel quantitative electrochemical-based immunoassay: the electric field-induced release and measurement (EFIRM) platform. The clinical utility was determined by measuring salivary anti-SSA/Ro-52 autoantibodies in patients with pSS and sicca (n = 34), patients without pSS with sicca (n = 35), and healthy subjects (n = 41). The statistical analysis of discrimination included the area under the receiver operating characteristic curve. Salivary anti-SSA/Ro-52 autoantibodies were measured in 94% (32 of 34) of patients with pSS with 85% (29 of 34) seropositivity. Four of the five seronegative patients with pSS had EFIRM-measurable anti-SSA/Ro-52 autoantibodies in saliva. Additionally, 60% (21 of 35) of the seronegative patients without pSS who had sicca had EFIRM-detectable SSA/Ro-52 autoantibodies in saliva, indicating the onset of autoimmune disease. Two of the 41 healthy control subjects had EFIRM-detectable SSA/Ro-52 autoantibodies in their saliva. Salivary SSA/Ro-52 autoantibodies significantly discriminated patients with pSS or patients with the initial stage of autoimmune disease from healthy subjects with an area under the receiver operating characteristic curve of 0.91. Our findings suggest that the proposed saliva SSA/Ro-52 immunoassay improves early and accurate diagnosis of seronegative patients with pSS and patients with early-onset autoimmune disease.

Two-year clinical outcomes after discontinuation of long-term golimumab therapy in Korean patients with rheumatoid arthritis.

BACKGROUND/AIMS: The aim of this study was to investigate long-term post-discontinuation outcomes in patients with rheumatoid arthritis (RA) who had been treated with tumor necrosis factor-α inhibitors (TNF-αi) which was then discontinued. METHODS: Sixty Korean patients with RA who participated in a 5-year GO-BEFORE and GO-FORWARD extension trials were included in this retrospective study. Golimumab was deliberately discontinued after the extension study (baseline). Patients were then followed by their rheumatologists. We reviewed their medical records for 2 years (max 28 months) following golimumab discontinuation. Patients were divided into a maintained benefit (MB) group and a loss-of-benefit (LB) group based on treatment pattern after golimumab discontinuation. The LB group included patients whose conventional disease-modifying antirheumatic drug(s) were stepped-up or added/switched (SC) and those who restarted biologic therapy (RB). RESULTS: The mean age of patients at baseline was 56.5 years and 55 (91.7%) were females. At the end of follow-up, 23 (38.3%) patients remained in the MB group. In the LB group, 75.7% and 24.3% were assigned into SC and RB subgroups, respectively. Fifty percent of patients lost MB after 23.3 months. Demographics and clinical variables at baseline were comparable between MB and LB groups except for age, C-reactive protein level, and corticosteroid use. Restarting biologic therapy was associated with swollen joint count (adjusted hazard ratio [HR], 1.90; 95% confidence interval [CI], 1.01 to 3.55) and disease duration (adjusted HR, 1.12; 95% CI, 1.02 to 1.23) at baseline. CONCLUSION: Treatment strategies after discontinuing TNF-αi are needed to better maintain disease control and quality of life of patients with RA.

Novel susceptibility alleles in HLA region for myositis and myositis specific autoantibodies in Korean patients.

OBJECTIVES: HLA genes are a major genetic risk factor for myositis and myositis specific antibodies (MSAs), exhibiting unique HLA backgrounds for myositis in different ethnic groups. This is the first large scale Korean study to genotype the HLA-DRB1 and -DPB1 alleles and to examine their association with myositis and MSAs. METHODS: HLA-DRB1 and HLA-DPB1 alleles and MSAs were examined in 179 patients with dermatomyositis (DM, n = 129) or polymyositis (PM, n = 50) and healthy controls (n = 800 for HLA-DRB1, n = 548 for HLA-DPB1). Associations between individual HLA alleles and myositis/MSA were examined. Bonferroni correction was applied for multiple testing comparing patients and controls. RESULTS: A total of 33 HLA-DRB1 and 24 HLA-DPB1 alleles were genotyped in patients and controls. MSAs were found in 67.0% of patients. Anti-MDA5 (26.8%) and anti-aminoacyl-tRNA synthetase antibodies (15.6%) were most common, followed by anti-Mi2 (9.5%) and anti-TIF1γ antibodies (8.9%). HLA-DRB1*12:02 and HLA-DRB1*14:03 were associated with DM and PM, respectively. HLA-DRB1*12:02 was associated with anti-MDA5, HLA-DRB1*08:03 with anti-ARS, HLA-DRB1*14:03 with anti-SRP, and HLA-DRB1*07:01 with anti-Mi2 antibodies. Although HLA-DRB1*13:01 was associated with anti-TIF1γ antibodies, the frequency of HLA-DRB1*13:01 was rare. HLA-DPB1*02:01 was negatively associated with myositis and PM while HLA-DPB1*17:01 was associated with anti-Mi2 positive DM. CONCLUSIONS: Unique immunogenetic background was observed for Korean patients with myositis. Novel myositis susceptibility alleles, HLA-DRB1*12:02 and HLA-DRB1*14:03, were identified, together with MSA-associated HLA alleles unique to Korean patients with myositis.

Combinatorial effects of an epigenetic inhibitor and ionizing radiation contribute to targeted elimination of pancreatic cancer stem cell.

Pancreatic cancer is associated with a high mortality rate, owing to de novo and acquired drug resistance, thereby leading to highly invasive and metastatic pancreatic cancer cells. Therefore, targeting pancreatic cancer stem cells (CSCs) may be a novel therapeutic strategy for the treatment of pancreatic cancer. Here, we combined a DNA methylation inhibitor (5-aza-2'-deoxycytidine; 5-aza-dC) and ionizing radiation (IR) to improve anti-cancer effects by inhibiting growth and proliferation and promoting apoptosis of pancreatic cancer cells in vitro and in vivo. Importantly, the combinatorial effect of 5-aza-dC with IR on sphere-forming pancreatic cancer cells was preferentially targeted toward CSCs through the downregulation of regulatory factors of self-renewal and CSC surface markers. We next performed the RNA sequencing to understand the underlying cellular mechanisms of the combined treatment with IR and 5-aza-dC in pancreatic cancer cells. Global transcriptome profiling indicated that the expression of the Oct4-centered transcriptional network of genes was significantly downregulated in cells with combination treatment. Our data suggested that combination treatment with DNA methylation inhibitor and IR may be a novel therapeutic strategy for pancreatic cancer. Overall, these findings support the use of epigenetic therapy in combination with radiotherapy to improve therapeutic efficacy by targeting and eradicating pancreatic CSCs.

Epigenetically altered miR-1247 functions as a tumor suppressor in pancreatic cancer.

Altered expression of microRNAs has been strongly implicated in human cancers, and growing evidence is emerging that a number of miRNAs are downregulated in cancer associated with CpG island hypermethylation. Although pancreatic cancer is one of the most malignant human cancers, the roles of miRNAs underlying the tumorigenesis of pancreatic cancer are still poorly understood. In the present study, we explored the molecular functional role of microRNA-1247 as tumor suppressor associated with epigenetic alteration in pancreatic cancer. CpG islands methylation of miR-1247 is frequently observed in various pancreatic cancer cell lines and in primary pancreatic tumors, but not in normal pancreatic tissue. Ectopic expression of miR-1247 in five pancreatic cancer cell lines results in suppressing of cell growth, proliferation, migration, and invasion in vitro and tumorigenicity of pancreatic cancer cells in vivo. Interestingly, we found one putative target gene of miR-1247, regulator of chromosome condensation 2 (RCC2), harbored miR-1247 target sequences in the 3' UTR of its mRNA. In functional studies in vitro to understand the interaction between miR-1247 and RCC2, decreasing of RCC2 gene expression by miR-1247 was observed by immunoblotting and immunohistochemistry at both mRNA and protein levels. Moreover, luciferase reporter assay confirmed that RCC2 was a direct target of miR-1247. Taken together, our data suggest that CpG island hypermethylation of miR-1247 is responsible for its downregulation in pancreatic cancer, and ectopic expression of miR-1247 functions as a potential tumor suppressor targeting RCC2 in pancreatic cancer cells.

Impact of Dose Tapering of Tumor Necrosis Factor Inhibitor on Radiographic Progression in Ankylosing Spondylitis.

OBJECTIVE: To investigate the impact of dose reduction of tumor necrosis factor inhibitor (TNFi) on radiographic progression in ankylosing spondylitis (AS). METHODS: One hundred and sixty-five patients treated with etanercept or adalimumab were selected from a consecutive single-center observational cohort based on the availability of radiographs at baseline and after two- and/or four-years of follow up. Radiographs were assessed by two blinded readers using the modified Stokes AS Spinal Score (mSASSS). Radiographic progression in patients treated with standard-dose TNFi (standard-dose group, n = 49) was compared with patients whose dosage was tapered during the treatment (tapering group, n = 116) using linear mixed models. RESULTS: Baseline characteristics between two groups were comparable except for higher BASDAI (7.1 vs. 6.3, p = 0.003) in the standard-dose group. At two years after the treatment, mean dose quotient (S.D.) of the tapering group was 0.59 (0.17). During follow up, rate of radiographic progression in overall patients was 0.90 mSASSS units/year. Radiographic progression over time between the two groups was similar at the entire group level. However, in the subgroup of patients with baseline syndesmophytes, progression occurred significantly faster in the tapering group after the adjustment for baseline status (1.23 vs. 1.72 mSASSS units/year, p = 0.023). Results were consistent when radiographic progression was assessed by the number of newly developed syndesmophytes (0.52 vs. 0.73/year, p = 0.047). Sensitivity analysis after multiple imputation of missing radiographs also showed similar results. CONCLUSION: A dose tapering strategy of TNFi is associated with more rapid radiographic progression in AS patients who have syndesmophytes at baseline.

Survival benefit associated with early cyclosporine treatment for dermatomyositis-associated interstitial lung disease.

Interstitial lung disease (ILD) is the most common cause of death in dermatomyositis (DM). Cyclosporine A (CsA) has shown to be effective in DM-associated ILD (DM-ILD). This study aimed to define the optimal time of CsA administration. A total of 47 patients with DM-ILD, who were treated with CsA at Seoul National University Hospital between January 1998 and June 2013, were enrolled. ILD was diagnosed based on typical chest high-resolution computed tomography (HRCT) findings. Patients with early and delayed CsA treatment were compared in regard to the mortality and ILD progression on HRCT. The early (n = 16) and the delayed treatment group (n = 31) did not differ in regard to baseline clinical characteristics including HRCT scores and pulmonary function. Patients with clinically amyopathic DM (CADM) were more common in the early treatment group. The mortality rate was significantly lower in the early treatment group than in the delayed treatment group (p = 0.009). The survival benefit of early CsA treatment remained significant even after adjusting for age, degree of dyspnea, CADM status, and the year of CsA treatment (hazard ratio 0.057, 95 % confidence interval 0.007-0.472). CsA stabilized disease progression on HRCT in the early treatment group (p = 0.738). Delay in CsA treatment is associated with a worse survival in patients with DM-ILD. Early CsA treatment should be considered at DM-ILD diagnosis especially in patients at a higher risk of developing a rapidly progressive ILD.

A decision model for the watch-and-wait strategy in systemic sclerosis-associated interstitial lung disease.

OBJECTIVE: To develop a decision model to identify SSc-associated interstitial lung disease (ILD) patients who are eligible for watchful waiting at ILD diagnosis. METHODS: One hundred and fifty-one SSc-ILD patients who received medical care at Seoul National University Hospital from 1986 to 2013 were enrolled in this retrospective cohort study. ILD was diagnosed by chest CT. Patients with and without immunosuppressive treatment were compared in terms of characteristics at ILD diagnosis to identify distinguishing variables. After multivariate analysis, a decision model for watchful waiting was formulated. Its validity was assessed by comparing the survival of patients whose management in real practice did and did not accord with the management recommended by the model. RESULTS: The untreated group had better survival than the immunosuppressive treatment group (P = 0.0316, by log-rank test). The untreated group was less likely to have gastrointestinal involvement (P = 0.008) and pulmonary arterial hypertension (PAH), as determined by echocardiography) (P = 0.015) and more likely to have favourable initial forced vital capacity (P = 0.0004), favourable initial lung diffusion capacity for carbon monoxide (P = 0.0002) and a low CT grade (P < 0.001). The final watchful waiting decision model included lack of PAH and limited ILD extent on CT. Application of the model to the cohort revealed that patients who were eligible for watchful waiting (as determined by the model) and underwent this management strategy had better survival than eligible patients who underwent immunosuppressive treatment (P = 0.048, by log-rank test). CONCLUSION: Watchful waiting may be effective for SSc-ILD patients who have minimal pulmonary involvement on CT and lack PAH on echocardiography at baseline.

An RNA aptamer that specifically binds to the glycosylated hemagglutinin of avian influenza virus and suppresses viral infection in cells.

The influenza virus surface glycoprotein hemagglutinin (HA) is responsible for viral attachment to sialic acid-containing host cell receptors and it facilitates the initial stage of viral infection. In the present study, we isolated an RNA aptamer specific to the glycosylated receptor-binding domain of the HA protein (gHA1) after 12 cycles of the systematic evolution of ligands by exponential enrichment procedure (SELEX), and we then investigated if the selected aptamer suppresses viral infection in host cells. Nitrocellulose filter binding and enzyme-linked immunosorbent assay (ELISA) experiments revealed that 1 RNA aptamer, HA12-16, bound specifically to the gHA1 protein. Cell viability assay showed that the HA12-16 RNA aptamer suppressed viral infection in host cells by enhancing cell viability. Immunofluorescence microscopic analysis further demonstrated that the HA12-16 RNA aptamer suppresses viral attachment to host cells by neutralizing the receptor-binding site of influenza virus HA. These results indicate that the isolated RNA aptamer can be developed as an antiviral reagent against influenza through appropriate therapeutic formulation.

Sniffing for gene-silencing efficiency of siRNAs in HeLa cells in comparison with that in HEK293T cells: correlation between knockdown efficiency and sustainability of sirnas revealed by FRET-based probing.

Investigation of the intracellular fate of small interfering RNAs (siRNAs) following their delivery into cells is of great importance to elucidate their dynamics in cytoplasm. Here we describe the use of an advanced fluorescence-based method to probe the dissociation and/or degradation of double-labeled siRNAs in HeLa cells in comparison with that in human embryonic kidney 293T (HEK293T) cells. This work was performed with three siRNAs labeled with fluorescence resonance energy transfer (FRET) dyes, allowing a non-destructive and non-invasive assessment of the dissociation and degradation state of siRNAs in cultured cells. Our FRET analysis not only shows the asymmetric degradation as well as the time-dependent dissociation of each siRNA strand during the measured time period, underlining the high intrinsic nuclease resistance of duplex siRNAs, but also reveals the longer sustainability of siRNAs in HeLa cells compared with that in HEK293T cells, explaining the gene silencing in HeLa cells is more efficient than that in HEK293T cells. In addition, our single-molecule FRET assays demonstrate the potential of the delineated fluorescence-based technique for future research on biological behavior of siRNAs even at the single-molecule level. The fluorescence-based method is a straightforward technique to gain direct information on siRNA integrity inside living cells, which can provide a detection tool for dynamics of biological molecules.

FRET-based probing to gain direct information on siRNA sustainability in live cells: Asymmetric degradation of siRNA strands.

Investigation of the intracellular fate of small interference RNA (siRNA) following their delivery into cells is of great interest to elucidate dynamics of siRNA in cytoplasm. However, its cellular delivery and sustainability should be understood at the molecular level and improved for the successful in vivo application of siRNA. Here we present a fluorescence resonance energy transfer (FRET) based method using oligonucleotide probes to study intracellular dissociation (or melting) and sustainability of siRNAs in live cells. The FRET probes were specifically designed to observe intracellular dissociation (or melting) and degradation of short synthetic RNAs in real-time, thus providing the desired kinetic information in cells. Intracellular FRET analysis shows that siRNA duplex is gradually diffused into cytosol, dissociated, and degraded for a duration of 3.5 h, which is confirmed by confocal microscopy colocalization measurements. In addition, our FRET assays reveal the asymmetric degradation as well as the time-dependent dissociation of each siRNA strand. The application of this FRET technique can allow for direct information on siRNA integrity inside living cells, providing a detection tool for dynamics of biological molecules.

Structural and functional characterization of a highly specific serpin in the insect innate immunity.

The Toll signaling pathway, an essential innate immune response in invertebrates, is mediated via the serine protease cascade. Once activated, the serine proteases are irreversibly inactivated by serine protease inhibitors (serpins). Recently, we identified three serpin-serine protease pairs that are directly involved in the regulation of Toll signaling cascade in a large beetle, Tenebrio molitor. Of these, the serpin SPN48 was cleaved by its target serine protease, Spätzle-processing enzyme, at a noncanonical P1 residue of the serpin's reactive center loop. To address this unique cleavage, we report the crystal structure of SPN48, revealing that SPN48 exhibits a native conformation of human antithrombin, where the reactive center loop is partially inserted into the center of the largest ß-sheet of SPN48. The crystal structure also shows that SPN48 has a putative heparin-binding site that is distinct from those of the mammalian serpins. Ensuing biochemical studies demonstrate that heparin accelerates the inhibition of Spätzle-processing enzyme by a proximity effect in targeting the SPN48. Our finding provides the molecular mechanism of how serpins tightly regulate innate immune responses in invertebrates.

Diversity of innate immune recognition mechanism for bacterial polymeric meso-diaminopimelic acid-type peptidoglycan in insects.

In Drosophila, the synthesis of antimicrobial peptides in response to microbial infections is under the control of the Toll and immune deficiency (Imd) signaling pathway. The Toll signaling pathway responds mainly to the lysine-type peptidoglycan of Gram-positive bacteria and fungal ß-1,3-glucan, whereas the Imd pathway responds to the meso-diaminopimelic acid (DAP)-type peptidoglycan of Gram-negative bacteria and certain Gram-positive bacilli. Recently we determined the activation mechanism of a Toll signaling pathway biochemically using a large beetle, Tenebrio molitor. However, DAP-type peptidoglycan recognition mechanism and its signaling pathway are still unclear in the fly and beetle. Here, we show that polymeric DAP-type peptidoglycan, but not its monomeric form, formed a complex with Tenebrio peptidoglycan recognition protein-SA, and this complex activated the three-step proteolytic cascade to produce processed Spätzle, a Toll receptor ligand, and induced Drosophila defensin-like antimicrobial peptide in Tenebrio larvae similarly to polymeric lysine-type peptidoglycan. Monomeric DAP-type peptidoglycan induced Drosophila diptericin-like antimicrobial peptide in Tenebrio hemocytes. In addition, both polymeric and monomeric DAP-type peptidoglycans induced expression of Tenebrio peptidoglycan recognition protein-SC2, which is DAP-type peptidoglycan-selective N-acetylmuramyl-l-alanine amidase that functions as a DAP-type peptidoglycan scavenger, appearing to function as a negative regulator of the DAP-type peptidoglycan signaling by cleaving DAP-type peptidoglycan in Tenebrio larvae. Taken together, these results demonstrate that molecular recognition mechanism for polymeric DAP-type peptidoglycan is different between Tenebrio larvae and Drosophila adults, providing biochemical evidences of biological diversity of innate immune responses in insects.

Cooperative translocation enhances the unwinding of duplex DNA by SARS coronavirus helicase nsP13.

SARS coronavirus encodes non-structural protein 13 (nsP13), a nucleic acid helicase/NTPase belonging to superfamily 1 helicase, which efficiently unwinds both partial-duplex RNA and DNA. In this study, unwinding of DNA substrates that had different duplex lengths and 5'-overhangs was examined under single-turnover reaction conditions in the presence of excess enzyme. The amount of DNA unwound decreased significantly as the length of the duplex increased, indicating a poor in vitro processivity. However, the quantity of duplex DNA unwound increased as the length of the single-stranded 5'-tail increased for the 50-bp duplex. This enhanced processivity was also observed for duplex DNA that had a longer single-stranded gap in between. These results demonstrate that nsP13 requires the presence of a long 5'-overhang to unwind longer DNA duplexes. In addition, enhanced DNA unwinding was observed for gapped DNA substrates that had a 5'-overhang, indicating that the translocated nsP13 molecules pile up and the preceding helicase facilitate DNA unwinding. Together with the propensity of oligomer formation of nsP13 molecules, we propose that the cooperative translocation by the functionally interacting oligomers of the helicase molecules loaded onto the 5'-overhang account for the observed enhanced processivity of DNA unwinding.

Oligodeoxyribozymes that cleave beta-catenin messenger RNA inhibit growth of colon cancer cells via reduction of beta-catenin response transcription.

Abnormal regulation of Wnt/beta-catenin signaling followed by increased levels of the beta-catenin protein have been identified in enhanced cellular proliferation and development of colon polyps and cancers. To inhibit beta-catenin gene expression in colon cancer cells, RNA-cleaving oligodeoxyribozyme (DNAzyme) was employed to destroy the beta-catenin mRNA. We designed a strategy to identify the cleavage sites in beta-catenin RNA with a pool of random sequences from a DNAzyme library and identified four potential DNAzyme-working sites. DNAzymes were constructed for the selected target sites and were tested for the ability to cleave beta-catenin RNA. When introduced into the cells, the selected DNAzymes decreased the expression of beta-catenin significantly as well as its downstream gene, cyclin D1. Additionally, we designed short hairpin RNA that targets the same cleavage site for the selected DNAzyme. The designed short hairpin RNA also inhibited beta-catenin gene expression in colon cancer cells. Our studies show that RNA-cleaving DNAzymes and RNA interference targeted to beta-catenin significantly reduced beta-catenin-dependent gene expression, resulting in inhibition of colon cancer cell growth. These results indicate that the functional antisense oligonucleotides directed against beta-catenin might have potential as a therapeutic intervention to treat colon cancer.

Crystallization and preliminary X-ray crystallographic analysis of a highly specific serpin from the beetle Tenebrio molitor.

The Toll signalling pathway, which is crucial for innate immunity, is transduced in insect haemolymph via a proteolytic cascade consisting of three serine proteases. The proteolytic cascade is downregulated by a specific serine protease inhibitor (serpin). Recently, the serpin SPN48 was found to show an unusual specific reactivity towards the terminal serine protease, Spätzle-processing enzyme, in the beetle Tenebrio molitor. In this study, the mature form of SPN48 was overexpressed in Escherichia coli and purified. The purified SPN48 protein was crystallized using 14% polyethylene glycol 8000 and 0.1 M 2-(N-morpholino)ethanesulfonic acid pH 6.0 as the precipitant. The crystals diffracted X-rays to 2.1 A resolution and were suitable for structure determination. The crystals belonged to space group P2(1). The crystal structure will provide information regarding how SPN48 achieves its unusual specificity for its target protease.

Three pairs of protease-serpin complexes cooperatively regulate the insect innate immune responses.

Serpins are known to be necessary for the regulation of several serine protease cascades. However, the mechanisms of how serpins regulate the innate immune responses of invertebrates are not well understood due to the uncertainty of the identity of the serine proteases targeted by the serpins. We recently reported the molecular activation mechanisms of three serine protease-mediated Toll and melanin synthesis cascades in a large beetle, Tenebrio molitor. Here, we purified three novel serpins (SPN40, SPN55, and SPN48) from the hemolymph of T. molitor. These serpins made specific serpin-serine protease pairs with three Toll cascade-activating serine proteases, such as modular serine protease, Spätzle-processing enzyme-activating enzyme, and Spätzle-processing enzyme and cooperatively blocked the Toll signaling cascade and beta-1,3-glucan-mediated melanin biosynthesis. Also, the levels of SPN40 and SPN55 were dramatically increased in vivo by the injection of a Toll ligand, processed Spätzle, into Tenebrio larvae. This increase in SPN40 and SPN55 levels indicates that these serpins function as inducible negative feedback inhibitors. Unexpectedly, SPN55 and SPN48 were cleaved at Tyr and Glu residues in reactive center loops, respectively, despite being targeted by trypsin-like Spätzle-processing enzyme-activating enzyme and Spätzle-processing enzyme. These cleavage patterns are also highly similar to those of unusual mammalian serpins involved in blood coagulation and blood pressure regulation, and they may contribute to highly specific and timely inactivation of detrimental serine proteases during innate immune responses. Taken together, these results demonstrate the specific regulatory evidences of innate immune responses by three novel serpins.

N-terminal GNBP homology domain of Gram-negative binding protein 3 functions as a beta-1,3-glucan binding motif in Tenebrio molitor.

The Toll signalling pathway in invertebrates is responsible for defense against Gram-positive bacteria and fungi, leading to the expression of antimicrobial peptides via NF-kappaB-like transcription factors. Gram-negative binding protein 3 (GNBP3) detects beta-1,3-glucan, a fungal cell wall component, and activates a three step serine protease cascade for activation of the Toll signalling pathway. Here, we showed that the recombinant N-terminal domain of Tenebrio molitor GNBP3 bound to beta-1,3-glucan, but did not activate down-stream serine protease cascade in vitro. Reversely, the N-terminal domain blocked GNBP3-mediated serine protease cascade activation in vitro and also inhibited beta-1,3-glucan-mediated antimicrobial peptide induction in Tenebrio molitor larvae. These results suggest that the N-terminal GNBP homology domain of GNBP3 functions as a beta-1,3-glucan binding domain and the C-terminal domain of GNBP3 may be required for the recruitment of immediate down-stream serine protease zymogen during Toll signalling pathway activation.