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PubMed1994

Immunological significances of invariant chain from the aspect of its structural homology with the cystatin family.

The primary structure of p31 of invariant chain (Ii-chain) shows about 50% homology with those of the cystatin family which are endogenous cysteine protease inhibitors. The binding domains between Ii-chain and HLA-DR-7 were estimated from the structural homology between cystatin and Ii-chain and also between cathepsins and DR-7, respectively. The QL64-71 and GS76-88 of Ii-chain were estimated to be the binding domains with GG45-51 and VS57-63 of HLA-DR7, respectively. The purified human Ii-chain from spleen is capable of forming four molecular forms from monomer to tetramer by redox-potential dependent disulfide bond formation. The Ii-chain inhibits cathepsin L and H competitively as a dimer and the K(i) value for cathepsin L was 4.1 x 10(-8) M, but cathepsin B was not inhibited at all. The Ii-chain showed mainly a dimer (60 kDa) under the assay condition of cathepsins with cysteine and was not degraded by these cathepsins. The Ii-chain may play an important role in the regulation of antigenic peptide presentation to MHC class II.

Amino Acid Sequence↗
PubMed1995

Temporary inhibition of papain by hairpin loop mutants of chicken cystatin. Distorted binding of the loops results in cleavage of the Gly(9)-Ala10 bond.

Temporary inhibition of the cysteine proteinases papain and cathepsin L was observed with several hairpin loop mutants of recombinant chicken cystatin at enzyme concentrations above nanomolar. Kinetic modelling of inhibition data, gel electrophoresis and amino acid sequencing revealed that reappearance of papain activity is due to selective cleavage of the Gly(9)-Ala10 bond in the N-terminal binding area of the chicken cystatin variants, resulting in truncated inhibitors of lower affinity. Cleavage of the same bond by contaminating papaya proteinase IV was ruled out by previous purification of papain and suitable control experiments. According to the proposed kinetic model, cleavage occurs within the enzyme-inhibitor complex with first order rate constants ktemp of 2.3 x 10(-3) up to 5 x 10(-1) s-1. A similar ktemp/Km ratio was found for all mutants (0.7 x 10(6)-2.1 x 10(6) s-1.M-1); it is almost identical with the kcat/Km ratio of the peptide substrate Z-Phe-Arg-NHMec. These results suggest that distorted contacts of one of the hairpin loops affect binding of the N-terminal contact area in a way that covalent interaction of the Gly(9)-Ala10 bond with the active-site Cys residue of papain can occur and the bond is cleaved in a substrate-like manner.

Alanine↗
PubMed1991

Isolation of three forms of cystatin from submandibular saliva of isoproterenol-treated rats, its properties and kinetic data.

Three rat salivary cystatins (designated as RSC-1, RSC-2 and RSC-3) induced by chronic isoproterenol (IPR) treatment, but not detected in normal rats, were purified from submandibular saliva of chronically IPR-treated rats by Mono-Q, hydroxyapatite and TSKgel Phenyl-5PW chromatographies. Their molecular weights (Mr) and isoelectric points (pI) differed from each other as follows: RSC-1 (Mr 16,500, pI 4.4), RSC-2 (Mr 15,500, pI 4.4) and RSC-3 (Mr 14,500, pI 4.5). The amino acid compositions of these inhibitors were very similar and the three forms showed complete immunological identity in a double immunodiffusion system. The partial amino acid sequence results showed that these inhibitors belonged to family 2 of the cystatin superfamily. These three forms strongly inhibited the enzyme activities of ficin and papain, but not of cathepsin B and trypsin. The inhibition constants (Ki) of RSC-1, RSC-2 and RSC-3 for ficin were 0.19, 0.50 and 0.012 nM, and for papain were 1.5, 0.93 and 0.03 nM, respectively. RSC-3 inhibited ficin and papain more strongly than did RSC-1 and RSC-2.

Amino Acid Sequence↗
PubMed2005

Isolation, characterization and kinetics of goat cystatins.

Two cysteine proteinase inhibitors I and II were purified from goat kidney using alkaline denaturation, ammonium sulphate fractionation, gel filtration on Sephadex G-75 and ion exchange chromatography on DEAE cellulose. The purified inhibitors were homogenous and showed a single band on SDS PAGE under reducing and non-reducing conditions with an apparent molecular mass of 67 kDa. The cystatin forms were stable in the range of pH 3-10 and up to 95 degrees C. Immunological identity with the sheep LMW kininogen was obtained suggesting that the inhibitor is closely related to kininogens. Spectral studies confirm that the inhibitors have predominantly an alpha-helical structure and undergo major conformational changes during complex formation with papain. The inhibitors had similar inhibitory activities on cysteine proteinases. Both inhibitors inhibited papain, ficin and bromelain competitively, with maximum affinity for papain. The overall lower affinity of these inhibitors to cysteine proteinases compared to other known cystatins can be attributed to the unusual N-terminal sequence where Leu is substituted by Ile. Furthermore, N-terminal sequence analysis revealed maximum homology to mammalian LMW kininogen.

Amino Acid Sequence↗
PubMed2005

T-kininogen, a cystatin-like molecule, inhibits ERK-dependent lymphocyte proliferation.

Plasma levels of kininogens increase with age in both rats and humans. Kininogens are inhibitors of cysteine proteinases, and filarial cysteine proteinase inhibitors (cystatins) reduce the proliferation of T cells. We evaluated whether T-kininogen (T-KG) might mimic this effect, and here we present data indicating that exposure of either rat splenocytes or Jurkat cells to purified T-KG results in inhibition of both ERK activation and [(3)H]-thymidine incorporation, both basal and in response to ConA or PHA. Interestingly, T-KG did not impair [(3)H]-thymidine incorporation in response to IL-2, which requires primarily the activation of the JNK and Jak/STAT pathways. These effects were neither the consequence of increased cell death, nor required the activity of kinin receptors. Furthermore, when T cell receptor proximal events were bypassed by the use of PMA plus Calcium ionophore, T-KG no longer inhibited ERK activation, suggesting that inhibition occurs upstream of these events, possibly at the level of membrane associated signal transduction molecules. We conclude that, like filarial cystatins, T-KG inhibits ERK-dependent T cell proliferation, and these observations suggest a possible role for T-KG in immunosenescence.

Animals↗
PubMed2002

A sugarcane cystatin: recombinant expression, purification, and antifungal activity.

Plants possess several defense mechanisms against pathogenic attack. One of these defenses is the use of protease inhibitor proteins, which interfere in the development and growth of pathogens. Sugarcane productivity can be impacted by the plant's susceptibility to fungal diseases that result in production losses. A relevant line of investigation, therefore, is into the plant's natural defense mechanisms for the control of phytopathogens using cystatins-proteins that specifically inhibit cysteine proteases. In this paper, we discuss the expression, in Escherichia coli, of a sugarcane cystatin, its purification, antifungal activity, and circular dichroism to monitor correct folding. These studies revealed a secondary structure similar to that of the oryzacystatin I of rice. Moreover, the purified protein proved capable of inhibiting the growth of the filamentous fungus Trichoderma reesei, suggesting that it can also be employed to inhibit the growth of pathogenic sugarcane fungi.

Amino Acid Sequence↗
PubMed1985

Cystatin, a protein inhibitor of cysteine proteases alters viral protein cleavages in infected human cells.

Chicken cystatin is a previously described small protein which has the property of inhibiting cysteine active site proteases. The protein, when added to cultured human cells, alters the intracellular proteolytic processing of poliovirus proteins, and causes a reduction in virus yield. It is suggested that the cystatin is able to penetrate to the cellular cytoplasm and inhibit the action of the poliovirus protease.

Animals↗
PubMed2000

Biotic and abiotic stress can induce cystatin expression in chestnut.

A cysteine proteinase inhibitor (cystatin) from chestnut (Castanea sativa) seeds, designated CsC, has been previously characterized. Its antifungal, acaricide and inhibitory activities have allowed to involve CsC in defence mechanisms. The CsC transcription levels decreased during seed maturation and increased throughout germination, an opposite behavior to that shown by most phytocystatins. No inhibition of endogenous proteinase activity by purified CsC was found during the seed maturation or germination processes. CsC message accumulation was induced in chestnut leaves after fungal infection, as well as by wounding and jasmonic acid treatment. Induction in roots was also observed by the last two treatments. Furthermore, CsC transcript levels strongly raised, both in roots and leaves, when chestnut plantlets were subjected to cold- and saline-shocks, and also in roots by heat stress. All together, these data suggest that chestnut cystatin is not only involved in defence responses to pests and pathogen invasion, but also in those related to abiotic stress.

Acetates↗
PubMed1998

Refolding of recombinant sulphonated procathepsin S and of reduced chicken cystatin; implications for renaturation experiments.

Kinetic stopped-flow measurements of refolding of the recombinant sulphonated procathepsin S from 6 M urea are presented. The experiments were performed using intrinsic tryptophan fluorescence and fluorescence of the hydrophobic probe 1-anilino-naphthalene-8-sulfonate (ANS). Initially, (t1/2 = 3 +/- 1 ms) an intermediate with increased ANS fluorescence and protected tryptophan environment is formed. Much later, a slow increase in ANS fluorescence occurs with no accompanying changes in tryptophan fluorescence. The reaction of the slow ANS fluorescence increase correlates with the rate of aggregation as shown by the size exclusion chromatography (SEC). For comparison, the folding reactions of the reduced chicken cystatin were measured, both, by intrinsic tryptophan and extrinsic ANS fluorescence. An early intermediate forms very fast in the refolding of reduced chicken cystatin on 6-fold dilution from 5.7 M GuHCl (t1/2 = 5 +/- 2 ms), similarly to that observed for the sulphonated procathepsin S. ANS fluorescence and tryptophan fluorescence decrease further (t1/2 = 100 +/- 50 ms) leading to a late, 'more structured' intermediate which is prone to dimerization.

Animals↗
PubMed1987

Collagenolytic cathepsins of rabbit spleen: a kinetic analysis of collagen degradation and inhibition by chicken cystatin.

We have investigated the steady state kinetics of the degradation of native fibrillar collagen at pH 3.4 by four collagenolytic cathepsins of rabbit spleen. For each enzyme, the dependence of initial velocity on collagen concentration was well described by the Michaelis-Menten mechanism. Km, expressed as the concentration of triple-helical chains, and kcat values were determined for cathepsins B, L, N and S. The ratio of Kcat to Km suggest that cathepsins L and N are far more effective at collagen solubilization than either cathepsins S or B. Ki values were determined for the inhibition of collagenolytic activity at pH 3.4 using cystatin, a naturally-occurring cysteine proteinase inhibitor. All four cysteine proteinases were inhibited by cystatin in this assay system, although it was found to be a tighter binding inhibitor of cathepsin L, than for cathepsins N and S (approximately 5-fold less), or cathepsin B (approximately 500-fold less).

Animals↗
PubMed2001

Bm-CPI-2, a cystatin homolog secreted by the filarial parasite Brugia malayi, inhibits class II MHC-restricted antigen processing.

While interference with the class I MHC pathway by pathogen-encoded gene products, especially those of viruses, has been well documented, few examples of specific interference with the MHC class II pathway have been reported. Potential targets for such interference are the proteases that remove the invariant chain chaperone and generate antigenic peptides. Indeed, recent studies indicate that immature dendritic cells express cystatin C to modulate cysteine protease activity and the expression of class II MHC molecules [1]. Here, we show that Bm-CPI-2, a recently discovered cystatin homolog produced by the filarial nematode parasite Brugia malayi (W. F. Gregory et al., submitted), inhibits multiple cysteine protease activities found in the endosomes/lysosomes of human B lymphocyte lines. CPI-2 blocked the hydrolysis of synthetic substrates favored by two different families of lysosomal cysteine proteases and blocked the in vitro processing of the tetanus toxin antigen by purified lysosome fractions. Moreover, CPI-2 substantially inhibited the presentation of selected T cell epitopes from tetanus toxin by living antigen-presenting cells. Our studies provide the first example of a product from a eukaryotic parasite that can directly interfere with antigen presentation, which, in turn, may suggest how filarial parasites might inactivate the host immune response to a helminth invader.

Animals↗
PubMed2001

Secretion of whey acidic protein and cystatin is down regulated at mid-lactation in the red kangaroo (Macropus rufus).

Milk collected from the red kangaroo (Macropus rufus) between day 100 and 260 of lactation showed major changes in milk composition at around day 200 of lactation, the time at which the pouch young begins to temporarily exit the pouch and eat herbage. The carbohydrate content of milk declined abruptly at this time and although there was only a small increase in total protein content, SDS PAGE analysis of milk revealed asynchrony in the secretory pattern of individual proteins. The levels of alpha-lactalbumin, beta-lactoglobulin, serum albumin and transferrin remain unchanged during lactation. In contrast, the protease inhibitor cystatin, and the putative protease inhibitor whey acidic protein (WAP) first appeared in milk at elevated concentrations after approximately 150 days of lactation and then ceased to be secreted at approximately 200 days. In addition, a major whey protein, late lactation protein, was first detected in milk around the time whey acidic protein and cystatin cease to be secreted and was present at least until day 260 of lactation. The co-ordinated, but asynchronous secretion of putative protease inhibitors in milk may have several roles during lactation including tissue remodelling in the mammary gland and protecting specific proteins in milk required for physiological development of the dependent young.

Amino Acid Sequence↗
PubMed2001

A constitutive cystatin-encoding gene from barley (Icy) responds differentially to abiotic stimuli.

A barley cDNA clone encoding a cysteine proteinase inhibitor was characterized. The deduced amino acid sequence of this barley cystatin (Hv-CPI) contains the motif QXVXG conserved among members of the cystatin superfamily. The gene (Icy), located on chromosome 2, was expressed in embryos, developing endosperms, leaves and roots as assessed by northern blot analysis. Western blot analysis detected a slightly retarded band in leaves that was not present in roots or seeds. In these two organs a more precise location of Hv-CPI was done by immuno-histochemical analysis, with polyclonal antibodies raised against the recombinant CPI protein expressed in Escherichia coli. This protein efficiently inhibited papain (Ki 2.0 x 10(-8) M) and ficin (Ki 2.2 x 10(-8) M) and, to a lesser extent, chymopapain (Ki 1.6 x 10(-7) M) and was inactive against bromelain. The Icy mRNA expression in vegetative tissues increased in response to anaerobiosis, dark and cold shock (6 degrees C).

Amino Acid Motifs↗
PubMed1984

Cystatin. Amino acid sequence and possible secondary structure.

The amino acid sequence of cystatin, the protein from chicken egg-white that is a tight-binding inhibitor of many cysteine proteinases, is reported. Cystatin is composed of 116 amino acid residues, and the Mr is calculated to be 13 143. No striking similarity to any other known sequence has been detected. The results of computer analysis of the sequence and c.d. spectrometry indicate that the secondary structure includes relatively little alpha-helix (about 20%) and that the remainder is mainly beta-structure.

Amino Acid Sequence↗
PubMed2003

AtCYS1, a cystatin from Arabidopsis thaliana, suppresses hypersensitive cell death.

In plants, cysteine protease inhibitors are involved in the regulation of protein turnover and play an important role in resistance against insects and pathogens. AtCYS1 from Arabidopsis thaliana encodes a protein of 102 amino acids that contains the conserved motif of cysteine protease inhibitors belonging to the cystatin superfamily (Gln-Val-Val-Ala-Gly). Recombinant A. thaliana cystatin-1 (AtCYS1) was expressed in Escherichia coli and purified. AtCYS1 inhibits the catalytic activity of papain (Kd = 4.0 x 10-2 micro m, at pH 7.0 and 25 degrees C), generally taken as a molecular model of cysteine proteases. The molecular bases for papain inhibition by AtCYS1 have been analysed taking into account the three-dimensional structure of the papain-stefin B complex. AtCYS1 is constitutively expressed in roots and in developing siliques of A. thaliana. In leaves, AtCYS1 is strongly induced by wounding, by challenge with avirulent pathogens and by nitric oxide (NO). The overexpression of AtCYS1 blocks cell death activated by either avirulent pathogens or by oxidative and nitrosative stress in both A. thaliana suspension cultured cells and in transgenic tobacco plants. The suppression of the NO-mediated cell death in plants overexpressing AtCYS1 provides the evidence that NO is not cytotoxic for the plant, indicating that NO functions as cell death trigger through the stimulation of an active process, in which cysteine proteases and theirs proteinaceous inhibitors appear to play a crucial role.

Amino Acid Sequence↗
PubMed1998

Testatin: a cystatin-related gene expressed during early testis development.

To isolate genes involved in morphogenic aspects of testis development, and which may act in cell signaling pathways downstream of the testis-determining gene Sry, we have developed a modified mRNA differential display method named signal peptide differential display. It was used to target those genes that encode proteins having a signal peptide sequence. By using this method, we isolated a gene named testatin. This gene was found to be related to a group of genes that encodes cysteine protease inhibitors known as cystatins. Cystatins and their target proteases have been associated with tumor formation and metastasis, but also are involved in natural tissue remodeling events such as bone resorption and embryo implantation. We show that testatin expression is restricted to fetal gonads and adult testis. Furthermore, testatin is expressed during testis cord formation in pre-Sertoli cells, believed to be the site of Sry action, at a time immediately after the peak of Sry expression. This finding suggests that testatin might be activated by transcription factors that are known to orchestrate the early testis development pathway. This gene therefore represents one of the putative downstream targets likely to have an essential role in tissue reorganization during early testis development.

Amino Acid Sequence↗
PubMed1995

Isolation and molecular cloning of a novel bone phosphoprotein related in sequence to the cystatin family of thiol protease inhibitors.

We describe here the isolation of a novel non-collagenous protein from the acid demineralization extract of bovine cortical bone. This 24-kDa protein is multiply phosphorylated at serine residues in Ser-X-Glu/Ser(P) sequences, a recognition motif for phosphorylation by the secretory pathway protein kinase, and we have termed this protein secreted phosphoprotein 24 (spp24). The cDNA structure of spp24 was determined by sequencing cDNA fragments obtained by reverse transcription-polymerase chain reaction, 3'-rapid amplification of cDNA ends, and screening a lambda gt11 cDNA library. This cDNA sequence predicts a 200-residue initial translation product which consists of a 20-residue signal sequence and the 180-residue mature spp24. Northern blot analysis using the spp24 cDNA showed that spp24 mRNA is in liver and bone but not in heart, lung, kidney, or spleen. A search of existing protein sequences revealed that the N-terminal 107 residues of mature spp24 are related in sequence to the cystatin family of thiol protease inhibitors, which suggests that spp24 could function to modulate the thiol protease activities that are known to be involved in bone turnover. Several of the proteins in the cystatin family that are most closely related to spp24 are not only thiol protease inhibitors but are also precursors to peptides with potent biological activity, peptides such as bradykinin and the neutrophil antibiotic peptides. It is therefore possible that the intact form of spp24 found in bone could also be a precursor to a biologically active peptide, a peptide which could coordinate an aspect of bone turnover.

Amino Acid Sequence↗
PubMed2006

Antiinflammatory and immunosuppressive activity of sialostatin L, a salivary cystatin from the tick Ixodes scapularis.

Here we report the ability of the tick Ixodes scapularis, the main vector of Lyme disease in the United States, to actively and specifically affect the host proteolytic activity in the sites of infestation through the release of a cystatin constituent of its saliva. The cystatin presence in the saliva was verified both biochemically and immunologically. We named the protein sialostatin L because of its inhibitory action against cathepsin L. We also show that the proteases it targets, although limited in number, have a prominent role in the proteolytic cascades that take place in the extracellular and intracellular environment. As a result, sialostatin L displays an antiinflammatory role and inhibits proliferation of cytotoxic T lymphocytes. Beyond unraveling another component accounting for the properties of tick saliva, contributing to feeding success and pathogen transmission, we describe a novel tool for studying the role of papain-like proteases in diverse biologic phenomena and a protein with numerous potential pharmaceutical applications.

Amino Acid Sequence↗