Bowman-Birk Trypsin Inhibitor Research Articles

The rice E3 ubiquitin ligase-transcription factor module targets two trypsin inhibitors to enhance broad-spectrum disease resistance

Broad-spectrum disease resistance (BSR) is crucial for controlling plant diseases and relies on immune signals that are subject to transcriptional and post-translational regulation. How plants integrate and coordinate these signals remains unclear. We show here that the rice really interesting new gene (RING)-type E3 ubiquitin ligase OsRING113 targets APIP5, a negative regulator of plant immunity and programmed cell death (PCD), for 26S proteasomal degradation. The osring113 mutants in Nipponbare exhibited decreased BSR, while the overexpressing OsRING113 plants showed enhanced BSR against Magnaporthe oryzae (M.oryzae) and Xanthomonas oryzae pv. oryzae (Xoo). Furthermore, APIP5 directly suppressed the transcription of the Bowman-Birk trypsin inhibitor genes OsBBTI5 and AvrPiz-t-interacting protein 4 (APIP4). Overexpression of these two genes, which are partially required for APIP5-mediated PCD and disease resistance, conferred BSR. OsBBTI5 and APIP4 associated with and stabilized the pathogenesis-related protein OsPR1aL, which promotes M.oryzae resistance. Our results identify an immune module with integrated and coordinated hierarchical regulations that confer BSR in plants.

Expression and Purification of Recombinant Bowman-Birk Trypsin Inhibitor from Foxtail Millet Bran and Its Anticolorectal Cancer Effect In Vitro and In Vivo.

Trypsin inhibitors derived from plants have various pharmacological activities and promising clinical applications. In our previous study, a Bowman-Birk-type major trypsin inhibitor from foxtail millet bran (FMB-BBTI) was extracted with antiatherosclerotic activity. Currently, we found that FMB-BBTI possesses a prominent anticolorectal cancer (anti-CRC) activity. Further, a recombinant FMB-BBTI (rFMB-BBTI) was successfully expressed in a soluble manner in host strain Escherichia coli. BL21 (DE3) was induced by isopropyl-β-d-thiogalactoside (0.1 mM) at 37 °C for 3.5 h by the pET28a vector system. Fortunately, a purity greater than 93% of rFMB-BBTI with anti-CRC activity was purified by nickel-nitrilotriacetic acid affinity chromatography. Subsequently, we found that rFMB-BBTI displays a strikingly anti-CRC effect, characterized by the inhibition of cell proliferation and clone formation ability, cell cycle arrest at the G2/M phase, and induction of cell apoptosis. It is interesting that the rFMB-BBTI treatment had no obvious effect on normal colorectal cells in the same concentration range. Importantly, the anti-CRC activity of rFMB-BBTI was further confirmed in the xenografted nude mice model. Taken together, our study highlights the anti-CRC activity of rFMB-BBTI in vitro and in vivo, uncovering the clinical potential of rFMB-BBTI as a targeted agent for CRC in the future.

The effect of radiofrequency heat treatment on trypsin inhibitor activity and in vitro digestibility of soybean varieties (Glycine max. (L.) Merr.)

Kunitz (KTI) and Bowman-Birk (BBI) trypsin inhibitors were characterized in soybean seeds. Cultivars having KTI/BBI (Pannónia Kincse, PK) or lacking KTI (Aries; Hilario; Bahia) were assessed with well-characterized soybean varieties having Ti-a or ti types of KTI mobility. The TIA values of Pannónia Kincse (9.8 ± 0.48 mg/g) were not significantly different (p ≤ 0.05) from Ti-a samples (10.07 ± 1.86 mg/g), while of Aires, Bahia, Hilario (6.19 ± 1.89) were identical (p ≤ 0.05) with ti samples (6.63 ± 1.99). Radiofrequency heat treatment (RF) decreased TIA values (p ≤ 0.05) at ≥ 100 °C. However, in the traditional soybean variety, the RF at 110 °C was more effective in eliminating the residual KTI activity. The remaining or the disapperaing bioactive form of trypsin inhibitors were succesfully characterized by the means of a standardized in vitro digestion model. It showed that residual BBI-originated trypsin inhibitor activity was in the stomach even after RF at 110 °C, whereas its chymotrypsin inhibitor activity was not detectable at all. Although PK and KTI null types of soybean seeds still required an energy-saving, gentle heat treatment to inactive the trypsin inhibitors before using them as food or feed, the physicochemical properties and processing quality of soybean products were protected, improved.

Determination of protease inhibitors, glycinin, and beta-conglycinin in soybeans and their relationships.

In order to search for suitable soybean varieties for different applications, the protein contents of Kunitz trypsin inhibitor (KTI), Bowman-Birk trypsin inhibitor (BBI), glycinin (11S), and β-conglycinin (7S) of 93 soybean samples from different sources and harvest years were quantified by sodium dodecyl sulfate polyacrylamidegelelectrophoresis. Meanwhile, the protease inhibitory activities against trypsin and chymotrypsin were determined. Results showed that the individual protein contents and trypsin inhibitor activities differed significantly (p<0.05) among soybean samples. KTI contents ranged from 5.25 to 14.60mg·g-1 ; BBI contents ranged from 1.81 to 5.74mg·g-1 ; 11S varied from 13.65% to 48.55% and 7S varied from 15.68% to 42.15% of total soluble protein; trypsin and chymotrypsin inhibitory activities were 8.93-20.95mg TI·g-1 and 4.18 -12.79mg CI·g-1 , respectively. Excellent linear relationships existed between trypsin inhibitor contents and their activities. The regression equations offer a rapid method for estimating the activity of KTI or BBI in raw soybeans. PRACTICAL APPLICATION: The regression equations established based on a large number of soybean varieties offered a rapid method to estimate the activity of trypsin inhibitors. The data presented here provided useful information for the food industry or breeders to select soybean varieties with different inhibitory activities or protein contents for different food processing applications.

Trypsin inhibitor content and activity of soaking water whey as waste in soy milk processing

Soybean soaking water whey (SWW) is obtained as the waste of soy milk production and mostly represents an environmental problem. The aim of this study was to assess the content of proteins and content and activity of trypsin inhibitors of fresh SWW, obtained during soy milk production. Two zones of Bowman-Birk trypsin inhibitors (BBI) were detected. One was identified as a monomeric form of BBI (0.61-2.93%) and the other one was identified as a polymeric form of BBI (0.45-3.33%). The degree of BBI extraction (1.88-5.49%) was influenced by the soybean genotype and the grain size, i.e. it increased with increasing grain size. Kunitz trypsin inhibitor was not detected. Total proteins were found in traces in SWW (0.03-0.06%). Low residual trypsin inhibitor activity (0.32-0.55%) suggested that SWW can potentially be applied for preparing food or feed. In that case it will not be waste but a cheap functional supplement with BBI as a biologically active component.

A fungal effector and a rice NLR protein have antagonistic effects on a Bowman-Birk trypsin inhibitor.

SummaryBowman–Birk trypsin inhibitors (BBIs) play important roles in animal and plant immunity, but how these protease inhibitors are involved in the immune system remains unclear. Here, we show that the rice (Oryza sativa) BBI protein APIP4 is a common target of a fungal effector and an NLR receptor for innate immunity. APIP4 exhibited trypsin inhibitor activity in vitro and in vivo. Knockout of APIP4 in rice enhanced susceptibility, and overexpression of APIP4 increased resistance to the fungal pathogen Magnaporthe oryzae. The M. oryzae effector AvrPiz‐t interacted with APIP4 and suppressed APIP4 trypsin inhibitor activity. By contrast, the rice NLR protein Piz‐t interacted with APIP4, enhancing APIP4 transcript and protein levels, and protease inhibitor activity. Our findings reveal a novel host defence mechanism in which a host protease inhibitor targeted by a fungal pathogen is protected by an NLR receptor.

In vivo anti-inflammatory efficacy of the combined Bowman-Birk trypsin inhibitor and genistein isoflavone, two biological compounds from soybean.

Soybean Bowman-Birk protease inhibitor (BBI) and genistein, two biological compounds from soybean, are well-known for their anti-inflammatory, antioxidant, and anticancer activities. The aim of this study was designing a BBI-genistein conjugate and then investigating its protective effect on lipopolysaccharide (LPS)-induced inflammation in BALB/c mice, compared with the effects of combination of BBI and genistein. BBI was purified from soybean and the BBI-genistein conjugate was synthesized. The BALB/c mice were intraperitoneally treated 2 hours before LPS induction. Our results showed that treatment with the combination of BBI and genistein greatly led to more reduced serum levels of tumor necrosis factor (TNF)-α and interferon (IFN)-γ compared with the treatments of BBI alone, the BBI-genistein conjugate, and genistein alone, respectively. Moreover, the expression of TNF-α and IFN-γ in the splenocytes was significantly downregulated along with improving host survival against the LPS-induced lethal endotoxemia in the same way. Our data support a new combined therapy using BBI and genistein, as natural anti-inflammatory agents, to develop a new drug for inflammatory diseases.

Insight into the inactivation mechanism of soybean Bowman-Birk trypsin inhibitor (BBTI) induced by epigallocatechin gallate and epigallocatechin: Fluorescence, thermodynamics and docking studies

Soybean Bowman-Birk trypsin inhibitor (BBTI), an antinutritional factor of soy products, could strongly inhibit the protein digestion. The inactivation effect and mechanism of BBTI induced by tea polyphenols (TPs) and its major components (EGCG and EGC), were investigated in this study using fluorescence, FTIR, CD spectroscopy, isothermal titration calorimetry (ITC) and molecular docking. EGCG and EGC interacted with BBTI via static quenching process and hydrophobic interaction, with binding constant (Ka) of 2.19 × 103 M−1 and 0.25 × 103 M−1 at 298 K, respectively. TPs, EGCG and EGC induced a transition of BBTI conformation from disorder to order. ITC analysis and molecular docking revealed the interaction of EGCG-BBTI and EGC-BBTI were spontaneous, and hydrophobic interactions and hydrogen bonds were the predominant forces. Overall, this study clearly suggested that EGCG could be a promising inactivating agent for BBTI, which could also improve the safety and nutritional value of soy products.

Soybean Trypsin Inhibitors: Selective Inactivation at Hydrolysis of Soybean Proteins by Some Enzymatic Complexes

Inactivation of the Kunitz and Bowman-Birk soybean trypsin inhibitors at hydrolysis of soybean proteins by enzymatic complexes was studied. These complexes are derived from king crab hepatopancreas, cod fish pyloric caeca, and a commercial enzymatic complex of protosubtilin. Analysis of the soybean protein hydrolysates showed that these enzymatic complexes completely digested the Kunitz trypsin inhibitor (60–70% of the total trypsin inhibitors) and had almost no effect on the Bowman-Birk trypsin and chymotrypsin inhibitor. All of the enzymatic complexes contain different sets of enzymes with different proteolytic specificity. This allow to make the conclusion that Bowman-Birk inhibitor is highly resistant to proteolysis and is not inactivated at enzymatic hydrolysis.

Peanut Seed Cultivars with Contrasting Resistance to Aspergillus parasiticus Colonization Display Differential Temporal Response of Protease Inhibitors.

Significant efforts are being made to minimize aflatoxin contamination in peanut seeds and one possible strategy is to understand and exploit the mechanisms of plant defense against fungal infection. In this study we have identified and characterized, at biochemical and molecular levels, plant protease inhibitors (PPIs) produced in peanut seeds of the resistant PI 337394 and the susceptible Forman cultivar during Aspergillus parasiticus colonization. With chromatographic methods and 2D-electrophoresis-mass spectrometry we have isolated and identified four variants of Bowman-Birk trypsin inhibitor (BBTI) and a novel Kunitz-type protease inhibitor (KPI) produced in response to A. parasiticus colonization. KPI was detected only in the resistant cultivar, while BBTI was produced in the resistant cultivar in a higher concentration than susceptible cultivar and with different isoforms. The kinetic expression of KPI and BBTI genes along with trypsin inhibitory activity was analyzed in both cultivars during infection. In the susceptible cultivar an early PPI activity response was associated with BBTI occurrence. Meanwhile, in the resistant cultivar a later response with a larger increase in PPI activity was associated with BBTI and KPI occurrence. The biological significance of PPI in seed defense against fungal infection was analyzed and linked to inhibitory properties on enzymes released by the fungus during infection, and to the antifungal effect of KPI.

Identification of peptides in the terminal ileum of broiler chickens fed diets based on maize and soybean meal using proteomics

A total of 160 Ross PM3 birds were used in a two treatment feeding study in order to explore the usefulness of proteomics to identify the origin of peptides in ileal digesta. Two diets were fed, one conventional maize/soy-based diet acted as a reference whereas a second diet, formulated to be nutritionally equivalent to the reference diet in protein and energy provision, contained 20% raw soy meal in order to (putatively) elicit changes in intestinal protein flow. Each diet was fed to 10 replicate cages of eight birds per cage from Day 1 to 21. Feed and water were available ad libitum and an indigestible marker was included for assessment of ileal digestibility. Weight gain and feed intake were monitored and at the end of the trial period birds were killed, pancreatic mass was measured and the ileum was excised and the contents were collected, immediately frozen in liquid nitrogen and were subsequently lyophilised. Protein from the ileal digesta was extracted and exposed to proteomic analysis with peptide fragments identified and compared with an amalgamated database containing protein sequences from chicken, soy and maize. Addition of 20% raw soy meal to the maize/soy-based diet resulted in a reduction in weight gain, feed intake and an increase in feed conversion ratio (P &amp;lt; 0.001). Pancreatic mass was significantly increased and the apparent ileal digestibility of protein was significantly decreased by raw soy meal inclusion. Overall, a total of 248 proteins were identified from endogenous origin, 336 from soy and 411 from maize. However, the relative abundance of these proteins were ~20–30% for endogenous protein, 65–75% for soy protein and ~2–4% for maize protein. The addition of 20% raw soy meal resulted in an increase in the relative abundance of endogenous protein and a reduction in the relative abundance of protein from soy with no measurable effect on the presence of protein from maize. Specifically, in the endogenous protein fraction, there was a significant reduction in the relative abundance of metalloendopeptidase, aminopeptidase and alkaline phosphatase and a significant increase in the relative abundance of colipase and trypsin, in response to raw soy meal inclusion. For proteins originating from soybean, the addition of raw soy meal to the diet resulted in a significant increase in the relative abundance of protein from the 2S albumin fraction, Kunitz and Bowman–Birk trypsin inhibitors and soybean agglutinin whereas there was a reduction in the relative abundance of globulin and glycinin. Addition of raw soy meal to the diet also resulted in a significant increase in the presence of maize prolamin in the lumen and a significant decrease in the presence of globulin-2, β-1–3-glucanase and cystatin. These results demonstrate considerable potential of proteomics technology to identify changes in the digestion and secretion of protein in the intestine of chickens. Although these data are preliminary and based on an animal model that included diets that were formulated to have a chronic effect on intestinal physiology it is evident that changes in diet composition can have a profound effect on the origin of protein that leaves the ileum.

The influence of soybean genotypes and HTC processing method on trypsin inhibitor activity of soymilk

Kunitz inhibitor (KTI) and Bowman-Birk trypsin inhibitor (BBI) are inhibitors of digestive enzymes in raw soybeans. Due to their antinutritive properties in the active state, their inactivation by heat treatment is commonly used. Soymilk is a turbid and stable colloidal solution, obtained by thermal treatment of soybean. In this study soymilk was made on a pilot-plant scale from six soybean cultivars using hydrothermal cooking (HTC) as the production method. This procedure is significantly different from the traditional one. The aim of this investigation was to evaluate the impact of the HTC processing for soymilk production and different soybean genotypes on trypsin inhibitor content and activity. Obtained soymilk contained BBI in trace amounts, in the BBI-polymeric forms. The BBI monomeric forms were not detected. The soymilk of the investigated soybean genotypes had very similar KTI levels (2.34-2.99%). Results have suggested that the soybean genotype does not have substantial effects on the levels of KTI, as well as on the value of residual trypsin inhibitor activity (rTIA). The total content of TI and rTIA showed a strong dependence (r=0.91; p&lt;0.05). HTC-soymilk rTIA was &lt;20% (7.15-19.89%). These results have indicated that HTC processed soymilk is applicable for human consumption.

Effects of Two Trypsin Inhibitors on Trypsin in Activity and Structure

Two reversible trypsin inhibitors, Kunitz trypsin inhibitor (KTI) and Bowman-Birk trypsin inhibitor (BBI) were compared to find the more optimal one as the inhibit factor during trypsin immobilization. Fluorescence spectroscopy, UV–visible absorption spectroscopy and circular dichroism (CD) spectroscopy were used to explore the effects of the two inhibitors on trypsin in activity and structure. The results showed that both inhibitors combined with trypsin in 1:1. CD circular dichroism spectroscopy showed that KTI and BBI led to different changes in trypsin second structure. The results can help us find out the mechanism between the two inhibitors and trypsin and select the more optimal inhibitor in trypsin immobilization.

Purification and Characterization of Bowman-Birk Trypsin Inhibitor from Soybean

In this paper, crude extract of a Bowman-Birk trypsin inhibitor from soybean meal was firstly isolated by a combination of , thermal denaturation, isoelectric precipitation and acetone precipitation. Then this extract was purified by DE-52 ion exchange and affinity chromatography. The results showed that soybean Bowman-Birk trypsin inhibitor (SBBI) was purified to 50.07-fold with trypsin activity of 822.31 U·mg-1. The purified SBBI gave a single protein band in SDS-PAGE electrophoresis. The accurate molecular mass of this inhibitor was determined to be 8837.46Da by MALDI-TOF. N-terminal sequence showed high homology with other serine proteinase inhibitors belonging to the Leguminosae family.

Identification of a Bioactive Bowman–Birk Inhibitor from an Insect-Resistant Early Maize Inbred

Breeding of maize, Zea mays, has improved insect resistance, but the genetic and biochemical basis of many of these improvements is unknown. Maize oligonucleotide microarrays were utilized to identify differentially expressed genes in leaves of three maize inbreds, parents Oh40B and W8 and progeny Oh43, developed in the 1940s. Oh43 had enhanced leaf resistance to corn earworm larvae, Helicoverpa zea, and fall armyworm larvae, Spodoptera frugiperda, compared to one or both parents. Among ca. 100 significantly differentially expressed genes, expression of a Bowman-Birk trypsin inhibitor (BBI) gene was at least ca. 8-fold higher in Oh43 than in either parent. The Oh43 BBI gene was expressed as a recombinant protein. Purified BBI inhibited trypsin and the growth of fall armyworm larvae when added to insect diet. These experiments indicate that comparative gene expression analysis combined with insect resistance measurements of early inbreds can identify previously unrecognized resistance genes.

The Bowman–Birk Trypsin Inhibitor IBP1 Interacts with and Prevents Degradation of IDEF1 in Rice

To acquire slightly soluble iron from the rhizosphere, plants transcriptionally induce genes involved in iron acquisition in response to low iron availability. The transcription factor IDEF1 plays an important role in the regulation of this response in graminaceous plants. By yeast two-hybrid screening, we identified IDEF1-binding proteins, designated IBP1.1 and IBP1.2, as two homologous Bowman–Birk trypsin inhibitors. Interaction between IDEF1 and IBP1.1 was also confirmed by pull-down assay. IBP1.1 and IBP1.2 expression showed induction in response to iron deficiency and IDEF1 dependence. IBP1.1 localized to the nucleus and the cytoplasm when transiently expressed in onion epidermal cells. Transgenic rice plants overexpressing IBP1.1 showed enhanced expression of the Fe(II)-nicotianamine transporter gene OsYSL2. IDEF1 protein is degraded in a 26S proteasome-dependent manner and this degradation was prevented by IBP1.1. These results suggest that induced expression of IBP1.1 under Fe-deficient conditions contributes to the IDEF1-mediated iron deficiency response by preventing the degradation of IDEF1.

Production and properties of recombinant glutenin-cleaving proteinases from Eurygaster integriceps Put

cDNAs coding for a mature form of glutenin-cleaving trypsin-like proteinase (referred to as glutenin-hydrolyzing proteinase 3 or GHP3) from the insect pest-Eurygaster integriceps Put. and a zymogen of this proteinase containing a signal peptide required for protein secretion were cloned into vectors pPIC9 and pPIC3.5, respectively. The constructs were used for protein expression in cells of the methylotrophic yeast Pichia pastoris. The recombinant protein corresponding to the mature form of the proteinase was secreted into the culture medium and possessed proteolytic activity, while the zymogen acquired activity after trypsin, treatment. Both recombinant enzymes hydrolyzed high-molecular weight glutenin subunits from wheat of the variety Ege-88 and a range of other soft and durum wheat varieties. Chymotrypsin inhibitor I from potatoes and related inhibitors from seeds of plants of the subclass Asteridae, the Kunitz-type trypsin inhibitor from soybeans, and bovine aprotinin had a weak inhibitory effect on the recombinant proteinases, while the Bowman-Birk trypsin and chymotrypsin inhibitor from soybeans did not interact with these enzymes:

Brown Kidney Bean Bowman–Birk Trypsin Inhibitor is Heat and pH Stable and Exhibits Anti-proliferative Activity

A trypsin inhibitor with a molecular mass around 17kDa was purified from the seeds of Phaseolus vulgaris cv. 'brown kidney bean'. The purification protocol involved, in sequence, affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on Q-Sepharose and Mono Q, and gel filtration on Superdex 75. The molecular size and N-terminal amino acid sequence of the trypsin inhibitor resembled leguminous Bowman-Birk protease inhibitors (BBIs), signifying that brown kidney bean trypsin inhibitor is a BBI. Brown kidney bean trypsin inhibitor exhibited pronounced thermostability and pH stability. Its trypsin inhibitory activity was retained at all pH values (0-14) and up to 90°C. The trypsin inhibitor also inhibited the proliferation of human breast cancer MCF7 cells with an IC(50) of 71.52μM. On the other hand, it only slightly inhibited proliferation of hepatoma HepG2 and embryonic liver WRL68 cells at a concentration over 110μM.

IMPROVEMENT OF PURIFICATION OF TRYPSIN INHIBITOR FROM WILD SOYBEAN (GLYCINE SOJA SIEB. &amp; ZUCC.) USING CHITOSAN RESIN-IMMOBILIZED TRYPSIN

Chitosan resin-trypsin was prepared for one-step affinity purification of trypsin inhibitor (TI) from a wild-type soybean (Glycine soja Sieb. & Zucc.). The influences of several parameters (cross-linking, chemical modifying and activating agent) on the chitosan resin-trypsin activity were investigated. The results showed that the chitosan resin-trypsin composite demonstrated considerable trypsin activity at levels of, 3.6% glutaraldehyde (cross-linking agent), 0.06 mol/L NaBH4 (chemical modifying agent) and 15% epichlorohydrin (activating agent). Furthermore, the trypsin activity assay indicated that chitosan resin-trypsin could tolerate relatively high temperature (65C) and wide pH range (5.0–9.0), compared with free trypsin (45C, pH 6.0–8.0). With chitosan resin-trypsin as matrix on affinity chromatography column, a Bowman-Birk trypsin inhibitor from a wild soybean was purified (adsorption capacity of chitosan resin-trypsin for TI was about 1.33 mg/g wet matrix), with homogeneous MW of 8.2 kDa estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The affinity chromatography purification method described for TI was 279-fold with a 58.3% recovery rate of TI, much improved over established procedures. PRACTICAL APPLICATIONS The use of chitosan resin as a support for enzymes in affinity chromatography has advantages over other materials: low cost, good stability and versatility. The covalent immobilization of trypsin on chitosan resin permits easy recovery and reuse of the enzyme, and produces a quick and effective tool to isolate the Bowman-Birk trypsin inhibitor form wild soybean. This method could be a competitive choice for large-scale purification of this functional molecule.

MSPL/TMPRSS13

Two variant cDNAs, mosaic seine protease large-form (MSPL) and transmembrane protease serine 13 (TMPRSS13), have been identified from a human lung cDNA library by polymerase chain reaction. The deduced amino acid sequences of these proteins show a type II transmembrane protein structure with a unique and long cytoplasmic tail containing tandem repeat phosphorylation motifs of protein kinases, a transmembrane domain, and a trypsin-like serine protease domain at the extracellular C-terminal side. These proteins have an identical serine protease sequence except for the C-terminal ends, and the consensus protease domain exhibits 42, 39 and 43% sequence identity with those of plasma kallikrein, hepsin and transmembrane protease serine 2 (TMPRSS2), respectively. Although both genes are widely expressed in various tissues, they are predominantly expressed in human lung, placenta, pancreas and prostate. TMPRSS13 is expressed higher than MSPL in thymus, spleen and peripheral blood lymphocytes, particularly in CD8+ cells and CD19+ cells. Enzymatic properties of the recombinant soluble MSPL and TMPRSS13 show that these enzymes preferentially recognize the sites consisting of paired basic amino acid residues, and are strongly inhibited by aprotinin, benzamidine and Bowman-Birk trypsin inhibitor, but poorly inhibited by alpha 1-antitrypsin and leupeptin. These properties raise the possibility that MSPL and TMPRSS13 play roles in the proteolytic processing of prohormones, precursors of growth factors, and also play roles in the pathogenicity of many viruses and bacteria in vivo.