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PubMed1986

Characterization of a new cysteine proteinase inhibitor of human saliva, cystatin SN, which is immunologically related to cystatin S.

A new cysteine proteinase inhibitor, cystatin SN, was purified from human whole saliva by chromatography with DE32, Sephacryl S200, and CM-Sepharose CL6B. Cystatin SN is immunologically related to cystatin S and both inhibitors have a similar molecular mass of about 13 kDa. The new inhibitor, however, was clearly distinguished from cystatin S by its much higher pI value. These inhibitors showed similar inhibitory activity for ficin, but cystatin SN was a much better inhibitor for papain and dipeptidyl peptidase I. The amino acid sequence of cystatin SN deduced in the light of the known structure of cystatin S indicates that they have 10 different amino acid residues in the sequence comprising in total 113 residues.

Amino Acid Sequence↗
PubMed2005

Reevaluation of the predicted gene structure of Dictyostelium cystatin A3 (cpiC) by nucleotide sequence determination of its cDNA* and its phylogenetic position in the cystatin superfamily.

Cystatins, cysteine protease inhibitors, are widely distributed among eukaryotes. We reevaluated the structure of the gene cpiC, a gene encoding the third identified member of cystatin family (cystatin A3) that was predicted in the genome database of the social amoeba Dictyostelium discoidium (dictyBase) but remained controversial. We determined the sequences of cDNA and PCR-amplified genomic DNA fragment and found a critical error in the registered nucleotide sequence. The corrected cystatin A3 gene has an open reading frame (ORF) without intron sequence interruption and encodes 94 amino acids (aa), in contrast to the previously predicted sequence of either 80, 82 or 118 aa. The cDNA has an unusual internal poly(A) sequence of 31 adenines, which immediately follows the translation termination codon (TAA) located 146 nucleotides upstream of the post-transcriptional polyadenylation site. The amino acid sequence of Dictyostelium cystatin A3 shows a high similarity to those of previously reported Dictyostelium cystatins as well as Family I cystatins of higher eukaryotes.

Amino Acid Sequence↗
PubMed1987

Characterization and amino acid sequence of a new acidic cysteine proteinase inhibitor (cystatin SA) structurally closely related to cystatin S, from human whole saliva.

A cysteine proteinase inhibitor (designated as cystatin SA) was isolated from human whole saliva by procedures including chromatography on DE 32 and DEAE-Sepharose CL-6B. The amino acid sequence determined by conventional methods showed sequence homology of 90 and 87% as compared with the sequences of cystatin S and cystatin SN, respectively, both of which are salivary inhibitors characterized previously. The new inhibitor consisted of 117 residues and had a pI value of 4.3. Cystatin SA inhibited ficin and papain more strongly than cystatin S or cystatin SN did. It also exhibited inhibitory activity toward dipeptidyl peptidase I but the activity was much weaker than those toward ficin and papain.

Amino Acid Sequence↗
PubMed2006

Extracellular production of human cystatin S and cystatin SA by Bacillus subtilis.

We herein describe the development of a Bacillus subtilis system that can be used to produce large quantities of recombinant (r-) human salivary cystatins, a cysteine protease inhibitor of family 2 in the cystatin superfamily. The B. subtilis that lacked the alkaline protease E gene (DeltaaprE type mutant strain) was prepared by homologous recombination. The cDNA fragments coding for mature cystatins (S and SA) were ligated in frame to the DNA segment for the signal peptide of endoglucanase in the pHSP-US plasmid vector that was then use to transform the DeltaaprE type mutant strain of B. subtilis. The transformants carrying the expression vectors were cultivated in 5-L jar fermenters for 3 days at 30 degrees C. Both r-cystatin S and r-cystatin SA were successfully expressed and secreted into the culture broth, and were purified using a fast performance liquid chromatography system. The first use of DeltaaprE type mutant strain of B. subtilis made it possible to obtain a high yield of secreted protein, which makes this system an improvement over expression in Escherichia coli. We conclude that this system has high utility for expression of commercial quantities of secreted proteins.

Bacillus subtilis↗
PubMed1998

The role of Gly-4 of human cystatin A (stefin A) in the binding of target proteinases. Characterization by kinetic and equilibrium methods of the interactions of cystatin A Gly-4 mutants with papain, cathepsin B, and cathepsin L.

The importance of the evolutionarily conserved Gly-4 residue for the affinity and kinetics of interaction of cystatin A with several cysteine proteinases was assessed by site-directed mutagenesis. Even the smallest replacement, by Ala, resulted in approximately 1000-, approximately 10- and approximately 6000-fold decreased affinities for papain, cathepsin L, and cathepsin B, respectively. Substitution by Ser gave further 3-8-fold reductions in affinity, whereas the largest decreases, >10(5)-fold, were observed for mutations to Arg and Glu. The kinetics of inhibition of papain by the mutants with small side chains, Ala and Ser, were compatible with a one-step bimolecular reaction similar to that with wild-type cystatin A. The decreased affinities of these mutants for papain and cathepsin L were due exclusively to increased dissociation rate constants, but the reduced affinities for cathepsin B were due also to decreased association rate constants. The latter finding indicates that the intact N-terminal region serves as a guide directing cystatin A to the active site of cathepsin B, as has been proposed for cystatin C. The kinetics of binding of the mutants with charged side chains, Arg and Glu, to papain were consistent with a two-step binding mechanism, in which the mutant side chains are accommodated in the complex by a conformational change. The NMR solution structure of the Ala and Trp mutants showed only minor changes compared with wild-type cystatin A, indicating that the large reductions in affinity for proteinases are not due to altered structures of the mutants. Instead, a side chain larger than a hydrogen atom at position 4 affects the interaction with the proteinase most likely by interfering with the binding of the N-terminal region.

Binding, Competitive↗
PubMed1998

Structure and transcriptional regulation of the human cystatin A gene. The 12-O-tetradecanoylphorbol-13-acetate (TPA) responsive element-2 site (-272 to -278) on cystatin A gene is critical for TPA-dependent regulation.

Cystatin A, a cysteine proteinase inhibitor, is one of the precursor proteins of cornified cell envelope of keratinocytes and is expressed during the late stage of keratinocyte differentiation. We have isolated and characterized the human cystatin A gene. The cystatin A gene consists of three exons and two introns. The first, the second, and the third exons consist of coding sequences that are 66, 102, and 126 base pairs in length, respectively. The first and the second introns consist of 14 and 3.6 kilobase pairs, respectively. The transcription initiation site was located 55 base pairs upstream from the first translation site. The fragment, +77 to -2595 in the 5'-flanking region of the human cystatin A gene, was subcloned into a chloramphenicol acetyltransferase (CAT) reporter vector. The expression vector, p2672CAT, produced a significant CAT activity in transiently transfected SV40-transformed human keratinocytes (SVHK cells), that were further stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent protein kinase C activator. Sequence analysis of the gene detected three TPA responsive elements (TRE-1, TRE-2, and TRE-3) and one AP-2 site on the 5' upstream promoter region. Deletion analyses of the p2672CAT vector demonstrated that TRE-2, which was located between -272 and -278, was critical for the regulation by TPA. Gel shift analyses revealed that c-Jun, JunD, and c-Fos bound to the TRE-2 region and that the p2672CAT activity level was elevated by co-transfection with c-Jun and c-Fos or with JunD and c-Fos expression vectors. Furthermore, co-transfection of SVHK cells with the protein kinase C-alpha expression vector and the p2672CAT expression vector also resulted in an increased CAT activity. These results indicate that the 5'-flanking region of the human cystatin A gene confers promoter activity and contains a TRE (TRE-2) that mediates, at least in part, the enhanced expression of this gene by TPA.

Amino Acid Sequence↗
PubMed1989

The human cystatin C gene (CST3), mutated in hereditary cystatin C amyloid angiopathy, is located on chromosome 20.

Hereditary cystatin C amyloid angiopathy has recently been shown to be caused by a point mutation in the cystatin C gene. To determine the chromosomal localization of the gene, 20 human-rodent somatic cell hybrids and a full-length cystatin C cDNA probe were used. Southern blot analysis of BamHI digested cell hybrid DNA revealed that the probe recognizes a 10.6 kb human specific fragment and that this fragment cosegregates with human chromosome 20. Therefore, the human cystatin C gene (CST3) was assigned to chromosome 20.

Amyloidosis↗
PubMed1995

Probing the functional role of the N-terminal region of cystatins by equilibrium and kinetic studies of the binding of Gly-11 variants of recombinant human cystatin C to target proteinases.

The interaction between cystatin C variants, in which the evolutionarily conserved Gly-11 residue was substituted by Ala, Glu or Trp, and the cysteine proteinases, papain, ficin, actinidin and cathepsin B, was characterized. The substitutions reduced the affinity of binding in a manner consistent with the Gly residue of the wild-type inhibitor, allowing the N-terminal region to adopt a conformation that was optimal for interaction with target proteinases. Replacement of Gly-11 by Ala resulted in only a 5- to 100-fold reduction in binding affinity. Comparison with the affinities of wild-type cystatin C lacking the N-terminal region indicated that even this small structural change affects the conformation of this region sufficiently to largely abolish its interaction with the weakly binding proteinases, actinidin and cathepsin B. However, the substitution allows interactions of appreciable strength between the N-terminal region and the tightly binding enzymes, papain or ficin. Replacement of Gly-11 with the larger Glu and Trp residues substantially decreased the affinity of binding to all enzymes, from 10(3)- to 10(5)-fold. These substitutions further affect the conformation of the N-terminal region, so that interactions of this region with papain and ficin are also essentially eliminated. The decreased affinities of the three cystatin C variants for papain, ficin and actinidin were due exclusively to increased dissociation rate constants. In contrast, the decreased affinity between cathepsin B and the Ala-11 variant, the only one for which rate constants could be determined with this enzyme, was due almost entirely to a decreased association rate constant. This behaviour is analogous to that observed for forms of cystatin C lacking the N-terminal region and supports the conclusion that the mode of interaction of this region with target proteinases varies with the enzyme as a result of structural differences in the active-site region of the latter.

Cathepsin B↗
PubMed1991

Gene organization of oryzacystatin-II, a new cystatin superfamily member of plant origin, is closely related to that of oryzacystatin-I but different from those of animal cystatins.

The gene structure of oryzacystatin-II, a new cystatin superfamily member of rice seed origin, was determined. It spans approximately 2.5 kbp and comprises 3 exons. The number of exons and the intron-breakpoints coincide with those of oryzacystatin-I, the first well-defined plant cystatin. However, no similar sequences were observed between the two oryzacystatin genes in 5'-upstream regulatory regions, even though both are expressed specifically during the ripening stage of rice seeds. The gene organization of these two plant cystatins is generally different from that of animal cystatins.

Amino Acid Sequence↗
PubMed1993

Structure of rat genes encoding androgen-regulated cystatin-related proteins (CRPs): a new member of the cystatin superfamily.

Cystatin-related proteins (CRPs) are abundant androgen-regulated secretory glycoproteins that are specifically synthesized in the ventral prostate and lachrymal gland of the rat. Two complete 6-kb genes, Crp1 and Crp2, have been cloned and characterized. They are differentially expressed and encode slightly different proteins. The genes each contain four exons which are interrupted by large introns. An alignment of their sequences demonstrates an overall homology of 90%. The 3' end of a third gene, Crp3, from which only a 1.5-kb fragment was isolated, displays a sequence identity of 84%. These data indicate the existence of a Crp multigene family. The 5' flanking regions of Crp1 and Crp2 are highly homologous and contain a GATAAA sequence 29 nt upstream from the transcription start point. This TATA-box-like element is also found in the promoters of the genes encoding cystatin type-2 proteins. No other recognizable transcription control elements can be detected. Potential binding sites (ARE) for the androgen receptor are scattered throughout the entire genes. The exon/intron organization of the genes encoding CRPs, the size of the exons and their encoding amino acid sequences exhibiting a characteristic spacing of the Cys residues are structural elements displaying a remarkable similarity with the corresponding elements in the genes encoding cystatin type-2 proteins. CRPs must therefore belong to the cystatin superfamily. However, due to their additional domain encoded in an extra exon 2, CRPs must be classified as a new family, type 5.

Amino Acid Sequence↗
PubMed2005

Bm-CPI-2, a cystatin from Brugia malayi nematode parasites, differs from Caenorhabditis elegans cystatins in a specific site mediating inhibition of the antigen-processing enzyme AEP.

The filarial parasite Brugia malayi survives for many years in the human lymphatic system. One immune evasion mechanism employed by Brugia is thought to be the release of cysteine protease inhibitors (cystatins), and we have previously shown that the recombinant cystatin Bm-CPI-2 interferes with protease-dependent antigen processing in the MHC class II antigen presentation pathway. Analogy with vertebrate cystatins suggested that Bm-CPI-2 is bi-functional, with one face of the protein blocking papain-like proteases, and the other able to inhibit legumains such as asparaginyl endopeptidase (AEP). Site-directed mutagenesis was carried out on Bm-CPI-2 at Asn-77, the residue on which AEP inhibition is dependent in vertebrate homologues. Two mutations at this site (to Asp and Lys) showed 10-fold diminished and ablated activity respectively, in assays of AEP inhibition, while blocking of papain-like proteases was reduced by only a small degree. Comparison of the B. malayi cystatins with two homologues encoded by the free-living model organism, Caenorhabditis elegans, suggested that while the papain site may be intact, the AEP site would not be functional. This supposition was tested with recombinant C. elegans proteins, Ce-CPI-1 (K08B4.6) and Ce-CPI-2 (R01B10.1), both of which block cathepsins and neither of which possess the ability to block AEP. Thus, Brugia CPI-2 may have convergently evolved to inhibit an enzyme important only in the mammalian environment.

Amino Acid Sequence↗
PubMed1991

The human cystatin gene family: cloning of three members and evolutionary relationship between cystatins and Bowman-Birk type proteinase inhibitors.

Three genes from the human cystatin gene family have been isolated from a bacteriophage lambda library containing Hind III digests of human genomic DNA. The cloned genes were identified with three DNA probes each containing exon 1, exon 2 and exon 3 of the CST1 gene for cystatin SN. The genes, which we name CST2B, CST4, and CST5, are 6.8 kb, 5.4 kb and 12.5 kb in size, respectively. Statistical analysis of DNA sequence homology elucidated that the second and third exons of cystatin (family II) genes and three cystatin (family II) gene like segments in the kininogen (family III) genes are significantly homologous to the gene segments coding for the inhibitory domains of Bowman-Birk type proteinase inhibitors.

Amino Acid Sequence↗
PubMed2001

Isolation of the protease component of maize cysteine protease-cystatin complex: release of cystatin is not crucial for the activation of the cysteine protease.

The maize cysteine protease complex, which required SDS for its activation in vitro, is a 179 kDa trimeric complex (P-I)3 of a cysteine protease (P) [EC 3.4.22] and a cysteine protease inhibitor (I), cystatin [Yamada et al. (1998) Plant Cell Physiol. 39: 106, Yamada et al. (2000) Plant Cell Physiol. 41: 185]. Here, we show the mechanism of the SDS-dependent activation of the trimeric (P-I) complex and stabilization of the activated protease by its specific substrates. The cystatin-free cysteine protease isolated by preparative SDS-PAGE was still specifically activated by SDS, and its profile of SDS-dependency was exactly the same as that of the trimeric (P-I) complex. It is, therefore, evident that an SDS-dependent conformational change of the protease itself, rather than the release of cystatin from the complex, is crucial for the activation. Pre-treatment analysis with SDS revealed that SDS was required for the initiation of the activation of the trimeric (P-I) complex. Furthermore, we found that once the protease was activated, if there was no substrate, it was rapidly inactivated under optimum conditions of proteolysis, and showed that such inactivation was not due to autolysis of the protease. In contrast, addition of specific substrates prevented the inactivation, and thus we presumed that the activity of the cysteine protease is regulated by both activation by conformational change and rapid inactivation after consumption of substrates.

Cystatins↗
PubMed2002

Cystatin 11: a new member of the cystatin type 2 family.

Cystatin (CST)11, a novel member of the CST type 2 family of cysteine protease inhibitors, was identified in Macaca mulatta epididymis by subtractive hybridization cloning. The human CST11 gene on chromosome 20p11.2 is located near three other CST genes expressed predominantly in the male reproductive tract. The CST11 gene spans three exons, a structure similar to that of other CST family 2 genes. An exon 2-deleted alternative transcript (CST11Delta2) was also identified. CST11 mRNA is expressed only in the epididymis as judged by Northern blot hybridization and is androgen regulated. The protein is most abundant in the initial segment, but is detected throughout the epididymis and on ejaculated human sperm. The calculated tertiary structure of CST11 reveals that the three regions corresponding to the protease inhibitory wedge of CST3 are similarly juxtaposed in CST11, consistent with protease inhibitor function. Intact and exon 2-deleted CST11 recombinant proteins were tested for antibacterial activity. After a 2-h incubation of Escherichia coli with 50 microg/ml recombinant CST11 or CST11Delta2, bacterial colony-forming units were reduced to 30% of control, indicating that both forms have antimicrobial activity.

Amino Acid Sequence↗
PubMed1999

Inhibition of mammalian legumain by some cystatins is due to a novel second reactive site.

We have investigated the inhibition of the recently identified family C13 cysteine peptidase, pig legumain, by human cystatin C. The cystatin was seen to inhibit enzyme activity by stoichiometric 1:1 binding in competition with substrate. The Ki value for the interaction was 0.20 nM, i.e. cystatin C had an affinity for legumain similar to that for the papain-like family C1 cysteine peptidase, cathepsin B. However, cystatin C variants with alterations in the N-terminal region and the "second hairpin loop" that rendered the cystatin inactive against cathepsin B, still inhibited legumain with Ki values 0.2-0.3 nM. Complexes between cystatin C and papain inhibited legumain activity against benzoyl-Asn-NHPhNO2 as efficiently as did cystatin C alone. Conversely, cystatin C inhibited papain activity against benzoyl-Arg-NHPhNO2 whether or not the cystatin had been incubated with legumain, strongly indicating that the cystatin inhibited the two enzymes with non-overlapping sites. A ternary complex between legumain, cystatin C, and papain was demonstrated by gel filtration supported by immunoblotting. Screening of a panel of cystatin superfamily members showed that type 1 inhibitors (cystatins A and B) and low Mr kininogen (type 3) did not inhibit pig legumain. Of human type 2 cystatins, cystatin D was non-inhibitory, whereas cystatin E/M and cystatin F displayed strong (Ki 0.0016 nM) and relatively weak (Ki 10 nM) affinity for legumain, respectively. Sequence alignments and molecular modeling led to the suggestion that a loop located on the opposite side to the papain-binding surface, between the alpha-helix and the first strand of the main beta-pleated sheet of the cystatin structure, could be involved in legumain binding. This was corroborated by analysis of a cystatin C variant with substitution of the Asn39 residue in this loop (N39K-cystatin C); this variant showed a slight reduction in affinity for cathepsin B (Ki 1.5 nM) but >>5,000-fold lower affinity for legumain (Ki >>1,000 nM) than wild-type cystatin C.

Amino Acid Sequence↗
PubMed2003

[Expression of Chinese sturgeon cystatin in yeast Pichia pastoris and its proteinase inhibitory activity analysis].

Cystatin, which widely distributed in both tissues and body fluids of animal and plant, was a superfamily of cysteine proteinase inhibitors. It could form activity-inhibitor complexes with cysteine proteinases to inhibit the hydrolytic activity of proteinases. Cystatin played important roles not only in the inhibition of the proteolytic degradation of fish muscle, but also in biological defense systems against invaders. To explore the functions of fish cystatin and the potential values in fish disease prevention and cure, as well as seafood processing, the recombinant yeast strains which could express Chinese sturgeon cystatin were constructed. First, the cystatin cDNA of Chinese sturgeon, which had been PCR modified, was subcloned into yeast integrated vector pPICZaA. After extracted and purified, the recombinant plasmids were linearized by Sac I. The yeast Pichia pastoris GS115 strain was transformed by use of the Lithium Chloride transformation method, and the recombinant cystatin yeast strains got. After 0.5% methanol induction, SDS-PAGE analysis of the culture supernatant indicated that the yield of recombinant cystatin was about 215mg x L(-1) with the percentage about 73.6%. The recombinant cystatin was purified through Q-Sepharose anion-exchange chromatography, and the purity reached about 94.2%. The inhibitory activity of recombinant cystatin was measured by inhibiting the proteinase activity of papain. The results showed that about 1 microg recombinant cystatin could inhibit the activity of 15 microg papain. Heat stability assay results showed that there was a decrease in inhibitory activity of cystatin with the increasing of temperature. When solution of recombinant cystatin was kept at 70 degrees C for 5min, the inhibitory activity reduced fast. While the recombinant cystatin was heated to 90 degrees C for 5min, the inhibitory activity of recombinant cystatin was undetected. The inhibitory activity for recombinant Chinese sturgeon cystatin was higher than that of CPI (cysteine proteinase inhibitor) from seeds of corn, that about 1 microg purified CIP could inhibited the activity of 0.278 microg papain. But the heat stability of recombinant cystatin is lower than that of the corn CPI. The expression level and the activity of recombinant cystatin from yeast Pichia pastoris were higher than those from E. coli. Moreover, recombinant cystatin from Pichia pastoris was easier to separate and purify. This paper reported that recombinant fish cystatin was produced in a highly efficient expression system based on the methylotrophic yeast, further work will focus on the function of recombinant Chinese sturgeon cystatin to resist fish disease and explore the value of cystatin as a food additive to inhibit cysteine proteinases during surimi processing.

Animals↗
PubMed1994

Cystatins S and C in human whole saliva and in glandular salivas in periodontal health and disease.

Cystatins are inhibitors of cysteine proteinases and could play a protective and regulatory role under inflammatory conditions. Since total cystatin activity of whole saliva was increased in periodontal patients (Henskens et al., 1993), we wanted to investigate the types or origins of cystatins involved in this increase. Distinct types of cystatins were identified by isoelectric focusing and immunoblotting with specific antibodies against one of the salivary acidic isoforms, cystatin S. and the widely distributed basic cystatin C. Clarified human whole saliva (CHWS) of healthy subjects contained cystatin S, whereas cystatin C was barely detectable. In contrast, in CHWS of gingivitis and periodontitis patients, both cystatin C and S levels were higher. The origin of cystatin activity was investigated by collecting submandibular (SM), sublingual (SL), and parotid (PAR) saliva from seven subjects with mild gingivitis. Total cystatin activity was about five times higher in SM saliva than in PAR saliva. In SM and SL saliva, both cystatins S and C were demonstrated. In contrast, in PAR samples, solely cystatin C was detectable. The introduction of experimental gingivitis in one periodontally healthy subject resulted in the appearance of a cystatin C band in PAR saliva and in an increase of cystatins S and C in SM saliva. We conclude that the previously observed increase of cystatin activity in whole saliva in inflammatory periodontal disease is, at least in part, due to an increased glandular output of both the isoform cystatin S (pI 4.7) and the basic cystatin C (pI 9.0).

Adult↗