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PubMed1995

Functional display and expression of chicken cystatin using a phagemid system.

A recombinant phage antibody system has been used to display functionally active chicken cystatin, a small protein-type inhibitor of papain-like cysteine proteinases. A synthetic gene, AEF-[S1M, M29I, M89L] chicken cystatin, was ligated into phagemid pCANTAB 5E, cloned, sequenced and displayed on the tips of filamentous phage as a fusion protein with protein III (gpIII). The gene was expressed also as soluble inhibitor in a non-suppressor E.coli strain. Recombinant phages were selected by binding to carboxymethylated-papain. The positive phages showed binding to papain in dose dependent manner. The chicken cystatin-gpIII fusion protein was detected with anti-chicken cystatin antibody and with anti-protein III antibody by western blot analysis. Unfused soluble inhibitor was identified in the periplasma and showed strong papain inhibitory activity. The results demonstrate that functionally active cystatin can be displayed on the phage surface. This technology can be employed further to select inhibitor variants which differ in their binding affinity to papain and papain-like enzymes.

Animals↗
PubMed1993

Conformational variability of chicken cystatin. Comparison of structures determined by X-ray diffraction and NMR spectroscopy.

The structural model derived from X-ray crystallography for unphosphorylated wild-type chicken cystatin is compared with two chicken cystatin structures derived from NMR spectroscopy: the phosphorylated wild-type and the genetically engineered variant AEF-SIM-M29I-M89L. The comparison shows the same overall fold, but also significant differences in structurally variable segments of the polypeptide chain. The largest such segment, comprising residues 71 to 89, is a region characteristic of the family 2 cystatin inhibitors which contains a disulphide bridge (71-81) and the phosphorylation site (Ser80) discussed in the accompanying article. In the crystal structure, the segment 71 to 76 is found as a flexible loop, 77 to 85 as an alpha-helical segment, and 86 to 89 is completely undefined. The solution NMR structures on the other hand are disordered in the initial segment 72 to 80, have an extended conformation at 81 to 83 in contact with the beta-sheet, and clearly show a beta-turn at residues 87 to 90. The segment comprising residues 53 to 57, with smaller variability, is of particular interest as the hairpin loop conserved throughout the cystatin superfamily which binds to the cysteine proteinase. In most of the solution NMR structures, this segment adopts a conformation more like that of stefin B, a family 1 cystatin inhibitor, as was observed in the crystal structure of its inhibitory complex with papain. The differences between the structures are rationalized by an examination of the crystal contacts generated by hypothetical crystal packing of the NMR structures. Additionally, the X-ray refinement shows evidence of conformational disorder in the crystal. Joint refinement with NOE restraints and reflection data does not produce a structure to satisfy the restraints of both methods.

Animals↗
PubMed2006

Effects of potato plants expressing a barley cystatin on the predatory bug Podisus maculiventris via herbivorous prey feeding on the plant.

The aim of this study was to assess the effects of potato plants expressing a barley cystatin on a potentially cystatin-susceptible natural enemy by predation on susceptible and non-susceptible preys feeding on the plant. We have focussed on the impact of the variant HvCPI-1 C68 --> G, in which the only cysteine residue was changed by a glycine, on the growth and digestive physiology of the Colorado potato beetle (CPB), Leptinotarsa decemlineata, and the Egyptian cotton leafworm (ECW), Spodoptera littoralis. Moreover, we have studied the prey-mediated effects of the barley cystatin at the third trophic level, using the predatory spined soldier bug (SSB), Podisus maculiventris, as a model. Feeding trials conducted with CPB larvae reared on transgenic potato plants expressing the C68 --> G variant resulted in significantly lower weight gains compared to those fed on non-transformed (NT) plants. On the contrary, larger weight gains were obtained when ECW larvae, that lack digestive cysteine proteases, were reared on transgenic potato expressing the cystatin, as compared to larvae fed on NT plants. No negative effects on survival and growth were observed when SSB nymphs were exposed to HvCPI-1 C68 --> G by predation on either CPB or ECW larvae reared on transgenic potato plants expressing the barley cystatin, despite the fact that the inhibitor suppressed in vitro gut proteolysis of the predatory bug. To investigate the physiological background, biochemical analysis were carried out on guts of insects dissected at the end of the feeding assays.

Amino Acid Substitution↗
PubMed1992

Levels of salivary cystatins in periodontally healthy and diseased older adults.

Cystatins are cysteine protease inhibitors present in a variety of tissues and body fluids, including saliva. One possible function of these molecules may be to modulate tissue destruction in periodontal diseases. To investigate the potential role of salivary cystatins in these events, the levels of cystatins in saliva from periodontally healthy and diseased individuals were measured by enzyme-linked immunosorbent assay. Flow rates and total protein content were determined in all the samples collected, while cysteine protease inhibitory activity was assessed in submandibular-sublingual secretions. Statistical analysis showed no significant differences in the levels and activity of salivary cystatins in periodontally healthy and diseased individuals. These findings suggest that comparing the levels of cystatins in glandular salivas may not be a suitable indicator of periodontal disease status.

Aged↗
PubMed1990

Sequence analysis, and chromosomal localization of a gene encoding a cystatin-like protein from Drosophila melanogaster.

Using polyclonal antibodies raised against a Drosophila Ca2(+)-binding protein (DCABP-23), clones were isolated from a Drosophila head cDNA library constructed in the expression vector lambda gt11. Two non-homologous clones have been isolated and are being subjected to sequence analysis. One of these clones, though not encoding DCABP-23, does encode a Drosophila cystatin-like protein. This presumed Drosophila cystatin shows homology to mammalian cystatins, chicken egg white cystatin and the rice oryzacystatin. The Drosophila cystatin has been mapped, by in situ hybridization, to region 88C on the right arm of the third chromosome.

Amino Acid Sequence↗
PubMed1994

Inhibition of growth and cysteine proteinase activity of Staphylococcus aureus V8 by phosphorylated cystatin alpha in skin cornified envelope.

The activity of a cysteine proteinase purified from Staphylococcus aureus V8 (SAV8) was inhibited by phosphorylated cystatin alpha (P-cystatin alpha) and by purified cornified envelope protein of newborn rat, a conjugated form of P-cystatin alpha. Immunohistochemical analysis demonstrated a marked decrease in P-cystatin alpha content in cornified envelope treated with sphingosine. The inhibition of papain activity by proteins from sphingosine-treated skin was much weaker than that exerted by proteins from the untreated skin. The suppression of SAV8 colony formation inoculated on the sphingosine-treated skin was examined. Colony formation on the sphingosine-treated skin was enhanced compared to that on normal skin. These findings suggest that P-cystatin alpha in the cornified envelope may have a bacteriostatic barrier function against bacterial infection, such as that with SAV8.

Animals↗
PubMedDate not supplied

Synthetic domains of cystatins linked to enkephalins are novel inhibitors of brain cathepsins L/B.

Cystatin domains or homologous sequences were synthesized and tested as inhibitors of papain, and rat brain cathepsins B and L. These domains included: I, an enzyme substrate binding site containing a -GG- cleavage site (YGGFL); II, known cystatin consensus sequences (-QVVAG- or -QLVSG-); and III, the proposed ancillary site for binding of chicken cystatin to papain (-IPWLN-). A Domain II analog QVVAG(K-NH2) inhibited cathepsin L and papain with Ki 1-4 X 10(-4) M but was inactive towards cathepsin B. A peptide containing Domains I and II, YGGFL-QVVAG(K-NH2), inhibited papain and cathepsin B with Ki 10(-4)-10(-5) M, and cathepsin L with Ki 10(-6) M. The presence of Domain III in the analog YGGFL-QVVAG-IPWLN(K-NH2) resulted in a 10-fold increase in potency towards papain. These data demonstrated that putative cystatin domains are: 1) probably proximal in the intact cystatins; 2) can be linked directly to each other to yield smaller peptides active as inhibitors; 3) showed some specificity towards the three cysteine proteinases.

Amino Acid Sequence↗
PubMed2005

Maize cystatins respond to developmental cues, cold stress and drought.

Comprehensive searches of maize EST data allowed us to identify 8 novel Corn Cystatin (CC) genes in addition to the previously known genes CCI and CCII. The deduced amino acid sequences of all 10 genes contain the typical cystatin family signature. In addition, they show an extended overall similarity with cystatins from other species that belong to several different phyto-cystatin subfamilies. To gain further insight into their respective roles in the maize plant, gene-specific expression profiles were established by semi-quantitative RT-PCR. While 7 CC genes were expressed in two or more tissues varying from gene to gene, CCI was preferentially expressed in immature tassels and CC8 and CC10 in developing kernels. As shown by in situ hybridisation of maize kernels, CC8 was specifically expressed in the basal region of the endosperm and CC10 both in the starchy endosperm and the scutellum of the embryo. The remaining, not kernel-specific genes, all had distinct expression kinetics during kernel development, generally with peaks during the early stages. In addition to developmental regulation, the effect of cold stress and water starvation were tested on cystatin expression. Two genes (CC8 and CC9) were induced by cold stress and 5 genes (CCII, CC3, CC4, CC5 and CC9) were down-regulated in response to water starvation. Taken together our data suggest distinct functions for CC genes in the maize plant.

Amino Acid Sequence↗
PubMed2005

RNAi-mediated gene silencing to assess the role of synaptobrevin and cystatin in tick blood feeding.

In addition to being the conduit for pathogens into hosts, tick saliva contains a broad array of secretory products that facilitate prolonged tick attachment and blood feeding. Proteins found in tick saliva modulate host hemostasis and immune responses. However, it is not clear whether ticks manipulate the immune responses of their hosts by disrupting the antigen-processing pathways of the hosts. Protein secretion into tick saliva from the salivary glands is due to exocytosis of vesicular membrane-bound granular material regulated by SNARE complex proteins. Proteins associated with vesicles (v-SNAREs) are essential components of the exocytotic process. In this study, we assessed the functional significance of synaptobrevin, a SNARE protein, and cystatin, a cysteine protease inhibitor to blood feeding success, in the lone star tick, Amblyomma americanum, using in vivo RNA interference. In separate experiments, tick salivary cystatin and synpatobrevin genes were silenced by injecting adult ticks with 500 ng of dsRNA complementing each gene sequence. Silencing was demonstrated by reduced transcript in midguts and salivary glands. Additionally, disrupting expression of cystatin and synaptobrevin by RNAi reduced the ability of ticks to feed successfully, as demonstrated by feeding inhibition and reduced engorgement weights. Moreover, normal ticks exposed to a rabbit previously exposed to cystatin-silenced ticks exhibited significant resistance to tick feeding. Based on these findings, ticks appear to skillfully evade the host immune system by secreting cystatin, which disrupts normal antigen processing in antigen-presenting cells of hosts.

Animals↗
PubMed2005

Detailed biochemical characterization of human placental cystatin (HPC).

A low molecular weight thiol protease inhibitor (12,500) purified from human placenta has been characterized in detail. Human placental cystatin (HPC) was found to be stable in the pH range 3.0-9.0 and temperature stability was between 40 and 100 degrees C. It does not have any disulphide groups and carbohydrate content. There was no cross-reaction between anti-HPC serum and other purified cystatins like HMW kininogens isolated from sheep plasma and phytocystatins isolated from Phaseolus mungo. The kinetics of inhibition of HPC was studied with ficin and bromelain and the comparison was made with our already reported results with papain. The respective K(i) values obtained for ficin and bromelain are 8.4 x 10(-8) M and 9.5 x 10(-8) M, respectively, whereas the value for papain was 5.5 x 10(-8) M. The values of association constants (K(ass)) for ficin and bromelain were 2.9 x 10(3) and 8.6 x 10(2) M(-1) s(-1), respectively, however, the value for papain was 3.4 x 10(4) M(-1) s(-1), the respective dissociation constant values for ficin and bromelain were 2.6 x 10(-5) and 2.1 x 10(-5) s(-1), respectively, and the value obtained for papain was 2.3 x 10(-5) s(-1). These kinetic parameters taken together along with t(1/2) values and IC(50) values imply that HPC binds more effectively to papain, then ficin and least with bromelain. Far-UV-CD analysis shows that HPC has 21.08% alpha-helical structure and significant amount of beta structure. Near-UV-CD spectra of HPC show positive peak at 280 nm indicating significant amount of tertiary interactions. The partial amino acid sequence analysis shows that HPC has highest sequence homology with chicken cystatin and Gly residue is present at position 11 rather than at conserved position 9, which has also been reported for human stefin A structure. The hydropathy plot of 1-30 amino acid residues shows that most amino acids of this stretch are present in the hydrophobic core of the protein. Owing to low molecular weight, absence of disulphide bonds and carbohydrate content HPC can be placed in type I cystatin family with some resemblance to chicken cystatin as shown by CD studies and amino acid sequence analysis.

Amino Acid Sequence↗
PubMed2003

Molecular cloning and characterization of a novel cystatin-like molecule, CLM, from human bone marrow stromal cells.

The cystatins are physiological cysteine proteinase inhibitors. Here we report the cloning of a novel human cystatin-like molecule (CLM) from human bone marrow stromal cell (BMSC) cDNA library. The putative CLM protein contained 159 residues with a 29-residue signal peptide. CLM protein was highly homologous to family 2 cystatins, especially mouse and human testatin. The CLM gene spanned two exons and was mapped on chromosome 20p11.2, among cystatin superfamily gene clusters. CLM mRNA was barely detected in most tumor cell lines except for breast adenocarcinoma MCF-7 cells and glioblastoma U251 cells, but after LPS or PMA stimulation, CLM expression was increased in myelogenous leukemia cell lines HL-60 and U-937. Northern blot analysis revealed CLM was ubiquitously expressed in normal tissues, which was clearly different from the testis-specific expression pattern of most family 2 cystatins. When overexpressed in 293 cells, GFP-fused CLM targeted extracellularly through secretory pathway by Golgi apparatus. The results indicated that the secreted CLM protein might play roles in hematopoietic differentiation or inflammation.

Adult↗
PubMed2000

Overexpression of the soluble form of chicken cystatin in Escherichia coli and its purification.

A cDNA encoding chicken cystatin was cloned into the pET-23a(+) expression vector and then transformed into Escherichia coli AD494(DE3)pLysS expression host. An active soluble form of cystatin was expressed in the cytoplasm of E. coli induced by isopropyl beta-D-thiogalactopyranoside. The recombinant chicken cystatin was purified to electrophoretic homogeneity by a simple and rapid method involving heat treatment and Sephacryl S-100 gel filtration chromatography. The recombinant cystatin behaved as a thermal-stable protein and exhibited papain-like protease inhibition activity comparable to the natural chicken cystatin.

Animals↗
PubMed2002

Plant seed cystatins and their target enzymes of endogenous and exogenous origin.

Cystatins are protein inhibitors of cysteine proteinases of the papain family, and those of animal origin have long been studied from medical and physiological aspects. In the meantime, oryzacystatin cloned from rice seeds in 1987 was recognized as the first well-defined cystatin of plant origin. Cloning studies followed to disclose various plant cytstatins including those of corn and soybean origin, their similarities to and differences from animal cystatins being analyzed in detail. Plant seed cystatins are now understood as factors controlling germination by inhibition of endogenous cysteine proteinases. They can also recognize insect midgut proteinases as exogenous target enzymes to control. This paper discusses chemical and phytophysiological relationships between cystatins and their targets.

Amino Acid Sequence↗
PubMed2002

Characterization of the expression of a wheat cystatin gene during caryopsis development.

A cDNA coding for phytocystatin, a protease inhibitor, was isolated from wheat embryos by differential display RT-PCR and the corresponding full-length cDNA (named WC5 for wheat cystatin gene 5) subsequently obtained by RACE. The deduced primary sequence of the protein suggests the presence of a 28 amino acid N-terminal signal sequence and a 100 amino acid mature protein containing the three consensus motifs known to interact with the active site of cysteine peptidases. Northern and western analysis revealed a spatio-temporal pattern of the cystatin gene expression during caryopse development. In the embryo, WC5 was only expressed during early embryogenesis whereas, in seed covering layers, WC5 expression was restricted to the maturation stage of grain development. In addition, immunolocalization experiments showed that cystatin accumulated in the aleurone layer of the maturating seed and in the parenchymal tissues of the embryo scutellum. A recombinant form of the wheat cystatin was shown to be able to inhibit peptidase activities present in whole seed protein extracts. In addition, immunological techniques allowed us to identify two putative target peptidases. The possible roles of the cystatin protein are discussed in relation with tissular localization and putative peptidase targets during seed maturation.

Amino Acid Sequence↗
PubMed2004

Prototype demonstration of transgenic resistance to the nematode Radopholus similis conferred on banana by a cystatin.

Cavendish banana was transformed using Agrobacterium tumefaciens to express a protein engineered rice cystatin (OcIdeltaD86) of value for control of plant parasitic nematodes. Expression for each line was under control of a constitutive promoter from the maize ubiquitin gene (UBI-1), a constitutive, chimeric promoter from the octopine and mannopine synthase genes of A. tumefaciens or a promoter from a root-preferentially expressed tubulin gene Arabidopsis (TUB-1). Lines were selected as of potential interest after 8 weeks challenge in containment if their mean R. similis/25 g roots for three sibling plants were more than 1 standard normal variate below the grand mean for all plants in c7-15 lines challenged concurrently. A total of 16 lines were selected on this basis as putative positives. Western blots confirmed that eight of these lines expressed cystatin with a mean of 0.08 +/- 0.04% tsp. All but two of 19 negatively selected lines from bioassays did not express cystatin. The mean resistance level of the confirmed positive lines was 69 +/- 17%. ELISA established the positive lines under control of UBI provided significantly higher expression levels of OcIdeltaD86 than recorded for the other two promoters. Lines of interest were confirmed as producing a transcript for OcIdeltaD86 by RT-PCR. Eight plants of one UBI promoter line expressing only 0.1 +/- 0.004% tsp as cystatin were re-challenged with R. similis and achieved a resistance of 70 +/- 10%. Subsequent repeat western blotting confirmed that this line still produced the cystatin after the trial. This is the first report of transgenic resistance against a major pest or disease of banana.

Animals↗
PubMed1983

Cystatin, a protein inhibitor of cysteine proteinases. Improved purification from egg white, characterization, and detection in chicken serum.

The protein from chicken egg white that inhibits cysteine proteinases, and has been named 'cystatin', was purified by ovomucin precipitation, affinity chromatography on carboxymethylpapain-Sepharose and chromatofocusing. The final purification step separated two major forms of the protein (pI 6.5 and 5.6), with a total recovery of about 20% from egg white. By use of affinity chromatography and immunodiffusion it was shown that the inhibitor is also present at low concentrations in the serum of male and female chickens. Tryptic peptide maps of the separated forms 1 and 2 of egg-white cystatin were closely similar, and each form had the N-terminal sequence Ser-Glx-Asx. The two forms showed complete immunological identity, and neither contained carbohydrate. Ki values for the inhibition of cysteine proteinases were as follows: papain (less than 1 X 10(-11)M), cathepsin B (8 X 10(-10)M), cathepsin H (about 2 X 10(-8)M) and cathepsin L (about 3 X 10(-12)M). Some other cysteine proteinases, and several non-cysteine proteinases, were found not to be significantly inhibited by cystatin. The inhibition of the exopeptidase dipeptidyl peptidase I by cystatin was confirmed and the Ki found to be 2 X 10(-10)M. Inhibitor complexes with active cysteine proteinases and the inactive derivatives formed by treatment with iodoacetate, E-64 [L-trans-epoxysuccinylleucylamido(4-guanidino)butane] and benzyloxycarbonylphenylalanylalanyldiazomethane were demonstrated by isoelectric focusing and cation-exchange chromatography. The complexes dissociated in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (with or without reduction) with no sign of fragmentation of the inhibitor. Cystatin was found not to contain a free thiol group, and there was no indication that disulphide exchange plays any part in the mechanism of inhibition.

Animals↗
PubMed2002

The role of the second binding loop of the cysteine protease inhibitor, cystatin A (stefin A), in stabilizing complexes with target proteases is exerted predominantly by Leu73.

The aim of this work was to elucidate the roles of individual residues within the flexible second binding loop of human cystatin A in the inhibition of cysteine proteases. Four recombinant variants of the inhibitor, each with a single mutation, L73G, P74G, Q76G or N77G, in the most exposed part of this loop were generated by PCR-based site-directed mutagenesis. The binding of these variants to papain, cathepsin L, and cathepsin B was characterized by equilibrium and kinetic methods. Mutation of Leu73 decreased the affinity for papain, cathepsin L and cathepsin B by approximately 300-fold, >10-fold and approximately 4000-fold, respectively. Mutation of Pro74 decreased the affinity for cathepsin B by approximately 10-fold but minimally affected the affinity for the other two enzymes. Mutation of Gln76 and Asn77 did not alter the affinity of cystatin A for any of the proteases studied. The decreased affinities were caused exclusively by increased dissociation rate constants. These results show that the second binding loop of cystatin A plays a major role in stabilizing the complexes with proteases by retarding their dissociation. In contrast with cystatin B, only one amino-acid residue of the loop, Leu73, is of principal importance for this effect, Pro74 assisting to a minor extent only in the case of cathepsin B binding. The contribution of the second binding loop of cystatin A to protease binding varies with the protease, being largest, approximately 45% of the total binding energy, for inhibition of cathepsin B.

Animals↗
PubMed2001

Cystatin capture enzyme-linked immunosorbent assay for serodiagnosis of human clonorchiasis and profile of captured antigenic protein of Clonorchis sinensis.

Enzyme-linked immunosorbent assay (ELISA) with crude extracts of adult Clonorchis sinensis has been reported to have a high degree of sensitivity with a moderate degree of specificity for the serodiagnosis of clonorchiasis. The cystatin capture ELISA was investigated for its usefulness for the serodiagnosis of human clonorchiasis. Cystatin bound specifically to cysteine proteinases in crude extracts of adult C. sinensis worms, and its binding capacity was not hindered competitively by the other proteinase inhibitors tested. The cystatin capture ELISA for clonorchiasis showed a higher degree of specificity than the conventional ELISA, which produced some cross-reactivities to sera from patients with cysticercosis, sparganosis, and opisthorchiasis. Immunoglobulin G antibodies to C. sinensis cysteine proteinases were produced in experimental rabbits at week 3, and their levels increased rapidly and remained at a plateau after 8 weeks of infection. Of the proteins from the C. sinensis crude extract captured with cystatin, seven proteins were reactive with the serum from patients with clonorchiasis. The cystatin capture ELISA is indicated to be a sensitive and highly specific immunodiagnostic assay for serodiagnosis of human clonorchiasis.

Animals↗