A novel Kazal-type trypsin inhibitor from the skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas

The chemical complexity of the defensive skin secretion of the red-eyed leaf frog, (Agalychnis callidryas), has not been elucidated in detail. During a systematic study of the skin secretion peptidomes of phyllomedusine frogs, we discovered a novel Kazal-type protein with potent trypsin inhibitory activity (Ki = 1.9 nM) that displays the highest degree of structural similarity with Kazal proteins from bony fishes. The protein was located in reverse-phase HPLC fractions following a screen of such for trypsin inhibition and subsequent partial Edman degradation of the peak active fraction derived the sequence: ATKPR-QYIVL-PRILRPV-GT. The molecular mass of the major component in this fraction was established by MALDI-TOF MS as 5893.09 Da. This partial sequence (assuming blank cycles to be Cys residues) was used to design a degenerate primer pool that was employed successfully in RACE-PCR to clone homologous precursor-encoding cDNA that encoded a mature Kazal protein of 52 amino acid residues with a computed molecular mass of 5892.82 Da. The protein was named A. callidryas Kazal trypsin inhibitor (ACKTI). BLAST analysis revealed that ACKTI contained a canonical Kazal motif (C-x(7)-C-x(6)-Y-x(3)-C-x(2,3)-C). This novel amphibian skin Kazal trypsin inhibitor adds to the spectrum of trypsin inhibitors of Kunitz- and Bowman Birk-type reported from this amphibian source.     

Purification, characterization and cDNA cloning of a trypsin from the hepatopancreas of snakehead (Channa argus)

A trypsin was purified from the hepatopancreas of snakehead (Channa argus) by ammonium sulfate fractionation and a series of column chromatographies including DEAE-Sepharose, Sephacryl S-200 HR and Hi-Trap Capto-Q. The molecular mass of the purified trypsin was about 22 kDa, as estimated by SDS-PAGE. The optimum pH and temperature of the purified trypsin were 9.0 and 40 °C, respectively. The trypsin was stable in the pH range of 7.5-9.5 and below 45 °C. The enzymatic activity was strongly inhibited by serine proteinase inhibitors, such as MBTI, Pefabloc SC, PMSF, LBTI and benzamidine. Peptide mass fingerprinting (PMF) of the purified protein obtained 2 peptide fragments with 25 amino acid residues and were 100% identical to the trypsinogen from pufferfish (Takifugu rubripes). The activation energy (Ea) of this enzyme was 24.65 kJ·M^− 1. Apparent K_m was 1.02 μM and k_cat was 148 S^− 1 for fluorogenic substrate Boc-Phe-Ser-Arg-MCA. A trypsinogen gene encoding 247 amino acid residues was further cloned on the basis of the sequence obtained from PMF and the conserved site peptide of trypsinogen together with 5′-RACE and 3′-RACE. The deduced amino acid sequence contains a signal peptide of 15 residues and an activation peptide of 9 amino acid residues with a mature protein of 223 residues. The catalytic triad His-64, Asp-107, Ser-201 and 12 Cys residues which may form 6 disulfide bonds were conserved. Compared with the PMF data, only 2 amino acid residues difference were identified, suggesting the cloned trypsinogen is quite possibly the precursor of the purified trypsin.     

MOLECULAR CLONING OF TRYPSIN AND THE EFFECT OFDIETARY PROTEIN LEVELS AND STARVATION ON TRYPSINmRNA EXPRESSION AND ENZYME ACTIVITY IN JUVENILECHINESE SUCKER (MYXOCYPRINUS ASIATICUS BLEEKER)

The effect of dietary protein and starvation on the expression of trypsin was evaluated in the Chinesesucker (Myxocyprinus asiaticus Bleeker). The complete trypsin cDNA was cloned from juvenile Chinese suckerpancreatic tissue by using RACE and PCR methods. We used semi-quantitative RT-PCR and enzymatic activitymeasurements to quantify mRNA expression and trypsin activity in fish that were either starved or fed differinglevels of dietary casein (35%, 45% and 55%). The results showed that the Chinese sucker trypsin cDNA sequencewas 912 bp in length. Trypsin activity and mRNA levels were higher in fish that were fed moderate (45% casein)levels of protein than those that were fed high or low levels. Starvation significantly decreased mRNA expressionlevel and trypsin activity. The changes in trypsin activity tended to lag behind the changes in mRNA levels. Therewas no direct relationship between the trypsin activity and mRNA level. Given this, the trypsin synthesis is acomplex process regulated by a balance of several factors in the Chinese sucker.     

The Expression and Activation of Protease‐Activated Receptor‐2 Correlate with Skin Color

Skin color results from the production and distribution of melanin in the epidermis. The protease‐activated receptor‐2 (PAR‐2), expressed on keratinocytes but not on melanocytes, is involved in melanosome uptake via phagocytosis, and modulation of PAR‐2 activation affects skin color. The pattern of melanosome distribution within the epidermis is skin color‐dependent. In vitro, this distribution pattern is regulated by the ethnic origin of the keratinocytes, not the melanocytes. Therefore, we hypothesized that PAR‐2 may play a role in the modulation of pigmentation in a skin type‐dependent manner. We examined the expression of PAR‐2 and its activator, trypsin, in human skins with different pigmentary levels. Here we show that PAR‐2 and trypsin are expressed in higher levels, and are differentially localized in highly pigmented, relative to lightly pigmented skins. Moreover, highly pigmented skins exhibit an increase in PAR‐2‐specific protease cleavage ability. Microsphere phagocytosis was more efficient in keratinocytes from highly pigmented skins, and PAR‐2 induced phagocytosis resulted in more efficient microsphere ingestion and more compacted microsphere organization in dark skin‐derived keratinocytes. These results demonstrate that PAR‐2 expression and activity correlate with skin color, suggesting the involvement of PAR‐2 in ethnic skin color phenotypes.     

Fluorescent diphenylphosphonate-based probes for detection of serine protease activity during inflammation

Activity-based probes are small molecules that covalently bind to the active site of a protease in an activity-dependent manner. We synthesized and characterized two fluorescent activity-based probes that target serine proteases with trypsin-like or elastase-like activity. We assessed the selectivity and potency of these probes against recombinant enzymes and demonstrated that while they are efficacious at labeling active proteases in complex protein mixtures in vitro, they are less valuable for in vivo studies. We used these probes to evaluate serine protease activity in two mouse models of acute inflammation, including pancreatitis and colitis. As anticipated, the activity of trypsin-like proteases was increased during pancreatitis. Levels of elastase-like proteases were low in pancreatic lysates and colonic luminal fluids, whether healthy or inflamed. Exogenously added recombinant neutrophil elastase was inhibited upon incubation with these samples, an effect that was augmented in inflamed samples compared to controls. These data suggest that endogenous inhibitors and elastase-degrading proteases are upregulated during inflammation.     

The cowpea trypsin inhibitor promoter drives expression in response to cellular maturation in Arabidopsis thaliana

The bowman-birk type trypsin inhibitors accumulate in high concentration in legume and cereal seeds, especially during seed maturation and are considered to be involved in insect tolerance. The 5′ flanking sequences of the trypsin inhibitor was isolated from cowpea genomic DNA using anchor PCR. Analysis of sequences showed presence of seed specific RY elements and also other elements associated with seed development such as abscisic acid responsive elements (ABA responsive elements; ABRE) and dehydration responsive elements (DRE). Spatial and temporal control of the promoter driven expression pattern was analyzed using gus as reporter. Expression was found to occur both in embryo and endosperm; starting from torpedo stage of embryogenesis and continuing till the stage of final maturation i.e. bent cotyledon stage. Additional expression analyses showed that the promoter actually drives expression in tissues like leaves, roots, stipules, etc., but followed a specific pattern. Comparative analysis of expression in seeds and other organs indicated that the promoter driven expression is in response to cellular maturation.     

决明胰蛋白酶抑制剂1活性相关残基的定点突变与抑制活性分析

决明胰蛋白酶抑制剂1（CoTI1）属于Kunitz胰蛋白酶抑制剂家族成员,通过序列比对预测Arg86、Leu84和Thr88等3个氨基酸残基可能是CoTI1发挥抑制作用的关键残基。通过定点突变的方法将Arg86、Leu84与Thr88残基分别突变为Asp残基,并考察各突变体对胰蛋白酶及棉铃虫等鳞翅目害虫消化酶的抑制作用。与CoTI1相比,CoTI1^R86D、CoTI1^T88D与CoTI1^L84D突变体对胰蛋白酶的抑制活性分别下降了93%、64%与59%;对棉铃虫、甜菜夜蛾、斜纹夜蛾等3种鳞翅目害虫消化酶的平均抑制活性分别下降了88.7%、57%与60.7%。以上结果表明Arg86、Leu84与Thr88是CoTI1发挥抑制作用的关键残基,这为CoTI1的抑制分子机制及抗虫研究提供了重要的理论依据。     

Quantitation of major protein constituents of murine intestinal fluid

The gastrointestinal tract is a hostile biological environment, yet not all ingested materials are destroyed. The minute differences that determine whether a substance persists or is digested, liberated, adsorbed, excreted, or taken up are still poorly understood. Most attempts to investigate the events occurring during an orogastrointestinal passage rely on simplified in vitro systems where an analyte is exposed to artificial intestinal fluids. To closely mimic the events in the gastrointestinal tract, the exact intestinal fluid composition and the in vivo concentration of its constituents must be known. The widely used lavage procedures, however, dilute the intestinal fluids to an extent that precludes recalculation to the original concentrations. Thus, we developed procedures with which undiluted murine intestinal fluid can be harvested; determined the in vivo concentrations of the digestive enzymes trypsin, chymotrypsin, and elastase and the adsorbents mucin and immunoglobulin A in small intestinal fluid of fasted and unfasted female Balb/c mice; and identified chymotrypsin and immunoglobulin A as valid endogenous dilution markers for the recalculation of aqueous lavages. With these technologies and information at hand, more reliable investigations on the fate of allergens, pathogens, food, and anthropogenic xenobiotics in the gastrointestinal tract will be possible.