Use of Cystatin C and Serum Creatinine for the Diagnosis of Contrast-Induced Nephropathy in Patients Undergoing Contrast-Enhanced Computed Tomography at an Oncology Centre

ObjectiveOur aim was to assess renal function using as laboratory measurements serum creatinine and cystatin C concentrations before and after administration of low-osmolarity (nonionic) iodinated contrast medium in patients with cancer undergoing computed tomography (CT).MethodsThis prospective study included 400 oncologic outpatients. Serum creatinine and cystatin C concentrations were measured before and 72 h after contrast administration. Glomerular filtration rates (GFRs) were estimated using serum creatinine–based [Modification of Diet in Renal Disease (MDRD) and Cockroft-Gault and cystatin C based (Larsson) equations. Exploratory data analysis was performed. The nonparametric Wilcoxon test was used to compare pre and post contrast of test results and estimated clearance. The confidence interval used in the analysis was 95%.ResultsCompared with the pre-contrast values, the mean serum creatinine concentration was significantly higher and average GFRs estimated using MDRD and Cockcroft-Gault equations were significantly lower after the administration of contrast (p <0.001). It was also observed a significant increase after contrast in the concentration of Cystatin C (p = 0.015). In addition, a decrease in GFR estimated using the average Larsson (p = 0.021) was observed between time points. However, none of the patients presented clinically significant nephropathy.ConclusionsAssessment using serum creatinine and cystatin C concentrations showed changes in renal function among patients with cancer undergoing contrast-enhanced CT examination in this study. No significant renal damage related to the use of low-osmolarity iodinated contrast medium of the type and dosage employed in this study was observed. This contrast medium is thus safe for use in patients with cancer.     

胱蛋白酶抑制剂C与肌酐评价儿童肾小球滤过功能中的作用比较

用胱蛋白酶抑制剂C与肌酐、内生肌酐清除率评价儿童肾小球滤过功能,并将其作用进行比较,确定胱蛋白酶抑制剂C在儿童中的正常参考范围。采用颗粒增强散射免疫比浊法检测150例出生后2d～13岁正常儿童及90例1～16岁患不同程度肾脏疾病的儿童血清中胱蛋白酶抑制剂C和血肌酐的浓度,并比较胱蛋白酶抑制剂C与血肌酐的相关性。结果发现胱蛋白酶抑制剂C在出生后四个月内水平明显高于成人,但在出生5个月以后下降至接近成人参考范围。血清胱蛋白酶抑制剂浓度C与尿素清除率之间有显著相关性(P<0.01)。此外,在内生肌酐清除率CCr>80(属于正常参考范围)的肾脏疾病的患儿中有56%胱蛋白酶抑制剂C异常,说明胱蛋白酶抑制剂C比血肌酐更能够敏感地反应儿童肾小球滤过功能的损伤,建议用胱蛋白酶抑制剂C作为儿童肾脏疾病的患者肾小球滤过功能的损伤指标。     

Distinct properties of wild-type and the amyloidogenic human cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type

Variant human cystatin C (L68Q) is an amyloidogenic protein. It deposits in the cerebral vasculature of Icelandic patients with cerebral amyloid angiopathy, leading to stroke. Wild-type and variant cystatin C are cysteine proteinase inhibitors which form concentration dependent inactive dimers; however, variant cystatin C dimerizes at lower concentrations and has an increased susceptibility to a serine protease. We studied the effect of the L68Q amino acid substitution on cystatin C properties, utilizing full length cystatin C purified in mild conditions from media of cells stably transfected with either the wild-type or variant cystatin C genes. The variant cystatin C forms fibrils in vitro detectable by electron microscopy in conditions in which the wild-type protein forms amorphous aggregates. We also show by circular dichroism, steady-state fluorescence and Fourier-transformed infrared spectroscopy that the amino acid substitution modifies cystatin C structure by destabilizing alpha-helical structures and exposing the tryptophan residue to a more polar environment, yielding a more unfolded molecule. These spectral changes demonstrate that variant cystatin C has a three-dimensional structure different from that of the wild-type protein. The structural differences between variant and wild-type cystatin C account for the susceptibility of the variant protein to unfolding, proteolysis and fibrillogenesis.     

Cystatin C,a novel urinary biomarker for sensitive detection of acute kidney injury during haemorrhagic fever with renal syndrome

To explore the value of cystatin C for evaluating acute kidney injury (AKI) in haemorrhagic fever with renal syndrome (HFRS), the concentrations of cystatin C in serum and urine samples from HFRS patients were determined. The serum and urinary cystatin C concentrations significantly increased in HFRS patients compared with normal controls (p?<?0.001). In the acute phase of HFRS, urinary cystatin C increased to higher levels than serum creatinine, especially in severe or critical cases in the oliguric stage. Furthermore, higher levels of urinary cystatin C in the acute phase positively correlated with increased severity of the subsequent kidney injury. In conclusion, urinary cystatin C is a more sensitive clinical marker for AKI in HFRS, which may enable us to initiate treatment measures as early as possible.     

养血清脑颗粒对老年脑梗死后抑郁患者血清胱抑素C及认知功能的影响

目的:探究养血清脑颗粒对老年脑梗死后抑郁患者血清胱抑素C水平的影响及认知功能水平的影响。方法:选取2015年6月到2016年6月我院神经内科收治的老年脑梗死后抑郁患者74例,根据随机数字对照表分为对照组与试验组,各37例。对照组采用口服盐酸帕罗西汀治疗,试验组联合给予养血清脑颗粒治疗,4周为一个疗程,共治疗一个疗程。比较两组患者临床疗效、血清胱抑素C水平及认知功能水平。结果:治疗结束后,与对照组相比,试验组临床总有效率较高(P0.05),治疗后两组血清胱抑素C水平较治疗前降低(P0.05),MMSE评分较治疗前升高(P0.05);与对照组相比,血清胱抑素C水平较低(P0.05),MMSE评分较高(P0.05)。结论:养血清脑颗粒治疗老年脑梗死后抑郁患者临床疗效显著,认知功能明显改善,推测其与血清胱抑素C水平降低有关。     

Cystatin C Is Downregulated in Prostate Cancer and Modulates Invasion of Prostate Cancer Cells via MAPK/Erk and Androgen Receptor Pathways

Cystatin C is believed to prevent tumor progression by inhibiting the activities of a family of lysosomal cysteine proteases. However, little is known about the precise mechanism of cystatin C function in prostate cancer. In the present study, we examined the expression of cystatin C and its association with matrix metalloproteinases 2 (MMP2) and androgen receptor (AR) in a tissue microarray comparing benign and malignant specimens from 448 patients who underwent radical prostatectomy for localized prostate cancer. Cystatin C expression was significantly lower in cancer specimens than in benign tissues (p<0.001) and there was a statistically significant inverse correlation between expression of cystatin C and MMP2 (r_s ² = −0.056, p = 0.05). There was a clear trend that patients with decreased level of cystatin C had lower overall survival. Targeted inhibition of cystatin C using specific siRNA resulted in an increased invasiveness of PC3 cells, whereas induction of cystatin C overexpression greatly reduced invasion rate of PC3 in vitro. The effect of cystatin C on modulating the PC3 cell invasion was provoked by Erk2 inhibitor that specifically inhibited MAPK/Erk2 activity. This suggests that cystatin C may mediate tumor cell invasion by modulating the activity of MAPK/Erk cascades. Consistent with our immunohistochemical findings that patients with low expression of cystatin C and high expression of androgen receptor (AR) tend to have worse overall survival than patients with high expression of cystatin C and high AR expression, induced overexpression of AR in PC3 cells expressing cystatin C siRNA greatly enhanced the invasiveness of PC3 cells. This suggests that there may be a crosstalk between cystatin C and AR-mediated pathways. Our study uncovers a novel role for cystatin C and its associated cellular pathways in prostate cancer invasion and metastasis.     

Cathepsin B, cathepsin H, cathepsin X and cystatin C in sera of patients with early-stage and inflammatory breast cancer

Numerous studies have linked cathepsins and their inhibitor cystatin C to tumor invasion and metastasis. We examined whether cathepsin B, cathepsin H, cathepsin X and cystatin C could be detected in sera from women with early stage or inflammatory breast cancer and whether they correlated with clinicopathological characteristics. Preoperative serum was obtained from 176 patients with early-stage breast cancer (tumor size 相似文献   

Potential Contribution of Adipose Tissue to Elevated Serum Cystatin C in Human Obesity

Cystatin C, an endogenous inhibitor of cathepsin proteases has emerged as a biomarker of cardiovascular risk and reduced renal function. Epidemiological studies indicate that serum cystatin C increased in human obesity. Here, we evaluated the contribution of adipose tissue to this elevation, based on our previous observation that cystatin C is produced by in vitro differentiated human adipocytes. We measured serum cystatin C in 237 nonobese (age: 51 ± 0.8 years; BMI: 22.8 ± 0.11 kg/m²) and 248 obese subjects (age: 50 ± 0.8 years; BMI: 34.7 ± 0.29 kg/m²). Creatinine‐based estimated glomerular filtration rate (eGFR) was calculated to account for renal status. Cystatin C gene expression and secretion were determined on surgical adipose tissue biopsies in a distinct group of subjects. Serum cystatin C is elevated in obese subjects of both genders, independently of reduced eGFR. Cystatin C mRNA is expressed in subcutaneous and omental adipose tissue, at twice higher levels in nonadipose than in adipose cells. Gene expression and cystatin C release by adipose tissue explants increase two‐ to threefold in obesity. These data confirm elevation of serum cystatin C in human obesity and strongly argue for a contribution of increased production of cystatin C by enlarged adipose tissue. Because cystatin C has the potential to affect adipose tissue and vascular homeostasis through local and/or systemic inhibition of cathepsins, this study adds a new factor to the list of adipose tissue secreted bioactive molecules implicated in obesity and obesity‐linked complications.     

Developmental Regulation of Synthesis and Dimerization of the Amyloidogenic Protease Inhibitor Cystatin C in the Hematopoietic System

The cysteine protease inhibitor cystatin C is thought to be secreted by most cells and eliminated in the kidneys, so its concentration in plasma is diagnostic of kidney function. Low extracellular cystatin C is linked to pathologic protease activity in cancer, arthritis, atherosclerosis, aortic aneurism, and emphysema. Cystatin C forms non-inhibitory dimers and aggregates by a mechanism known as domain swapping, a property that reportedly protects against Alzheimer disease but can also cause amyloid angiopathy. Despite these clinical associations, little is known about the regulation of cystatin C production, dimerization, and secretion. We show that hematopoietic cells are major contributors to extracellular cystatin C levels in healthy mice. Among these cells, macrophages and dendritic cells (DC) are the predominant producers of cystatin C. Both cell types synthesize monomeric and dimeric cystatin C in vivo, but only secrete monomer. Dimerization occurs co-translationally in the endoplasmic reticulum and is regulated by the levels of reactive oxygen species (ROS) derived from mitochondria. Drugs or stimuli that reduce the intracellular concentration of ROS inhibit cystatin C dimerization. The extracellular concentration of inhibitory cystatin C is thus partly dependent on the abundance of macrophages and DC, and the ROS levels. These results have implications for the diagnostic use of serum cystatin C as a marker of kidney function during inflammatory processes that induce changes in DC or macrophage abundance. They also suggest an important role for macrophages, DC, and ROS in diseases associated with the protease inhibitory activity or amyloidogenic properties of cystatin C.     

血清胱抑素C 水平与糖尿病心室重构关系的临床研究

目的:研究血清胱抑素C水平与糖尿病心室重构的关系。方法:选择2013年10月~2015年10月在我院进行诊治的糖尿病患者90例,检测血清胱抑素C水平,按照糖尿病患者血清胱抑素C水平的中位数,分为正常组(胱抑素C水平1.65mg/L)和升高组(胱抑素C水平1.65mg/L)。行超声心动图检测左室舒张末内径、左房内径、左室舒张末容积、室间隔厚度和左室后壁厚度,并计算出左室质量指数。对两组的这些指标进行比较,并分析血清胱抑素C与糖尿病心室重构的相关性。结果:与正常组相比,升高组的胱抑素C、左室舒张末内径、左房内径、室间隔厚度、左室后壁厚度、左室质量指数和脑钠肽水平均明显增高(P0.05);经过相关性分析,血清胱抑素C水平与左室舒张末内径、左房内径、室间隔厚度、左室后壁厚度、左室质量指数和脑钠肽均呈正相关(P0.05);Logistic回归分析显示左室舒张末内径、左房内径、室间隔厚度、左室后壁厚度、左室质量指数和脑钠肽等是胱抑素C水平升高的危险因素。结论:血清胱抑素C水平与糖尿病患者的心功能和心室重构具有明显相关性,可作为衡量糖尿病患者心室重构程度的一项参考指标。     

前列地尔联合参芪扶正注射液对IgA肾病的临床疗效观察

目的:探讨前列地尔联合参芪扶正注射液治疗Ig A肾病的临床疗效。方法:选取2011年3月~2015年7月于我院就诊的Ig A肾病患者60例,按照随机数字表法将所有患者分为2组,每组各30例。对照组患者给予前列地尔,实验组患者在对照组的基础上联合使用参芪注射液。比较治疗前后两组患者血清胱抑素C、血肌酐、血尿素氮、血浆白蛋白及24小时尿蛋白水平,同时比较治疗结束后两组患者的临床总有效率。结果:治疗前相比,两组患者治疗后的血清胱抑素C、血肌酐、24小时尿蛋白水平均降低(P0.05);血浆白蛋白水平均升高(P0.05),且实验组患者血清胱抑素C、血肌酐、血尿素氮及24小时尿蛋白水平较对照组更低(P0.05),血浆白蛋白水平更高(P0.05)。与对照组相比,实验组患者临床总有效率较高(P0.05)。结论:前列地尔联合参芪扶正注射液能够提高Ig A肾病患者的临床总有效率,可能与升高血浆白蛋白水平有关。     

左西孟旦联合脑心通胶囊治疗急性心力衰竭的疗效及对血清NT-proBNP，Galectin-3，ET-1及CystatinC水平的影响

目的:分析左西孟旦联合脑心通胶囊治疗急性心力衰竭的临床疗效及对血清氨基末端B型钠尿肽前体(NT-proBNP)、半乳糖凝集素(Galectin-3)、内皮素-1(ET-1)、半胱氨酸蛋白酶抑制剂C(CystatinC)水平的影响。方法:选取2015年3月至2016年6月我院收治的90例急性心力衰竭患者,按抽签法将患者随机分为观察组(n=45)和对照组(n=45),对照组患者给予单纯左西孟旦治疗,观察者患者在此基础上联合应用脑心通胶囊治疗。比较两组患者的临床疗效、心功能指标、血压、心率、不良反应的发生情况及治疗前后血清NT-proBNP、Galectin-3、ET-1和CystatinC水平的变化。结果:治疗后,观察组总有效率为93.33%,比对照组显著升高(77.78%)(P0.05)。两组治疗后患者的左室部分缩短(LVFS)、左心射血分数(LVEF)、每搏输出量(SV)水平均较治疗前增高,血压、心率及血清NT-proBNP、Galectin-3、ET-1、CystatinC水平均较治疗前明显下降,且观察组患者的LVFS、LVEF、SV水平明显高于对照组(P0.05),血压、心率及血清NT-proBNP、Galectin-3、ET-1、CystatinC水平明显低于对照组(P0.05)。结论:左西孟旦联合脑心通胶囊治疗能提高急性心力衰竭的治疗效果,显著降低血清NT-proBNP、Galectin-3、ET-1和CystatinC水平,安全性较高。     

Control and expression of cystatin C by mouse decidual cultures.

During mouse embryo implantation, trophoblast invasion is controlled in part by a balance of trophoblast-derived proteinases and uterine decidual proteinase inhibitors. Our work has focused on cystatin C, the secreted inhibitor of cathepsins B and L. We have previously shown that cystatin C is synthesized by the uterine decidua and localized to the cells in close contact with the trophoblast during implantation in vivo. In the work reported here we have established that decidualizing cultures show a similar upregulation of cystatin C. Using Northern and Western blotting and immunolocalization techniques both cystatin C mRNA and secreted protein increased with the morphological differentiation of stromal or decidual capsule cultures. In an effort to understand the regulation of cystatin C expression, decidual cells were analyzed under various culture conditions. Cystatin C expression was upregulated by increased cell density and by the presence of serum in the media. The growth factors TGF-beta(1) and EGF were found to induce cystatin C to levels comparable to serum stimulation. Co-culture with ectoplacental cones (EPCs) likewise induced expression and resulted in the localization of cystatin at the decidua:trophoblast interface. This work shows that decidualizing cultures are a good system to study cystatin C expression and that the expression is controlled in part by TGF-beta(1) and EGF signaling.     

The affinity and kinetics of inhibition of cysteine proteinases by intact recombinant bovine cystatin C.

Recent studies have shown that the bovine cysteine proteinase inhibitor, cystatin C, is synthesized as a preprotein containing a 118-residue mature protein. However, the forms of the inhibitor isolated previously from bovine tissues had shorter N-terminal regions than expected from these results, and also lower affinity for proteinases than human cystatin C. In this work, we report the properties of recombinant, full-length bovine cystatin C having a complete N-terminal region. The general characteristics of this form of the inhibitor, as reflected by the isoelectric point, the far-ultraviolet circular dichroism spectrum, the thermal stability and the changes of tryptophan fluorescence on interaction with papain, resembled those of human cystatin C. The affinity and kinetics of inhibition of papain and cathepsins B, H and L by the bovine inhibitor were also comparable with those of the human inhibitor, although certain differences were apparent. Notably, the affinity of bovine cystatin C for cathepsin H was somewhat weaker than that of human cystatin C, and bovine cystatin C bound to cathepsin L with about a four-fold higher association rate constant than the human inhibitor. This rate constant is comparable with the highest values reported previously for cystatin-cysteine proteinase reactions. The full-length, recombinant bovine cystatin C bound appreciably more tightly to proteinases than the shorter form characterized previously. Digestion of the recombinant inhibitor with neutrophil elastase resulted in forms with truncated N-terminal regions and appreciably decreased affinity for papain, consistent with the forms of bovine cystatin C isolated previously having arisen by proteolytic cleavage of a mature, full-length inhibitor.     

Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids

Six cysteine proteinase inhibitors were isolated from human urine by affinity chromatography on insolubilized carboxymethylpapain followed by ion-exchange chromatography and immunosorption. Physicochemical and immunochemical measurements identified one as cystatin A, one as cystatin B, one as cystatin C, one as cystatin S, and one as low molecular weight kininogen. The sixth inhibitor displayed immunochemical cross-reactivity with salivary cystatin S but had a different pI (6.85 versus 4.68) and a different (blocked) N-terminal amino acid. This inhibitor was tentatively designated cystatin SU. The isolated inhibitors accounted for nearly all of the cysteine proteinase inhibitory activity of the urinary pool used as starting material. The enzyme inhibitory properties of the inhibitors were investigated by measuring inhibition and rate constants for their interactions with papain and human cathepsin B. Antisera raised against the inhibitors were used in immunochemical determinations of their concentrations in several biological fluids. The combined enzyme kinetic and concentration data showed that several of the inhibitors have the capacity to play physiologically important roles as cysteine proteinase inhibitors in many biological fluids. Cystatin C had the highest molar concentration of the inhibitors in seminal plasma, cerebrospinal fluid, and milk; cystatin S in saliva and tears; and kininogen in blood plasma, synovial fluid, and amniotic fluid.     

乙型肝炎患者凝血功能与胱抑素C 联合检测的临床意义

目的:探讨乙型肝炎患者凝血功能与血清胱抑素C联合检测的临床诊断价值。方法:收集260例乙型肝炎患者为实验组及健康者70例为对照组,采用全自动凝血分析仪进行活化部分凝血酶时间(APTT)、凝血酶原时间(PT)、纤维蛋白原(FIB)的测定,采用全自动生化分析仪进行血清胱抑素C、丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、血清γ谷氨酰转肽酶(GGT)的检测。结果:除急性肝炎组外,其他各组乙型肝炎患者的APTT、PT值均高于对照组(P<0.05)。重型肝炎和肝炎肝硬化组FIB值均低于对照组(P<0.05);对于血清胱抑素C水平,除急性肝炎组外,其他各组值均明显高于健康对照组(P<0.05),且肝炎肝硬化组依次高于重型肝炎组和慢性肝炎组。各实验组ALT和AST水平均明显高于对照组(P<0.05),而对于GGT水平,重型肝炎组和肝炎肝硬化组明显高于对照组(P<0.05)。结论:联合检测APTT、PT、FIB凝血功能指标与血清胱抑素C水平,对临床判断乙型肝炎患者病变程度及预后具有重要意义。     

Cathepsins and their endogenous inhibitors cystatins: expression and modulation in multiple sclerosis

Cathepsins are involved in a variety of physiological processes including antigen processing and presentation and extracellular matrix degradation. In the present study, we evaluated whether expression levels of cathepsins S and B and their inhibitors cystatins B and C are affected by multiple sclerosis (MS) disease state (relapse and remission) and therapies (interferon-β [IFN-β] and the glucocorticoid [GC] methylprednisolone), and whether they are associated with the IFN-β response phenotype. Real-time PCR was employed to compare RNA expression levels in peripheral blood leucocytes (PBLs) and ELISA to determine serum protein levels of MS patients and matched healthy individuals. Cathepsin S RNA was higher in MS patients in the relapse state compared to controls (by 74%, P = 3 × 10(-5), n = 30 versus n = 18) with a similar increase observed in serum (66%, P = 0.002, n = 18 versus n = 20). GC treatment reduced cathepsin S levels in PBL RNA (by 44%, P = 6 × 10(-6), n = 27) and serum proteins (by 27%, P = 1 × 10(-5), n = 26), reduced the serum protein levels of pro-cathepsin B (by 8%, P = 0.0007, n = 23), and in parallel increased the serum levels of their inhibitor cystatin C (by 82%, P = 8 × 10(-6), n = 26). IFN-β therapy significantly elevated the RNA levels (n = 16) of cathepsin B (by 16%, P = 0.03), cystatin B (44%, P = 0.004) and cystatin C (48%, P = 0.011). In the serum, only cathepsin S levels were reduced by IFN-β (16%, P = 0.006, n = 25). Interestingly, pre-treatment serum cathepsin S/cystatin C ratio was higher in 'good responders' to IFN-β therapy compared to patients without a good response (by 94%, P = 0.003). These results suggest that cathepsin S and cystatin C may contribute to disease activity in MS, specifically in a subgroup of patients that are responsive to IFN-β therapy, and that these proteins should be further evaluated as biomarkers in MS.     

The association between renal function and tooth loss in Japanese community-dwelling postmenopausal women

doi: 10.1111/j.1741‐2358.2011.00481.x
The association between renal function and tooth loss in Japanese community‐dwelling postmenopausal women Objectives: This study examined whether low renal function is associated with the number of remaining teeth among community‐dwelling elderly Japanese. Background data: Many elderly individuals display both low renal function and tooth loss. Materials and Methods: Subjects comprised 405 randomly selected women (55–74 years old). Serum cystatin C level was used to assess renal dysfunction. Multiple linear regression analysis was used to evaluate the relationship between number of remaining teeth and serum cystatin C level, with number of remaining teeth as the dependent variable. Six variables were selected as independent variables in the final model: serum cystatin C; age; mean clinical attachment level; serum cross‐linked N‐telopeptide of type I collagen level; body mass index and smoking habits. Results: Multiple linear regression analysis revealed a significant relationship between number of remaining teeth and serum cystatin C level. The beta value for serum cystatin C level for the number of remaining teeth was ?0.11 (p = 0.018). Conclusion: This study indicates a relationship between serum cystatin C level and number of remaining teeth, suggesting that low renal function could be associated with tooth loss.     

Fertility Defects in Mice Expressing the L68Q Variant of Human Cystatin C: A ROLE FOR AMYLOID IN MALE INFERTILITY*

Hereditary cystatin C amyloid angiopathy is an autosomal dominant disorder in which a variant form of cystatin C (L68Q) readily forms amyloid deposits in cerebral arteries in affected individuals resulting in early death. L68Q protein deposits in human cystatin C amyloid angiopathy patients have also been found in tissues outside of the brain including the testis, suggesting possible effects on fertility. Heterozygous transgenic mice (L68Q) that express the human L68Q variant of cystatin C under the control of the mouse cystatin C promoter were unable to generate offspring, suggesting the presence of L68Q cystatin C amyloid affected sperm function. In vitro studies showed that epididymal spermatozoa from L68Q mice were unable to fertilize oocytes and exhibited poor sperm motility. Furthermore, spermatozoa from L68Q mice exhibited reduced cell viability compared with wild type (WT) spermatozoa and often were detected in large agglutinated clumps. Examination of the epididymal fluid and spermatozoa from L68Q mice showed increased levels and distinct forms of cystatin C amyloid that were not present in WT mice. The addition of epididymal fluid from L68Q mice to WT spermatozoa resulted in a recapitulation of the L68Q phenotype in that WT spermatozoa showed reduced cell viability and motility compared with WT spermatozoa incubated in epididymal fluid from WT mice. L68Q epididymal fluid that was depleted of cystatin C amyloids, however, did not impair the motility of WT spermatozoa. Taken together these studies suggest that amyloids in the epididymal fluid can be cytotoxic to the maturing spermatozoa resulting in male infertility.     

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.