Expression of LEKTI domains 6-9' in the baculovirus expression system: recombinant LEKTI domains 6-9' inhibit trypsin and subtilisin A

The precursor lympho-epithelial Kazal-type-related inhibitor (LEKTI), containing two Kazal-type and 13 nonKazal-type domains, is an efficient inhibitor of multiple serine proteinases, among them plasmin, subtilisin A, cathepsin G, elastase, and trypsin. To gain insight into the structure and function of some of these domains, a portion of the cDNA coding for LEKTI domains 6-9' was cloned and expressed in Sf9 cells using the baculovirus expression vector system (BEVS). Through a single purification step using a Co2+ column, 3-4 mg of purified recombinant LEKTI-domains 6-9' (rLEKTI6-9') with the predicted molecular mass of 34.6 kDa was obtained from the cell pellet of a 1-L culture. Unlike full-length LEKTI, rLEKTI6-9' inhibited trypsin and subtilisin A but not plasmin, cathepsin G, or elastase. The inhibition of trypsin and subtilisin A by rLEKTI6-9' occurred through a noncompetitive mechanism, with inhibitory constants (Ki) of 356 +/- 12 and 193 +/- 10 nM, respectively. On the basis of the Ki values, rLEKTI6-9' was determined to be a more potent trypsin inhibitor and a less potent subtilisin A inhibitor than the full-length LEKTI. In contrast to LEKTI domains 6-9', recombinant LEKTI domain 6 does not inhibit subtilisin A but competitively inhibited trypsin with a Ki of 200 +/- 10 nM. Taking LEKTI6-9' as an example, the BEVS should facilitate the structure-function analysis of naturally occurring processed LEKTI forms that have physiological relevance.     

Inhibition of the cysteine proteinases cathepsins K and L by the serpin headpin (SERPINB13): a kinetic analysis

Headpin (SERPINB13) is a novel member of the serine proteinase inhibitor (Serpin) gene family that was originally cloned from a keratinocyte cDNA library. Western blot analysis using a headpin-specific antiserum recognized a protein with the predicted M(r) of 44kDa in lysates derived from a transformed keratinocyte cell line known to express headpin mRNA. Similarity of the reactive-site loop (RSL) domain of headpin, notably at the P1-P1(') residues, with other serpins that inhibit cysteine and serine proteinases suggests that headpin may inhibit similar proteinases. This study demonstrates that recombinant headpin indeed inhibits cathepsins K and L, but not chymotrypsin, elastase, trypsin, subtilisin A, urokinase-type plasminogen activator, plasmin, or thrombin. The second-order rate constants (k(a)) for the inhibitory reactions of rHeadpin with cathepsins K and L were 5.1+/-0.6x10(4) and 4.1+/-0.8x10(4)M(-1)s(-1), respectively. Headpin formed SDS-stable complexes with cathepsins K and L, a characteristic property of inhibitory serpins. Interactions of the RSL domain of headpin with cathepsins K and L were indicated by cleavage of headpin near the predicted P1-P1(') residues by these proteinases. These results demonstrate that the serpin headpin possesses specificity for inhibiting lysosomal cysteine proteinases.     

Sequencing and characterization of the citrus weevil, Diaprepes abbreviatus, trypsin cDNA. Effect of Aedes trypsin modulating oostatic factor on trypsin biosynthesis.

Trypsin mRNA from the citrus weevil, Diaprepes abbreviatus, was reverse transcribed and amplified by PCR. A cDNA species of 513 bp was cloned and sequenced. The 3' and 5' ends of the gene (262 bp and 237 bp, respectively) were amplified by rapid amplification of cDNA ends, cloned and sequenced. The deduced sequence of the trypsin cDNA (860 bp) encodes for 250 amino acids including 11 amino acids of activation and signal peptides and exhibited 16.8% identity to trypsin genes of selected Lepidoptera and Diptera. A three-dimensional model of Diaprepes trypsin contained two domains of beta-barrel sheets as has been found in Drosophila and Neobellieria. The catalytic active site is composed of the canonical triad of His41, Asp92 and Ser185 and a specificity pocket occupied by Asp179 with maximal activity at pH 10.4. Southern blot analysis indicated that at least two copies of the gene are encoded by Diaprepes midgut. Northern blot analysis detected a single RNA band below 1.35 kb at different larval ages (28-100 days old). The message increased with age and was most abundant at 100 days. Trypsin activity, on the other hand, reached a peak at 50 days and fell rapidly afterwards indicating that the trypsin message is probably regulated translationally. Feeding of soybean trypsin inhibitor and Aedes aegypti trypsin modulating oostatic factor affected trypsin activity and trypsin biosynthesis, respectively. These results indicate that Diaprepes regulates trypsin biosynthesis with a trypsin modulating oostatic factor-like signal.     

Differences in midgut serine proteinases from larvae of the bruchid beetles Callosobruchus maculatus and Zabrotes subfasciatus

Proteinase activities in the larval midguts of the bruchids Callosobruchus maculatus and Zabrotes subfasciatus were investigated. Both midgut homogenates showed a slightly acidic to neutral pH optima for the hydrolysis of fluorogenic substrates. Proteolysis of epsilon-aminocaproil-Leu-Cys(SBzl)-MCA was totally inhibited by the cysteine proteinase inhibitors E-64 and leupeptin, and was activated by 1.5 mM DTT in both insects, while hydrolysis of the substrate Z-ArgArg-MCA was inhibited by aprotinin and E-64, which suggests that it is being hydrolysed by serine and cysteine proteinases. Gel assays showed that the proteolytic activity in larval midgut of C. maculatus was due to five major cysteine proteinases. However, based on the pattern of E-64 and aprotinin inhibition, proteolytic activity in larval midgut of Z. subfasciatus was not due only to cysteine proteinases. Fractionation of the larval midgut homogenates of both bruchids through ion-exchange chromatography (DEAE-Sepharose) revealed two peaks of activity against Z-ArgArg-MCA for both bruchid species. The fractions from C. maculatus have characteristics of cysteine proteinases, while Z. subfasciatus has one non-retained peak of activity containing cysteine proteinases and another eluted in a gradient of 250-350 mM NaCl. The proteolytic activity of the retained peak is higher at pH 8.8 than at pH 6.0 and corresponds with a single peak that is active against N-p-tosyl-GlyGlyArg-MCA, and sensitive to 250 microM aprotinin (90% inhibition). The peak contains a serine proteinase which hydrolyzes alpha-amylase inhibitor 1 from the common bean (Phaseolus vulgaris). Arch.     

Human complement factor I: analysis of cDNA-derived primary structure and assignment of its gene to chromosome 4

Factor I is a serine proteinase of complement which together with one of several specific cofactors cleaves activation products of the third and fourth components of complement (C3b and C4b) and modulates the activity of C3 convertase. A heterodimer glycoprotein (Mr = 88,000), factor I is synthesized as a single-chain precursor, prepro-I, which undergoes intracellular proteolytic processing. The human hepatoma line HepG2, however, secretes predominantly the single-chain precursor pro-I. In order to determine the molecular basis for this apparent processing defect, factor I cDNA clones were isolated from a HepG2 mRNA-derived library. Sequencing of the largest insert, HI1971, revealed that it contains 14 base pairs of 5' untranslated region, the complete coding sequence for the 583-residue prepro-I (NH2-signal peptide-heavy chain-linking peptide-light chain-COOH), two polyadenylation signals within the 200-base pair 3' untranslated region, and a portion of poly(A) tail. Analysis of the derived protein structure 1) reveals a mosaic multidomain structure of the heavy chain; 2) demonstrates structural similarity between intracellular conversion of pro-I and activation of other serine proteinase zymogens; and 3) indicates that the light chain of factor I resembles most closely the active subunit of tissue plasminogen activator among all serine proteinases and factor D among complement proteinases. Furthermore, this protein sequence was compared to the sequences of factor I cDNA clones isolated from normal human liver libraries and found to be identical. By exclusion, this defines as cellular the basis for the inefficient processing of pro-I by the HepG2 line. Chromosomal localization by the somatic cell hybrid method maps the factor I gene to chromosome 4.     

Molecular cloning and partial characterization of a trypsin-like protein in salivary glands of Lygus hesperus (Hemiptera: Miridae)

Trypsin-like enzymes from the salivary gland complex (SGC) of Lygus hesperus Knight were partially purified by preparative isoelectric focusing (IEF). Enzyme active against Nalpha-benzoyl-L-arginine-p-nitroanilide (BApNA) focused at approximately pH 10 during IEF. This alkaline fraction gave a single activity band when analyzed with casein zymograms. The serine proteinase inhibitors, phenylmethylsulfonyl fluoride (PMSF) and lima bean trypsin inhibitor, completely inhibited or suppressed the caseinolytic activity in the crude salivary gland extract as well as the IEF-purified sample. Chicken egg white trypsin inhibitor also inhibited the IEF-purified sample but was not effective against a major caseinolytic band in the crude salivary gland extract. These data indicated the presence of serine proteinases in the SGC of L. hesperus. Cloning and sequencing of a trypsin-like precursor cDNA provided additional direct evidence for serine proteinases in L. hesperus. The encoded trypsin-like protein included amino acid sequence motifs, which are conserved with five homologous serine proteinases from other insects. Typical features of the putative trypsin-like protein from L. hesperus included residues in the serine proteinase active site (His(89), Asp(139), Ser(229)), conserved cysteine residues for disulfide bridges, residues (Asp(223), Gly(252), Gly(262)) that determine trypsin specificity, and both zymogen signal and activation peptides.     

Structure-function analysis of the reactive site in the first Kunitz-type domain of human tissue factor pathway inhibitor-2

Human tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type proteinase inhibitor that regulates a variety of serine proteinases involved in coagulation and fibrinolysis through their non-productive interaction with a P(1) residue (Arg-24) in its first Kunitz-type domain (KD1). Previous kinetic studies revealed that TFPI-2 was a more effective inhibitor of plasmin than several other serine proteinases, but the molecular basis for this specificity was unclear. In this study, we employed molecular modeling and mutagenesis strategies to produce several variants of human TFPI-2 KD1 in an effort to identify interactive site residues other than the P(1) Arg that contribute significantly to its inhibitory activity and specificity. Molecular modeling of KD1 based on the crystal structure of bovine pancreatic trypsin inhibitor revealed that KD1 formed a more energetically favorable complex with plasmin versus trypsin and/or the factor VIIa-tissue factor complex primarily due to strong ionic interactions between Asp-19 (P(6)) and Arg residues in plasmin (Arg-644, Arg-719, and Arg-767), Arg-24 (P(1)) with Asp-735 in plasmin, and Arg-29 (P(5)') with Glu-606 in plasmin. In addition, Leu-26 through Leu-28 (P(2)'-P(4)') in KD1 formed strong van der Waals contact with a hydrophobic cluster in plasmin (Phe-583, Met-585, and Phe-587). Mutagenesis of Asp-19, Tyr-20, Arg-24, Arg-29, and Leu-26 in KD1 resulted in substantial reductions in plasmin inhibitory activity relative to wild-type KD1, but the Asp-19 and Tyr-20 mutations revealed the importance of these residues in the specific inhibition of plasmin. In addition to the reactive site residues in the P(6)-P(5)' region of KD1, mutation of a highly conserved Phe at the P(18)' position revealed the importance of this residue in the inhibition of serine proteinases by KD1. Thus, together with the P(1) residue, the nature of other residues flanking the P(1) residue, particularly at P(6) and P(5)', strongly influences the inhibitory activity and specificity of human TFPI-2.     

Identification of potent inhibitors of Helicoverpa armigera gut proteinases from winged bean seeds

Dry mature seeds of winged bean (Psophocarpus tetragonolobus L., DC.) (WB) contain several proteinase inhibitors. Two-dimensional gel analysis of WB seed protein followed by activity visualization using a gel-X-ray film contact print technique revealed at least 14 trypsin inhibitors (TIs) in the range of 28-6 kD. A total of seven inhibitors (WBTI-1 to 7) were purified by heat treatment and gel filtration followed by elution from preparative native gels. Based on their biochemical characterization such as molecular mass, pI, heat stability, and susceptibility to inactivation by reducing agents, WBTI-1 to 4 are Kunitz type inhibitors while WBTI-5 to 7 are classified as Bowman-Birk type serine proteinase inhibitors. Although Kunitz type TIs (20-24 kD) of WB have been reported, the smaller TIs that belong to the Bowman-Birk type have not been previously characterized. Seven major TIs isolated from WB seed were individually assessed for their potential to inhibit the gut proteinases (HGP) of Helicoverpa armigera, a pest of several economically important crops, which produces at least six major and several minor trypsin/chymotrypsin/elastase-like serine proteinases in the gut. WBTI-1 (28 kD) was identified as a potent inhibitor of HGP relative to trypsin and among the other WBTIs; it inhibited 94% of HGP activity while at the same concentration it inhibited only 22% of trypsin activity. WBTI-2 (24 kD) and WBTI-4 (20 kD) inhibited HGP activity greater than 85%. WBTI-3,-5,-6 and-7 showed limited inhibition of HGP as compared with trypsin. These results indicate that WBTIs have different binding potentials towards HGP although most of the HGP activity is trypsin-like. We also developed a simple and versatile method for identifying and purifying proteinase inhibitors after two-dimensional separation using the gel-X-ray film contact print technique.     

Effects of a potato cysteine proteinase inhibitor on midgut proteolytic enzyme activity and growth of the southern corn rootworm, Diabrotica undecimpunctata howardi (Coleoptera: Chrysomelidae)

The major proteinase activity in extracts of larval midguts from the southern corn rootworm (SCR), Diabrotica undecimpunctata howardi, was identified as a cysteine proteinase that prefers substrates containing an arginine residue in the P1 position. Gelatin-zymogram analysis of the midgut proteinases indicated that the artificial diet-fed SCR, corn root-fed SCR, and root-fed western corn rootworms (Diabrotica virgifera virgifera) possess a single major proteinase with an apparent molecular mass of 25kDa and several minor proteinases. Similar proteinase activity pH profiles were exhibited by root-fed and diet-fed rootworms with the optimal activity being slightly acidic. Rootworm larvae reared on corn roots exhibited significantly less caseinolytic activity than those reared on the artificial diet. Midgut proteolytic activity from SCR was most sensitive to inhibition by inhibitors of cysteine proteinases. Furthermore, rootworm proteinase activity was particularly sensitive to inhibition by a commercial protein preparation from potato tubers (PIN-II). One of the proteins, potato cysteine proteinase inhibitor-10', PCPI-10', obtained from PIN-II by ion-exchange chromatography, was the major source of inhibitory activity against rootworm proteinase activity. PCPI-10' and E-64 were of comparable potency as inhibitors of southern corn rootworm proteinase activity (IC(50) =31 and 35nM, respectively) and substantially more effective than chicken egg white cystatin (IC(50) =121nM). Incorporation of PCPI-10' into the diet of SCR larvae in feeding trials resulted in a significant increase in mortality and growth inhibition. We suggest that expression of inhibitors such as PCPI-10' by transgenic corn plants in the field is a potentially attractive method of host plant resistance to these Diabrotica species.     

Capsicum annuum dehydrin,an osmotic-stress gene in hot pepper plants

Osmotic stress-related genes were selected from an EST database constructed from 7 cDNA libraries from different tissues of the hot pepper. A full-length cDNA of Capsicum annuum dehydrin (Cadhn), a late embryogenesis abundant (lea) gene, was selected from the 5' single pass sequenced cDNA clones and sequenced. The deduced polypeptide has 87% identity with potato dehydrin C17, but very little identity with the dehydrin genes of other organisms. It contains a serine-tract (S-segment) and 3 conserved lysine-rich domains (K-segments). Southern blot analysis showed that 2 copies are present in the hot pepper genome. Cadhn was induced by osmotic stress in leaf tissues as well as by the application of abscisic acid. The RNA was most abundant in green fruit. The expression of several osmotic stress-related genes was examined and Cadhn proved to be the most abundantly expressed of these in response to osmotic stress.     

Human pepsinogen C (progastricsin). Isolation of cDNA clones, localization to chromosome 6, and sequence homology with pepsinogen A

The entire pepsinogen C (PGC) coding sequence was determined by analysis of a series of five overlapping cDNA clones identified in a library constructed from human gastric mucosa poly(A+) RNA. A partial cDNA clone was initially identified using a 256-fold degenerate oligonucleotide probe for amino acid residues 4-12 of pepsin C, and subsequently 4 additional clones were identified upon rescreening with a probe complementary to the 5' region of the original cDNA clone. Northern analysis of gastric mucosa poly(A+) RNA with a PGC cDNA probe revealed an mRNA 1.5-kilobase species that was indistinguishable from that detected with a human pepsinogen A (PGA) cDNA probe. In contrast, the PGC and PGA cDNA probes detected distinct genomic restriction fragments indicating there was no detectable cross-hybridization under high stringency conditions. The PGC gene was localized to human chromosome 6 by analysis of a panel of human x mouse somatic cell hybrids. The regions containing the active site aspartyl groups of PGC are conserved in relationship to several other aspartic proteinases. We propose that the absence of detectable immunologic cross-reactivity between the two groups of human pepsinogens, A and C, results from divergent evolution of sequences located on the surface of the zymogens in contrast to the strongly conserved active site regions located within the binding cleft of the enzymes that are inaccessible for antigenic recognition.     

Molecular cloning of two cysteine proteinases from paw-paw (Carica papaya).

Two cDNA clones for plant cysteine proteinases have been isolated from a Carica papaya (paw-paw, papaya) leaf tissue cDNA library by using a mixture of 16 synthetic oligodeoxyribonucleotides as a hybridization probe. The inserted regions are 311 and 440 base-pairs in length and have the potential to encode a region corresponding to the C-terminal region of two proteins which are homologous with the known plant cysteine proteinases and the mammalian thiol cathepsins. One of the sequences shows a high (greater than 77%) homology with the plant cysteine proteinase papain, the other is closely related to papaya chymopapain. One sequence contains all, and the other most, of the 3' untranslated region of the mRNA. The inserts were used as specific probes in Northern Blot analyses giving an estimated size for the two mRNA species of 1.45 kilobases.     

Isolation and nucleotide sequence of cDNA clone for bovine pancreatic anionic trypsinogen. Structural identity within the trypsin family.

A cDNA clone encoding an anionic form of bovine trypsinogen was isolated from a pancreatic cDNA library. The corresponding 855-nucleotide mRNA contains a short 5' noncoding region of 8 nucleotides and a long 3' noncoding region of 56 nucleotides in addition to a poly(A) tail of at least 50 nucleotides. The deduced amino acid sequence for the anionic pretrypsinogen (247 residues) includes the N-terminal 15-amino-acid signal peptide followed by an 8-amino-acid activation peptide. The zymogen (232 residues) contains an additional C-terminal serine, compared with the amino acid sequence of bovine cationic trypsinogen. The identity between the anionic and cationic forms of bovine trypsinogen (65%) is lower than that existing between the anionic protein and other mammalian anionic trypsinogens (73-85%), suggesting that trypsin gene duplication in mammals occurred prior to the evolutionary events responsible for the species divergence. Bovine pancreatic anionic trypsin possesses all the key amino acids characteristic of the serine protease family.     

Enzyme specificity of proteinase inhibitor region in amyloid precursor protein of Alzheimer's disease: different properties compared with protease nexin I

Senile plaques, often surrounded by abnormally grown neurites, are characteristic of Alzheimer's diseased brain. The core of the plaque is mainly composed of amyloid beta protein (beta-AP), two of whose three precursors (APP) have serine proteinase inhibitor regions (APPI). APPI derivatives containing 60, 72 or 88 amino-acid fragments (APPI-60, APPI-72 and APPI-88, respectively) of the longest APP were produced in COS-1 cell culture medium, with the APPI cDNA ligated to the signal sequence of tissue plasminogen activator. The secreted APPIs were purified by sequential acetone precipitation followed by affinity chromatography using immobilized trypsin. These three APPIs and O-glycosylation-site-mutated APPI showed similar inhibitory activity against trypsin, chymotrypsin and plasmin. The purified APPI-72 was found to inhibit trypsin (Ki = 1.1 x 10(-10) M) and chymotrypsin (Ki = 5.8 x 10(-9) M) most strongly, and to inhibit leukocyte elastase (Ki = 7.9 x 10(-7) M) and several blood coagulation proteinases (Ki = 0.46-12 x 10(-7) M), but not urokinase or thrombin. The observed inhibition pattern was quite different from that of protease nexin I, one of serine proteinase inhibitors possessing neurite outgrowth activity. This suggests that the physiological roles of APPI are different from those of protease nexin I, and that APPI could not cause aberrant growth of neurite into the plaque. The presence of APPI having strong inhibitory activity in the brain might lead to the formation of amyloid deposits by preventing complete degradation of APPs.