Dietary regulation of serine proteinases that are resistant to serine proteinase inhibitors

Ingestion of Kunitz soybean trypsin inhibitor (STI) by larval Helicoverpa zea, Agrotis ipsilon, and Trichoplusia ni extended the retention time of food in the digestive tract and increased the level of activity of proteolytic enzymes that were not susceptible to inhibition by STI. The level of enhancement of activity of STI-resistant (STI-R) enzyme(s) was directly influenced by the dosage and timing of exposure to STI. However, not all proteinase inhibitors (PIs) enhanced the level of proteinase inhibitor resistant (PI-R) enzymes, even if those PIs inhibited a significant proportion of enzyme activity. These findings suggest that a complex system may be responsible for the regulation of proteolytic enzymes in the midgut of larval Lepidoptera, and one hypothesis for this regulation is proposed.     

Origin of the murine implantation serine proteinase subfamily

The S1 serine protease family is one of the largest gene families known. Within this family there are several subfamilies that have been grouped together as a result of sequence comparisons and substrate identification. The grouping of related genes allows for the speculation of function for newly found members by comparison and for novel subfamilies by contrast. Analysis of the evolutionary patterns of genes indicates whether or not orthologs are likely to be identified in other species as well as potentially indicating that hypothesized orthologs are in fact not. Looking at subtle differences between subfamily members can reveal intricacies about function and expression. Previously, we have described genes encoding two novel serine proteinases, ISP1 and ISP2, which are most closely related to tryptases. The ISP1 gene encodes the embryo-derived enzyme strypsin, which is necessary for blastocyst hatching and invasion in vitro. Additionally both ISP1 and ISP2 are co-expressed in the endometrial gland during the time of hatching, suggesting that they may also both participate in zona lysis from within the uterine lumen. Here, we demonstrate that the ISPs are tandemly linked within the tryptase cluster on 17A3.3. We suggest that remarkable similarities within the 5'-untranslated and first intron regions of ISP1 and ISP2 may explain their intimate co-regulation in uterus. We also suggest that ISP genes have evolved through gene duplication and that the ISP1 gene has also begun to adopt an additional new function in the murine preimplantation embryo.     

Implantation-associated proteinase in mouse uterine fluid

Proteinase activity was detected in mouse uterine fluid by means of a new casein-substrate assay. The activity was found to be generally low in diestrous and proestrous stages of the estrous cycle and was more variable in proestrous. During pregnancy, activity was very low on day 1 (counting the plug date as day 0). By day 3, proteinase activity (expressed as Pronase equivalents/mg protein of uterine fluid) increased more than 100-fold, and then declined on day 4. Peak activity thus coincides with initiation of embryo implantation, which occurs on day 3 of pregnancy in the strain tested. The results provide direct biochemical support for previous indirect bioassay indications of the presence in uterine fluid of a proteolytic factor of uterine origin. The quantitative changes observed here are also consistent with previous bioassay observations and with the hypothesis that the uterine proteinase may mediate initial attachment of the blastocyst to the uterine wall. These and other data are used to formulate a 2-stage hypothesis of implantation, according to which uterine and trophoblast proteinases act sequentially to cause attachment and invasion, respectively.     

Substrate specificity determination of mouse implantation serine proteinase and human kallikrein-related peptidase 6 by phage display

We constructed a random library of hexapeptides displayed on the surface of bacteriophage T7 to determine the substrate specificity of proteinases. The phage-displayed library was subjected to repeated rounds of biopanning with native implantation serine proteinase and recombinant human kallikrein-related peptidase 6 (KLK6) followed by selection and identification of putative substrates. For both enzymes, the results obtained demonstrate a preference for arginine and lysine at multiple positions in the recognition cleavage motif, confirming their previously reported trypsin-like substrate specificity. In the case of KLK6, there is also a pronounced presence of tryptophan within the cleaved peptide sequences, indicating its potential dual substrate specificity, acting as both a trypsin and chymotrypsin-like enzyme.     

A novel inhibitor protein for Bombyx cysteine proteinase is homologous to propeptide regions of cysteine proteinases

A cDNA clone for an inhibitor of Bombyx cysteine proteinase was isolated and sequenced. Active inhibitor proteins were expressed in Escherichia coli using the cDNA. The open reading frame of the cDNA encodes a 105 residues protein with 19 residues of a signal sequence. The inhibitor has amino acid sequences homologous to several cysteine proteinases, but only to their propeptide sequences. The results suggest that some cysteine proteinase proregions may have evolved as autonomous modules and become inhibitor proteins for cysteine proteinases.     

The picornaviral 3C proteinases: cysteine nucleophiles in serine proteinase folds.

The 3C proteinases are a novel group of cysteine proteinases with a serine proteinase-like fold that are responsible for the bulk of polyprotein processing in the Picornaviridae. Because members of this viral family are to blame for several ongoing global pandemic problems (rhinovirus, hepatitis A virus) as well as sporadic outbreaks of more serious pathologies (poliovirus), there has been continuing interest over the last two decades in the development of antiviral therapies. The recent determination of the structure of two of the 3C proteinases by X-ray crystallography opens the door for the application of the latest advances in computer-assisted identification and design of anti-proteinase therapeutic/chemoprophylactic agents.     

Refolding of serine proteinases

Bovine trypsinogen and chymotrypsinogen were successfully refolded as the mixed disulfide of glutathione using cysteine as the disulfide interchange catalyst. The native structures were regenerated with yields of 40%-50% at pH 8.6 and 4 degrees C, and the half-time for the refolding was approximately 60-75 min. We then refolded threonine-neochymotrypsinogen, which is a two-chain structure held together by disulfide bonds and produced on cleavage of Tyr 146-Thr 147 in native chymotrypsinogen [Duda CT, Light A, J Biol Chem 257 9866-9871, 1982]. Neochymotrypsinogen was denatured and fully reduced, and the thiols were converted to the mixed disulfide of glutathione. The two polypeptide fragments, representing the amino- and carboxyl-terminal domains, were separated on Sephadex G-75. Mixtures of the polypeptide fragments varying in the ratio of their concentration from 1:5 to 5:1 were refolded with yields of 21-28%. The lack of dependence on the concentration of either fragment and the relatively high yields suggest independent folding of the amino- and carboxyl-terminal domains. When the globular structures of the domains formed, they then interacted with one another and produced the native intermolecular disulfide bridge and the proper geometry of the active site.     

Characterization of secretory leukocyte protease inhibitor as an inhibitor of implantation serine proteinases

We have recently identified and characterized two implantation serine proteinase genes, ISP1 and ISP2, which give rise to a dimeric proteinase, ISP that facilitates embryo invasion during peri-implantation period. As many proteinases have cognate serpins that regulate their proteolytic activity, we have been investigating anti-tryptases, expressed during this window of implantation. Here, we report the differential expression of secretory leukocyte protease inhibitor (SLPI) in uterine endometrium around the implantation period. The co-localization of SLPI and ISP suggests the possibility that SLPI is an ISP serpin and that expression of SLPI may lead to a reduction in ISP activity. The expression of SLPI is down regulated during the window of embryo-uterine receptivity. Our results are consistent with a model suggesting that the drop in SLPI expression may help to refine the opening of the window of implantation, by allowing the proteolytic activity of embryo invasive serine proteinases such as the ISPs.     

Protein inhibitors of serine proteinases

Serine proteinases and their natural protein inhibitors belong to the most intensively studied models of protein-protein recognition. Protein inhibitors do not form a single group but can be divided into about 20 different families. Global structures of proteins representing different inhibitor families are completely different and comprise alpha-helical proteins, beta-sheet proteins, alpha/beta-proteins and different folds of small disulfide-rich proteins. Three different types of inhibitors can be distinguished: canonical (standard mechanism) inhibitors, non-canonical inhibitors, and serpins. The canonical inhibitor binds to the enzyme through the exposed and convex binding loop, which is complementary to the active site of the enzyme. The mechanism of inhibition in this group is consistently very similar and resembles that of an ideal substrate. Non-canonical inhibitors, originating from blood sucking organisms, specifically block enzymes of the blood clotting cascade. The interaction is mediated through inhibitor N-terminus which binds to the proteinase forming a parallel beta-sheet. There are also extensive secondary interactions which provide an additional buried area and contribute significantly to the strength and specificity of recognition. Serpins are major proteinase inhibitors occurring in plasma. Similarly to canonical inhibitors, serpins interact with their target proteinases in a substrate-like manner. However, in the case of serpins, cleavage of a single peptide bond in a flexible and exposed binding loop leads to dramatic structural changes.     

Labeled proteinase inhibitors: versatile tools for the characterization of serine proteinases in solid-phase assays.

Peptidyl chloromethyl ketones were used for the specific labeling of proteinases by attaching a biotin group to the N-terminal end of the peptide. Such labeled peptide inhibitors allowed the detection and quantitation of proteolytic enzymes immobilized on the plastic surface of a microtiter plate, as well as on nitrocellulose. The validity of these solid-phase assays was demonstrated using subtilisin Carlsberg as a model enzyme and biotinyl-epsilon-aminocaproyl-L-alanyl-L-alanyl-L-propyl-L-phenylal++ + anyl- chloromethyl ketone as a specific reagent. In addition to being usable for the screening of a particular proteinase in a large number of samples, these assays can be adapted for the analysis of specific proteolytic enzyme present in complex mixtures.     

Photochemical oxidation of snake gourd proteinase A2, a serine proteinase

Snake gourd proteinase A₂ was rapidly inactivated by methylene blue catalysed photooxidation at pH 7.8 and 25°. The rate of inactivation was pH-dependent and became slower at lower pH values, suggesting the involvement of some histidine residues in the inactivation. Changes in amino acid composition occurred only with histidine residues. One mole or more of histidine residues in the molecule are of essential importance in the catalytic function of snake gourd proteinase A₂.     

Bombyx cysteine proteinase inhibitor (BCPI) homologous to propeptide regions of cysteine proteinases is a strong, selective inhibitor of cathepsin L-like cysteine proteinases.

Bombyx cysteine proteinase inhibitor (BCPI) is a novel cysteine proteinase inhibitor. The protein sequence is homologous to the proregions of certain cysteine proteinases. Here we report the mechanism of its inhibition of several cysteine proteinases. BCPI strongly inhibited Bombyx cysteine proteinase (BCP) activity with a K(i) = 5.9 pM, and human cathepsin L with a K(i) = 36 pM. The inhibition obeyed slow-binding kinetics. The inhibition of cathepsin H was much weaker (K(i) = 82 nM), while inhibition of papain (K(i) > 1 microM) and cathepsin B (K(i) > 4 microM) was negligible. Following incubation with BCP, BCPI was first truncated at the C-terminal end, and then gradually degraded over time. The truncation mainly involved two C-terminal amino acid residues. Recombinant BCPI lacking the two C-terminal amino acid residues still retained substantial inhibitory activity. Our results indicate that BCPI is a stable and highly selective inhibitor of cathepsin L-like cysteine proteinases.     

Identification of four distinct serine proteinase inhibitors in rat skeletal muscle

The serine proteinase inhibitory capacity in the cytosolic fraction of rat skeletal muscle tissue is accounted for by several discrete inhibitory activities. Three of these activities are identical with the proteinase inhibitors α₁-proteinase inhibitor, rat proteinase inhibitor I and rat proteinase inhibitor I I respectively, which have been recently characterized as major serine proteinase inhibitors in rat serum (Kuehn, L., Rutschmann, M., Dahlmann, B. and Reinauer, H. (1984) Biochem. J. $\text{218}$, in the press). The other inhibitor molecule, having an M_r of about 15 000, appears to be an endogeneous inhibitor.     

Ingensin, a fatty acid-activated serine proteinase from rat liver cytosol

The enzyme responsible for the succinylleucylleucylvalyltyrosine methylcoumarylamide- (SLLVT-) degrading activity was purified from the postmitochondrial supernatant of rat liver (Yamamoto, T., Nojima, M., Ishiura, S. and Sugita, H. (1986) Biochim. Biophys. Acta 882, 297-304). The enzyme, named ingensin, was activated by saturated fatty acids, especially myristic acid, as well as by unsaturated linoleic acid and arachidonic acid. Although 2-mercaptoethanol activated ingensin 2-fold and p-chloromercuribenzoate and HgCl2 completely inhibited its peptide-hydrolyzing activity, the enzyme is activated by the addition of a thiol-blocking reagent, monoiodoacetic acid. Ingensin was also inhibited by a specific serine proteinase inhibitor, diisopropyl fluorophosphate, but not by a specific cysteine proteinase inhibitor, E-64-c. These results suggest that the enzyme is a serine proteinase with an active thiol group(s) near the active site. We have found that the addition of glycerol and nordihydroguaiaretic acid lowered the extent of its activation by fatty acids as well as its intrinsic peptide-hydrolyzing activity.     

Expression of a cGMP compatible Lucilia sericata insect serine proteinase debridement enzyme

Previously, we demonstrated the effectiveness of a research grade recombinant chymotrypsin, derived from the larvae of Lucilia sericata, in "debriding" slough/eschar from venous leg ulcers ex vivo. Furthermore, we were able to formulate this enzyme for successful delivery to in vitro wound healing assays, from a prototype hydrogel wound dressing, and showed that enzyme delivered in this way could degrade wound tissue ex vivo. Recently, to progress biotechnological development of the enzyme as a potential therapeutic product, we explored expression using current good manufacturing practice (cGMP) guidelines, and now report that a recombinant chymotrypsin I zymogen from L. sericata can be expressed in the cGMP acceptable strain of Escherichia coli (BLR-DE3). In addition, the conditions required for purification, refolding and activation of the chymotrypsinogen have been determined. The activated enzyme was stable, and effective in digesting wound slough/eschar tissue. To summarise, we have successfully initiated the production and characterisation of a novel cGMP compatible product for use in future clinical trials.     

Sequences homologous to the rat ID element in mouse DNA

Sequences homologous to the rat frain specific identifier sequences were found in the mouse genome. These sequences were defined by dot- and blotting-hybridisation in different DNA and RNA preparations. It was shown that there are (1-2) X 10(3) copies of these sequences per genome. They are not transcribed in mouse brain and possibly are not connected with mouse brain specific genes expression.