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.     

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.     

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.     

Expression of a barley cystatin gene in maize enhances resistance against phytophagous mites by altering their cysteine-proteases

Phytocystatins are inhibitors of cysteine-proteases from plants putatively involved in plant defence based on their capability of inhibit heterologous enzymes. We have previously characterised the whole cystatin gene family members from barley (HvCPI-1 to HvCPI-13). The aim of this study was to assess the effects of barley cystatins on two phytophagous spider mites, Tetranychus urticae and Brevipalpus chilensis. The determination of proteolytic activity profile in both mite species showed the presence of the cysteine-proteases, putative targets of cystatins, among other enzymatic activities. All barley cystatins, except HvCPI-1 and HvCPI-7, inhibited in vitro mite cathepsin L- and/or cathepsin B-like activities, HvCPI-6 being the strongest inhibitor for both mite species. Transgenic maize plants expressing HvCPI-6 protein were generated and the functional integrity of the cystatin transgene was confirmed by in vitro inhibitory effect observed against T. urticae and B. chilensis protein extracts. Feeding experiments impaired on transgenic lines performed with T. urticae impaired mite development and reproductive performance. Besides, a significant reduction of cathepsin L-like and/or cathepsin B-like activities was observed when the spider mite fed on maize plants expressing HvCPI-6 cystatin. These findings reveal the potential of barley cystatins as acaricide proteins to protect plants against two important mite pests.     

The human cystatin C gene (CST3) is a member of the cystatin gene family which is localized on chromosome 20

The fourth gene from the human cystatin gene family of salivary-type cysteine-proteinase inhibitors has been isolated and partially characterized by DNA analysis. The gene, which we name CST3, codes for human cystatin C, and has the same organization as the CST1 gene for cystatin SN and the CST2 gene for cystatin SA. Southern analysis of EcoR I digested DNAs from 32 independent somatic cell hybrid clones hybridized to a probe from CST1 demonstrated that all members of the cystatin gene family segregate with human chromosome 20. These results indicate that the genes for salivary-type cystatins and cystatin C are members of a multigene family--the cystatin gene family.     

Comparative phylogenetic analysis of cystatin gene families from arabidopsis,rice and barley

The plant cystatins or phytocystatins comprise a family of specific inhibitors of cysteine proteinases. Such inhibitors are thought to be involved in the regulation of several endogenous processes and in defence against pests and pathogens. Extensive searches in the complete rice and Arabidopsis genomes and in barley EST collections have allowed us to predict the presence of twelve different cystatin genes in rice, seven in Arabidopsis, and at least seven in barley. Structural comparisons based on alignments of all the protein sequences using the CLUSTALW program and searches for conserved motifs using the MEME program have revealed broad conservation of the main motifs characteristic of the plant cystatins. Phylogenetic analyses based on their deduced amino acid sequences have allowed us to identify groups of orthologous cystatins, and to establish homologies and define examples of gene duplications mainly among the rice and barley cystatin genes. Moreover, the absence of a counterpart between the two monocots, as well as strong variations in the motifs that interact with the cysteine proteinases, may be related to a species-specific evolutionary process. This cystatin classification should facilitate the assignment of proteinase specificities and functions to other cystatins as new information is obtained.Electronic Supplementary Material Supplementary material is available for this article at     

Molecular cloning and characterization of a novel cystatin-like molecule,CLM, from human bone marrow stromal cells

The cystatins are physiological cysteine proteinase inhibitors. Here we report the cloning of a novel human cystatin-like molecule (CLM) from human bone marrow stromal cell (BMSC) cDNA library. The putative CLM protein contained 159 residues with a 29-residue signal peptide. CLM protein was highly homologous to family 2 cystatins, especially mouse and human testatin. The CLM gene spanned two exons and was mapped on chromosome 20p11.2, among cystatin superfamily gene clusters. CLM mRNA was barely detected in most tumor cell lines except for breast adenocarcinoma MCF-7 cells and glioblastoma U251 cells, but after LPS or PMA stimulation, CLM expression was increased in myelogenous leukemia cell lines HL-60 and U-937. Northern blot analysis revealed CLM was ubiquitously expressed in normal tissues, which was clearly different from the testis-specific expression pattern of most family 2 cystatins. When overexpressed in 293 cells, GFP-fused CLM targeted extracellularly through secretory pathway by Golgi apparatus. The results indicated that the secreted CLM protein might play roles in hematopoietic differentiation or inflammation.     

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.     

Purification, molecular cloning, and sequencing of salivary cystatin SA-1

A "long form" salivary thiol protease inhibitor, designated cystatin SA-I, was purified to homogeneity from human submandibular-sublingual saliva by sequential gel filtration and ion-exchange chromatography. Automated peptide sequencing data revealed that cystatin SA-I shares sequence homologies with salivary cystatin SN, except that it contains an additional octapeptide at its NH2 terminus. To further characterize the molecular basis of salivary cystatin diversity, a mixed-base oligonucleotide probe corresponding to a region within the NH2-terminal sequence of the salivary cystatins was synthesized. This probe was used to screen a portion of a human submandibular gland cDNA library. The cDNA insert of a clone, designated pBR HSMSF 10G5.1, carried the entire peptide coding sequence of cystatin SA-I. The secretory peptide signal coding sequence was immediately followed by a sequence encoding the eight amino acid residues found at the NH2 terminus of purified cystatin SA-I. To estimate the number of genes encoding cystatins in the human genome, fragments of the pBR HSMSF 10G5.1 insert were used as probes in Southern blot analyses of human genomic DNA. These analyses revealed that the human genome carries 4-7 homologous cystatin genes. Collectively, our data suggest that some of the diversity in salivary cystatins could be generated by expression of different members of a multigene family and by posttranslational proteolytic cleavage of NH2-terminal regions (cystatin SA-I to cystatin SN).     

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 cystatains 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.     

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.     

Cloning of a novel gene,<Emphasis Type="Italic">Cymg1</Emphasis>, related to family 2 cystatins and expressed at specific stages of mouse testis development

We have cloned a novel gene,Cymg1 (GenBank accession number AY600990), from a mouse testis cDNA library.Cymg1 is located in 2G3 of mouse chromosome 2. The cDNA includes an open reading frame that encodes 141 amino acid residues. The encoded polypeptide has a cysteine protease inhibitor domain found in the family 2 cystatins but lacks critical consensus sites important for cysteine protease inhibition. These characteristics are seen in the proteins of the CRES subfamily of the family 2 cystatins which are expressed specifically in the reproductive tract. CYMG1 protein shows 44% identity with mouse CRES and 30% identity with mouse cystatin C. Northern blot analysis showed that theCymg1 gene was specifically expressed in adult mouse testis. RT-PCR also showed thatCymg1 was expressed in testis and spermatogonial cells.Cymg1 expression level varied in the different developmental stages of mouse testis, and were coincidental with spermatogenesis and sex maturation. These results indicate thatCymg1 may play important roles in mouse spermatogenesis and sex maturation     

Expression of type 2 cystatin genes CST1-CST5 in adult human tissues and the developing submandibular gland

Type 2 cystatins comprise a class of cysteine peptidase inhibitor presumed to mediate protective functions at various locations, including the oral cavity. Seven cystatin genes are clustered within a 300-kb region of human 20p11.2. "Salivary" cystatins, encoded by CST1, 2, 4, and 5, are present in saliva at significant levels but have also been reported in other secretions, such as tears, suggesting that during their evolution, these genes have acquired mechanisms directing differential tissue-specific expression. However, their patterns of expression, which might also provide additional clues to their individual functions, have not been determined. Gene-specific RNase protection assays were used to examine the qualitative and quantitative distribution of expression of these seven genes within a collection of 23 adult human tissues. The CST3 gene, encoding cystatin C, was expressed at modest levels in all tissues examined. The presumptive pseudogenes CSTP1 and CSTP2 were not expressed at detectable levels in any tissue. The CST1, 2, 4, and 5 genes were expressed in differential, tissue-specific patterns. Expression of CST2 and CST5 was restricted to the submandibular and parotid glands, while CST1 and CST4 were expressed in these tissues and in the lacrimal gland. Immunohistochemistry studies localized expression to the serous-type secretory end pieces. Coexpression of CST1 and CST4 was also observed in the epithelial lining of the gallbladder and seminal vesicle. The CST1 product was detected in the tracheal glands and CST4 in the kidney and prostate. Despite their different adult patterns of expression, analysis of CST1, 2, 4, and 5 mRNA levels in infant submandibular glands demonstrated a coordinate upregulation of expression of between 3.5 and 9 months of age. The patterns of cystatin gene expression are consistent with several proposed oral functions of the salivary cystatins but also suggest they are important in other locations and that, despite their close sequence similarity, they are individually specialized.     

Gene Duplication and Positive Selection Explains Unusual Physiological Roles of the Relaxin Gene in the European Rabbit

The relaxin gene family is a group of genes involved in different physiological roles, most of them related to reproduction. In vertebrates the genes in this family are located in three separate chromosomal locations, and have been called relaxin family locus (RFL) A, B, and C. Among mammals the RFLA and RFLC are the most conserved as no gene copy-number variation has been observed thus far. The RFLB locus is also conserved on most mammals other than primates, where there are several gene gains and losses. Interestingly, the relaxin gene found on the RFLB locus in the European rabbit has acquired a novel role. In addition to the classical reproductive roles, this gene is expressed in tracheobronchial epithelial cells and its expression has been linked to squamous differentiation. We reconstructed the evolutionary history of the European rabbit RFLB locus using the tools of comparative genomics and molecular evolution. We found that the European rabbit possess a RFLB locus which is unique among mammals in that there are five tandemly arranged relaxin gene copies, which contrast with the single relaxin copy gene found in most mammals. In addition we also found that the ancestral pre-duplication gene was subject to the action of positive selection, and several amino acid sites were identified under the action of natural selection including the sites B12 and B13 which are part of the receptor recognition and binding site.     

Cystatins

Chicken egg white cystatin was first described in the late 1960s. Since then, our knowledge about a superfamily of similar proteins present in mammals, birds, fish, insects, plants and some protozoa has expanded, and their properties as potent peptidase inhibitors have been firmly established. Today, 12 functional chicken cystatin relatives are known in humans, but a few evolutionarily related gene products still remain to be characterized. The type 1 cystatins (A and B) are mainly intracellular, the type 2 cystatins (C, D, E/M, F, G, S, SN and SA) are extracellular, and the type 3 cystatins (L- and H-kininogens) are intravascular proteins. All true cystatins inhibit cysteine peptidases of the papain (C1) family, and some also inhibit legumain (C13) family enzymes. These peptidases play key roles in physiological processes, such as intracellular protein degradation (cathepsins B, H and L), are pivotal in the remodelling of bone (cathepsin K), and may be important in the control of antigen presentation (cathepsin S, mammalian legumain). Moreover, the activities of such peptidases are increased in pathophysiological conditions, such as cancer metastasis and inflammation. Additionally, such peptidases are essential for several pathogenic parasites and bacteria. Thus cystatins not only have capacity to regulate normal body processes and perhaps cause disease when down-regulated, but may also participate in the defence against microbial infections. In this chapter, we have aimed to summarize our present knowledge about the human cystatins.     

Bactericidal effect of rat cystatin S on an oral bacterium Porphyromonas gingivalis

We tested antibacterial and antiviral activities of rat cystatin S, a cysteine proteinase inhibitor, belonging to the family 2 cystatins against 18 different bacterial species and poliovirus type 1 (Sabin). Rat cystatin S specifically inhibited the growth of a human oral anaerobic bacterium Porphyromonas gingivalis due to a bactericidal effect.