Circadian expression and induction by wounding of tobacco genes for cysteine proteinase

Two sets of clones were isolated from a tobacco cDNA library, utilizing as a probe a PCR fragment obtained from tomato cDNA using a degenerate primer based on the sequence of tomato systemin. Contrary to expectation, the clones did not correspond to tobacco homologues of tomato pro-systemin. However, the cDNAs encoded two highly similar proteins with extensive structural homology to cysteine proteinases from a wide range of plant and animal species. Northern blot analyses showed that in unstressed tobacco leaf the genes for the putative proteinases are expressed according to a circadian rhythm. Furthermore, incision wounding enhances the expression approximately six-fold. Other forms of stress, such as infection with tobacco mosaic virus, treatment with ethephon or UV light do not result in induced expression of the tobacco cysteine proteinase genes.     

Peptidoglycan inducible expression of a serine proteinase homologue from kuruma shrimp (Marsupenaeus japonicus)

A cDNA encoding a serine proteinase homologue of kuruma shrimp (Marsupenaeus japonicus) was cloned. The 1257 bp cDNA encodes a 339 amino acid putative peptide, with a signal sequence of 16 amino acid residues. The deduced amino acid sequence is 42-67% similar to the immune-related serine proteinases and serine proteinase homologues of arthropods. It contains catalytic triad residues in the putative catalytic domain except for one substitution of Ser by a Gly residue. The six cysteine residues that form three disulphide bridges in most serine proteinases were conserved. The M. japonicus serine proteinase homologue was mainly expressed in haemocytes, in which expression dramatically increased after 3 days feeding with peptidoglycan at 0.2 mg kg(-1) shrimp body weight per day.     

Isolation and characterization of a cDNA encoding a serine proteinase from the root-knot nematode Meloidogyne incognita

This report describes the first serine proteinase gene isolated from the sedentary nematode Meloidogyne incognita. Using degenerate primers, a 1372bp cDNA encoding a chymotrypsin-like serine proteinase (Mi-ser1) was amplified from total RNA of adult females by RT-PCR and 5' and 3' rapid amplification of cDNA ends. The deduced amino acid sequence of Mi-ser1 encoded a putative signal peptide and a prodomain of 22 and 33 amino acids, respectively, and a mature proteinase of 341 amino acids with a predicted molecular mass of 37,680Da. Sequence identity with the top serine proteinases matches from the databases ranged from 23 to 27%, including sequences from insects, mammals, and other nematodes. Southern blot analysis suggested that Mi-ser1 is encoded by a single or few gene copies. The pattern of developmental expression analyzed by Northern blot and RT-PCR indicated that Mi-ser1 was transcribed mainly in females. The domain architecture composed of a single chymotrypsin-like catalytic domain and the detection of a putative signal peptide suggested a digestive role for Mi-ser1.     

Cloning and sequencing of cDNA encoding a rat salivary cysteine proteinase inhibitor inducible by beta-adrenergic agonists

The beta-adrenergic agonist isoproterenol induces a unique secretory protein (LM) in the salivary glands of developing and adult rats. In order to study the regulation of growth and gene expression by catecholamines, we have isolated and sequenced several cDNA clones encoding the LM protein. Each of the LM cDNA clones described identifies, by Northern blot analyses, a single mRNA species of approximately 900 bases in size. The mRNA encoding this secreted protein was not detected in submandibular glands or brains of untreated adult rats. Sequence analyses of the LM cDNA clones revealed a striking similarity to the family 2 of cysteine proteinase inhibitors. Furthermore, when purified LM protein was used to assay for inhibition of cysteine proteinases, the data demonstrated that it is indeed a type of cysteine proteinase inhibitor. This inhibitor, termed rat cystatin S, provides the first example of cysteine proteinase inhibitors that can be induced by beta-adrenergic agonists.     

Nucleotide and protein sequences of a proteinase inhibitor from the vitelline envelope of dace (Tribolodon hakonensis) eggs

Our experimental purpose is to probe the structure(s) of the chorionic proteinase inhibitor and its cDNA sequence(s) and to develop the application of safe medicines for protection of human and other animal bodies from pathogenic microbe attacks. In this study, chorionic proteinase inhibitor protein was isolated, sequenced and used to base the design of PCR primers, which were then used to amplify DNA using RT-PCR. A cDNA clone of the protein which inhibited the activities of serine proteinases and thermolysin was obtained on the basis of mRNA extracted from ovarian tissue of dace, Tribolodon hakonensis, and the deduced amino acid sequence was determined. Chorionic proteinase inhibitor (TribSPI) peptides of about 9.0 kDa (TribSPI) and 14 kDa (TribSPI-S) were purified from vitelline envelope extracts by thermolysin-immobilized affinity-chromatography. The cloned TribSPI cDNA was 1806 bp in length, and the open reading flame (ORF) was 915 bp encoding a protein of 305 amino acid residues. The inhibitor protein had a molecular mass of 33,550 daltons and was composed of five similar domains. Each domain contained eight cysteine residues, and it's deduced amino acid sequence was only 33 approximately 34% identical to those of human and porcine antileukoproteinases (hALP and pALP, respectively). A possible binding-site for serine proteinases, Arg-Ile, was contained in three domains.     

Cloning and characterization of a mouse cysteine proteinase

cDNA clones encoding a mouse cysteine proteinase were isolated from a cDNA library constructed from mRNA derived from the macrophage-like cell line J774. The DNA sequence predicts a protein that is closely related to, but distinct from, the lysosomal enzyme cathepsin H. Alignment of the predicted amino acid sequence with the known protein sequences for seven other cysteine proteinases suggests that the cloned DNA encodes a 334-residue protein containing both a 17-amino acid pre-region and a 96-amino acid pro-region. Consistent with this prediction, antiserum raised to a recombinant fusion protein expressed in Escherichia coli immunoprecipitated multiple forms of the cysteine proteinase in mouse peritoneal macrophages and fibroblasts. In pulse-chase experiments, a 36-kDa precursor, presumedly the pro-form, was converted intracellularly into a 28-kDa protein and subsequently into a 21-kDa protein. Indirect immunofluorescence microscopy results suggested that the cysteine proteinase was localized to lysosomes. Western blot analysis detected significantly more of the proteinase in thioglycolate-elicited peritoneal macrophages than in resident peritoneal macrophages. Northern blot analysis revealed that several cell lines failed to express mouse cysteine proteinase mRNA.     

A unique 33-kD cysteine proteinase accumulates in response to larval feeding in maize genotypes resistant to fall armyworm and other Lepidoptera

Plants respond to insect feeding with a number of defense mechanisms. Using maize genotypes derived from Antiquan germ plasm that are resistant to Lepidoptera, we have demonstrated that a unique 33-kD cysteine proteinase accumulates in the whorl in response to larval feeding. The abundance of the proteinase increased dramatically at the site of larval feeding after 1 hr of infestation and continued to accumulate for as long as 7 days. The 33-kD cysteine proteinase was most abundant in the yellow-green portion of the whorl-the normal site of larval feeding and the tissue that has the greatest inhibitory effect on larval growth in bioassays. The proteinase was expressed in response to wounding and was found in senescent leaves. It may be a marker of programmed cell death. The gene coding for the proteinase, mir1, has been transformed into Black Mexican Sweet callus. When larvae were reared on callus expressing the proteinase, their growth was inhibited approximately 60 to 80%. The expression of a cysteine proteinase, instead of a cysteine proteinase inhibitor, may be a novel insect defense mechanism in plants.     

Characterisation of cysteine proteinases responsible for digestive proteolysis in guts of larval western corn rootworm (Diabrotica virgifera) by expression in the yeast Pichia pastoris

Cysteine proteinases are the major class of enzymes responsible for digestive proteolysis in western corn rootworm (Diabrotica virgifera), a serious pest of maize. A larval gut extract hydrolysed typical cathepsin substrates, such as Z-phe-arg-AMC and Z-arg-arg-AMC, and hydrolysis was inhibited by Z-phe-tyr-DMK, specific for cathepsin L. A cDNA library representing larval gut tissue mRNA contained cysteine proteinase-encoding clones at high frequency. Sequence analysis of 11 cysteine proteinase cDNAs showed that 9 encoded cathepsin L-like enzymes, and 2 encoded cathepsin B-like enzymes. Three enzymes (two cathepsin L-like, DvRS5 and DvRS30, and one cathepsin B-like, DvRS40) were expressed as recombinant proteins in culture supernatants of the yeast Pichia pastoris. The cathepsin L-like enzymes were active proteinases, whereas the cathepsin B-like enzyme was inactive until treated with bovine trypsin. The amino acid residue in the S2 binding pocket, the major determinant of substrate specificity in cathepsin cysteine proteinases, predicted that the two cathepsin L-like enzymes, DvRS5 and DvRS30, should differ in substrate specificity, with the latter resembling cathepsin B in hydrolysing substrates with a positively charged residue at P2. This prediction was confirmed; DvRS5 only hydrolysed Z-phe-arg-AMC and not Z-arg-arg-AMC, whereas DvRS30 hydrolysed both substrates. The enzymes showed similar proteolytic activity towards peptide substrates.     

Identification and cloning of an aspartyl proteinase from Coccidioides immitis

A 45 kDa protein was isolated from a soluble vaccine prepared from formaldehyde-killed spherules of Coccidioides immitis. From the N-terminal amino acid sequence, the protein yielded a 17-amino-acid peptide that was homologous to sequences of other fungal aspartyl proteinases. The coccidioidal cDNA encoding the proteinase was amplified using oligonucleotide primers designed from the 45 kDa N-terminal amino acid sequence and a fungal aspartyl proteinase consensus amino acid sequence. The PCR product was cloned and sequenced, and the remaining 5' upstream and 3' downstream cDNA was amplified, cloned, and sequenced. The cDNA encoding the coccidioidal aspartyl proteinase open reading frame was cloned and the fusion protein containing a C-terminal His-tag expressed in E. coli. The recombinant aspartyl proteinase was purified by immobilized metal affinity chromatography. This recombinant protein will be used for further studies to evaluate its antigenicity, including protective immunogenicity.     

Association of a 33-kilodalton cysteine proteinase found in corn callus with the inhibition of fall armyworm larval growth.

Protein patterns of callus from corn (Zea mays L.) inbreds that are either resistant or susceptible to fall armyworm (Spodoptera frugiperda [J.E. Smith]) were analyzed by two-dimensional electrophoresis. Fall armyworm larvae reared on callus initiated from resistant inbreds were significantly smaller than those reared on callus of susceptible inbreds. A 33-kD protein found in callus from the resistant inbreds Mp704 and Mp708 was absent in callus from the susceptible inbreds Tx601 and Ab24E. However, a 36-kD protein found in Ab24E callus immunoreacted with polyclonal antibody raised against the 33-kD protein. When Mp704 nonfriable callus changed to friable, larval growth was not inhibited and the 33-kD protein was absent. There was a significant negative correlation between the concentration of the 33-kD protein in the callus and the weight of the larvae feeding on the callus in the F2 progeny of Mp704 x Tx601. The N-terminal amino acid sequence of the 33-kD protein suggested that it was cysteine proteinase. Purification of the 33- (Mp708) and 36-kD (Ab24E) proteins indicated that they were both cysteine proteinases. The 33-kD cysteine proteinase had 7-fold higher specific activity than the 36-kD enzyme.     

A new type of cysteine proteinase inhibitor--the salarin gene from Atlantic salmon (Salmo salar L.) and Arctic charr (Salvelinus alpinus)

Salarin is a 43 kDa glycoprotein which is found so far only in salmonid fish species. It is a strong inhibitor of cysteine proteinases. Here we characterised the salarin gene from Atlantic salmon and cDNA from Arctic charr. The salarin gene has 13 exons and 1026 bp long coding sequence. The translated amino acid sequence has four similar domains. The sequence resembles the proregion of cathepsins, known to inhibit cysteine proteinases. Salarin can be a new type of cysteine proteinase inhibitor.     

Differential in vitro activation and deactivation of cysteine proteinases isolated during spore germination and vegetative growth of Dictyostelium discoideum.

Acid-activatable cysteine proteinases of Dictyostelium discoideum were first identified in spore extracts of strain SG1 using gelatin/SDS/PAGE, followed by acid treatments. Here we utilized the technique of acid activation to identify cryptic cysteine proteinases throughout auto-induced and heat-induced spore germination of D. discoideum strain SG2 and SG1. The major acid-activatable cysteine proteinase identified in SG2 and SG1 spore extracts was ddCP38 (D. discoideum cysteine proteinase with a molecular mass of 38 kDa) and ddCP48, respectively. Further investigation of these enzymes revealed that they were also base deactivatable with a treatment of ammonium chloride directly following acid activation. However, the most intriguing observation was the reversibility of the effects of base deactivation on the enzymes following a second treatment with acetic acid. Thus, we hypothesize that, unlike most mammalian cysteine proteinases which generally require the cleavage of a pro-peptide region for activation, these cysteine proteinases of D. discoideum likely undergo reversible conformational changes between latent and active forms. Moreover, we were able to detect these cryptic cysteine proteinases in the vegetative cells and early aggregates of both strains SG1 and SG2. Studies using 4-[(2S, 3S)-3-carboxyoxiran-2-ylcarbonyl-L-leucylamido]buty lguanidine, a cysteine proteinase inhibitor, revealed that acid activation of a portion of these proteinases was still achievable even after incubation with the inhibitor, further supporting the concept of two stable and reversible conformational arrangements of the enzymes. Thus, we speculate that the pH shuffles that modulate proteinase conformation and activity in vitro may be a reflection of the in vivo regulation of these enzymes via H+-ATPases and ammonia.