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PubMed1998

Structural basis for different inhibitory specificities of human cystatins C and D.

Human cystatins C and D share almost identical primary structures of two out of the three segments proposed to be of importance for enzyme interactions but have markedly different profiles for inhibition of the target cysteine peptidases, cathepsins B, H, L, and S. To investigate if the N-terminal binding regions of the inhibitors are responsible for the different inhibition profiles, and thereby confer biological selectivity, two hybrid cystatins were produced in Escherichia coli expression systems. In one hybrid, the N-terminal segment of cystatin C was placed on the framework of cystatin D, and the second was engineered with the N-terminal segment of cystatin D on the cystatin C scaffold. Truncated cystatin C and D variants, devoid of their N-terminal segments, were obtained by incubation with glycyl endopeptidase and isolated, in a second approach to assess the importance of the N-terminal binding regions for cystatin function and specificity. The affinities of the four cystatin variants for cathepsins B, H, L, and S were measured. By comparison with corresponding results for wild-type cystatins C and D, it was concluded (1) that both the N-terminal and framework part of the molecules significantly contribute to the observed differences in inhibitory activities of cystatins C and D and (2) that the N-terminal segment of cystatin C increases the inhibitory activity of cystatin D against cathepsin S and cathepsin L but results in decreased activity against cathepsin H. These differences in specificity were explained by the residues interacting with the S2 subsite of peptidases (Val- and Ala-10 in cystatin C and D, respectively). Also, removal of the N-terminal segment results in total loss of enzyme affinity for cystatin D but not for cystatin C. Therefore, structural differences in the framework parts, as well as in the N-terminal segments, are critical for both inhibitory specificity and potency. Homology modeling was used to identify residues likely responsible for the generally reduced inhibitory potency of cystatin D.

Amino Acid Sequence↗
PubMed2002

Regulated expression and intracellular localization of cystatin F in human U937 cells.

Cystatin F is a cysteine peptidase inhibitor recently discovered in haematopoietic cells by cDNA cloning. To further investigate the expression, distribution and properties of the native human inhibitor the promyeloid cell line U937 has been studied. The cells expressed relatively large quantities of cystatin F, which was found both secreted and intracellularly. The intracellular levels were unusually high for a secreted cystatin ( approximately 25% of the cystatin F in 2- or 4-day culture medium). By contrast, U937 cells contained only 3-4% of the related inhibitor, cystatin C. Cystatin F purified from lysates of U937 cells showed three major forms carrying two, one or no carbohydrate chains. Immunocytochemistry demonstrated a marked cytoplasmic cystatin F staining in a granular pattern. Double staining with a marker for endoplasmic reticulum revealed no colocalization for cystatin F. Analysis of the promoter region of the cystatin F gene (CST7) showed that it, like that of the cystatin C gene (CST3), is devoid of typical TATA- and CAAT-box elements. In contrast to the cystatin C promoter, it does not contain multiple Sp1 binding sites, but has a unique site for C/EBPalpha, possibly explaining the restricted expression of the cystatin F gene. Cells stimulated with all-trans retinoic acid to differentiate them towards a granulocytic pathway, showed a strong ( approximately 18-fold) down-regulation of intracellular cystatin F and almost abolished secreted levels of the inhibitor. Stimulation with tetradecanoyl phorbol acetate, causing monocytic differentiation, also resulted in down-regulation (two fold to threefold) of cystatin F expression, whereas the cystatin C expression was essentially unaltered in both experiments. The results suggest that cystatin F as an intracellular cysteine peptidase inhibitor with readily regulated expression, may be a candidate to control the cysteine peptidase activity known to be essential for antigen presentation in different blood cell lineages.

Binding Sites↗
PubMed1998

Intracellular accumulation of the amyloidogenic L68Q variant of human cystatin C in NIH/3T3 cells.

AIM: To study the cellular transport of L68Q cystatin C, the cystatin variant causing amyloidosis and brain haemorrhage in patients suffering from hereditary cystatin C amyloid angiopathy (HCCAA). METHODS: Expression vectors for wild-type and L68Q cystatin C were constructed and used to transfect mouse NIH/3T3 cells. Stable cell clones were isolated after cotransfection with pSV2neo. Clones expressing human wild-type and L68Q cystatin C were compared with respect to secreted cystatin C by enzyme linked immunosorbent assay (ELISA), and for intracellular cystatin C by western blotting and immunofluorescence cytochemistry. Colocalisation studies in cells were performed by double staining with antibodies against human cystatin C and marker proteins for lysosomes, the Golgi apparatus, or the endoplasmic reticulum, and evaluated by confocal microscopy. RESULTS: Concentrations of human cystatin C secreted from transfected NIH/3T3 cells were similar to those secreted from human cells in culture. In general, clones expressing the gene encoding L68Q cystatin C secreted slightly lower amounts of the protein than clones expressing wild-type human cystatin C. Both immunofluorescence cytochemistry and western blotting experiments showed an increased accumulation of cystatin C in cells expressing the gene encoding L68Q cystatin C compared with cells expressing the gene for the wild-type protein. The intracellularly accumulating L68Q cystatin C was insoluble and located mainly in the endoplasmic reticulum. CONCLUSIONS: The cellular transport of human cystatin C is impeded by the pathogenic amino acid substitution Leu68-->Gln. The resulting intracellular accumulation and increased localised concentration of L68Q cystatin C might be an important event in the molecular pathophysiology of amyloid formation and brain haemorrhage in patients with HCCAA.

Animals↗
PubMed1990

Evolution of proteins of the cystatin superfamily.

We have examined the amino acid sequences of a number of proteins that have been suggested to be related to chicken cystatin, a protein from chicken egg white that inhibits cysteine proteinases. On the basis of statistical analysis, the following proteins were found to be members of the cystatin superfamily: human cystatin A, rat cystatin A(alpha), human cystatin B, rat cystatin B(beta), rice cystatin, human cystatin C, ox colostrum cystatin, human cystatin S, human cystatin SA, human cystatin SN, chicken cystatin, puff adder cystatin, human kininogen, ox kininogen, rat kininogen, rat T-kininogens 1 and 2, human alpha 2HS-glycoprotein, and human histidine-rich glycoprotein. Fibronectin is shown not to be a member of this superfamily, and the c-Ha-ras oncogene protein p21 (Val-12) probably is not a member also. It was convenient to divide members of the superfamily into four types on the basis of the presence of one, two, or three copies of cystatin-like segments and the presence or absence of disulfide bonds. Evolutionary dendrograms were calculated by three methods, and from these we have constructed a scheme depicting the sequence of events in the evolution of these proteins. We suggest that about 1000 million years ago a precursor containing disulfide loops appeared, and that all disulfide-containing cystatins are derived from this. We follow the evolution of the proteins of the superfamily along four main lineages, with special attention to the part that duplication of segments has played in the development of the more complex molecules.

Amino Acid Sequence↗
PubMed1998

Cysteine proteinase inhibitor cystatin A in breast cancer.

Cystatin A (acid cysteine proteinase inhibitor; ACPI) is a natural inhibitor of cysteine proteinases. It has been suggested that an inverse correlation exists between cystatin A and malignant progression. We wanted to assess the biological and clinical significance of cystatin A in infiltrative breast carcinoma by immunohistochemical staining. Formalin-fixed paraffin-embedded material from 440 cases treated during the years 1988-1991 was used in the study. After exclusion of patients with disseminated disease at diagnosis, previous contralateral breast carcinoma, and absence of follow-up data, 384 patients could be included in the survival analysis. For immunohistochemical analysis of cystatin A, we used monoclonal cystatin A antibody WR-23/2/3/3, the binding of which was detected by the avidin-biotin-peroxidase method. Immunohistochemical analysis of Bcl-2 and p53 was also done, and mitotic activity was evaluated. Positive staining for cystatin A was found in 52 of 440 cases. The staining was irregular but showed irrefutably positive areas within neoplastic tissue. Most of the positive tumors were of the ductal infiltrative type, but two were mucinous carcinomas, one medullary and one squamous cell carcinoma. No lobular carcinomas showed positive staining. Focal cystatin A positivity was seen in myoepithelial cells of benign ducts. Occasional apoptotic bodies within the neoplasm showed strong positivity for cystatin A. Tumors positive for cystatin A were of larger size and had higher mitotic activity than cystatin A-negative tumors. Cystatin A was associated with negative Bcl-2 staining, but there was no statistically significant association between axillary lymph node status or p53 immunostaining. The risk for breast cancer-related death was significantly higher in patients with cystatin A-positive tumors than in those with cystatin A-negative ones. The risk increase was significant also in lymph node-negative patients. After adjusting for the effect of tumor size, histological grade, and lymph node status, cystatin A-positive patients still had a higher risk of death. Patients with cystatin A and p53 coexpression had a higher risk of death than the other patients. The findings reveal a new variant of aggressive breast cancer. This type of carcinoma may develop during tumor progression through genetic instability that allows cystatin A expression and gives growth advantage to a clone of tumor cells.

Adenocarcinoma, Mucinous↗
PubMed1994

Expression of the cysteine proteinase inhibitor cystatin C mRNA in rat eye.

BACKGROUND: Cystatin C, a naturally occurring inhibitor of cysteine proteinases, belongs to family 2 of the cystatin superfamily. While cystatins in general, and cystatin C specifically, are expressed in various cell types and found in biological fluids, cystatins in ocular structures have not been investigated. In the present study, the expression of cystatin C mRNA in the eye of the rat was studied. METHODS: Total RNA was extracted from eyes as well as from pooled corneae, retinas, lenses, sclerae, and corneae of adult rats. Cystatin C mRNA was detected in the RNA samples by reverse transcriptase--polymerase chain reaction and Northern blot hybridization. In addition, in situ hybridizations of formalin-fixed cryostat sections were carried out using a digoxigenin-labeled cystatin C probe. RESULTS: Cystatin C mRNA was demonstrated in total RNAs extracted from the eye, sclera, and retina, but not in RNAs isolated from the cornea and lens. In situ hybridizations revealed cystatin C mRNA in most of the stromal cells of the sclera. In the retina, a strong signal was localized in the outer nuclear layer. The distribution of the reaction product suggested that in the retina Müller cells and rod cells are the primary sites of expression of cystatin C. In addition, some glial cells in the inner nuclear and ganglion cell layers were stained. No specific signal for cystatin C mRNA was detected in the cornea, lens, iris, ciliary body, and choroid. CONCLUSIONS: In the eye of the rat, significant levels of cystatin C mRNA are detected in the sclera and retina. In the sclera cystatin C may play a role in modulating the activities of cysteine proteinases, mostly cathepsins, involved in the turnover and remodeling of the stroma. In the retina, cystatins synthesized and presumably released by Müller cells and rod cells may have a protective function against the harmful effects of cysteine proteinases released under physiologic and pathologic conditions.

Animals↗
PubMed2001

Comparison in localization between cystatin C and cathepsin K in osteoclasts and other cells in mouse tibia epiphysis by immunolight and immunoelectron microscopy.

We compared the distribution of a cysteine proteinase inhibitor, cystatin C, with that of cathepsin K in osteoclasts of the mouse tibia by immunolight and immunoelectron microscopy. Light microscopically, strong immunoreactivity for cystatin C was found extracellularly along the resorption lacuna and intracellularly in the organelles of osteoclasts. In serial sections, various patterns of cystatin C and cathepsin K localization were seen, specifically: (1) some resorption lacuna were positive for both cystatin C and cathepsin K; (2) others were positive for either cystatin C or cathepsin K, but not both; and (3) some lacuna were negative for both. In osteoclasts, the localization of cystatin C was similar to that of cathepsin K. Furthermore, cystatin C immunoreactivity was detected in preosteoclasts and osteoblasts, whereas cathepsin K was seen only in preosteoclasts. Electron microscopically, cystatin C immunoreactive products were found in the rough endoplasmic reticulum (ER), Golgi apparatus, vesicles, granules, and vacuoles of osteoclasts. These cystatin C-positive vesicles had fused or were in the process of fusion with the ampullar vacuoles (extracellular spaces) containing cystatin C-positive, fragmented, fibril-like structures. The extracellular cystatin C was deposited on and between the cytoplasmic processes of ruffled borders, and on and between type I collagen fibrils. In the basolateral region of osteoclasts, cystatin C-positive vesicles and granules also fused with vacuoles that contained cystatin C-positive or negative fibril-like structures. These results indicate that osteoclasts not only synthesize and secrete cathepsin K from the ruffled border into the bone resorption lacunae, but also a cysteine proteinase inhibitor, cystatin C. Therefore, it is suggested that cystatin C regulates the degradation of bone matrix by cathepsin K, both extracellularly and intracellularly.

Animals↗
PubMed2002

Cystatin M / E expression in inflammatory and neoplastic skin disorders.

BACKGROUND: Cystatins are natural and specific inhibitors of endogenous mammalian lysosomal cysteine proteinases and exogenous microbial cysteine proteinases. Cystatins were shown to provide regulatory and protective functions against uncontrolled proteolysis in several disease processes. Recently we reported that cystatin M/E, which is a novel member of the cystatin gene family, has an unusually restricted expression pattern that is limited to skin. Although cystatin M/E possesses two distinct biochemical properties (it is a proteinase inhibitor and a substrate for transglutaminase) its physiological function is unknown. Disturbance of the balance between proteinases and their inhibitors can lead to irreversible damage as in chronic inflammatory reactions and tumour invasion. OBJECTIVES: To examine the expression pattern of cystatin M/E in inflammatory conditions and neoplastic skin disorders in order to obtain possible clues on its function. Furthermore, we wished to determine whether cystatin M/E expression could discriminate between various types of neoplasia. METHODS: Biopsy material of normal skin, atopic dermatitis and psoriatic lesional skin, healing excisional wounds in healthy volunteers, and several types of epidermal neoplasia (keratoacanthoma, actinic keratosis, basal cell carcinoma and squamous cell carcinoma) were used in this study. For comparison we studied the expression of cystatin M/E in squamous neoplasias from non-cutaneous origin. Affinity-purified polyclonal antibodies against cystatin M/E were used for immunohistochemical detection. RESULTS: Cystatin M/E is constitutively expressed in the stratum granulosum of normal skin, sebaceous glands, eccrine sweat glands and the infundibular epithelium of hair follicles. Expression in atopic dermatitis and psoriasis was found to extend to several layers of the stratum spinosum. In wound healing, cystatin M/E was not found in the edge of migrating keratinocytes, but it was strongly expressed in the suprabasal layers of the neo-epidermis. In epidermal neoplasias cystatin M/E expression was only found in differentiated cells and keratinized cell nests. CONCLUSIONS: Inflammation causes cystatin M/E to be expressed in the spinous cell layers where it colocalizes with transglutaminase for which it serves as a substrate. Speculatively, increased expression of cystatin M/E is compatible with a role in controlling increased levels of cysteine proteinases during inflammation and infection. Cystatin M/E expression in neoplastic epidermis is confined to well-differentiated cells and as such does not discriminate between benign and (pre)malignant epidermal neoplasias.

Carcinoma, Squamous Cell↗
PubMed1998

Purification and characterization of a new cystatin inhibitor from Taiwan cobra (Naja naja atra) venom.

Cobra cystatin, a new cysteine-proteinase inhibitor of the cystatin superfamily, was isolated from the venom of the Taiwan cobra (Naja naja atra) by affinity chromatography on S-carboxymethylpapain-Sepharose and reverse-phase chromatography. The venom contained two forms of the inhibitor, one of 11870 Da and the other of 12095 Da, as determined by MS, and pI values of 6.2 and 6.1. Cobra cystatin strongly inhibits cysteine proteinases of the papain family, but not calpain. Papain, cathepsin L, cathepsin B and cathepsin S are inhibited with Ki values of 0.19, 0.1, 2.5 and 1.2 nM respectively. The amino acid sequence of cobra cystatin shows that it is a Type 2 cystatin. The amino acid sequence is 73% identical with that of the cystatin in African-puff-adder (Bitis arietans) venom, with which it shares a unique six-residue insertion in a region opposite the reactive inhibitory site. Cobra cystatin is 25-42% identical with other Type 2 cystatins, the most closely related being the recently described human cystatin M, which also has a similar five-residue insertion starting at position 76 (chicken cystatin numbering). A molecular phylogenetic tree of 16 representative members of Family 2 cystatins was constructed by parsimony analysis; it suggests that snake cystatins, together with Tachypleus tridentatus (Japanese horseshoe crab) cystatin and human cystatin M, form a new subfamily within cystatin Family 2.

Amino Acid Sequence↗
PubMed1991

Cystatins in human tear fluid.

The activities of cysteine proteinases which include several lysosomal cathepsins are controlled by naturally occurring inhibitory proteins termed cystatins. Cystatins occur both intracellularly and extracellularly in various tissue fluids including tears. Tears were collected by the Schirmer paper strip method from healthy volunteers who had no history or signs of external ocular disease. The tear components were extracted from the filter papers, and used to determine the apparent free cystatin activity and cystatin levels of tears, and for immunoblots. Tears were also collected using capillary tubes for the measurements of cystatins. By titrating papain, a cysteine proteinase, of known specific activity with tear fluid, relatively high levels of apparent free cystatin activity were demonstrated in tears: 28.8 +/- 3.47 (S.E.M.) pmols papain inhibited per mg tear protein (n = 9). The concentrations of cystatins in tear samples were measured by an indirect enzyme-linked immunosorbent assay (ELISA) using antibodies against human salivary cystatin S and purified cystatin S as standard. The ELISAS revealed that tears contain high levels of cystatin-like immunoreactive material, amounting to about 10% of tear proteins. In microgram cystatin S/mg protein the values were: right eye: 94.7 +/- 9.9; left eye: 115.5 +/- 14.8; n = 12. Cystatin levels of tears collected using capillary tubes were comparable: 120.7 +/- 19 micrograms/mg protein (n = 10). Immunoblots of tear fluids revealed a protein of about 14,000 molecular weight which reacted with antihuman cystatin SN monoclonal antibodies. Protein(s) of similar molecular weight were visualized using antibodies against human cystatins S and C. Less abundant additional cystatin-like immuno-reactive proteins were detected by using the two latter antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Structure and expression of the gene encoding cystatin D, a novel human cysteine proteinase inhibitor.

A new member of the human cystatin multigene family has been cloned from a genomic library using a cystatin C cDNA probe. The complete nucleotide sequence of a 4.3-kilobase DNA segment, containing a complete gene with structure very similar to those of known Family 2 cystatin genes, was determined. The novel gene, called CST4, is composed of three exons and two introns. It contains the coding information for a protein of 142 amino acid residues, which has been tentatively called cystatin D. The deduced amino acid sequence includes a putative signal peptide and presents 51-55% identical residues with the sequences of either cystatin C or the secretory gland cystatins S, SN, or SA. The cystatin D sequence contains all regions of relevance for cysteine proteinase inhibitory activity and also the 4 cysteine residues that form disulfide bridges in the other members of cystatin Family 2. Northern blot analysis revealed that the cystatin D gene is expressed in parotid gland but not in seminal vesicle, prostate, epididymis, testis, ovary, placenta, thyroid, gastric corpus, small intestine, liver, or gall-bladder tissue. This tissue-restricted expression is in marked contrast with the wider distribution of all the other Family 2 cystatins, since cystatin C is expressed in all these tissues and the secretory gland cystatins are present in saliva, seminal plasma, and tears. Cystatin D, being the first described member of a third subfamily within the cystatin Family 2, thus appears to have a distinct function in the body in contrast to other cystatins.

Amino Acid Sequence↗
PubMed1997

Cystatin A in gingival crevicular fluid of periodontal patients.

Cystatins are physiological inhibitors of cysteine proteinases which are widely distributed in human tissues and fluids. In the present study we analysed both the cystatin activity and the different cystatin isoforms in gingival crevicular fluid and saliva samples of nine periodontitis patients. All crevicular fluid samples, which were collected with filter paper points, showed cystatin activity ranging from 7-67 units/mg protein. The mean cystatin activity (24 units/mg protein) was significantly lower (p < 0.05) than that of the saliva samples (mean 93 units/mg protein). The cystatin isoforms in the crevicular fluid were further characterized by immunoblotting with specific antibodies against cystatin C, S, SN and A. While they were clearly present in saliva, cystatin C, cystatin S and cystatin SN could not be detected in any of the crevicular fluid samples. Remarkably, cystatin A was found in all the crevicular fluids as well as in the saliva samples. It is concluded that the cystatin activity found in crevicular fluid is caused, at least partially, by cystatin A. Furthermore, the gingival crevicular fluid is not a major contributor of cystatin C, S and SN activity in saliva.

Adult↗
PubMedDate not supplied

Cystatin C is highly expressed in the human male reproductive system.

Cystatin C displays the strongest inhibitory activity of all cystatins toward lysosomal cysteine proteases in general and has a widespread distribution in human tissues and body fluids, including seminal plasma. The aim of this study was to investigate the distribution of cystatin C in the male reproductive system. Immunohistochemistry revealed a widespread distribution of cystatin C in normal tissues from the testis, epididymis, vas deferens, seminal vesicle, and prostate gland. Immunoreactive cystatin C was localized in basal and secretory epithelial cells, but also in neuroendocrine cells in the prostate, identified by immunostaining for chromogranin A. On adjacent tissue sections, we demonstrated local production of cystatin C utilizing nonradioactive in situ hybridization with a 201-base-long digoxigenin-labeled antisense RNA probe specific for the cystatin C transcript. Staining patterns obtained by immunohistochemistry and in situ hybridization correlated well. Enzyme-linked immunosorbent assay for quantitative analysis of cystatin C demonstrated that cystatin C was present at high concentrations in tissue homogenates from all locations investigated, compared to liver, muscle, spleen, and other general tissues. Western blotting of tissue homogenates revealed a predominant 15-kd cystatin C immunoreactive component in accordance with previous findings in other organs. Quantitative real-time polymerase chain reaction analysis to determine messenger RNA levels in whole tissue extracts showed that the cystatin C gene is highly expressed in the seminal vesicles and the prostate gland, indicating that the major amount of cystatin C in the male reproductive organs and seminal plasma is produced by cells in these 2 tissues. It is concluded that cystatin C is highly expressed and widely distributed throughout the male genital tract, suggesting that cystatin C is an important regulator for normal and pathological proteolysis in the male reproductive system.

Cystatin C↗
PubMed2005

Murine monoclonal antibody which can distinguish cystatins SA1 and SA2.

To develop a diagnostic trial enabling the selective examination for a target cystatin in human body fluids, we attempted to prepare monoclonal antibodies against human cystatin SA1 (originally cystatin SA) and its variant form (cystatin SA2). BALB/c mice were immunized with recombinant (r-) cystatins SA1 and SA2. Two monoclonal antibodies designated Cys3F11 and Cys2E5 were selected. By ELISA analyses, the Cys2E5 was shown to react with r-cystatin SA2 but also somewhat with r-cystatin SA1 (22% cross-reactivity) and with plasma cystatin C (18% cross-reactivity), indicating a high specificity for cystatin SA2. The Cys3F11 reacted not only with r-cystatin SA1 but also with r-cystatin SA2 (89% cross-reactivity) and plasma cystatin C (47% cross-reactivity). This finding was further emphasized by immunoblotting of human submandibular-sublingual saliva samples. ELISA additivity test suggests that the two monoclonal antibodies bind to distinct epitopes. In conclusion, we have succeeded in producing two antibodies that discriminate the structural differences between salivary cystatins S and SN, which share more than 90% identity in amino acid sequence with cystatin SA.

Amino Acid Sequence↗
PubMed1998

Cystatin F is a glycosylated human low molecular weight cysteine proteinase inhibitor.

A previously undescribed human member of the cystatin superfamily called cystatin F has been identified by expressed sequence tag sequencing in human cDNA libraries. A full-length cDNA clone was obtained from a library made from mRNA of CD34-depleted cord blood cells. The sequence of the cDNA contained an open reading frame encoding a putative 19-residue signal peptide and a mature protein of 126 amino acids with two disulfide bridges and enzyme-binding motifs homologous to those of Family 2 cystatins. Unlike other human cystatins, cystatin F has 2 additional Cys residues, indicating the presence of an extra disulfide bridge stabilizing the N-terminal region of the molecule. Recombinant cystatin F was produced in a baculovirus expression system and characterized. The mature recombinant protein processed by insect cells had an N-terminal segment 7 residues longer than that of cystatin C and displayed reversible inhibition of papain and cathepsin L (Ki = 1.1 and 0.31 nM, respectively), but not cathepsin B. Like cystatin E/M, cystatin F is a glycoprotein, carrying two N-linked carbohydrate chains at positions 36 and 88. An immunoassay for quantification of cystatin F showed that blood contains low levels of the inhibitor (0.9 ng/ml). Six B cell lines in culture secreted barely detectable amounts of cystatin F, but several T cell lines and especially one myeloid cell line secreted significant amounts of the inhibitor. Northern blot analysis revealed that the cystatin F gene is primarily expressed in peripheral blood cells and spleen. Tissue expression clearly different from that of the ubiquitous inhibitor, cystatin C, was also indicated by a high incidence of cystatin F clones in cDNA libraries from dendritic and T cells, but no clones identified by expressed sequence tag sequencing in several B cell libraries and in >600 libraries from other human tissues and cells.

Amino Acid Sequence↗
PubMed2004

Factors influencing serum cystatin C levels other than renal function and the impact on renal function measurement.

BACKGROUND: It is well known that serum creatinine may be used as a marker of renal function only if taking into account factors that influence creatinine production, such as age, gender, and weight. Serum cystatin C has been proposed as a potentially superior marker than serum creatinine, because serum cystatin C level is believed to be produced at a constant rate and not to be affected by such factors. However, there are limited data on factors that may influence serum cystatin C levels, and there are limited data comparing cystatin C-based estimates of renal function with creatinine-based estimates that adjust for such factors, especially in individuals with normal, or mildly reduced, renal function. METHODS: This was a cross-sectional study of 8058 inhabitants of the city of Groningen, The Netherlands, 28 to 75 years of age. Serum cystatin C and serum creatinine levels were measured, and creatinine clearance was determined from the average of two separate 24-hour urine collections. We performed multivariate analyses to identify factors independently associated with serum cystatin C levels after adjusting for creatinine clearance. Then, partial Spearman correlations were obtained after adjusting for factors that may influence serum cystatin C and creatinine levels. We also compared the goodness-of-fit (R(2)) of different multivariate linear regression models including serum cystatin C level and serum creatinine level for the outcome of creatinine clearance. RESULTS: Older age, male gender, greater weight, greater height, current cigarette smoking, and higher serum C-reactive protein (CRP) levels were independently associated with higher serum cystatin C levels after adjusting for creatinine clearance. After adjusting for age, weight, and gender, the partial Spearman correlations between creatinine and, respectively, serum cystatin C level and serum creatinine level were -0.29 (P < 0.001) and -0.42 (P < 0.001), respectively. The R(2) values for serum cystatin C level and serum creatinine level adjusted for age, weight, and gender were 0.38 and 0.42, respectively. The addition of cigarette smoking and serum CRP levels did not improve the R(2) value for the multivariate serum cystatin C-based model. CONCLUSION: Serum cystatin C appears to be influenced by factors other than renal function alone. In addition, we found no evidence that multivariate serum cystatin C-based estimates of renal function are superior to multivariate serum creatinine-based estimates.

Adult↗
PubMed2005

Serum cystatin C in pregnant women: reference values, reliable and superior diagnostic accuracy.

BACKGROUND: A simple, endogenous, accurate and minimally invasive marker of glomerular filtration rate (GFR) is much desired in clinical nephrology. Cystatin C fulfills all criteria to be a marker for GFR. For early detection of renal impairment in pregnant women, it is necessary to determine serum cystatin C reference values and the correlation with GFR. The present study was therefore undertaken. METHOD: Healthy pregnant women were followed during pregnancy and the postnatal period. Patient demographics included age, height, weight, BMI, parity, total blood count, LFT, urea, creatinine, Na, K, and blood sugar. Serum cystatin C was estimated using particle enhanced nephlo-immunoassay method. All the parameters were recorded at the start of pregnancy and then in each trimester and the postnatal period. Regression analysis correlation coefficient, ANOVA and the Student's t-test were used for analysis using the SPSS statistical package. RESULTS: A total of 197 pregnant women were included. Mean serum cystatin C for all the women was 0.82 +/- 0.184 mg/l. Serum cystatin C levels were high -0.89 +/- 0.12 mg/l in the first trimester, decreased significantly to 0.651 +/- 0.14 mg/l during the second trimester (p = 0.0000 compared to first trimester), and increased again to 0.82 +/- 0.191 mg/l in the third trimester. After delivery the level rose to 0.94 +/- 0.12 mg/l. A strong correlation was found between serum cystatin C and serum creatinine. A strong negative correlation was found between GFR and cystatin C values in the women (r = -0.546, p = 0.000). A linear relationship was found between GFR and cystatin C levels. A significant increase in the GFR was noted with the progression of pregnancy from 128.06 +/- 29.7 ml/min in the first trimester to 155.2 +/- 29.59 ml/min during second trimester (p = 0.006). A decline in the level of cystatin C exactly parallel to the increase in the GFR was noted with the progression of pregnancy. Interestingly cystatin C was found to have a strong negative correlation with gestational age (r = -0.663, p = 0.000). CONCLUSION: Our results indicate that the mean serum cystatin C levels reflect changes in the GFR during the entire pregnancy and also in the postnatal period. Moreover, serum cystatin C levels are independent of age, height, weight, or blood sugar level. Cystatin C can be used for close supervision and early diagnosis of renal impairment in pregnant patients. Cystatin C is a reliable, useful and promising marker of GFR in pregnant women.

Adolescent↗
PubMed1995

Characterization by spectroscopic, kinetic and equilibrium methods of the interaction between recombinant human cystatin A (stefin A) and cysteine proteinases.

The near-UV spectroscopic changes induced by the binding of recombinant human cystatin A to papain were appreciably different from those induced by cystatin C, reflecting mainly interactions involving the single tryptophan of cystatin C, Trp-106. Cystatin A bound tightly and rapidly to papain and cathepsin L, with dissociation equilibrium constants of approximately 10(-11)-10(-13) M and association rate constants of 3 x 10(6)-5 x 10(6) M-1.s-1. These affinities are at least 50-100-fold higher than previously reported values. The kinetics of binding to papain were consistent with a simple reversible bimolecular reaction mechanism, indicating that cystatin A, like chicken cystatin and cystatin C, binds to papain with no appreciable conformational adaptation of either reacting protein. Cystatin A bound more weakly to actinidin and cathepsins B, C and H, with dissociation equilibrium constants of 10(-8)-10(-9) M. The weaker binding to cathepsin B was largely due to a considerably reduced association rate constant (approximately 4 x 10(4) M-1.s-1), consistent with the 'occluding loop' of cathepsin B markedly restricting the access of cystatin A to the active site. The lower affinities for actinidin and cathepsins C and H were due partly to lower association rate constants (2 x 10(5)-6 x 10(5) M-1.s-1) but primarily to higher dissociation rate constants. The mode of binding of cystatin A to inactivated papains indicated that there is appreciably less space around the active-site cysteine of papain in the complex with cystatin A than in the complexes with chicken cystatin and cystatin C. An N-terminally truncated form of cystatin A, lacking the first six residues, had considerably lower affinity for papain than the full-length inhibitor, consistent with an intact N-terminal region being of importance for proteinase binding.

Amino Acid Sequence↗