Cloning and characterization of Litopenaeus vannamei cystainB-like in WSSV infection.

Cystatins B is an endogenous cysteine cathepsin inhibitor. In shrimp, cystatins B-like (CSTB-L) has not been characterized and its role in WSSV infection is largely unknown. In this study, a full-length 699 bp CSTB-L sequence with 291 bp open reading frame encoding a 96 amino acid from L.vannamei (Lv) was first cloned. The tissue distribution assay indicated that LvCSTB-L presented ubiquitous expression in most examined tissues, with the most predominant expression in the hepatopancreas and the weakest expression in the muscles. LvCSTB-L transcripts could be induced in the intestine and hepatopancreas by WSSV challenge. The relative expression level of IE1 and VP28 in the LvCSTB-L knockdown shrimp were increased significantly. In addition, the shrimp cumulative mortality was remarkably (p < 0.01) increased after LvCSTB-L knockdown. Moreover, following the LvCSTB-L silencing, significant decreases in the mRNA levels of p53, p38, caspase3, STAT and ERK were also observed. The results suggested that LvCSTB-L could play positively roles in antiviral immune response by JAK-STAT, MAPK and apoptotic pathway. These findings would further our understanding of shrimp antiviral response, and therefore help for virus control and prevention.

Identification and characterization of cystatin B from black rockfish, Sebastes schlegelii, indicating its potent immunological importance.

Cystatins represent a large superfamily of proteins involved in the competitive reversible inhibition of C1 class cysteine proteases. Plant-derived papain proteases and cysteine cathepsins are the major cysteine proteases that interact with cystatins. The cystatin superfamily can be further classified into three groups: stefins, cystatins, and kininogens. Among these, cystatin B is categorized under stefins. Cystatin B lacks a signal sequence, disulfide bonds, and carbohydrate groups. However, it contains the conserved cystatin family signature, including a single cystatin-like domain, cysteine protease inhibitory signature concealing pentapeptide (QXVXG) consensus sequence, and two conserved neighboring glycine (8GG9) residues at the N-terminal. In the current study, a member of cystatin B was identified from Korean black rockfish (Sebastes schlegeli) using a cDNA database and designated as RfCytB. The full-length cDNA of RfCytB was 573 bp long, with a coding region of 294 bp. The 5'-untranslated region (UTR) comprised 55 bp, and the 263-bp-long 3'-UTR included a polyadenylation signal sequence and a poly-A tail. The coding sequence encodes a polypeptide comprising 97 amino acids, with a predicted molecular weight of 11 kDa and theoretical isoelectric point of 6.3. RfCytB shared homology features with similar molecules from other teleost and vertebrate species, and was clustered with Cystatin family 1 in our phylogenetic reconstruction. RfCytB was ubiquitously expressed in all tissue types of healthy animals, with the highest levels of expression observed in gill and spleen. Temporal expression of RfCytB displayed significant up-regulation upon infection with Aeromonas salmonicida. Recombinantly expressed RfCytB showed a concentration-dependent inhibitory activity towards papain, with a high thermal stability. Transient expression of RfCytB in LPS activated murine macrophages, thereby inducing the expression of genes related to pro-inflammatory conditions, such as iNOS and TNF α. These results provide evidence for its protease inhibitory and immunity relevant roles in hosts.

A novel zebrafish model to emulate lung injury by folate deficiency-induced swim bladder defectiveness and protease/antiprotease expression imbalance.

Lung injury is one of the pathological hallmarks of most respiratory tract diseases including asthma, acute respiratory distress syndrome (ARDS) and chronic obstructive pulmonary disease (COPD). It involves progressive pulmonary tissue damages which are usually irreversible and incurable. Therefore, strategies to facilitate drug development against lung injury are needed. Here, we characterized the zebrafish folate-deficiency (FD) transgenic line that lacks a fully-developed swim bladder. Whole-mount in-situ hybridization revealed comparable distribution patterns of swim bladder tissue markers between wild-type and FD larvae, suggesting a proper development of swim bladder in early embryonic stages. Unexpectedly, neutrophils infiltration was not observed in the defective swim bladder. Microarray analysis revealed a significant increase and decrease of the transcripts for cathepsin L and a cystatin B (CSTB)-like (zCSTB-like) proteins, respectively, in FD larvae. The distribution of cathepsin L and the zCSTB-like transcripts was spatio-temporally specific in developing wild-type embryos and, in appropriate measure, correlated with their potential roles in maintaining swim bladder integrity. Supplementing with 5-formyltetrahydrofolate successfully prevented the swim bladder anomaly and the imbalanced expression of cathepsin L and the zCSTB-like protein induced by folate deficiency. Injecting the purified recombinant zebrafish zCSTB-like protein alleviated FD-induced swim bladder anomaly. We concluded that the imbalanced expression of cathepsin L and the zCSTB-like protein contributed to the swim bladder malformation induced by FD and suggested the potential application of this transgenic line to model the lung injury and ECM remodeling associated with protease/protease inhibitor imbalance.

Prolines Affect the Nucleation Phase of Amyloid Fibrillation Reaction; Mutational Analysis of Human Stefin B.

Proline residues play a prominent role in protein folding and aggregation. We investigated the influence of single prolines and their combination on oligomerization and the amyloid fibrillation reaction of human stefin B (stB). The proline mutants influenced the distribution of oligomers between monomers, dimers, and tetramers as shown by the size-exclusion chromatography. Only P74S showed higher oligomers, reminiscent of the molten globule reported previously for the P74S of stB-Y31 variant. The proline mutants also inhibited to various degree the amyloid fibrillation reaction. At 30 and 37 °C, inhibition was complete for the P74S single mutant, two double mutants (P6L P74S and P74S P79S), and for the triple mutant P6L P11S P74S. At 30 °C the single mutant P6L completely inhibited the reaction, while P11S and P79S formed amyloid fibrils with a prolonged lag phase. P36D did not show a lag phase, reminiscent of a downhill polymerization model. At 37 °C in addition to P36D, P11S, and P79S, P6L and P11S P74S also started to fibrillate; however, the yield of the fibrils was much lower than that of the wild-type protein as judged by transmission electron microscopy. Thus, Pro 74 cis/trans isomerization proves to be the key event, acting as a switch toward an amyloid transition. Using our previous model of nucleation and growth, we simulated the kinetics of all the mutants that exhibited sigmoidal fibrillation curves. To our surprise, the nucleation phase was most affected by Pro cis/trans isomerism, rather than the fibril elongation phase.

A five-residue motif for the design of domain swapping in proteins.

Domain swapping is the process by which identical monomeric proteins exchange structural elements to generate dimers/oligomers. Although engineered domain swapping is a compelling strategy for protein assembly, its application has been limited due to the lack of simple and reliable design approaches. Here, we demonstrate that the hydrophobic five-residue 'cystatin motif' (QVVAG) from the domain-swapping protein Stefin B, when engineered into a solvent-exposed, tight surface loop between two ß-strands prevents the loop from folding back upon itself, and drives domain swapping in non-domain-swapping proteins. High-resolution structural studies demonstrate that engineering the QVVAG stretch independently into various surface loops of four structurally distinct non-domain-swapping proteins enabled the design of different modes of domain swapping in these proteins, including single, double and open-ended domain swapping. These results suggest that the introduction of the QVVAG motif can be used as a mutational approach for engineering domain swapping in diverse ß-hairpin proteins.

Proline Residues as Switches in Conformational Changes Leading to Amyloid Fibril Formation.

Here we discuss studies of the structure, folding, oligomerization and amyloid fibril formation of several proline mutants of human stefin B, which is a protein inhibitor of lysosomal cysteine cathepsins and a member of the cystatin family. The structurally important prolines in stefin B are responsible for the slow folding phases and facilitate domain swapping (Pro 74) and loop swapping (Pro 79). Moreover, our findings are compared to ß2-microglobulin, a protein involved in dialysis-related amyloidosis. The assessment of the contribution of proline residues to the process of amyloid fibril formation may shed new light on the critical molecular events involved in conformational disorders.

Putative alternative functions of human stefin B (cystatin B): binding to amyloid-beta, membranes, and copper.

We describe studies performed thus far on stefin B from the family of cystatins as a model protein for folding and amyloid fibril formation studies. We also briefly mention our studies on aggregation of some of the missense EPM1 mutants of stefin B in cells, which mimic additional pathological traits (gain in toxic function) in selected patients with EPM1 disease. We collected data on the reported interactors of stefin B and discuss several hypotheses of possible cytosolic alternative functions.

Protein Domain-Swapping Can Be a Consequence of Functional Residues.

Monomer topology has been implicated in domain-swapping, a potential first step on the route to disease-causing protein aggregation. Despite having the same topology (ß1-α1-ß2-ß3-ß4-ß5), the cysteine protease inhibitor stefin-B domain swaps more readily than a single-chain variant of the heterodimeric sweet protein monellin (scMn). Here, we computationally study the folding of stefin-B and scMn in order to understand the molecular basis for the difference in their domain-swapping propensities. In agreement with experiments, our structure-based simulations show that scMn folds cooperatively without the population of an intermediate while stefin-B populates an equilibrium intermediate state. Since the simulation intermediate has only one domain structured (ß3-ß4-ß5), it can directly lead to domain-swapping. Using computational variants of stefin-B, we show that the population of this intermediate is caused by regions of stefin-B that have been implicated in protease inhibition. We also find that the protease-binding regions are located on two structural elements and localized in space. In contrast, the residues that contribute to the sweetness of monellin are not localized to a few structural elements but are distributed over the protein fold. We conclude that the distributed functional residues of monellin do not induce large local perturbations in the protein structure, eliminating the formation of folding intermediates and in turn domain-swapping. On the other hand, the localized protease-binding regions of stefin-B promote the formation of a folding intermediate which can lead to domain-swapping. Thus, domain-swapping can be a direct consequence of the constraints that function imposes on the protein structure.

Efficient expression and purification of biologically active human cystatin proteins.

Cystatins are reversible cysteine protease inhibitor proteins. They are known to play important roles in controlling cathepsins, neurodegenerative disease, and in immune system regulation. Production of recombinant cystatin proteins is important for biochemical and function characterization. In this study, we cloned and expressed human stefin A, stefin B and cystatin C in Escherichia coli. Human stefin A, stefin B and cystatin C were purified from soluble fraction. For cystatin C, we used various chaperone plasmids to make cystatin C soluble, as it is reported to localize in inclusion bodies. Trigger factor, GroES-GroEL, DnaK-DnaJ-GrpE chaperones lead to the presence of cystatin C in the soluble fraction. Immobilized metal affinity chromatography, glutathione sepharose and anion exchange chromatography techniques were employed for efficient purification of these proteins. Their biological activities were tested by inhibition assays against cathepsin L and H3 protease.

Assembly of Stefin B into Polymorphic Oligomers Probed by Discrete Molecular Dynamics.

Assembly of an amyloidogenic protein stefin B into molten globule oligomers is studied by efficient discrete molecular dynamics. Consistent with in vitro findings, tetramers form primarily through dimer association, resulting in a decreased trimer abundance. Oligomers up to heptamers display elongated rod-like morphologies akin to protofibrils, whereas larger oligomers, decamers through dodecamers, form elongated, branched, as well as annular structures, providing structural insights into pore forming ability and toxicity of amyloidogenic proteins.

Limited Proteolysis Reveals That Amyloids from the 3D Domain-Swapping Cystatin B Have a Non-Native ß-Sheet Topology.

3D domain-swapping proteins form multimers by unfolding and then sharing of secondary structure elements, often with native-like interactions. Runaway domain swapping is proposed as a mechanism for folded proteins to form amyloid fibres, with examples including serpins and cystatins. Cystatin C amyloids cause a hereditary form of cerebral amyloid angiopathy whilst cystatin B aggregates are found in cases of Unverricht-Lundborg Syndrome, a progressive form of myoclonic epilepsy. Under conditions that favour fibrillisation, cystatins populate stable 3D domain-swapped dimers both in vitro and in vivo that represent intermediates on route to the formation of fibrils. Previous work on cystatin B amyloid fibrils revealed that the α-helical region of the protein becomes disordered and identified the conservation of a continuous 20-residue elongated ß-strand (residues 39-58), the latter being a salient feature of the dimeric 3D domain-swapped structure. Here we apply limited proteolysis to cystatin B amyloid fibrils and show that not only the α-helical N-terminal of the protein (residues 1-35) but also the C-terminal of the protein (residues 80-98) can be removed without disturbing the underlying fibril structure. This observation is incompatible with previous models of cystatin amyloid fibrils where the ß-sheet is assumed to retain its native antiparallel arrangement. We conclude that our data favour a more generic, at least partially parallel, arrangement for cystatin ß-sheet structure in mature amyloids and propose a model that remains consistent with available data for amyloids from either cystatin B or cystatin C.

Gain in toxic function of stefin B EPM1 mutants aggregates: correlation between cell death, aggregate number/size and oxidative stress.

EPM1 is a rare progressive myoclonus epilepsy accompanied by apoptosis in the cerebellum of patients. Mutations in the gene of stefin B (cystatin B) are responsible for the primary defect underlying EPM1. Taking stefin B aggregates as a model we asked what comes first, protein aggregation or oxidative stress, and how these two processes correlate with cell death. We studied the aggregation in cells of the stefin B wild type, G4R mutant, and R68X fragment before (Ceru et al., 2010, Biol. Cell). The present study was performed on two more missense mutants of human stefin B, G50E and Q71P, and they similarly showed numerous aggregates upon overexpression. Mutant- and oligomer-dependent increase in oxidative stress and cell death in cells bearing aggregates was shown. On the other hand, there was no correlation between the size and number of the aggregates and cell death. We suggest that differences in toxicity of the aggregates depend on whether they are in oligomeric/protofibrillar or fibrillar form. This in turn likely depends on the mutant's 3D structure where unfolded proteins show lower toxicity. Imaging by transmission electron microscopy showed that the aggregates in cells are of different types: bigger perinuclear, surrounded by membranes and sometimes showing vesicle-like invaginations, or smaller, punctual and dispersed throughout the cytoplasm. All EPM1 mutants studied were inactive as cysteine proteases inhibitors and in this way contribute to loss of stefin B functions. Relevance to EPM1 disease by gain in toxic function is discussed.

Enzyme kinetics studies on 29-kDa human liver cathepsin L.

Enzyme kinetics studies reported in the literature showed that human liver Cathepsin L is active only at lysosomal acidic pH values, while biochemical studies in living cells showed that the enzyme works even at neutral pH values (in a condition compatible with the extracellular compartment). Such an apparent ambiguity highlighted the need of analysing in depth the kinetics of ~29-kDa Cathepsin L, which is the form commonly used in experiments. The stability and catalytic activity of this enzyme were investigated at different pH values, reducing and non-reducing environments, presence of copper, iron and zinc ions, and presence of the natural modulator/inhibitor cystatin B. Our experiments showed that ~29-kDa human liver Cathepsin L is stable and catalytically functional even at neutral pH values and under non-reducing conditions, which simulate the extracellular compartment. Under these conditions, Cathepsin L was also proved to interact with cystatin B, being also modulated by physiological concentrations of Cu(++) , Fe(++) and Zn(++) . This paper suppose an advance in the comprehension of the catalytic properties of human liver Cathepsin L, its implications in different physiological processes and its potential use within a drug screening programme in which agents acting extracellularly are being considered.

Partial rotational lattice order-disorder in stefin B crystals.

At present, the determination of crystal structures from data that have been acquired from twinned crystals is routine; however, with the increasing number of crystal structures additional crystal lattice disorders are being discovered. Here, a previously undescribed partial rotational order-disorder that has been observed in crystals of stefin B is described. The diffraction images revealed normal diffraction patterns that result from a regular crystal lattice. The data could be processed in space groups I4 and I422, yet one crystal exhibited a notable rejection rate in the higher symmetry space group. An explanation for this behaviour was found once the crystal structures had been solved and refined and the electron-density maps had been inspected. The lattice of stefin B crystals is composed of five tetramer layers: four well ordered layers which are followed by an additional layer of alternatively placed tetramers. The presence of alternative positions was revealed by the inspection of electron-density score maps. The well ordered layers correspond to the crystal symmetry of space group I422. In addition, the positions of the molecules in the additional layer are related by twofold rotational axes which correspond to space group I422; however, these molecules lie on the twofold axis and can only be related in a statistical manner. When the occupancies of alternate positions and overlapping are equal, the crystal lattice indeed fulfills the criteria of space group I422; when these occupancies are not equal, the lattice only fulfills the criteria of space group I4.

The role of initial oligomers in amyloid fibril formation by human stefin B.

Oligomers are commonly observed intermediates at the initial stages of amyloid fibril formation. They are toxic to neurons and cause decrease in neural transmission and long-term potentiation. We describe an in vitro study of the initial steps in amyloid fibril formation by human stefin B, which proved to be a good model system. Due to relative stability of the initial oligomers of stefin B, electrospray ionization mass spectrometry (ESI MS) could be applied in addition to size exclusion chromatography (SEC). These two techniques enabled us to separate and detect distinguished oligomers from the monomers: dimers, trimers, tetramers, up to decamers. The amyloid fibril formation process was followed at different pH and temperatures, including such conditions where the process was slow enough to detect the initial oligomeric species at the very beginning of the lag phase and those at the end of the lag phase. Taking into account the results of the lower-order oligomers transformations early in the process, we were able to propose an improved model for the stefin B fibril formation.

Expression profile of cystatin B ortholog from Manila clam (Ruditapes philippinarum) in host pathology with respect to its structural and functional properties.

Cystatins are a well-characterized group of cysteine protease inhibitors, which play crucial roles in physiology and immunity. In the present study, an invertebrate ortholog of cystatin B was identified in Manila clam (Ruditapes philippinarum) (RpCytB) and characterized at the molecular level, demonstrating its inhibitory activity against the well-known cysteine protease, papain. The complete coding sequence of RpCytB (297 bp in length) encodes a 99 amino acid peptide with a calculated molecular mass of 11 kDa and a theoretical isoelectric point of 5.9. The derived peptide was found to harbor typical features of cystatin proteins, including the 'Q-X-V-X-G' motif, which was identified as QLVAG in RpCytB. Phylogenetic analysis of RpCytB revealed close evolutionary relationships with its invertebrate counterparts, especially those from mollusks. Recombinant RpCytB (rRpCytB) was overexpressed as a fusion with maltose binding protein (MBP) in Escherichia coli BL21 (DE3) cells. Purified rRpCytB fusion protein exhibited a detectable inhibitory activity against papain, while the control MBP showed an almost constant negligible activity. While quantitative RT-PCR detected ubiquitous RpCytB expression in all tissues examined, the expressions in hemocytes and gills were relatively higher. Upon in vivo immune challenge with lipopolysaccharide (LPS), the expression of RpCytB in gills and hemocytes was down-regulated. Similar challenges with poly I:C and intact Vibrio tapetis bacteria revealed a complicated transcriptional regulation, wherein mRNA expression levels fluctuated over time of exposure. Moreover, a precise induction of RpCytB expression after bacterial infection was detected in gills by in situ hybridization. Collectively, our findings in this study indicate that RpCytB expression is sensitive to host pathological conditions and may contribute cysteine protease inhibitory activity to modulate the immune response.

Influence of partial unfolding and aggregation of human stefin B (cystatin B) EPM1 mutants G50E and Q71P on selective cleavages by cathepsins B and S.

Human stefins and cystatins are physiologically important cysteine proteinase inhibitors, acting as a first line of defense against undesirable proteolysis. Mutations in the cystatin B gene cause a rare form of epilepsy EPM1. Its two missense mutants, G50E and Q71P, lack the inhibitory activity and are partially unfolded, which leads to changes in their aggregation behavior, both in vitro and in the cell. SDS-PAGE and MALDI-TOF mass spectrometry were used to follow the hydrolysis of human stefin B wild type, G50E and Q71P, by cathepsins B and S in vitro. Cathepsin S was found to degrade both mutants, with Q71P being degraded faster. This correlates with the openness of the protein structure, Q71P having more exposed hydrophobic surfaces. Cathepsin B acted more selectively, degrading G50E into smaller fragments, while still leaving a portion of the full-length protein intact. Q71P was cleaved only at the exposed N-terminal end. The co-localization of stefin B wild type and EPM1 mutants with cathepsins showed that cathepsins accumulate around the aggregates formed by the EPM1 mutants. We hypothesize that the aggregation of both full-length mutants prevents the cathepsin molecule from accessing the substrate protein's core, whereas the cleaved fragments would be expected to aggregate stronger.

Genomic characterization and expression profiles upon bacterial infection of a novel cystatin B homologue from disk abalone (Haliotis discus discus).

Cystatins are a large family of cysteine proteinase inhibitors which are involved in diverse biological and pathological processes. In the present study, we identified a gene related to cystatin superfamily, AbCyt B, from disk abalone Haliotis discus discus by expressed sequence tag (EST) analysis and BAC library screening. The complete cDNA sequence of AbCyt B is comprised of 1967 nucleotides with a 306 bp open reading frame (ORF) encoding for 101 amino acids. The amino acid sequence consists of a single cystatin-like domain, which has a cysteine proteinase inhibitor signature, a conserved Gly in N-terminal region, QVVAG motif and a variant of PW motif. No signal peptide, disulfide bonds or carbohydrate side chains were identified. Analysis of deduced amino acid sequence revealed that AbCyt B shares up to 44.7% identity and 65.7% similarity with the cystatin B genes from other organisms. The genomic sequence of AbCyt B is approximately 8.4 Kb, consisting of three exons and two introns. Phylogenetic tree analysis showed that AbCyt B was closely related to the cystatin B from pacific oyster (Crassostrea gigas) under the family 1.Functional analysis of recombinant AbCyt B protein exhibited inhibitory activity against the papain, with almost 84% inhibition at a concentration of 3.5 µmol/L. In tissue expression analysis, AbCyt B transcripts were expressed abundantly in the hemocyte, gill, mantle, and digestive tract, while weakly in muscle, testis, and hepatopancreas. After the immune challenge with Vibrio parahemolyticus, the AbCyt B showed significant (P<0.05) up-regulation of relative mRNA expression in gill and hemocytes at 24 and 6 h of post infection, respectively. These results collectively suggest that AbCyst B is a potent inhibitor of cysteine proteinases and is also potentially involved in immune responses against invading bacterial pathogens in abalone.

Cystatin B homolog from rock bream Oplegnathus fasciatus: genomic characterization, transcriptional profiling and protease-inhibitory activity of recombinant protein.

Cysteine proteases are present in all living organisms and, in animals, function in a vast array of physiological and pathological processes. Cysteine protease inhibitors act upon the cysteine proteases to regulate their activity. The cystatin superfamily of cysteine protease inhibitors has members represented in all living organisms studied to date. Here, we report the identification of a new member of the family 1 cystatin in Oplegnathus fasciatus rock bream (denoted as RbCyt B) and the characterization at the molecular level. The complete genomic sequence of RbCyt B consists of three exons and a promoter region. The open reading frame (ORF) encodes for a 100 amino acids length polypeptide with a single cystatin-like domain and a cysteine protease inhibitor signature motif. The conserved N-terminal glycine, glutamine-valine-glycine motif, QxVxG, and a variant of the proline-tryptophan, PW, motif were identified. RbCyt B showed closest phylogenetic distance to Dicentrarchus labrax cystatin B, and shared up to 73% amino acid identity and 90% amino acid similarity with known cystatin B genes. RbCyt B mRNA expression was detected in nine different tissues and was highly expressed in liver, spleen, gill, brain, intestine, kidney, head kidney, and blood, as compared with muscle. In vivo immune stimulation with Edwardsiella tarda bacteria caused significant up-regulation of RbCyt B mRNA in head kidney and spleen at 24h post-infection (P<0.05). Recombinant RbCyt B was expressed in Escherichia coli, and the purified protein demonstrated 82% papain inhibitory activity at 500 × 10(-3) µg µL(-1) in a concentration-dependent manner. These results suggest that RbCyt B is a member of family 1 cystatin with high homology to cystatin B, and is a biologically active protein possessing papain inhibitory activity and potentially involved in immune responses against invading Gram-negative bacteria in rock bream.

RP-HPLC-ESI-MS evidenced that salivary cystatin B is detectable in adult human whole saliva mostly as S-modified derivatives: S-Glutathionyl, S-cysteinyl and S-S 2-mer.

An HPLC-ESI-MS analysis of adult human whole saliva evidenced three protein masses (M average 11,487±2, 11,301±2 and 22,362±3Da) eluting in the 32.5-35.0min range. Treatment in reducing conditions allowed establishing that they are S-derivatives of N-terminal acetylated cystatin B, namely its S-glutathionyl, S-cysteinyl and S-S dimer. The identification was confirmed by high resolution HPLC-ESI-MS-MS experiments on the intact naturally occurring proteins and their tryptic digests. S-unmodified cystatin B is rarely detectable in whole saliva of healthy adults (5 subjects out of 65) and its percentage does not overcome approximately 20% of total cystatin B (11±9%). In the majority of subjects (60 out of 65) the mean percentages of the S-modified derivatives were S-glutathionyl 53±13%, S-cysteinyl 15±5%, S-S 2-mer 32±13%. Variations of the percentages of these S-modified derivatives of cystatin B could be indicative of oral oxidative stress. As we are aware, this is the first time that S-glutathionylation and S-cysteinylation were described as extensive PTM of a salivary protein and the first time that these PTMs were detected in naturally occurring cystatin B.