An explorative study of inflammation-related proteins associated with kidney injury in male heat-stressed workers.

Chronic kidney disease of non-traditional origin (CKDnt) and acute kidney injury (AKI) often affect heat-stressed Mesoamerican manual workers. Inflammation occurs concurrently with AKI in this population, but its role remains unknown. To explore links between inflammation and kidney injury in heat stress, we compared levels of inflammation-related proteins in cutters with and without increasing serum creatinine levels during sugarcane harvest. These sugarcane cutters have previously been identified to be repeatedly exposed to severe heat stress during the five month harvest season. A nested case-control study was conducted among male Nicaraguan sugarcane cutters in a CKDnt hotspot. Cases (n = 30) were defined as having an increase in creatinine of ≥0.3 mg/dL across the five-month harvest. Controls (n = 57) had stable creatinine levels. Ninety-two inflammation-related proteins in serum were measured before and after harvest using Proximity Extension Assays. Mixed linear regression was used to identify differences in protein concentrations between cases and controls before harvest, differential trends during harvest, and association between protein concentrations and the urine kidney injury markers Kidney Injury Molecule (KIM)-1, Monocyte Chemoattractant Protein (MCP)-1 and albumin. One protein, chemokine (C-C motif) ligand 23 (CCL23), was elevated among cases at pre-harvest. Changes in seven inflammation-related proteins (CCL19, CCL23, colony-stimulating factor 1 [CSF1], hepatocyte and fibroblast growth factors [HGF and FGF23], and tumor necrosis factor beta [TNFB] and TNF-related activation-induced cytokine [TRANCE]) were associated with case status and at least two out of three urine kidney injury markers (KIM-1, MCP-1 and albumin). Several of these have been implicated in myofibroblast activation, which likely is an important step in kidney interstitial fibrotic disease such as CKDnt. This study provides an initial exploration of immune system determinants of, and activation during, kidney injury experienced during prolonged heat stress.

The complexity of kidney disease and diagnosing it - cystatin C, selective glomerular hypofiltration syndromes and proteome regulation.

Estimation of kidney function is often part of daily clinical practice, mostly done by using the endogenous glomerular filtration rate (GFR)-markers creatinine or cystatin C. A recommendation to use both markers in parallel in 2010 has resulted in new knowledge concerning the pathophysiology of kidney disorders by the identification of a new set of kidney disorders, selective glomerular hypofiltration syndromes. These syndromes, connected to strong increases in mortality and morbidity, are characterized by a selective reduction in the glomerular filtration of 5-30 kDa molecules, such as cystatin C, compared to the filtration of small molecules <1 kDa dominating the glomerular filtrate, for example water, urea and creatinine. At least two types of such disorders, shrunken or elongated pore syndrome, are possible according to the pore model for glomerular filtration. Selective glomerular hypofiltration syndromes are prevalent in investigated populations, and patients with these syndromes often display normal measured GFR or creatinine-based GFR-estimates. The syndromes are characterized by proteomic changes promoting the development of atherosclerosis, indicating antibodies and specific receptor-blocking substances as possible new treatment modalities. Presently, the KDIGO guidelines for diagnosing kidney disorders do not recommend cystatin C as a general marker of kidney function and will therefore not allow the identification of a considerable number of patients with selective glomerular hypofiltration syndromes. Furthermore, as cystatin C is uninfluenced by muscle mass, diet or variations in tubular secretion and cystatin C-based GFR-estimation equations do not require controversial race or sex terms, it is obvious that cystatin C should be a part of future KDIGO guidelines.

Large difference but high correlation between creatinine and cystatin C estimated glomerular filtration rate in Mesoamerican sugarcane cutters.

OBJECTIVES: To explore the relationship between creatinine and cystatin C based estimated glomerular filtration rate (eGFR) in actively working sugarcane cutters. METHODS: This cohort study included 458 sugarcane cutters from Nicaragua and El Salvador. Serum samples were taken before and at end of harvest seasons and analysed for creatinine and cystatin C. Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formulas were used to calculate eGFRs based on creatinine (eGFRcr), cystatin C (eGFRcys) and both creatinine and cystatin C (eGFRcrcys) at each time point. Bland-Altman plots and paired t-tests were used to compare the difference between eGFRcr and eGFRcys, and the difference in eGFRs between before and at end of the harvest seasons. RESULTS: The mean eGFRcr was higher than eGFRcys in both cohorts; absolute difference 22 mL/min/1.73 m2 (95% CI 21 to 23) in Nicaragua and 13 mL/min/1.73 m2 (95% CI 11 to 15) in El Salvador. Correlations between eGFRcr and eGFRcys were high, with r=0.69, 0.77 and 0.67 in Nicaragua at pre-harvest, end-harvest and cross-harvest, and r=0.89, 0.89 and 0.49 in El Salvador. CONCLUSIONS: Creatinine increases among heat-stressed workers reflect reduced glomerular filtration as estimated using eGFRcys, a marker independent of muscle mass and metabolism. The discrepancy between eGFRcr and eGFRcys may indicate reduced glomerular filtration of larger molecules and/or systemic bias in CKD-EPI performance in this population.

Externally added cystatin C reduces growth of A375 melanoma cells by increasing cell cycle time.

Some secreted cysteine protease inhibitors of the cystatin family appear to affect intracellular proteolysis and growth of human cells, as a result of internalization. Here, we studied the effects of external addition of the most abundant human cystatin, cystatin C, on viability and proliferation of cancer cells in culture. A dose-dependent decrease in viable cells was seen for A375 melanoma, MCF-7 breast cancer, and PC-3 prostate cancer cells cultured in 1-5 µm cystatin C after 24 h. Real-time assessment of growth rates in A375 cell cultures for 48 h by digital holographic microscopy showed an increased doubling time for cells cultured in the presence of 5 µm cystatin C (20.1 h) compared with control cells (14.7 h). A prolonged doubling time was already observed during the first 12 h, indicating a rapid general decrease in cell proliferation at the population level. Tracking of individual cells in phase holographic images showed that dividing cells incubated with 5 µm cystatin C underwent fewer mitoses during 48 h than control cells. In addition, the time between cell divisions was longer, especially for the first cell cycle. Incubation with the variant W106F-cystatin C (with high cellular uptake rate) resulted in a lower number of viable cells and a prolonged doubling time than when cells were incubated with wild-type cystatin C, but no effect was observed for (R24A,R25A)-cystatin C (low cellular uptake). Thus, cystatin C causes prolonged cell division leading to decreased proliferation of melanoma cells, and internalization seems to be a prerequisite for this effect.

Secreted cystatins decrease proliferation and enhance apoptosis of human leukemic cells.

Cysteine proteases are implicated in proteolysis events favoring cancer cell growth, spread, and death by apoptosis. Herein, we have studied whether the net growth and survival of the leukemic cell lines Jurkat, U937, and HL-60 are affected by external addition of five proteins acting as natural cysteine protease inhibitors. None of the cystatins examined (A, C, D, and E/M) or chagasin showed consistent effects on Fas-induced apoptosis when evaluated at 1 µm. In contrast, when the intrinsic apoptosis pathway was activated by hydrogen peroxide, addition of cystatin D augmented caspase-3-like activity within all three cell lines. Flow cytometric analysis of U937 cells also showed increased numbers of annexin V-positive cells when hydrogen peroxide was used to initiate apoptosis and cells were cultured in the presence of cystatin D or C. Moreover, stimulation of hydrogen peroxide-induced apoptotic U937 cells with either cystatin C or D resulted in a dose-dependent decrease in the number of cells. Cell viability was also decreased when U937 cells were cultured in the presence of cystatin C or D (1-9 µm) only, demonstrating that these cystatins can reduce cell proliferation by themselves in addition to enhancing apoptosis induced by oxidative stress. These effects on U937 cells were paralleled by internalization of cystatins C and D, indicating these effects are caused by downregulation of intracellular proteolysis. External addition of cystatins C and D to HL-60 and Jurkat cells demonstrated similar degrees of cystatin D uptake and decreased viability as for U937 cells, indicating that these effects are general for leukemic cells.

Shrunken pore syndrome and mortality: a cohort study of patients with measured GFR and known comorbidities.

Shrunken pore syndrome (SPS) is defined by a cystatin C-based estimation of glomerular filtration rate (eGFRCYS) being less than 60% or 70% of a creatinine-based GFR estimation (eGFRCR) in the absence of extrarenal influences on cystatin C or creatinine concentrations. SPS has been associated with a substantial increase in mortality or morbidity in all investigated populations. However, in these studies, neither the diagnoses, nor causes of death were described, and only estimated GFR was available. The present study concerns 2781 individuals with measured GFR (mGFR), known diagnoses, and known causes of death during 5.6 years in median. Cox multivariate proportional hazards regression model was used to estimate hazard ratios (HR) for all-cause and cancer, cardiovascular, diabetes or chronic kidney disease (CKD) as cause-specific mortality among patients with SPS. At an eGFRCYS/eGFRCR-ratio <0.70, the adjusted SPS death risk in the total cohort (HR 3.0, 95% CI 2.4-3.7) was clearly higher than that for the other diagnosis groups. In a sub-cohort of 1300 persons with or without diagnosis, but with normal mGFR, the all-cause mortality of SPS was markedly increased (HR 4.1, 95% CI 2.6-6.5). In a sub-cohort of 567 persons with normal mGFR and no diagnosis, the all-cause mortality of SPS was even more increased (HR 7.3, 95% CI 2.3-23). The prevalence of SPS in the total cohort was 23% and in the sub-cohorts 17 and 12%, respectively. As SPS is associated with a high mortality, occurs in the absence of reduced mGFR and albuminuria, it expands the spectrum of kidney disorders.

Insufficient mixing of thawed serum samples leading to erroneous results - experience from a field study and use of a correction procedure.

Incorrect analysis results that are close to expected might not be recognized in scientific studies or routine patient care. In two field studies we obtained unexpected results in a large number of samples. The present study aimed to identify the source of error in the samples from these studies and to validate a method to obtain correct results. Pre-analytical procedures were scrutinized, giving no indications of inappropriate pre-analytical sample handling in the field or during transport in a tropical climate. Using a new set of samples from volunteers in simulation experiments, we observed the known concentration gradient of analytes sampled in gel as well as plain tubes after freezer storage and thawing. Experiments demonstrated that mixing of samples by vortexing alone was not sufficient to disrupt the gradient formed by freezing and thawing, which appeared to cause the problem encountered when we in field studies analyzed and biobanked large sample sets by robot pipetting. A correction procedure was introduced, in which the obtained value of an analyte was multiplied by a correction factor calculated for each sample using the expected sodium level (140 mmol/L) divided by the measured sodium value. When it was validated on results from the simulation experiments, we repeatedly found that the correction lead to results very close to true values for analytes of different size and charge. Usefulness of the procedure was demonstrated when applied to a large set of field study results.

Shrunken Pore Syndrome Is Associated With Increased Levels of Atherosclerosis-Promoting Proteins.

INTRODUCTION: Shrunken pore syndrome (SPS), originally defined by cystatin C-based estimated glomerular filtration rate (eGFRcystatin C) being less than 60% of creatinine-based estimated glomerular filtration rate (eGFRcreatinine) in the absence of extrarenal influences on the plasma levels of cystatin C or creatinine, is associated with a high increase in mortality, even in the absence of reduced glomerular filtration rate (GFR). The objective of the present study was to determine whether the proteome of patients with SPS shows differences from that of patients with normal or reduced measured GFR (mGFR) without SPS. METHODS: Four patient cohorts were included: 1 cohort with normal mGFR without SPS, 1 with normal mGFR with SPS, 1 with reduced mGFR without SPS, and 1 with reduced mGFR with SPS. The plasma levels of 177 selected proteins were analyzed. RESULTS: Differences in the levels of 30 proteins were specific for SPS; 31 differences were specific for patients with both SPS and reduced mGFR; and 27 were specific for reduced mGFR. Eighteen of the differences specific for SPS concerned proteins described as promoting, or being associated with, atherosclerosis. Twelve of the differences specific for patients with both SPS and reduced mGFR and 10 of the differences specific for reduced mGFR also concerned proteins described as promoting, or being associated with, atherosclerosis. Almost all (82 of 88) of the concentration differences represented increased levels. For SPS, but not for reduced mGFR, a correlation between protein size and increase in level was observed, with smaller proteins being associated with higher levels. CONCLUSION: The high mortality in shrunken pore syndrome might be caused by the accumulation of atherosclerosis-promoting proteins in this condition.

Low-level internalization of cystatin E/M affects legumain activity and migration of melanoma cells.

The ratio between proteases and their inhibitors is unbalanced in cancer. The cysteine protease inhibitor cystatin C is internalized by some cancer cells, which affects cellular properties. Here we aimed to investigate if uptake of cystatin C and the related inhibitor cystatin E/M occur in melanoma cell lines and to evaluate to what extent the uptake affects the legumain activity that is typically increased in melanoma. First we studied the basic expression, secretion, and intracellular content of all type 2 cystatins as well as expression and activity of their possible target enzymes legumain and cathepsin B in MDA-MB-435S, A375, and C8161 melanoma cells. Legumain activity was measureable in all cell lines, and of the potential legumain inhibitors, cystatin C, E/M, and F, cystatin C was the one mainly produced. All cells internalized cystatin C added to culture media, leading to increased intracellular cystatin C levels by 120-200%. Cystatin E/M was internalized as well but at a modest rate. The effects on intracellular legumain activity were nevertheless pronounced, probably because the cells lacked this inhibitor, and its affinity for legumain is 100-fold higher than that of cystatin C. Likewise, the low-degree uptake resulted in reduced migration and invasion of A375 cells in Matrigel to an extent comparable with the W106F variant of cystatin C with optimal uptake properties and resulting in much higher intracellular levels. Thus, cystatin E/M appears to be a good candidate to efficiently down-regulate the increased legumain activity, possibly important for the malignant phenotype of melanoma cells.

Estimating glomerular filtration rate (GFR) in children. The average between a cystatin C- and a creatinine-based equation improves estimation of GFR in both children and adults and enables diagnosing Shrunken Pore Syndrome.

Estimating glomerular filtration rate (GFR) in adults by using the average of values obtained by a cystatin C- (eGFRcystatin C) and a creatinine-based (eGFRcreatinine) equation shows at least the same diagnostic performance as GFR estimates obtained by equations using only one of these analytes or by complex equations using both analytes. Comparison of eGFRcystatin C and eGFRcreatinine plays a pivotal role in the diagnosis of Shrunken Pore Syndrome, where low eGFRcystatin C compared to eGFRcreatinine has been associated with higher mortality in adults. The present study was undertaken to elucidate if this concept can also be applied in children. Using iohexol and inulin clearance as gold standard in 702 children, we studied the diagnostic performance of 10 creatinine-based, 5 cystatin C-based and 3 combined cystatin C-creatinine eGFR equations and compared them to the result of the average of 9 pairs of a eGFRcystatin C and a eGFRcreatinine estimate. While creatinine-based GFR estimations are unsuitable in children unless calibrated in a pediatric or mixed pediatric-adult population, cystatin C-based estimations in general performed well in children. The average of a suitable creatinine-based and a cystatin C-based equation generally displayed a better diagnostic performance than estimates obtained by equations using only one of these analytes or by complex equations using both analytes. Comparing eGFRcystatin and eGFRcreatinine may help identify pediatric patients with Shrunken Pore Syndrome.

Inhibition of lipopolysaccharide-induced osteoclast formation and bone resorption in vitro and in vivo by cysteine proteinase inhibitors.

Inflammation-induced bone destruction is a major treatment target in many inflammatory skeletal diseases. The aim of this study was to investigate if the cysteine proteinase inhibitors cystatin C, fungal cysteine proteinase inhibitor (E-64), and N-benzyloxycarbonyl-arginyl-leucyl-valyl-glycyl-diazomethane acetate (Z-RLVG-CHN2) can inhibit LPS-induced osteoclast formation. Mouse bone marrow macrophages (BMMs) were isolated and primed with receptor activator of NF-κB ligand (RANKL) for 24 h, followed by stimulation with LPS, with and without inhibitors. Adult mice were injected locally with LPS and then treated with E-64 and osteoclast formation assessed by the number of cathepsin K+ multinucleated cells. Cystatin C inhibited LPS-induced osteoclast formation time and concentration dependently (IC50 = 0.3 µM). The effect was associated with decreased mRNA and protein expression of tartrate-resistant acid phosphatase (TRAP) and cathepsin K and of the osteoclastogenic transcription factors c-Fos and NFATc1. LPS-induced osteoclast formation on bone slices was also inhibited by cystatin C, resulting in decreased pit formation and release of bone matrix proteins. Similar data were obtained with E-64 and Z-RLVG-CHN2 Cystatin C was internalized in BMMs stimulated by LPS but not in unstimulated BMMs. Osteoclast formation induced by LPS was dependent on TNF-α, and the 3 inhibitors abolished LPS-induced TNF superfamily 2 (gene encoding TNF-α; Tnfsf2) mRNA expression without affecting Il1b, Il6, or oncostatin M (Osm) expression. Formation of osteoclasts in the skull bones after local LPS stimulation was inhibited by E-64. It is concluded that cysteine proteinase inhibitors effectively inhibit LPS-induced osteoclast formation in vivo and in vitro by inhibition of TNF-α expression. The targeting of cysteine proteinases might represent a novel treatment modality for prevention of inflammatory bone loss.

Calcium Regulates the Activity and Structural Stability of Tpr, a Bacterial Calpain-like Peptidase.

Porphyromonas gingivalis is a peptide-fermenting asaccharolytic periodontal pathogen. Its genome contains several genes encoding cysteine peptidases other than gingipains. One of these genes (PG1055) encodes a protein called Tpr (thiol protease) that has sequence similarity to cysteine peptidases of the papain and calpain families. In this study we biochemically characterize Tpr. We found that the 55-kDa Tpr inactive zymogen proteolytically processes itself into active forms of 48, 37, and 33 kDa via sequential truncations at the N terminus. These processed molecular forms of Tpr are associated with the bacterial outer membrane where they are likely responsible for the generation of metabolic peptides required for survival of the pathogen. Both autoprocessing and activity were dependent on calcium concentrations >1 mm, consistent with the protein's activity within the intestinal and inflammatory milieus. Calcium also stabilized the Tpr structure and rendered the protein fully resistant to proteolytic degradation by gingipains. Together, our findings suggest that Tpr is an example of a bacterial calpain, a calcium-responsive peptidase that may generate substrates required for the peptide-fermenting metabolism of P. gingivalis. Aside from nutrient generation, Tpr may also be involved in evasion of host immune response through degradation of the antimicrobial peptide LL-37 and complement proteins C3, C4, and C5. Taken together, these results indicate that Tpr likely represents an important pathogenesis factor for P. gingivalis.

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.

Stabilization, characterization, and selective removal of cystatin C amyloid oligomers.

The pathophysiological process in amyloid disorders usually involves the transformation of a functional monomeric protein via potentially toxic oligomers into amyloid fibrils. The structure and properties of the intermediary oligomers have been difficult to study due to their instability and dynamic equilibrium with smaller and larger species. In hereditary cystatin C amyloid angiopathy, a cystatin C variant is deposited in arterial walls and cause brain hemorrhage in young adults. In the present investigation, we use redox experiments of monomeric cystatin C, stabilized against domain swapping by an intramolecular disulfide bond, to generate stable oligomers (dimers, trimers, tetramers, decamers, and high molecular weight oligomers). These oligomers were characterized concerning size by gel filtration, polyacrylamide gel electrophoresis, and mass spectrometry, shape by electron and atomic force microscopy, and, function by assays of their capacity to inhibit proteases. The results showed the oligomers to be highly ordered, domain-swapped assemblies of cystatin C and that the oligomers could not build larger oligomers, or fibrils, without domain swapping. The stabilized oligomers were used to induce antibody formation in rabbits. After immunosorption, using immobilized monomeric cystatin C, and elution from columns with immobilized cystatin C oligomers, oligomer-specific antibodies were obtained. These could be used to selectively remove cystatin C dimers from biological fluids containing both dimers and monomers.

Cystatin C properties crucial for uptake and inhibition of intracellular target enzymes.

To elucidate the molecular requirements for cancer cell internalization of the extracellular cysteine protease inhibitor cystatin C, 12 variants of the protein were produced and used for uptake experiments in MCF-7 cells. Variants with alterations in the cysteine cathepsin binding region ((Δ1-10)-, K5A-, R8G-, (R8G,L9G,V10G)-, (R8G,L9G,V10G,W106G)-, and W106G-cystatin C) were internalized to a very low extent compared with the wild-type inhibitor. Substitutions of N39 in the legumain binding region (N39K- and N39A-cystatin C) decreased the internalization and (R24A,R25A)-cystatin C, with substitutions of charged residues not involved in enzyme inhibition, was not taken up at all. Two variants, W106F- and K75A-cystatin C, showed that the internalization can be positively affected by engineering of the cystatin molecule. Microscopy revealed vesicular co-localization of internalized cystatin C with the lysosomal marker proteins cathepsin D and legumain. Activities of both cysteine cathepsins and legumain, possible target enzymes associated with cancer cell invasion and metastasis, were down-regulated in cell homogenates following cystatin C uptake. A positive effect on regulation of intracellular enzyme activity by a cystatin variant selected from uptake properties was illustrated by incubating cells with W106F-cystatin C. This resulted in more efficient down-regulation of intracellular legumain activity than when cells were incubated with wild-type cystatin C. Uptake experiments in prostate cancer cells corroborated that the cystatin C internalization is generally relevant and confirmed an increased uptake of W106F-cystatin C, in PC3 cells. Thus, intracellular cysteine proteases involved in cancer-promoting processes might be controled by cystatin uptake.

Cysteine proteinase inhibitors regulate human and mouse osteoclastogenesis by interfering with RANK signaling.

The cysteine proteinase inhibitor cystatin C inhibited RANKL-stimulated osteoclast formation in mouse bone marrow macrophage cultures, an effect associated with decreased mRNA expression of Acp5, Calcr, Ctsk, Mmp9, Itgb3, and Atp6i, without effect on proliferation or apoptosis. The effects were concentration dependent with half-maximal inhibition at 0.3 µM. Cystatin C also inhibited osteoclast formation when RANKL-stimulated osteoclasts were cultured on bone, leading to decreased formation of resorption pits. RANKL-stimulated cells retained characteristics of phagocytotic macrophages when cotreated with cystatin C. Three other cysteine proteinase inhibitors, cystatin D, Z-RLVG-CHN2 (IC50 0.1 µM), and E-64 (IC50 3 µM), also inhibited osteoclast formation in RANKL-stimulated macrophages. In addition, cystatin C, Z-RLVG-CHN2, and E-64 inhibited osteoclastic differentiation of RANKL-stimulated CD14(+) human monocytes. The effect by cystatin C on differentiation of bone marrow macrophages was exerted at an early stage after RANKL stimulation and was associated with early (4 h) inhibition of c-Fos expression and decreased protein and nuclear translocation of c-Fos. Subsequently, p52, p65, IκBα, and Nfatc1 mRNA were decreased. Cystatin C was internalized in osteoclast progenitors, a process requiring RANKL stimulation. These data show that cystatin C inhibits osteoclast differentiation and formation by interfering intracellularly with signaling pathways downstream RANK.

Intra- and extracellular regulation of activity and processing of legumain by cystatin E/M.

Legumain, an asparaginyl endopeptidase, is up-regulated in tumour and tumour-associated cells, and is linked to the processing of cathepsin B, L, and proMMP-2. Although legumain is mainly localized to the endosomal/lysosomal compartments, legumain has been reported to be localized extracellularly in the tumour microenvironment and associated with extracellular matrix and cell surfaces. The most potent endogenous inhibitor of legumain is cystatin E/M, which is a secreted protein synthesised with an export signal. Therefore, we investigated the cellular interplay between legumain and cystatin E/M. As a cell model, HEK293 cells were transfected with legumain cDNA, cystatin E/M cDNA, or both, and over-expressing monoclonal cell lines were selected (termed M38L, M4C, and M3CL, respectively). Secretion of prolegumain from M38L cells was inhibited by treatment with brefeldin A, whereas bafilomycin A1 enhanced the secretion. Cellular processing of prolegumain to the 46 and 36 kDa enzymatically active forms was reduced by treatment with either substance alone. M38L cells showed increased, but M4C cells decreased, cathepsin L processing suggesting a crucial involvement of legumain activity. Furthermore, we observed internalization of cystatin E/M and subsequently decreased intracellular legumain activity. Also, prolegumain was shown to internalize followed by increased intracellular legumain processing and activation. In addition, in M4C cells incomplete processing of the internalized prolegumain was observed, as well as nuclear localized cystatin E/M. Furthermore, auto-activation of secreted prolegumain was inhibited by cystatin E/M, which for the first time shows a regulatory role of cystatin E/M in controlling both intra- and extracellular legumain activity.

Proteolysis of cystatin C by cathepsin D in the breast cancer microenvironment.

The aspartic protease cathepsin D, a poor prognostic indicator of breast cancer, is abundantly secreted as procathepsin D by human breast cancer cells and self-activates at low pH in vitro, giving rise to catalytically active cathepsin D. Due to a lower extracellular pH in tumor microenvironments compared to normal tissues, cathepsin D may cleave pathophysiological substrates contributing to cancer progression. Here, we show by yeast 2-hybrid and degradomics analyses that cystatin C, the most potent natural secreted inhibitor of cysteine cathepsins, both binds to and is a substrate of extracellular procathepsin D. The amount of cystatin C in the extracellular environment is reduced in the secretome of mouse embryonic fibroblasts stably transfected with human cathepsin D. Cathepsin D extensively cleaved cystatin C in vitro at low pH. Cathepsin D secreted by breast cancer cells also processed cystatin C at the pericellular pH of tumors and so enhancing extracellular proteolytic activity of cysteine cathepsins. Thus, tumor derived cathepsin D assists breast cancer progression by reducing cystatin C activity, which, in turn, enhances cysteine cathepsin proteolytic activity, revealing a new link between protease classes in the protease web.

Cleavage of IgG1 and IgG3 by gingipain K from Porphyromonas gingivalis may compromise host defense in progressive periodontitis.

Degradation of immunoglobulins is an effective strategy of bacteria to evade the immune system. We have tested whether human IgG is a substrate for gingipain K of Porphyromonas gingivalis and found that the enzyme can hydrolyze subclass 1 and 3 of human IgG. The heavy chain of IgG(1) was cleaved at a single site within the hinge region, generating Fab and Fc fragments. IgG(3) was also cleaved within the heavy chain, but at several sites around the CH2 region. Investigation of the enzyme kinetics of IgG proteolysis by gingipain K, using FPLC- and isothermal titration calorimetry-based assays followed by Hill plots, revealed non-Michaelis-Menten kinetics involving a mechanism of positive cooperativity. In ex vivo studies, it was shown that gingipain K retained its IgG hydrolyzing activity in human plasma despite the high content of natural protease inhibitors; that IgG(1) cleavage products were detected in gingival crevicular fluid samples from patients with severe periodontitis; and that gingipain K treatment of serum samples from patients with high antibody titers against P. gingivalis significantly hindered opsonin-dependent phagocytosis of clinical isolates of P. gingivalis by neutrophils. Altogether, these findings underline a biological function of gingipain K as an IgG protease of pathophysiological importance.

Cystatins--Extra- and intracellular cysteine protease inhibitors: High-level secretion and uptake of cystatin C in human neuroblastoma cells.

Cystatins are present in mammals, birds, fish, insects, plants, fungi and protozoa and constitute a large protein family, with most members sharing a cysteine protease inhibitory function. In humans 12 functional cystatins exist, forming three groups based on molecular organisation and distribution in the organism. The type 1 cystatins (A and B) are known as intracellular, type 2 cystatins (C, D, E/M, F, G, S, SN and SA) extracellular and type 3 cystatins (L- and H-kininogen) intravascular proteins. The present paper is focused on the human cystatins and especially those of type 2, which are directed (with signal peptides) for cellular export following translation. Results indicating existence of systems for significant internalisation of type 2 cystatins from the extracellular to intracellular compartments are reviewed. Data showing that human neuroblastoma cell lines generally secrete high levels, but also contain high amounts of cystatin C are presented. Culturing of these cells in medium containing cystatin C at concentrations found in body fluids resulted in increased intracellular cystatin C, as a result of an uptake process. At immunofluorescence cytochemistry a pronounced vesicular cystatin C staining was observed. The simplistic denotation of the type 2 cystatins as extracellular inhibitors is thus challenged, and possible biological functions of the internalised cystatins are discussed. To illustrate the special case of high cellular cystatin content seen in cells of patients with hereditary cystatin C amyloid angiopathy, expression vectors for wild-type and L68Q mutated cystatin C were used to transfect SK-N-BE(2) cells. Clones overexpressing the two variants showed increased secreted levels of cystatin C. Within the cells the L68Q variant appeared to mainly localise to the endoplasmic reticulum rather than to acidic vesicular organelles, indicating limitations in the transport out from the cell rather than increased uptake as explanation for the elevated cellular cystatin levels seen in hereditary cystatin C amyloid angiopathy.