The structure and function of Iristatin, a novel immunosuppressive tick salivary cystatin.

To successfully feed, ticks inject pharmacoactive molecules into the vertebrate host including cystatin cysteine protease inhibitors. However, the molecular and cellular events modulated by tick saliva remain largely unknown. Here, we describe and characterize a novel immunomodulatory cystatin, Iristatin, which is upregulated in the salivary glands of feeding Ixodes ricinus ticks. We present the crystal structure of Iristatin at 1.76 Å resolution. Purified recombinant Iristatin inhibited the proteolytic activity of cathepsins L and C and diminished IL-2, IL-4, IL-9, and IFN-γ production by different T-cell populations, IL-6 and IL-9 production by mast cells, and nitric oxide production by macrophages. Furthermore, Iristatin inhibited OVA antigen-induced CD4+ T-cell proliferation and leukocyte recruitment in vivo and in vitro. Our results indicate that Iristatin affects wide range of anti-tick immune responses in the vertebrate host and may be exploitable as an immunotherapeutic.

Bioinformatics and expression analyses of the Ixodes scapularis tick cystatin family.

The cystatins are inhibitors of papain- and legumain-like cysteine proteinases, classified in MEROPS subfamilies I25A-I25C. This study shows that 84 % (42/50) of tick cystatins are putatively extracellular in subfamily I25B and the rest are putatively intracellular in subfamily I25A. On the neighbor joining phylogeny guide tree, subfamily I25A members cluster together, while subfamily I25B cystatins segregate among prostriata or metastriata ticks. Two Ixodes scapularis cystatins, AAY66864 and ISCW011771 that show 50-71 % amino acid identity to metastriata tick cystatins may be linked to pathways that are common to all ticks, while ISCW000447 100 % conserved in I. ricinus is important among prostriata ticks. Likewise metastriata tick cystatins, Dermacentor variabilis-ACF35512, Rhipicephalus microplus-ACX53850, A. americanum-AEO36092, R. sanguineus-ACX53922, D. variabilis-ACF35514, R. sanguineus-ACX54033 and A. maculatum-AEO35155 that show 73-86 % amino acid identity may be essential to metastriata tick physiology. RT-PCR expression analyses revealed that I. scapularis cystatins were constitutively expressed in the salivary glands, midguts and other tissues of unfed ticks and ticks that were fed for 24-120 h, except for ISCW017861 that are restricted to the 24 h feeding time point. On the basis of mRNA expression patterns, I. scapularis cystatins, ISCW017861, ISCW011771, ISCW002215 and ISCW0024528 that are highly expressed at 24 h are likely involved in regulating early stage tick feeding events such as tick attachment onto host skin and creation of the feeding lesion. Similarly, ISCW018602, ISCW018603 and ISCW000447 that show 2-3 fold transcript increase by 120 h of feeding are likely associated with blood meal up take, while those that maintain steady state expression levels (ISCW018600, ISCW018601 and ISCW018604) during feeding may not be associated with tick feeding regulation. We discuss our findings in the context of advancing our knowledge of tick molecular biology.

Cysteine peptidases and their inhibitors in Tetranychus urticae: a comparative genomic approach.

BACKGROUND: Cysteine peptidases in the two-spotted spider mite Tetranychus urticae are involved in essential physiological processes, including proteolytic digestion. Cystatins and thyropins are inhibitors of cysteine peptidases that modulate their activity, although their function in this species has yet to be investigated. Comparative genomic analyses are powerful tools to obtain advanced knowledge into the presence and evolution of both, peptidases and their inhibitors, and could aid to elucidate issues concerning the function of these proteins. RESULTS: We have performed a genomic comparative analysis of cysteine peptidases and their inhibitors in T. urticae and representative species of different arthropod taxonomic groups. The results indicate: i) clade-specific proliferations are common to C1A papain-like peptidases and for the I25B cystatin family of inhibitors, whereas the C1A inhibitors thyropins are evolutionarily more conserved among arthropod clades; ii) an unprecedented extensive expansion for C13 legumain-like peptidases is found in T. urticae; iii) a sequence-structure analysis of the spider mite cystatins suggests that diversification may be related to an expansion of their inhibitory range; and iv) an in silico transcriptomic analysis shows that most cathepsin B and L cysteine peptidases, legumains and several members of the cystatin family are expressed at a higher rate in T. urticae feeding stages than in embryos. CONCLUSION: Comparative genomics has provided valuable insights on the spider mite cysteine peptidases and their inhibitors. Mite-specific proliferations of C1A and C13 peptidase and I25 cystatin families and their over-expression in feeding stages of mites fit with a putative role in mite's feeding and could have a key role in its broad host feeding range.

Cystatins of parasitic organisms.

The cystatin superfamily comprises several groups of protease inhibitors. In this chapter we will focus on I25 family members, which consist predominantly of the type 2 cystatins. Recently, a wealth of information on these molecules and their activities has been described. Parasite cystatins are shown to have dual functions via interaction with both parasite and host proteases. Thereby, parasite cystatins are not only essentially involved in the regulation of physiological processes during parasite development, but also represent important pathogenicity factors. Interestingly, some studies indicate that parasite cystatins evolved exceptional immuno-modulatory properties. these capacities could be exploited to interfere with unwanted immune responses in unrelated human inflammatory diseases. We highlight the different biological roles of parasite cystatins and the anticipated future developments.

Normal sexual development and fertility in testatin knockout mice.

The testatin gene was previously isolated in a screen focused on finding novel signaling molecules involved in sex determination and differentiation. testatin is specifically upregulated in pre-Sertoli cells in early fetal development, immediately after the onset of Sry expression, and was therefore considered a strong candidate for involvement in early testis development. testatin expression is maintained in the adult Sertoli cell, and it can also be found in a small population of germ cells. Testatin shows homology to family 2 cystatins, a group of broadly expressed small secretory proteins that are inhibitors of cysteine proteases in vitro but whose in vivo functions are unclear. testatin belongs to a novel subfamily among the cystatins, comprising genes that all show expression patterns that are strikingly restricted to reproductive tissue. To investigate a possible role of testatin in testis development and male reproduction, we have generated a mouse with targeted disruption of the testatin gene. We found no abnormalities in the testatin knockout mice with regard to fetal and adult testis morphology, cellular ultrastructure, body and testis weight, number of offspring, spermatogenesis, or hormonal parameters (testosterone, luteinizing hormone, and follicle-stimulating hormone).

Cloning, characterization and identification of Rcet1-v1 and Rcet1-v2, two novel splice variants of mouse Rcet1 related to Cres subgroup of family 2 cystatins.

Cystatins are physiological cysteine proteinase inhibitors. We used digital differential display (DDD) to clone two novel splice variants Rcet1-v1 and Rcet1-v2 which were isolated from adult mouse testis cDNA library. Sequence analysis revealed that Rcet1-v1 and Rcet1-v2 cDNAs are 454 and 610 bp in length, respectively, and each has four exons, but the lengths of their second and third exons are different, with the results that these cDNAs encoded two different putative proteins. The deduced proteins were 88 amino acid residues (RCET1-v1) and 140 residues (RCET1-v2) in length and have one potential signal peptide and one cystatin domain, respectively, but lack part critical consensus sites important for cysteine protease inhibition. These characteristics are seen in CRES subgroup, which related to the family 2 cystatins and primarily expressed in reproductive tract. RT-PCR analysis showed that Rcet1-v1 and Rcet1-v2 were specifically expressed in adult mouse testis, epididymis and cerebrum, but higher in testis than in epididymis and cerebrum. RT-PCR analysis also showed that Rcet1-v1 and Rcet1-v2 were specifically expressed in adult mouse pituitary and spermatogonium, but not expressed in spermatozoa. Results of in situ hybridization showed that Rcet1 gene expressed abundantly in mouse spermatogonium, spermatocytes and round spermatids; did not expressed in spermatozoa. At mouse testis different development stages, Rcet1-v1 and Rcet1-v2 were expressed very low from postnatal 1 day to postnatal 3 weeks; after postnatal 4 weeks, expressed steadily increased from postnatal 4 to 7 weeks, highest in postnatal 7 to 8 weeks, then keeping on the expressing level of postnatal 6 weeks in postnatal 13-57 weeks. All these indicated that Rcet1-v1 and Rcet1-v2 primarily expressed in mouse male reproductive tract and may play important roles in mouse spermatocytes and round spermatid development. Rcet1-v1 and Rcet1-v2 may be new members of Cres subgroup of the family 2 cystatins.

Towards novel anti-cancer strategies based on cystatin function.

Cystatins have recently emerged as important players in a multitude of physiological and patho-physiological settings that range from cell survival and proliferation, to differentiation, cell signaling and immunomodulation. This group of cysteine protease inhibitors forms a large super-family of proteins composed of one, two, three, and, in some species, more than three cystatin domains. Over the last 20 years or so, members of the cystatin super-family have been primarily explored with respect to their capacity to inhibit intracellular cysteine proteases. Yet, this classical mode of action does not fully explain their remarkably diverse biological functions. Due to the space limitations, the author will discuss here the most recent findings that suggest that some of the single-domain, cytoplasmic and cell-secreted cystatins may play important roles in the promotion or suppression of tumor growth, invasion and metastasis. Based on the present understanding of cystatin function, novel avenues for anti-cancer strategies are proposed.

Pig leukocyte cysteine proteinase inhibitor (PLCPI), a new member of the stefin family.

A new stefin type low-M(r) cysteine proteinase inhibitor (PLCPI) was isolated from pig polymorphonuclear leukocytes as a contaminant of the cathelin sample. The inhibitor consists of 103 amino acids, and its M(r) was calculated to be 11,768. The inhibitor exhibits considerable sequence identity with inhibitors from the stefin family, particularly with human stefin A. The PLCPI is a fast acting inhibitor of papain and cathepsins L and S (k(ass) > or = 1 x 10(6) M-1 x s-1) and forms very tight complexes with these enzymes (Ki < or = 190 pM). The affinity for cathepsins B and H (Ki > or = 125 nM) was lower. These results also show that the inhibitory activity previously ascribed to cathelin was due to the presence of PLCPI.

cDNA cloning, identification and characterization of a novel cystatin from the tentacle of Cyanea capillata.

Cystatin is of interest from biochemical and evolutionary prospective, and also has been applied in biotechnology. In this paper, a novel cystatin was found by EST sequence analysis of the cDNA library of Cyanea capillata tentacle. The sequence of a full-length cDNA clone contained an open reading frame encoding a putative 18-residue signal peptide and a mature protein of 113 amino acids, which showed only 26% identities to Family 2 cystatins and had its own characteristic enzyme-binding motifs, Ser(97)-Trp(98), which had not been found in any other known cystatins. Thus, the novel cystatin cloned from jellyfish was designated as cystatin J, which may belong to a new family of cystatin, called Family 4. The mature cystatin J was produced in Escherichia coli as a thioredoxin (Trx) fusion protein using the pET expression system and purified by affinity and cation exchange chromatography. The recombinant cystatin J of approximately M(r) = 12,800 displayed an obvious inhibition of papain (K(i) value below 0.5 nM), in competition with substrate. Thus, the recombinant cystatin J was a functional cystatin in spite of relatively lower sequence similarity with other cystatins. Activity of the novel cystatin was stable at pH 4-11 at 4 degrees C, but unstable at neutral pH at >50 degrees C.

A new subgroup of the family 2 cystatins.

The cystatins are a superfamily of cysteine protease inhibitors. Several genes including Cres (cystatin-related epididymal spermatogenic), testatin, and cystatin T, have been identified that are related to the family 2 cystatins but lack critical consensus sites important for cysteine protease inhibition. In addition, these genes are primarily expressed in the reproductive tract suggesting they may have evolved to perform tissue-specific functions distinct from that of the typical cystatins. This review describes the CRES subgroup within the family 2 cystatins including potential new members and their putative functions in the reproductive tract.

Degradation of antimicrobial histatin-variant peptides in Staphylococcus aureus and Streptococcus mutans.

Histidine-free variants of salivary histatin 5 have a broad antimicrobial activity against various bacteria. In relation to a possible therapeutic application, we were interested in the susceptibility of these small peptides (14 amino acids long) to microbial proteinases and whether this affects their antimicrobial activity. Analyses by SDS-PAGE of supernatants of peptide-bacteria incubation showed a reduction in protein bands within 15 minutes' incubation, as a result of cellular internalization. Degradation products of dhvar1 and dhvar2 appeared within one hour in the supernatants of Streptococcus mutans and Staphylococcus aureus. In contrast, the variants dhvar3 and dhvar4 were more resistant to degradation under the same conditions. MALDI-TOF analyses identified cleavage of dhvar1 and dhvar2 at Glu(6). The N-terminal peptide part (1-6) of dhvar1 and 2 showed no bactericidal activity, while peptide fragment (7-14) showed a highly reduced bactericidal activity.

Neutrophil chemotactic activity is modulated by human cystatin C, an inhibitor of cysteine proteases.

Cystatin C, a cysteine proteinase inhibitor has recently been suggested to be a potent regulator of inflammatory processes and may act in defense against viral and bacterial infections. Two common forms of the protein were purified from the urine of a patient having received a renal transplant. The slow form of cystatin C possessed the N-terminal tetrapeptide Lys Pro Pro Arg, which was cleaved in the fast form. This peptide sequence, called postin, was synthesized. The three molecules, slow and fast forms of cystatin and the synthetic peptide, were tested for their effects on the migration activity of human polymorphonuclear neutrophils (PMNs). The slow form was found to display both chemotactic and chemokinetic activities, while the fast form and postin were only chemokinetic. Nevertheless, all the substances could induce a "motile" morphology. In addition, the two forms of cystatin C were powerful inhibitors of PMN chemotaxis induced by complement-derived chemotactic factors. This suggests that cystatin C in its two different cleaved forms and the N-terminal tetrapeptide can modulate PMN locomotion. Cysteine proteases may therefore play a role in neutrophil migration activity.

Purification and characterization of two cysteine proteinase inhibitors from the skin of Atlantic salmon (Salmo salar L.).

Two cysteine proteinase inhibitors, designated Tromsin I and II, were purified from the skin of Atlantic salmon, using three steps of chromatography, including affinity, anion exchange and gelfiltration. The two cysteine proteinase inhibitors were both of the high molecular weight type, with apparent MW 49 and 76 kDa. The isoelectric points (pI) of Tromsin I and II were estimated to be 4.5 and 5.2, respectively. The inhibitors were both stained by the PAS reaction for carbohydrates, and showed a remarkable heat stability. Western blotting revealed that the inhibitors also could be found in significant amounts in serum. Tromsin I and II share many common features with members of the family 3 cystatins, i.e. mammalian kininogen, such as molecular weight, papain inhibition and tissue distribution. Based on N-terminal sequence from Tromsin II however, no homology with known cysteine proteinase inhibitors can be found. This does not exclude that the inhibitors belong to the family 3 cystatins, because the N-terminal amino acid sequences of known cysteine proteinase inhibitors show very low homology.

[Cres (cystatin-related epididymal spermatogenic) gene regulation and function].

The CRES (cystatin-related epididymal spermatogenic) protein defines a new subgroup in the family 2 cystatins of the cystatin superfamily of cysteine protease inhibitors. However, unlike the ubiquitous expression of cystatin C, the Cres gene is preferentialy expressed in postmeiotic germ cells, the proximal caput epididymidis, and anterior pituitary gonadotrophs. Furthermore, CRES protein lacks two of the three consensus sites necessary for the cystatin inhibition of C1 cysteine proteases. Therefore, CRES may perform unique and tissue-specific functions in the reproductive and neuroendocrine systems. In the present review, we describe our studies on: 1. the Cres gene promoter and the transcriptional regulatory protein and their associated DNA binding sites that may be important for tissue-specific expression; and 2. the biochemical function of CRES protein. In brief, Northern blot, gel shift analyses, and transient transfection assays demonstrated that the C/EBP beta (CCAAT/enhancer binding protein) transcription factor is the predominant C/EBP family member expressed in the epididymis and gonadotroph cells and is necessary for high levels of Cres expression in these two tissues. In other studies, analyses of transgenic mice expressing a CAT reporter gene driven by 1.6 kb of Cres promoter revealed CAT mRNA and protein only in the germ cells. These studies suggest that the 1.6 kb of Cres 5' flanking sequence contains the required DNA elements for expression in the testis, but lacks the elements to correctly target expression of the reporter gene in the epididymis. Alternatively, repressor elements may be present. Finally, in vitro protease assays were performed to determine if CRES functions as a protease inhibitor. In contrast to cystain C, CRES did not inhibit the C1 cysteine protease papain but rather inhibited at nanomolar concentrations the serine protease PC2, a prohormone processing enzyme. Therefore, CRES is a new cross-class inhibitor that may regulate PC2 of PC2-like proteases and suggests a role for CRES in the regulation of prohormone and proprotein processing.

Antiinflammatory and immunosuppressive activity of sialostatin L, a salivary cystatin from the tick Ixodes scapularis.

Here we report the ability of the tick Ixodes scapularis, the main vector of Lyme disease in the United States, to actively and specifically affect the host proteolytic activity in the sites of infestation through the release of a cystatin constituent of its saliva. The cystatin presence in the saliva was verified both biochemically and immunologically. We named the protein sialostatin L because of its inhibitory action against cathepsin L. We also show that the proteases it targets, although limited in number, have a prominent role in the proteolytic cascades that take place in the extracellular and intracellular environment. As a result, sialostatin L displays an antiinflammatory role and inhibits proliferation of cytotoxic T lymphocytes. Beyond unraveling another component accounting for the properties of tick saliva, contributing to feeding success and pathogen transmission, we describe a novel tool for studying the role of papain-like proteases in diverse biologic phenomena and a protein with numerous potential pharmaceutical applications.