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PubMed2000

A plant defensive cystatin (soyacystatin) targets cathepsin L-like digestive cysteine proteinases (DvCALs) in the larval midgut of western corn rootworm (Diabrotica virgifera virgifera).

Feeding bioassay results established that the soybean cysteine proteinase inhibitor N (soyacystatin N, scN) substantially inhibits growth and development of western corn rootworm (WCR), by attenuating digestive proteolysis [Zhao, Y. et al. (1996) Plant Physiol. 111, 1299-1306]. Recombinant scN was more inhibitory than the potent and broad specificity cysteine proteinase inhibitor E-64. WCR digestive proteolytic activity was separated by mildly denaturing SDS-PAGE into two fractions and in-gel assays confirmed that the proteinase activities of each were largely scN-sensitive. Since binding affinity to the target proteinase [Koiwa, H. et al. (1998) Plant J. 14, 371-380] governs the effectiveness of scN as a proteinase inhibitor and an insecticide, five peptides (28-33 kDa) were isolated from WCR gut extracts by scN affinity chromatographic separation. Analysis of the N-terminal sequence of these peptides revealed similarity to a cathepsin L-like cysteine proteinase (DvCAL1, Diabrotica virgifera virgifera cathepsin L) encoded by a WCR cDNA. Our results indicate that cathepsin L orthologs are pivotal digestive proteinases of WCR larvae, and are targets of plant defensive cystatins (phytocystatins), like scN.

Amino Acid Sequence↗
PubMed2002

Genes encoding two cystatins in the flesh fly Sarcophaga crassipalpis and their distinct expression patterns in relation to pupal diapause.

Two genes encoding cystatins, cysteine proteinase inhibitors, were cloned from the flesh fly Sarcophaga crassipalpis. One, sarcocystatin A (abbreviated Scys-A and SCYS-A for the gene and protein, respectively) was previously cloned from Sarcophaga peregrina, but the other is novel. Here the novel gene is named sarcocystatin B (abbreviated Scys-B and SCYS-B for the gene and protein, respectively). Tissue distribution of the two sarcocystatins differs: the fat body is the major site of Scys-A expression, while Scys-B is present in both the fat body and gut. Both Scys-A and -B are developmentally regulated, but their expression patterns also differ. Scys-A transcripts are present in both diapause- and non-diapause-destined third instar wandering larvae, and are then downregulated throughout pupal diapause. By contrast, Scys-B transcripts are only weakly expressed during the third larval instar but are highly upregulated in early diapause. The upregulation of Scys-B in early diapause suggests a possible role for this proteinase inhibitor in halting development.

Amino Acid Sequence↗
PubMed2005

Inhibition of protease in intact fish fillets by soaking in or injection of recombinant soy cystatin or bovine plasma.

Arrowtooth flounder (AF) fillets are known to contain a heat-activated cysteine protease similar to that found in Pacific whiting, which results in soft texture upon cooking. A crude recombinant soy cystatin (CRSC) produced by Escherichia coli, which has been shown to inhibit the protease(s) in Pacific whiting, was introduced into AF fillets by immersion or injection at one of three levels of inhibitory activity: 10 times less than, equal to, or 10 times greater than that of a 20% bovine plasma protein (BPP) solution, a known inhibitor of AF protease(s). Fillets treated with CRSC or BPP at equal inhibitory strength subsequently exhibited the same degree of protection against textural degradation during cooking. Fillets treated with CRSC at lesser or greater levels of inhibitory activity than those of BPP exhibited lesser or higher protection, accordingly. As revealed by SDS-PAGE, the outer portion of fillets soaked with inhibitory solutions was more effectively protected than the inner portion. Such differences between the outer and inner portions of the fillets were not evident when inhibitory solutions were injected into the fillets.

Animals↗
PubMed2004

Association analysis of cystatin A and zinc finger protein 148, two genes located at the psoriasis susceptibility locus PSORS5.

Psoriasis is a multifactorial hereditary skin disease. The searches for causative DNA variations have generated several susceptibility loci, but at present, the gene(s) involved has not been identified. In this article, we investigated whether cystatin A, an upregulated gene in psoriatic plaques and located at chromosome 3q21, is the disease-causing gene at the psoriasis susceptibility locus PSORS5. We also investigated association to a second gene located in this region, zinc finger protein 148. The two genes have been sequenced in a small case/control set in search for SNP markers, followed by family-based association analysis using the transmission disequilibrium test. We did not detect association with either of the genes.

Chromosomes, Human, Pair 3↗
PubMed1997

Resistance to both cyst and root-knot nematodes conferred by transgenic Arabidopsis expressing a modified plant cystatin.

Plant nematodes are major pests of agriculture. Transgenic plant technology has been developed based on the use of proteinase inhibitors as nematode anti-feedants. The approach offers prospects for novel plant resistance and reduced use of environmentally damaging nematicides. A modified rice cystatin, Oc-I delta D86, expressed as a transgene in Arabidopsis thaliana, has a profound effect on the size and fecundity of females for both Heterodera schachtii (beet-cyst nematode) and Meloidogyne incognita (root-knot nematode). No females of either species achieved the minimum size they require for egg production. Ingestion of Oc-I delta D86 from the plant was correlated with loss of cysteine proteinase activity in the intestine thereby suppressing normal growth, as required of an effective antifeedant plant defence.

Animals↗
PubMed2003

Cystatin 10, a novel chondrocyte-specific protein, may promote the last steps of the chondrocyte differentiation pathway.

This study attempts to characterize cystatin 10 (Cst10), which we recently identified as a novel protein implicated in endochondral ossification. Expression of Cst10 was specific to cartilage, localized in the cytosol of prehypertrophic and hypertrophic chondrocytes of the mouse growth plate. In the mouse chondrogenic cell line ATDC5, Cst10 expression preceded type X collagen expression and increased in synchrony with maturation. When we compared ATDC5 cells transfected with Cst10 cDNA with cells transfected with a mock vector, hypertrophic maturation and mineralization of chondrocytes were promoted by Cst10 gene overexpression in that type X collagen expression was observed earlier, and alizarin red staining was stronger. On the other hand, type II collagen expression and Alcian blue staining, both of which are markers of the early stage of chondrocyte differentiation, were similar in both cells. Overexpression of the Cst10 gene also caused fragmentation of nuclei, the appearance of annexin V, a change in the mitochondrial membrane potential, and activation of caspases. These results strongly suggest that Cst10 may play an important role in the last steps of the chondrocyte differentiation pathway as an inducer of maturation, followed by apoptosis of chondrocytes.

Alcian Blue↗
PubMed2003

A comparison between cystatin C, plasma creatinine and the Cockcroft and Gault formula for the estimation of glomerular filtration rate.

BACKGROUND: In clinical practice, the glomerular filtration rate (GFR) is often estimated from plasma creatinine. Several studies have shown cystatin C (cys C) to be a better parameter for the diagnosis of impaired renal function. No data are available, however, on the performance of cys C in follow-up of patients, compared with creatinine. Also, comparisons of cys C with the Cockcroft and Gault (C&G) formula for estimation of GFR are few. METHODS: Plasma samples were obtained from 93 consecutive patients seen for GFR determination and from 30 patients with diabetes mellitus type 2, of whom 23 were investigated a second time after 2 years. GFR was determined with [125I]iothalamate. Plasma creatinine was determined enzymatically and the creatinine clearance calculated according to C&G. Cys C was measured with a particle-enhanced immunonephelometric method. RESULTS: GFR correlated with 1/cys C (r = 0.873) as well as with C&G (r = 0.876). The area under the curve (AUC) of the receiver operating curves (ROCs), a measure of diagnostic accuracy, for cys C (0.931) and C&G (0.938) were equal (P = 0.815) and both better than the creatinine AUC (0.848; P = 0.006). Bland and Altman analysis showed that the simple formula GFR = -4.32 + 80.35 x 1/cys C, derived from our data, gave more accurate (P < 0.0001) and more precise (P = 0.024) GFR estimates than obtained with the C&G formula. The day-to-day variation (biological +analytical) for cys C was small (3.1%, SD 2.51%) in diabetic patients. In the follow-up study in diabetic patients, cys C was the parameter which had the best correlation (r = 0.66) with changes in GFR. CONCLUSIONS: Cys C shows a high correlation with GFR. With a very simple formula, cys C gives a good estimate of GFR, more accurate and precise than C&G. Because biological variation is low, cys C gives also a good assessment of GFR changes during follow-up. Cys C is the preferred endogenous parameter for GFR.

Adolescent↗
PubMed1990

Purification and complex formation analysis of a cysteine proteinase inhibitor (cystatin) from seeds of Wisteria floribunda.

Seeds of Wisteria floribunda contain several kinds of cysteine proteinase inhibitor (cystatin). We purified and characterized one of these inhibitors, named WCPI-3. The molecular weight of WCPI-3 was estimated to be 17,500 and 15,700 by gel filtration and SDS-PAGE, respectively. The isoelectric point was 5.7. WCPI-3 formed an equimolar complex with native papain and the dissociation constant was estimated to be 6.1 nM. Complex formation between WCPI-3 and Cys25-modified papain, such as S-carboxy-methylated or S-carbamoylmethylated papain, could not be observed by gel filtration or native PAGE analysis. A peptide fragment derived from WCPI-3 digested by Achromobacter proteinase (lysyl endopeptidase) had the amino acid sequence of VVAGVNYRFVLK. The VVAG sequence in this fragment corresponds to the conserved sequence QVVAG which is considered to be one of binding regions to cysteine proteinases. The amino acid sequence of the amino-terminal portion (34 residues) of WCPI-3 was highly homologous to that of oryzacystatin from rice seeds.

Amino Acid Sequence↗
PubMed2002

Identification and characterization of cystatin-related epididymal spermatogenic protein in human spermatozoa: localization in the equatorial segment.

Our earlier studies in mouse have shown that the cystatin-related epididymal spermatogenic (CRES) protein is highly expressed in elongating spermatids in the testis and is present in mouse sperm acrosomes, suggesting specific roles in sperm function, fertilization, or both. However, whether the human CRES gene is similar to that of the mouse and is expressed in germ cells has not yet been determined. Therefore, the present study was undertaken to characterize the human ortholog of mouse CRES: Northern blot and in situ hybridization experiments showed that CRES is highly expressed in the human testis, specifically within clusters of round spermatids. Furthermore, reverse transcription-polymerase chain reaction detected CRES mRNA in the epididymis. Western blot analysis of protein lysates prepared from human testis and ejaculated spermatozoa showed a predominant 19-kDa protein and a minor 14-kDa protein. However, in contrast to the acrosomal localization of CRES protein in mouse spermatozoa, indirect immunofluorescence of human spermatozoa treated with methanol/acetic acid using anti-human CRES antibodies revealed that CRES was strictly localized to the equatorial segment. Furthermore, the same staining was observed in both capacitated and acrosome-reacted spermatozoa. To determine whether CRES was associated with the plasma membrane, live spermatozoa were incubated with CRES antibody after capacitation and acrosome reaction. Only acrosome-reacted spermatozoa showed a weak but specific equatorial staining. Taken together, these studies show that CRES protein is present in the sperm equatorial segment and becomes accessible to the extracellular environment during fertilization.

Acrosome Reaction↗
PubMed2001

CCAAT/enhancer binding protein beta regulates expression of the cystatin-related epididymal spermatogenic (Cres) gene.

The CRES protein is a member of the cystatin superfamily of cysteine protease inhibitors with restricted expression in stage-specific germ cells, proximal caput epididymidis, and anterior pituitary gonadotroph cells. To elucidate the molecular mechanisms regulating the highly restricted expression of the cres gene, we have sequenced 1.6 kilobases of mouse cres 5' flanking sequence and performed studies to examine the cres gene promoter. Two putative CCAAT/enhancer binding protein (C/EBP) transcription factor binding motifs exist within the first 135 base pairs of cres promoter. Furthermore, our studies demonstrate that cres mRNA levels are dramatically reduced in the epididymides of C/EBP beta-deficient mice. These data suggest that the C/EBP family of transcription factors, in particular C/EBP beta, plays a role in the regulation of cres gene expression. In support of this finding, Northern blot analysis showed that C/EBP beta is the predominant C/EBP family member expressed in the L beta T2 gonadotroph cell line and the proximal caput epididymidis. Also, gel shift and supershift assays demonstrated that C/EBP beta protein in nuclear extracts from L beta T2 gonadotroph cells and epididymal cells bound to the two C/EBP sites in the cres promoter. Finally, to test the in vivo function of the C/EBP sites in cres gene expression, transfection studies were performed in L beta T2 gonadotroph cells and two heterologous cell systems. These experiments showed a significant reduction of cres transactivation when either C/EBP sites were mutated, and no transC/EBP activation of the cres promoter when both C/EBP sites were mutated. Taken together, these studies demonstrate that the C/EBP beta transcription factor is necessary for high levels of cres gene expression in the proximal caput epididymidis and anterior pituitary gonadotroph cells.

Animals↗
PubMed2006

A Phytophthora infestans cystatin-like protein targets a novel tomato papain-like apoplastic protease.

There is emerging evidence that the proteolytic machinery of plants plays important roles in defense against pathogens. The oomycete pathogen Phytophthora infestans, the agent of the devastating late blight disease of tomato (Lycopersicon esculentum) and potato (Solanum tuberosum), has evolved an arsenal of protease inhibitors to overcome the action of host proteases. Previously, we described a family of 14 Kazal-like extracellular serine protease inhibitors from P. infestans. Among these, EPI1 and EPI10 bind and inhibit the pathogenesis-related (PR) P69B subtilisin-like serine protease of tomato. Here, we describe EPIC1 to EPIC4, a new family of P. infestans secreted proteins with similarity to cystatin-like protease inhibitor domains. Among these, the epiC1 and epiC2 genes lacked orthologs in Phytophthora sojae and Phytophthora ramorum, were relatively fast-evolving within P. infestans, and were up-regulated during infection of tomato, suggesting a role during P. infestans-host interactions. Biochemical functional analyses revealed that EPIC2B interacts with and inhibits a novel papain-like extracellular cysteine protease, termed Phytophthora Inhibited Protease 1 (PIP1). Characterization of PIP1 revealed that it is a PR protein closely related to Rcr3, a tomato apoplastic cysteine protease that functions in fungal resistance. Altogether, this and earlier studies suggest that interplay between host proteases of diverse catalytic families and pathogen inhibitors is a general defense-counterdefense process in plant-pathogen interactions.

Algal Proteins↗
PubMed2003

Staphostatins resemble lipocalins, not cystatins in fold.

Staphostatins are the endogenous inhibitors of the major secreted cysteine proteases of Staphylococcus aureus, the staphopains. Here, we present the 1.4 A crystal structure of staphostatin B and show that the fold can be described as a fully closed, highly sheared eight-stranded beta-barrel. Thus, staphostatin B is related to beta-barrel domains that are involved in the inhibition or regulation of proteases of various catalytic types and to the superfamily of lipocalins/cytosolic fatty acid binding proteins. Unexpectedly for a cysteine protease inhibitor, staphostatin B is not significantly similar to cystatins.

Carrier Proteins↗
PubMed1991

Discrimination between rat thiostatin (T-kininogen) and one of its cystatin-like inhibitory fragments by a monoclonal antibody, and localization of the epitope.

A monoclonal antibody (mAb D3) raised against rat thiostatin (T-kininogen) strongly interacted with a fragment, identified as cystatin-like domain 3, which inhibits cysteine proteinases but did not recognize intact, native thiostatin. The antigen-antibody reaction requires cleavage of the single peptide chain of thiostatin in its inter-domain 2-3 region. This mAb can also differentiate between the two molecular varieties of thiostatin, reacting only with immobilized domain 3 from T1 thiostatin, which differs from the T2 variety by only 10 out of 125 residues. mAb D3 did not react with an N-terminally truncated domain 3 of T1 thiostatin prepared by submaxillary gland kallikrein k10 proteolysis. This suggests that the epitope, or an essential part of it, is located on a stretch of 12 residues at the N-terminal of the T1 thiostatin domain 3. This sequence in T1 thiostatin differs from that in T2 thiostatin by four amino acids, two of which are arginyl residues in T1. Chemical modification of these residues located at positions 246 and 250 decreased the reactivity of T1 domain 3 towards the antibody, suggesting that at least one of them is a critical residue of the epitope. Arginine 246 is part of a small disulfide loop between cysteines 245 and 248 which is also necessary for antibody recognition. This antibody does not change the inhibitory properties of purified domain 3 towards papain or rat liver cathepsin L, indicating that the N-terminal part of domain 3 is not involved in inhibition. mAb D3 was used to demonstrate the presence of inhibitory thiostatin fragments in ascites fluid but not in plasma from normal or turpentine-injected rats.

Amino Acid Sequence↗
PubMed2006

Effect of polyols and salts on the acid-induced state of human placental cystatin.

Polyols (glycerol and sorbitol) and salts (magnesium sulfate, sodium sulfate, and magnesium chloride) have been used to study the refolding of the acid-induced state of human placental cystatin (HPC), which is a low molecular weight (12,500 daltons) thiol proteinase inhibitor, in terms of CD spectroscopy, binding of hydrophobic dye 1-anilinonaphthalene-8-sulfonic acid (ANS), and intrinsic fluorescence measurements. The helical content of acid-denatured HPC increased with increase in glycerol concentration (0-80%). At 80% glycerol concentration, the secondary structural features observed in the far UV-CD region are similar to those of the native state (pH 6.0). The intrinsic fluorescence and near UV-CD studies showed that this 80% glycerol-induced state has a significant amount of tertiary structure with decreased ANS binding compared to the acid-denatured state. It was found that glycerol is more effective in stabilizing the acid-denatured state of HPC as compared to sorbitol. Among salts the stability effect was more for MgCl(2) (used up to concentration of 3 M) compared to MgSO(4) and Na(2)SO(4) (used up to the concentration of 1.5 M due to restricted solubility of HPC at higher sulfate salt concentrations) as determined by CD studies and fluorescence measurements, which showed secondary and tertiary structural resemblance of this MgCl(2)-induced state close to native state and showed overall spectral features in between the native state and the acid-denatured state. This MgCl(2) (3 M)-induced state showed decreased ANS fluorescence as compared to the acid-denatured state but more than that of the native state. The results taken together suggest that the acid-denatured state of HPC in the presence of 80% glycerol or 3 M MgCl(2) has a conformation in between that of the native state (pH 6.0) and the acid-induced state at pH 2.0.

Circular Dichroism↗
PubMed2006

Molten globule state of human placental cystatin (HPC) at low pH conditions and the effects of trifluoroethanol (TFE) and methanol.

Acid-induced conformational changes were studied in human placental cystatin (HPC) in terms of circular dichroism (CD) spectroscopy, the binding of hydrophobic dye 1-anilinonapthalene-8-sulphonic acid (ANS), and intrinsic fluorescence measurements. Our results show the formation of an acid-induced molten globule state at pH 2.0, with significant secondary and tertiary interactions that resemble the native state, exposed hydrophobic regions and the effects of trifluoroethanol (TFE) and methanol in conversion of the acid-denatured state of HPC to the alcohol-induced state, which is characterized by increased helical content, disrupted tertiary structure, and the absence of hydrophobic clusters. Alcohol-induced formation of alpha-helical structures at pH 2.0 is evident from the increase in the ellipticity values at 222 nm, with native-like secondary structural features at 40% TFE. The increase in helical content was observed up to 80% TFE concentration. The ability of TFE (40%) to refold acid-denatured HPC to native-state conformation is also supported by intrinsic and ANS fluorescence measurements.

Anilino Naphthalenesulfonates↗
PubMed1995

The genes coding for rat cystatin-related prostate protein (Cstrp) map to chromosome 3q41.

Two genes encoding rat cystatin-related prostate protein (Cstrp), previously called CRP (Devos et al., 1993), were mapped to chromosome 3q41 by fluorescent in situ hybridization. The results were confirmed using a panel of mouse-rat hybrids that segregate rat chromosomes. Analysis of genomic DNA indicates that the Cstrp locus comprises probably more than three very similar genes.

Animals↗
PubMed1995

Three members of the human cystatin gene superfamily, AHSG, HRG, and KNG, map within one megabase of genomic DNA at 3q27.

While constructing a contig in the human chromosome region 3q27, we identified two YAC clones that were positive for the polymorphic marker D3S1602. One of these clones was also positive for a sequence-tagged site derived from the kininogen (KNG) gene. Because of the known evolutionary and structural relationship of KNG to other members of the cystatin gene superfamily, we tested the physical linkage of the genes encoding alpha-2HS-glycoprotein (AHSG), KNG, and histidine-rich glycoprotein (HRG), all of which were previously mapped to the long arm of chromosome 3. Our results show the colocalization of the three genes in two independent, partially overlapping YAC clones. The genomic inserts of the two clones were 1 Mb and 1.3 Mb in size, indicating that the three genes map within 1 Mb of DNA. The largest YAC was also positive for the polymorphic marker D3S1262, substantiating previously reported data of genetic linkage between this marker and HRG. Fluorescence in situ hybridization localized the two YAC clones to chromosome band 3q27.

Blood Proteins↗
PubMed1993

An effect of androgens on the length of the poly(A)-tail and alternative splicing cause size heterogeneity of the messenger ribonucleic acids encoding cystatin-related protein.

The 22-kilodalton glycoprotein, expressed in the rat ventral prostate under the influence of androgens, is structurally a cystatin-related protein (CRP), as has been shown by copy DNA sequencing. In fact, two slightly different forms (CRP-1 and CRP-2) are expressed in the prostate; one of them (CRP-1) is also expressed in the exorbital lacrymal gland. In both glands, the CRP-1 messenger RNA (mRNA)s are androgen regulated. Moreover, androgens also influence the size of these mRNAs, which show marked heterogeneity (from 760-950 nucleotides). Indeed, the smaller forms are predominant in castrated animals, whereas the large forms are observed immediately after androgen induction. Hybridization with oligo(dT) followed by ribonuclease H treatment revealed that differences in length of the poly(A)-tail are responsible for this effect of androgens. Indeed, two well defined forms of CRP mRNA subsisted after removal of the poly(A)-tail by this treatment. In the less abundant shorter form (CRP-1 delta), 123 nucleotides are deleted by alternative splicing at the junction between the third and the fourth exon. The variant mRNA encodes a truncated protein, wherein the last 27 amino acids are replaced by a hydrophobic stretch of 8 amino acids. No alternative splicing was observed for the CRP-2 mRNA.

Alternative Splicing↗