Wild-type Cystatin Research Articles

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.

The Influence of the Mixed DPC:SDS Micelle on the Structure and Oligomerization Process of the Human Cystatin C.

Human cystatin C (hCC), a member of the superfamily of papain-like cysteine protease inhibitors, is the most widespread cystatin in human body fluids. Physiologically active hCC is a monomer, which dimerization and oligomerization lead to the formation of the inactive, insoluble amyloid form of the protein, strictly associated with cerebral amyloid angiopathy, a severe state causing death among young patients. It is known, that biological membranes may accelerate the oligomerization processes of amyloidogenic proteins. Therefore, in this study, we describe an influence of membrane mimetic environment—mixed dodecylphosphocholine:sodium dodecyl sulfate (DPC:SDS) micelle (molar ratio 5:1)—on the effect of the hCC oligomerization. The hCC–micelle interactions were analyzed with size exclusion chromatography, circular dichroism, and nuclear magnetic resonance spectroscopy. The experiments were performed on the wild-type (WT) cystatin C, and two hCC variants—V57P and V57G. Collected experimental data were supplemented with molecular dynamic simulations, making it possible to highlight the binding interface and select the residues involved in interactions with the micelle. Obtained data shows that the mixed DPC:SDS micelle does not accelerate the oligomerization of protein and even reverses the hCC dimerization process.

Distinct structural changes in wild-type and amyloidogenic chicken cystatin caused by disruption of C95–C115 disulfide bond

Human cystatin C (HCC) amyloid angiopathy (HCCAA) is characterized by tissue deposition of amyloid fibrils in blood vessels, which can lead to recurrent hemorrhagic stroke. Wild-type HCC forms part of the amyloid deposits in brain arteries of elderly people with amyloid angiopathy. A point mutation causing a glutamine to a leucine substitution at residue 68 in the HCC polypeptide chain greatly increases the amyloidogenic propensity of HCC and causes a more severe cerebral hemorrhage and premature death in young adults. In this study, we used molecular dynamics simulations to assess the importance of disulfide bridge formation upon the stability of chicken cystatin and how this may influence the propensity for amyloid formation. We found that disulfide bridge formation between Cys95 and Cys115 in human cystatin played a critical role in overall protein stability. Importantly, Cys95–Cys115 influenced cystatin structure in regions of the protein that play key roles in the protein-folding transitions that occur, which enable amyloid fibril formation. We hypothesized that correct disulfide bridge formation is a critical step in stabilizing cystatin toward its native conformation. Disrupting Cys95–Cys115 disulfide bridge formation within cystatin appears to significantly enhance the amyloidogenic properties of this protein. In addition, by combining in silico studies with our previous experimental results on Eps1, a molecular chaperone of the PDI family, we proposed that age-related HCCAA, may possess a different pathogenic mechanism compared with its amyloidogenic counterpart, the early onset amyloidogenic cystatin-related CAA.

Structural Studies of the Oligomerization Process of Human Cystatin C Variants

Human cystatin C (HCC), an inhibitor of cysteine proteases, is also involved in amyloidogenic processes within the human body. In the crystal structure, as in solution, wild-type (WT) cystatin C dimerizes via a domain swapping mechanism [1,2]. The same process has also been shown to be important for protein oligomerization [2]. Aggregation of HCC results in a heterogeneous mixture of species, both oligomeric and fibrillar, with varying shapes, sizes and molecular weights.The goal of our study was to characterize the assembly process of potentially neurotoxic oligomers of WT and variant HCC in solution. The oligomerization processes of several point mutation variants of HCC (V57N, V57P, V57D, V57G and L68V), with specific dimerization propensities [3], were compared to the WT protein. Prepared oligomers were purified by size-exclusion chromatography and using TEM and AFM studies, we observed characteristic donut-like structures along with fibrils and we determined the size distribution of cystatin C oligomers obtained from the WT and variant forms. Independently, the oligomerization process was analyzed by fluorescence spectroscopy and visualized by native electrophoresis. Additionally, induced secondary structure changes were characterised by circular dichroism (CD) and infrared (FTIR) spectroscopies. Finally, the structural studies were supplemented with the modelling of the larger HCC oligomers using molecular dynamics simulations, allowing the identification of the most stable oligomeric forms of HCC to be determined.This research project was financed by the National Science Centre (Poland), decision no. 2012/06/M/ST4/00036.[1] Janowski R, Kozak M, Jankowska E et al. (2001). Nature Struct. Mol. Biol. 8, 316.[2] Wahlbom M, Wang X, Lindstrom V, et al. (2007). J. Biol. Chem. 282, 18318.[3] Szymanska A, Jankowska E, Orlikowska M, et al. (2012) Front. Mol. Neurosci., 5, 82

Spectroscopic and SAXS Studies of Human Cystatin C Mutants - Early Stages of Amyloid Formation Process

Human cystatin C (HCC) is a cysteine protease inhibitor. This protein in pathological conditions, forms dimers via a “domain swapping” mechanism. HCC is also associated with two types of amyloid deposition diseases - hereditary amyloid angiopathy (related to the Leu68Gln mutation) and wild-type cystatin C co-precipitation.The aim of our studies was the characterisation of the self-assembling properties of native and mutated (at positions 57 or 68) forms of human cystatin C in solution. The structure, overall conformation and secondary structure changes in solution were studied by Fourier transformed infrared spectroscopy (FTIR), circular dichroism spectroscopy (CD), dynamic light scattering (DLS) and time resolved small angle scattering of synchrotron radiation (TR-SAXS).SAXS data for native and mutated HCC were subjected to analysis by using SVD and MCR-ALS methods as well as the low resolution structure determination. Besides the monomeric forms of human cystatin C, also dimers and higher oligomers were formed even after short (50-ms) exposure on synchrotron radiation. In addition we observed for first time, formation of domain swapped dimers of human cystatin C induced by irradiation. The spectroscopic studies confirmed conformational changes.Authors acknowledge also the partial support by HARMONIA3 grant (Project No. 2012/06/M/ST4/00036) from National Science Centre (Poland).

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.

Structural and Dynamic properties of a new Amyloidogenic chicken cystatin Mutant I108T

Chicken cystatin variant I108T is a mutant in the hydrophobic core of the molecule. It has shown many amyloid-prone characteristics in our previous experimental study. To explore the detailed structural and dynamic properties of the amyloidogenic mutant I108T, 10 ns molecular dynamic simulations of the I108T mutant and wild-type chicken cystatins were performed in this study. Our results suggested that the I108T mutant, which exhibited larger secondary structural fluctuations and hydrophobic core expanding tendency compared with the wild-type chicken cystatin, is a new amyloidogenic form of chicken cystatin, and therefore supported the hypothesis to some extent that site mutations in the hydrophobic core might induce the domain swapping.

Involvement of cystatin C in pathophysiology of CNS diseases

Cystatin C Leu68Gln variant is known to induce amyloid deposition in cerebral arterioles, resulting in Icelandic type cerebral amyloid angiopathy (CAA). Wild-type cystatin C is also observed in solitary CAA involving amyloid beta protein (Abeta), and accelerates the amyloidogenicity of Abeta in vitro. In neurological inflammatory diseases and leptomeningeal metastasis, low cystatin C levels are accompanied with high activities of cathepsins in the cerebrospinal fluid. Among the cells in CNS, astrocytes appear to secrete cystatin C in response to various proteases and cytokines. Co-localization of Abeta and cystatin C in the brains of Alzheimer's disease (AD) led to the hypothesis that cystatin C is involved in the disease process. We demonstrated that cystatin C microinjection into rat hippocampus induced neuronal cell death in dentate gyrus. Furthermore, apoptotic cell death was observed in neuronal cells treated with cystatin C in vitro. Up-regulation of cystatin C was observed in glial cells with neuronal cell death in vivo. These findings indicate the involvement of cystatin C in the process of neuronal cell death.

A dual Golgi- and mitochondria-localised Ala25Ser precursor cystatin C: An additional tool for characterising intracellular mis-localisation leading to increased AMD susceptibility

An artificial mutant Ala25Ser precursor cystatin C was created to help elucidate the cause of intracellular mis-localisation of the biochemically related variant B (Ala25Thr) precursor cystatin C to the mitochondria. Homozygotes of variant B precursor cystatin C were reported to carry an increased susceptibility to developing the exudative form of AMD. Ala25Ser precursor cystatin C shows a dual distribution to the Golgi apparatus and to the mitochondria. This localisation is thus intermediary between that of wild-type cystatin C (targeted to ER/Golgi compartment) and that of variant B precursor cystatin C. Furthermore, the level of secretion of Ala25Ser cystatin C by RPE cells is intermediary between wild type and variant B cystatin C. Ala25Ser precursor cystatin C thus represents a biochemical intermediate between the wild type and the AMD-associated cystatin C and as such, is a novel tool for the investigation of the mechanism of intracellular mis-localisation of variant B cystatin C. Our findings further support the hypothesis that substitution of the alanine residue in the penultimate position of precursor cystatin C signal sequence with a less hydrophobic amino acid residue, such as threonine (as in variant B cystatin C) or serine is sufficient to impair the intracellular trafficking and processing of the protein.

Checking the conformational stability of cystatin C and its L68Q variant by molecular dynamics studies: Why is the L68Q variant amyloidogenic?

Human L68Q cystatin C is one of the known human amyloidogenic proteins. In its native state it is a monomer with α/β structure. Experimental evidence suggests that L68Q variant associates into dimeric intermediates and that the dimers subsequently self-assemble to form amyloid deposits and insoluble fibrils. Details of the pathway of L68Q mutant amyloid formation are unclear; however, different experimental approaches with resolutions at molecular level have provided some clues. Probably, the stability and flexibility of monomeric L68Q variant play essential roles in the early steps of amyloid formation; thus, it is necessary to characterize early conformational changes of L68Q cystatin C monomers. In this paper, we demonstrate the possibility that the differences between the monomeric forms of wild-type (wt) cystatin C and its L68Q variant are responsible for higher tendency of the L68Q cystatin C amyloidogenesis. We started our studies with the simulations of wt and L68Q cystatin C monomers. Nanosecond time scale molecular dynamics simulations at 308 K were performed using AMBER7.0 program. The results show that the structure of the L68Q monomer was changed, relative to the wt cystatin C structure. The results support earlier speculation that the L68Q point mutation would easily lead to dimer formation.

Different Cysteine Proteinases Involved in Bone Resorption and Osteoclast Formation

Cysteine proteinases, especially cathepsin K, play an important role in osteoclastic degradation of bone matrix proteins and the process can, consequently, be significantly inhibited by cysteine proteinase inhibitors. We have recently reported that cystatin C and other cysteine proteinase inhibitors also reduce osteoclast formation. However, it is not known which cysteine proteinase(s) are involved in osteoclast differentiation. In the present study, we compared the relative potencies of cystatins C and D as inhibitors of bone resorption in cultured mouse calvariae, osteoclastogenesis in mouse bone marrow cultures, and cathepsin K activity. Inhibition of cathepsin K activity was assessed by determining equilibrium constants for inhibitor complexes in fluorogenic substrate assays. The data demonstrate that whereas human cystatins C and D are equipotent as inhibitors of bone resorption, cystatin D is 10-fold less potent as an inhibitor of osteoclastogenesis and 200-fold less potent as an inhibitor of cathepsin K activity. A recombinant human cystatin C variant with Gly substitutions for residues Arg8, Leu9, Val10, and Trp106 did not inhibit bone resorption, had 1,000-fold decreased inhibitory effect on cathepsin K activity compared to wildtype cystatin C, but was equipotent with wildtype cystatin C as an inhibitor of osteoclastogenesis. It is concluded that (i) different cysteine proteinases are likely to be involved in bone resorption and osteoclast formation, (ii) cathepsin K may not be an exclusive target enzyme in any of the two systems, and (iii) the enzyme(s) involved in osteoclastogenesis might not be a typical papain-like cysteine proteinase.

Unexpected intracellular localization of the AMD-associated cystatin C variant.

Cystatin C is abundantly expressed by the retinal pigment epithelium (RPE) of the eye. Targeting of cystatin C to the Golgi apparatus and processing through the secretory pathway of RPE cells are dependent upon a 26-amino acid signal sequence of precursor cystatin C. A variant with an alanine (A) to threonine (T) mutation in the penultimate amino acid of the signal sequence (A25T) was recently correlated with increased risk of developing exudative age-related macular degeneration. The biochemical consequence of the A25T mutation upon targeting of the protein is reported here. Targeting and trafficking of full-length mutant (A25T) precursor cystatin C-enhanced green fluorescent protein fusion protein were studied in living, cultured retinal pigment epithelial and HeLa cells. Confocal microscopy studies were substantiated by immunodetection. In striking contrast to wild-type precursor cystatin C fusion protein conspicuously targeted to the Golgi apparatus, the threonine variant was associated principally with mitochondria. Some diffuse fluorescence was also observed throughout the cytoplasm and nucleus (but not nucleoli). Secretion of fusion protein derived from the threonine variant was reduced by approximately 50% compared with that of the wild-type cystatin C fusion protein. Expression of the variant fusion protein did not appear to impair expression or secretion of endogenous cystatin C.

In vitro anti-cancer activities in Caco-2 and HCT-116 cells of recombinant cystatin C prepared by a Pichia expression system.

Caco-2 and HCT-116 cells were used to access growth-inhibition and anti-invasion activity of recombinant cystatin C expressed in Pichia pastoris X33, G12W/H86V. The mutant G12W/H86V prepared by a pilot plant production system showed more than 10% growth inhibition of Caco-2 cells at 0.56-56 nM concentrations. Growth-inhibited cells had lower cathepsin L activity than the control cells that were not treated with the inhibitor. Conversely, the cathepsin B activity was not changed by treatment with G12W/H86V. The in vitro anti-invasion test using HCT-116 cells showed that G12W/H86V suppressed the cell invasion by 15%, while its wild-type cystatin, aspartic protease inhibitor pepstatin A, and matrix metalloproteinase (MMP) inhibitor MMP-2/MMP-9 inhibitor III did not suppress cell invasion. These results indicate that the recombinant cystatin C with higher protease inhibitory activity effectively retards the growth and invasiveness of human colon carcinoma cells.

Enhancement of proteinase inhibitory activity of recombinant human cystatin C using random-centroid optimization

We had previously written a random-centroid optimization computer program for genetics (RCG) to optimize protein engineering, which was successfully applied to modify single site of the 16 amino acid residues at the active site of B. stearothermophilys neutral protease for improving thermostability [J. Agric. Food Chem., 46 (1998) 1655]. The same program was applied in this study to double-site mutation of the entire sequence of human cystatin C (HCC) with 120 residues for improving its protease inhibitory activity. The RCG program selected two sites simultaneously and amino acid residues to replace the sites selected in the sequence in order to find the best papain-inhibitory activity and stability of the protease inhibitor. Twenty-three double mutants and twenty-two single mutants were expressed by Pichia pastoris. Of the total 45 mutants, G12W/H86V mutant showed a 5-fold increase in the bioactivity over the recombinant wild-type (WT) cystatin. Also, P13F mutant exhibited a half-life temperature ( T 1/2) 5.2 °C higher than 68.2 °C of WT in addition to a 56% greater papain inhibitory activity. Mutation for diminishing β-sheet content reduced polymerization of cystatin C, thus improving papain-inhibitory activity. The approach using RCG was able to improve the functional properties of cystatin by least relying on the prior knowledge of its molecular structure.

Physico-chemical properties of the N-terminally truncated L68Q cystatin C found in amyloid deposits of brain haemorrhage patients.

Cystatin C, a major extracellular cysteine proteinase inhibitor, is deposited as amyloid in brain haemorrhage patients with hereditary cystatin C amyloid angiopathy (HCCAA). A disease-causing mutation on the genetic level results in the substitution Leu68-->Gln (L68Q) in cystatin C, which causes protein instability. Besides carrying the L68Q substitution, cystatin C in amyloid deposits isolated from patients is N-terminally truncated by 10 amino acids. To elucidate the role of the N-terminal truncation for protein stability and aggregation properties, (delta1-10,L68Q)-cystatin C was produced in an Escherichia coli expression system and characterised. Unlike wild-type cystatin C, this variant rapidly dimerised under physiological conditions. Two unfolding intermediates of (delta1-10,L68Q)-cystatin C were identified, under the same pH and ionic strength conditions as required to form intermediates of full-length L68Q cystatin C. No evidence was found that the N-terminal truncation per se alters protein stability and leads to higher forms of aggregation. Monomeric as well as dimeric L68Q cystatin C incubated with neutrophil elastase was truncated as in HCCAA patients' amyloid. A protein variant with a thrombin cleavage site placed in front of residue Gly11 in L68Q cystatin C was constructed and used to confirm that the N-terminal segment is similarly accessible to proteinases in the monomeric and dimeric states of L68Q cystatin C. Thus, the N-terminal segment of L68Q cystatin C is exposed to proteolytic attack and does not seem to be involved in intramolecular contacts leading to dimerisation or higher-order aggregation. We conclude that the N-terminal truncation likely is an event secondary to amyloid formation, and of no relevance for the development of HCCAA.

Role of the single cysteine residue, Cys 3, of human and bovine cystatin B (stefin B) in the inhibition of cysteine proteinases

Cystatin B is unique among cysteine proteinase inhibitors of the cystatin superfamily in having a free Cys in the N-terminal segment of the proteinase binding region. The importance of this residue for inhibition of target proteinases was assessed by studies of the affinity and kinetics of interaction of human and bovine wild-type cystatin B and the Cys 3-to-Ser mutants of the inhibitors with papain and cathepsins L, H, and B. The wild-type forms from the two species had about the same affinity for each proteinase, binding tightly to papain and cathepsin L and more weakly to cathepsins H and B. In general, these affinities were appreciably higher than those reported earlier, perhaps because of irreversible oxidation of Cys 3 in previous work. The Cys-to-Ser mutation resulted in weaker binding of cystatin B to all four proteinases examined, the effect varying with both the proteinase and the species variant of the inhibitor. The affinities of the human inhibitor for papain and cathepsin H were decreased by threefold to fourfold and that for cathepsin B by approximately 20-fold, whereas the reductions in the affinities of the bovine inhibitor for papain and cathepsins H and B were approximately 14-fold, approximately 10-fold and approximately 300-fold, respectively. The decreases in affinity for cathepsin L could not be properly quantified but were greater than threefold. Increased dissociation rate constants were responsible for the weaker binding of both mutants to papain. By contrast, the reduced affinities for cathepsins H and B were due to decreased association rate constants. Cys 3 of both human and bovine cystatin B is thus of appreciable importance for inhibition of cysteine proteinases, in particular cathepsin B.

The cerebral hemorrhage-producing cystatin C variant (L68Q) in extracellular fluids

A variant of the normal extracellular cysteine protease inhibitor cystatin C (L68Q-cystatin C), is the amyloid precursor in hereditary cystatin C amyloid angiopathy (HCCAA). It has been suggested that the mutation causes cellular entrapment ofL68Q-cystatin C in vivo and that the variant protein is not secreted to extracellular fluids. In order to test this hypothesis, we used matrix-assisted laser desorption ionization time-of-flight mass spectrometry in an effort to demonstrate the presence ofL68Q- along with wildtype cystatin C in plasma and cerebrospinal fluid (CSF) of HCCAA-patients. Plasma from all five investigated HCCAA-patients contained both L68Q- and wildtype cystatin C. The presence of approximately equal amounts of cystatin C dimers and monomers was demonstrated in plasma from HCCAA-patients, whereas only monomers could be found in normal plasma. L68Q-wildtype-cystatin C heterodimers seem to be present in the dimeric cystatin C population. CSF from six HCCAA-patients also contained cystatin C-dimers and monomers, but the dimeric fraction was minute. CSF from control patients did not contain dimeric cystatin C These results suggest that the milieu of L68Q-cystatin C is important for its stability and dimerization status and that certain milieus might hinder its further development into oligomers, amyloid fibrils and other precipiting aggregates.

A Novel Type of Bifunctional Inhibitor Directed against Proteolytic Activity and Receptor/Ligand Interaction: CYSTATIN WITH A UROKINASE RECEPTOR BINDING SITE

Cancer invasion and metastasis is a process requiring a coordinated series of (anti-)adhesive, migratory, and pericellular proteolytic events involving various proteases such as urokinase-type plasminogen activator (uPA)/plasmin, cathepsins B and L, and matrix metalloproteases. Novel types of double-headed inhibitors directed to different tumor-associated proteolytic systems were generated by substitution of a loop in chicken cystatin, which is nonessential for cysteine protease inhibition, with uPA-derived peptides covering the human uPA receptor binding sequence uPA-(19-31). The inhibition constants of these hybrids toward cysteine proteases are similar to those of wild-type cystatin (K(i), papain (pm), 1.9-2.4; K(i), cathepsin B (nm), 1.0-1.7; K(i), cathepsin L (pm), 0.12-0.61). FACS analyses revealed that the hybrids compete for binding of uPA to the cell surface-associated uPA receptor (uPAR) expressed on human U937 cells. The simultaneous interaction of the hybrid molecules with papain and uPAR was analyzed by surface plasmon resonance. The measured K(D) value of a papain-bound cystatin variant harboring the uPAR binding sequence of uPA (chCys-uPA-(19-31)) and soluble uPAR was 17 nm (K(D) value for uPA/uPAR interaction, 5 nm). These results indicate that cystatins with a uPAR binding site are efficient inhibitors of cysteine proteases and uPA/uPAR interaction at the same time. Therefore, these compact and small bifunctional inhibitors may represent promising agents for the therapy of solid tumors.

A peptidyl derivative structurally based on the inhibitory center of cystatin C inhibits bone resorption in vitro

Human cystatin C is a cysteine proteinase inhibitor belonging to the cystatin superfamily, which previously has been shown to inhibit bone resorption in bone organ culture. The aminoterminal segment, Arg 8-Leu 9-Val 10-Gly 11 (RLVG), of the single polypeptide chain of cystatin C constitutes an essential part of its inhibitory center. In the present study, the effect of benzyloxycarbonyl-Arg 8-Leu 9-Val 10-Gly 11-diazomethane (Z-RLVG-CHN 2) on bone resorption in vitro was compared with the effects of cystatin C and calcitonin. Bone resorption was assessed by the release of 45Ca and 3H from mouse calvarial bones prelabeled with [ 45Ca]CaCl 2 and [ 3H]-proline, respectively. Z-RLVG-CHN 2 concentration-dependently inhibited the release of 45Ca and 3H in bones stimulated by parathyroid hormone (PTH), with half-maximal inhibition obtained at 1 μmol/L. The inhibitory actions of Z-RLVG-CHN 2 and cystatin C were persistent, whereas action induced initially by calcitonin was lost with time. The inhibition caused by Z-RLVG-CHN 2 and cystatin C on PTH-stimulated 45Ca release was observed after 6 h, whereas inhibition by calcitonin was seen already after 2 h. In contrast, the inhibitory effects of Z-RLVG-CHN 2 and cystatin C, as well as that of calcitonin, on 3H release was seen already after 2 h. Z-RLVG-CHN 2, in which the reactive carboxyterminal diazomethane was substituted by nonreactive groups [−OH, −NH 2, or −N(CH 3) 2], resulted in peptidyl derivatives, which, in contrast to Z-RLVG-CHN 2 and cystatin C, inhibited neither cysteine proteinases nor bone resorption. In contrast to wild-type cystatin C, recombinant human cystatin C with Gly substitutions for residues Arg 8, Leu 9, Val 10, and Trp 106, and with low or nonexistent affinity for cysteine proteinases, did not display any inhibitory effect on bone resorption. These data strongly indicate that Z-RLVG-CHN 2 inhibits bone resorption in vitro by a mechanism that seems primarily to be due to an inhibition of bone matrix degradation via cysteine proteinases. The data also corroborate the hypothesis that cystatin C inhibits bone resorption by virtue of its cysteine proteinase inhibitory capacity.

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.