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.     

Interactions of papaya proteinase IV with inhibitors.

Papaya proteinase IV (PPIV) is not inhibited by chicken cystatin, or human cystatins A or C, unlike most other proteinases of the papain superfamily. The enzyme inactivates chicken cystatin and human cystatin C by limited proteolysis of the glycyl bond previously shown to be involved in the inhibitory inactivity of the cystatins, but has no action on cystatin A. Contamination of commercial crystalline papain with PPIV accounts for the limited proteolysis of cystatins by 'papain' reported previously. PPIV is slowly bound by human alpha 2-macroglobulin. The enzyme is irreversibly inactivated by E-64, and by peptidyl diazomethanes containing glycine in P1 and a hydrophobic side-chain in P2. The reaction of PPIV with iodoacetate is extremely slow. PPIV is inhibited by peptide aldehydes despite the presence of bulky sidechains in P1, suggesting that these reversible inhibitors do not bind as substrate analogues.     

Cytokine-inducing activity of family 2 cystatins

Cystatins are physiological cysteine proteinase inhibitors. Here we report a novel function for some family 2 cystatins that is not related to these activities. The release of interleukin-6 (IL-6) and interleukin-8 (IL-8) by the gingival fibroblasts and that of IL-6 by murine splenocytes were measured using ELISA systems specific for these cytokine molecules. Family 2 cystatins, including cystatins C, SA1, SA2, S, and egg white cystatin, upregulated the IL-6 production by two-lasts at physiological concentrations. After complete saturation with papain, those family 2 cystatins still upregulated IL-6 production, suggesting that the papain-inhibitory site was not involved in the cytokine-inducing activity.     

Identification of full-sized forms of salivary (S-type) cystatins (cystatin SN, cystatin SA, cystatin S, and two phosphorylated forms of cystatin S) in human whole saliva and determination of phosphorylation sites of cystatin S.

Our recent work on the gene structures for human salivary (S-type) cystatins [Saitoh, E. et al. (1987) Gene 61, 329-338] has suggested that the structures of cystatins which we determined previously at the protein level lack N-terminal peptide portions of the full-sized intact forms. In the present study, attempts were made to isolate full-sized S-type cystatins by introducing methanol fractionation into the purification steps to suppress the enzymatic activity present in saliva. Full-sized cystatin SN and two phosphorylated forms of full-sized cystatin S were thus isolated. Analysis of one fraction indicated that this was a mixture of full-sized cystatin SA and non-phosphorylated cystatin S. The phosphorylation sites of cystatin S were determined to be Ser-Ser-Ser1(P)-Lys-Glu-Glu- for monophosphorylated cystatin S and Ser1(P)-Ser-Ser3(P)-Lys-Glu-Glu- for diphosphorylated cystatin S. Immunoblotting analysis with anti-cystatin S antiserum revealed that tears and seminal plasma also contained S-type cystatins, but diphosphorylated cystatin S was detected neither in tears nor in seminal plasma and no cystatin SN was found in seminal plasma. These data indicate that S-type cystatins are secreted into the oral cavity without significant degradation in salivary glands or ducts and that they are expressed tissue specifically.     

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

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

Identification of the human salivary cystatin complex by the coupling of high-performance liquid chromatography and ion-trap mass spectrometry

Human salivary cystatins, five major (S, S1, S2, SA, SN) and two minor (C and D), are multifunctional proteins playing a different role in the oral environment. Salivary cystatin SN is able to effectively inhibit lysosomal cathepsins B, C, H and L and cystatin SA inhibits cathepsins C and L in vitro. These activities suggest, particularly for cystatin SN, an important role in the control of proteolytic events in vivo. Differently, cystatins S are involved, together with statherin, in the mineral balance of the tooth. Due to their distinct role, a reliable method for identification and quantification of the different cystatins, as well as of possible truncated and derived forms, could be helpful for the assessment of the status of the oral cavity. To this purpose high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI MS) was applied to the analysis of human saliva obtained from healthy subjects. All known salivary cystatins, with the exception of cystatin C, were detected. Strong evidence was also obtained for the presence in saliva of post-translational modified isoforms of cystatins, which may be related to donor habits. Cystatin SN and cystatins S, S1 and S2 were well separated by HPLC-ESI MS coupling from other components and thus this approach can be successfully applied to their quantification.     

Cystatins as calpain inhibitors: engineered chicken cystatin- and stefin B-kininogen domain 2 hybrids support a cystatin-like mode of interaction with the catalytic subunit of mu-calpain

Within the cystatin superfamily, only kininogen domain 2 (KD2) is able to inhibit mu- and m-calpain. In an attempt to elucidate the structural requirements of cystatins for calpain inhibition, we constructed recombinant hybrids of human stefin B (an intracellular family 1 cystatin) with KD2 and deltaL110 deletion mutants of chicken cystatin-KD2 hybrids. Substitution of the N-terminal contact region of stefin B by the corresponding KD2 sequence resulted in a calpain inhibitor of Ki = 188 nM. Deletion of L110, which forms a beta-bulge in family 1 and 2 cystatins but is lacking in KD2, improved inhibition of mu-calpain 4- to 8-fold. All engineered cystatins were temporary inhibitors of calpain due to slow substrate-like cleavage of a single peptide bond corresponding to Gly9-Ala10 in chicken cystatin. Biomolecular interaction analysis revealed that, unlike calpastatin, the cystatin-type inhibitors do not bind to the calmodulin-like domain of the small subunit of calpain, and their interaction with the mu-calpain heterodimer is completely prevented by a synthetic peptide comprising subdomain B of calpastatin domain 1. Based on these results we propose that (i) cystatin-type calpain inhibitors interact with the active site of the catalytic domain of calpain in a similar cystatin-like mode as with papain and (ii) the potential for calpain inhibition is due to specific subsites within the papain-binding regions of the general cystatin fold.     

Three-dimensional solution structure of oryzacystatin-I, a cysteine proteinase inhibitor of the rice, Oryza sativa L. japonica

The three-dimensional structure of oryzacystatin-I, a cysteine proteinase inhibitor of the rice, Oryza sativa L. japonica, has been determined in solution at pH 6.8 and 25 degrees C by (1)H and (15)N NMR spectroscopy. The main body (Glu13-Asp97) of oryzacystatin-I is well-defined and consists of an alpha-helix and a five-stranded antiparallel beta-sheet, while the N- and C-terminal regions (Ser2-Val12 and Ala98-Ala102) are less defined. The helix-sheet architechture of oryzacystatin-I is stabilized by a hydrophobic cluster formed between the alpha-helix and the beta-sheet and is considerably similar to that of monellin, a sweet-tasting protein from an African berry, as well as those of the animal cystatins studied, e.g., chicken egg white cystatin and human stefins A and B (also referred to as human cystatins A and B). Detailed structural comparison indicates that oryzacystatin-I is more similar to chicken cystatin, which belongs to the type-2 animal cystatins, than to human stefins A and B, which belong to the type-1 animal cystatins, despite different loop length.     

Identification of the probable inhibitory reactive sites of the cysteine proteinase inhibitors human cystatin C and chicken cystatin

When an excess of human cystatin C or chicken cystatin was mixed with papain, an enzyme-inhibitor complex was formed immediately. The residual free cystatin was then progressively converted to a form with different electrophoretic mobility and chromatographic properties. The modified cystatins were isolated and sequenced, showing that there had been cleavage of a single peptide bond in each molecule: Gly11-Gly12 in cystatin C, and Gly9-Ala10 in chicken cystatin. The residues Gly11 (cystatin C) and Gly9 (chicken cystatin) are among only three residues conserved in all known sequences of inhibitory cystatins. The modified cystatins were at least 1000-fold weaker inhibitors of papain than the native cystatins. An 18-residue synthetic peptide corresponding to residues 4-21 of cystatin C did not inhibit papain but was cleaved at the same Gly-Gly bond as cystatin C. When iodoacetate or L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guanidin o)butane was added to the mixtures of either cystatin with papain, modification of the excess cystatin was blocked. Papain-cystatin complexes were stable to prolonged incubation, even in the presence of excess papain. We conclude that the peptidyl bond of the conserved glycine residue in human cystatin C and chicken cystatin probably is part of a substrate-like inhibitory reactive site of these cysteine proteinase inhibitors of the cystatin superfamily and that this may be true also for other inhibitors of this superfamily. We also propose that human cystatin C and chicken cystatin, and probably other cystatins as well, inhibit cysteine proteinases by the simultaneous interactions with such proteinases of the inhibitory reactive sites and other, so far not identified, areas of the cystatins. The cleavage of the inhibitory reactive site glycyl bond in mixtures of papain with excess quantities of cystatins is apparently due to the activity of a small percentage of atypical cysteine proteinase molecules in the papain preparation that form only very loose complexes with cystatins under the conditions employed and degrade the free cystatin molecules.     

Comparative Analysis of Cystatin Superfamily in Platyhelminths

The cystatin superfamily is comprised of cysteine proteinase inhibitors and encompasses at least 3 subfamilies: stefins, cystatins and kininogens. In this study, the platyhelminth cystatin superfamily was identified and grouped into stefin and cystatin subfamilies. The conserved domain of stefins (G, QxVxG) was observed in all members of platyhelminth stefins. The three characteristics of cystatins, the cystatin-like domain (G, QxVxG, PW), a signal peptide, and one or two conserved disulfide bonds, were observed in platyhelminths, with the exception of cestodes, which lacked the conserved disulfide bond. However, it is noteworthy that cestode cystatins had two tandem repeated domains, although the second tandem repeated domain did not contain a cystatin-like domain, which has not been previously reported. Tertiary structure analysis of Taenia solium cystatin, one of the cestode cystatins, demonstrated that the N-terminus of T. solium cystatin formed a five turn α-helix, a five stranded β-pleated sheet and a hydrophobic edge, similar to the structure of chicken cystatin. Although no conserved disulfide bond was found in T. solium cystatin, the models of T. solium cystatin and chicken cystatin corresponded at the site of the first disulfide bridge of the chicken cystatin. However, the two models were not similar regarding the location of the second disulfide bridge of chicken cystatin. These results showed that T. solium cystatin and chicken cystatin had similarities and differences, suggesting that the biochemistry of T. solium cystatin could be similar to chicken cystatin in its inhibitory function and that it may have further functional roles. The same results were obtained for other cestode cystatins. Phylogenetic analysis showed that cestode cystatins constituted an independent clade and implied that cestode cystatins should be considered to have formed a new clade during evolution.     

Cystatins from filarial parasites: evolution, adaptation and function in the host-parasite relationship

Cystatins, together with stefins and kininogens, are members of the cystatin superfamily of cysteine protease inhibitors (CPI) present across the animal and plant kingdoms. Their role in parasitic organisms may encompass both essential developmental processes and specific interactions with the parasite's vector and/or final host. We summarise information gathered on three cystatins from the human filarial nematode Brugia malayi (Bm-CPI-1, -2 and -3), and contrast them those expressed by other parasites and by the free-living nematode Caenorhabditis elegans. Bm-CPI-2 differs from C. elegans cystatin, having acquired the additional function of inhibiting asparaginyl endopeptidase (AEP), in a manner similar to some human cystatins. Thus, we propose that Bm-CPI-2 and orthologues from related filarial parasites represent a new subset of nematode cystatins. Bm-CPI-1 and CPI-3 share only 25% amino acid identity with Bm-CPI-2, and lack an evolutionarily conserved glycine residue in the N-terminal region. These sequences group distantly from the other nematode cystatins, and represent a second novel subset of filarial cystatin-like genes. Expression analyses also show important differences between the CPI-2 and CPI-1/-3 groups. Bm-cpi-2 is expressed at all time points of the parasite life cycle, while Bm-cpi-1 and -3 expression is confined to the late stages of development in the mosquito vector, terminating within 48h of infection of the mammalian host. Hence, we hypothesise that CPI-2 has evolved to block mammalian proteases (including the antigen-processing enzyme AEP) while CPI-1 and -3 function in the milieu of the mosquito vector necessary for transmission of the parasite.     

Evolution of C,D and S-Type Cystatins in Mammals: An Extensive Gene Duplication in Primates

Cystatins are a family of inhibitors of cysteine peptidases that comprises the salivary cystatins (D and S-type cystatins) and cystatin C. These cystatins are encoded by a multigene family (CST3, CST5, CST4, CST1 and CST2) organized in tandem in the human genome. Their presence and functional importance in human saliva has been reported, however the distribution of these proteins in other mammals is still unclear. Here, we performed a proteomic analysis of the saliva of several mammals and studied the evolution of this multigene family. The proteomic analysis detected S-type cystatins (S, SA, and SN) in human saliva and cystatin D in rat saliva. The evolutionary analysis showed that the cystatin C encoding gene is present in species of the most representative mammalian groups, i.e. Artiodactyla, Rodentia, Lagomorpha, Carnivora and Primates. On the other hand, D and S-type cystatins are mainly retrieved from Primates, and especially the evolution of S-type cystatins seems to be a dynamic process as seen in Pongo abelii genome where several copies of CST1-like gene (cystatin SN) were found. In Rodents, a group of cystatins previously identified as D and S has also evolved. Despite the high divergence of the amino acid sequence, their position in the phylogenetic tree and their genome organization suggests a common origin with those of the Primates. These results suggest that the D and S type cystatins have emerged before the mammalian radiation and were retained only in Primates and Rodents. Although the mechanisms driving the evolution of cystatins are unknown, it seems to be a dynamic process with several gene duplications evolving according to the birth-and-death model of evolution. The factors that led to the appearance of a group of saliva-specific cystatins in Primates and its rapid evolution remain undetermined, but may be associated with an adaptive advantage.     

Cloning and characterisation of novel cystatins from elapid snake venom glands

Snake venoms contain a complex mixture of polypeptides that modulate prey homeostatic mechanisms through highly specific and targeted interactions. In this study we have identified and characterised cystatin-like cysteine-protease inhibitors from elapid snake venoms for the first time. Novel cystatin sequences were cloned from 12 of 13 elapid snake venom glands and the protein was detected, albeit at very low levels, in a total of 22 venoms. One highly conserved isoform, which displayed close sequence identity with family 2 cystatins, was detected in each elapid snake. Crude Austrelaps superbus (Australian lowland copperhead) snake venom inhibited papain, and a recombinant form of A. superbus cystatin inhibited cathepsin L ≅ papain > cathepsin B, with no inhibition observed for calpain or legumain. While snake venom cystatins have truncated N-termini, sequence alignment and structural modelling suggested that the evolutionarily conserved Gly-11 of family 2 cystatins, essential for cysteine protease inhibition, is conserved in snake venom cystatins as Gly-3. This was confirmed by mutagenesis at the Gly-3 site, which increased the dissociation constant for papain by 10⁴-fold. These data demonstrate that elapid snake venom cystatins are novel members of the type 2 family. The widespread, low level expression of type 2 cystatins in snake venom, as well as the presence of only one highly conserved isoform in each species, imply essential housekeeping or regulatory roles for these proteins.     

Silencing barley cystatins HvCPI‐2 and HvCPI‐4 specifically modifies leaf responses to drought stress

Protein breakdown and mobilization are some of the major metabolic features associated with abiotic stresses, essential for nutrient recycling and plant survival. Genetic manipulation of protease and/or protease inhibitors may contribute to modulate proteolytic processes and plant responses. The expression analysis of the whole cystatin family, inhibitors of C1A cysteine proteases, after water deprivation in barley leaves highlighted the involvement of Icy‐2 and Icy‐4 cystatin genes. Artificial microRNA lines independently silencing the two drought‐induced cystatins were generated to assess their function in planta. Phenotype alterations at the final stages of the plant life cycle are represented by the stay‐green phenotype of silenced cystatin 2 lines. Besides, the enhanced tolerance to drought and differential responses to water deprivation at the initial growing stages are observed. The mutual compensating expression of Icy‐2 and Icy‐4 genes in the silencing lines pointed to their cooperative role. Proteolytic patterns by silencing these cystatins were concomitant with modifications in the expression of potential target proteases, in particular, HvPap‐1, HvPap‐12, and HvPap‐16 C1A proteases. Metabolomics analysis lines also revealed specific modifications in the accumulation of several metabolites. These findings support the use of plants with altered proteolytic regulation in crop improvement in the face of climate change.     

Molecular cloning, chromosome mapping and characterization of a testis-specific cystatin-like cDNA, cystatin T

The cystatin superfamily of cysteine proteinase inhibitors consists of three major families. In the present study, we report the cloning of the cDNA for mouse cystatin T, which is related to family 2 cystatins. The deduced amino acid sequence of cystatin T contains regions of significant sequence homology including the four highly conserved cysteine residues in exact alignment with all cystatin family 2 members. However, cystatin T lacks some of the conserved motifs believed to be important for inhibition of cysteine proteinase activity. These characteristics are seen in two other recently cloned genes, CRES and Testatin. Thus, cystatin T appears to be the third member of the CRES/Testatin subgroup of family 2 cystatins. The mouse cystatin T gene was mapped on a region of chromosome 2 that contains a cluster of cystatin genes, including cystatin C and CRES. Northern blot analysis demonstrated that expression of mouse cystatin T is highly restricted to the mouse testis. Thus, a shared characteristic of the cystatin family 2 subgroup members is an expression pattern limited primarily to the male reproductive tract.     

Down-regulation of human extracellular cysteine protease inhibitors by the secreted staphylococcal cysteine proteases, staphopain A and B

Of seven human cystatins investigated, none inhibited the cysteine proteases staphopain A and B secreted by the human pathogen Staphylococcus aureus. Rather, the extracellular cystatins C, D and E/M were hydrolyzed by both staphopains. Based on MALDI-TOF time-course experiments, staphopain A cleavage of cystatin C and D should be physiologically relevant and occur upon S. aureus infection. Staphopain A hydrolyzed the Gly11 bond of cystatin C and the Ala10 bond of cystatin D with similar Km values of approximately 33 and 32 microM, respectively. Such N-terminal truncation of cystatin C caused >300-fold lower inhibition of papain, cathepsin B, L and K, whereas the cathepsin H activity was compromised by a factor of ca. 10. Similarly, truncation of cystatin D caused alleviated inhibition of all endogenous target enzymes investigated. The normal activity of the cystatins is thus down-regulated, indicating that the bacterial enzymes can cause disturbance of the host protease-inhibitor balance. To illustrate the in vivo consequences, a mixed cystatin C assay showed release of cathepsin B activity in the presence of staphopain A. Results presented for the specificity of staphopains when interacting with cystatins as natural protein substrates could aid in the development of therapeutic agents directed toward these proteolytic virulence factors.     

Cystatin SA, a cysteine proteinase inhibitor, induces interferon-gamma expression in CD4-positive T cells

Recently, it has been demonstrated that family 2 cystatins upregulate interleukin-6 production by human gingival fibroblasts. In the present study, we investigated the effects of cystatin SA on cytokine production by helper T cells. Human CD4-positive T cells were cultured with phytohemagglutinin in the presence or absence of 0.1 microM recombinant cystatin SA1 or SA2. When the amounts of interleukin-4 (IL-4) and interferon-gamma (IFNgamma) were analyzed in an ELISA system after stimulation with either cystatin, no significantly increased levels of IL-4 were detected. However, the amounts of IFNgamma were significantly increased after stimulation with the cystatins. Our results suggest that salivary family 2 cystatins are involved in immune responses through the cytokine network.     

A new multi-domain member of the cystatin superfamily expressed by Fasciola hepatica

Cystatins are cysteine protease inhibitors that are widespread in the plant and animal kingdoms. Cystatins are expressed by helminth parasites that may employ these proteins to regulate parasite cysteine protease activity and to modulate host immune responses. Here, we describe the cloning of a cDNA encoding a high molecular weight protein of Fasciola hepatica that contains two domains with significant identity to the cardinal cystatin signatures and four domains with degenerated cystatin signatures. This is the first report of a multi-domain cystatin in an invertebrate species. While cystatins are divided into three evolutionary related families, our phylogenetic analysis shows that all cystatin domains within this protein, like several other helminth cystatins, belong to the cystatin family 2. The DNA region encoding the domain 4 that is the best conserved at the level of its cystatin signatures was expressed in Drosophila cells and a recombinant protein was produced and purified. This protein was a potent inhibitor of the papain and of the major cysteine protease of F. hepatica, the cathepsin L1.     

Histidine-rich glycoprotein is evolutionarily related to the cystatin superfamily. Presence of two cystatin domains in the N-terminal region

A new member of the cystatin superfamily is introduced. Human plasma histidine-rich glycoprotein (HRG) was found to contain 2 cystatin-like sequences in tandem in the N-terminal region. Domain 1 (residues 1-112) was most homologous to domain 1 of the heavy chain of human kininogen and domain 2 (residues 113-225) was most homologous to human cystatin S as well as other cystatins and domain 3 of the heavy chain of kininogen, suggesting that the cystatin domains of HRG may represent a hitherto unknown binary form (or intermediate molecule) composed of 2 cystatin domains, and evolutionarily intermediate between the cystatin and the kininogen families.