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.     

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.     

Two secreted cystatins of the soft tick Ornithodoros moubata: differential expression pattern and inhibitory specificity

Two genes coding for cysteine peptidase inhibitors of the cystatin family (Om-cystatin 1 and 2) were isolated from a gut-specific cDNA library of the soft tick Ornithodoros moubata. Both cystatins were clearly down-regulated after a blood meal. Om-cystatin 1 is mainly expressed in the tick gut, while Om-cystatin 2 mRNA was also found in other tick tissues. Authentic Om-cystatin 2 was significantly more abundant than Om-cystatin 1 in the gut contents of fasting ticks and was associated with hemosome-derived residual bodies accumulated in the gut lumen. Om-cystatin 2 was also expressed by type 2 secretory cells in the salivary glands of unfed ticks. The inhibitory specificity of recombinant Om-cystatins 1 and 2 was tested with mammalian cysteine peptidases, as well as endogenous cysteine peptidases present in the tick gut. Both cystatins efficiently inhibited papain-like peptidases, including cathepsin B and H, but differed significantly in their affinity towards cathepsin C and failed to block asparaginyl endopeptidase. Our results suggest that the secreted cystatin isoinhibitors are involved in the regulation of multiple proteolytic targets in the tick digestive system and tick-host interaction.     

Expression and induction by beta-adrenergic agonists of the cystatin S gene in submandibular glands of developing rats.

Repeated administration of the beta-adrenergic agonist isoprenaline (isoproterenol, IPR), which produces hypertrophic/hyperplastic enlargements of rat submandibular and parotid glands, induces synthesis of a secretory protein shown to be a cysteine proteinase inhibitor, rat cystatin S. In the current study, Northern blot and hybridizations in situ were carried out to establish the developmental and beta-adrenergic regulation of the expression of the cystatin S gene. Cystatin S mRNA was not detected in submandibular glands of 20-day-old fetuses, nor in the glands of newborn or 10-day-old rats. However, steady-state levels of cystatin S mRNA increased between 21 and 28 days, reaching a conspicuously high concentration at 28 days; cystatin S mRNA then declined rapidly to a barely detectable level in glands of 32-day-old rats. IPR administration for 4 days induced high levels of cystatin S mRNA in submandibular glands of developing and adult rats. In both prepubertal and mature animals, induction of cystatin S mRNA in submandibular glands was more pronounced in female than in male animals. Hybridizations in situ revealed cystatin S mRNA only in acinar but not in duct cells of the submandibular gland. Developmentally, expression of the cystatin S gene coincided with acinar cell differentiation. These data suggest a complex neural, hormonal and developmental regulation of salivary cystatin genes.     

Human cysteine-proteinase inhibitors: nucleotide sequence analysis of three members of the cystatin gene family

Three genes from the human cystatin gene family of cysteine-proteinase inhibitors have been isolated from a bacteriophage lambda library containing HindIII digests of human genomic DNA. Two of the genes code for salivary cystatin SN and SA, the third is a pseudogene. The cloned genes were identified with a probe made from a salivary cystatin cDNA. The complete nucleotide sequence of the gene that codes for the precursor form of the neutral salivary protein, cystatin SN, was determined. The gene, which we name CST1, contains three exons and two intervening sequences. The expected CAT and ATA boxes are present in the 5'-flanking region of the gene. Partial nucleotide sequence determination of a second gene revealed that it codes for the precursor form of the acidic salivary protein, cystatin SA. This gene, which we name CST2, has the same gene organization as CST1. The complete nucleotide sequence of a third gene was determined. It does not contain a typical ATA box, and in addition, a premature stop codon and a frameshift deletion mutation occur within the gene. These inactivation mutations show that this gene, which we name CSTP1, is a cystatin pseudogene. These data combined with our genomic Southern-blot analyses show that the cystatin genes form a multigene family with at least seven members.     

Sympathetic and parasympathetic regulation of cystatin S gene expression

The autonomic nervous system plays a regulatory role in the differentiation and growth of salivary glands, and in the expression of salivary specific genes. Cystatin S, a member of the evolutionarily conserved family 2 of the cysteine proteinase inhibitor superfamily, expressed in submandibular and parotid glands of rats during development, can be induced in adults by the beta-adrenergic agonist isopreterenol (IPR). It was shown previously that unilateral sympathectomy or bilateral parasympathectomy reduces IPR-induced cystatin S expression. The present experiments demonstrate that IPR-induced cystatin S gene expression in submandibular glands is reduced as early as 3 days post bilateral denervation of both branches of the autonomic nervous system. The reduction is nearly equal to that of either sympathectomy or parasympathectomy alone, suggesting that factor(s) in both sympathetic and parasympathetic fibers are simultaneously required for IPR-induced cystatin S gene expression.     

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).     

Maize cystatins respond to developmental cues, cold stress and drought

Comprehensive searches of maize EST data allowed us to identify 8 novel Corn Cystatin (CC) genes in addition to the previously known genes CCI and CCII. The deduced amino acid sequences of all 10 genes contain the typical cystatin family signature. In addition, they show an extended overall similarity with cystatins from other species that belong to several different phyto-cystatin subfamilies. To gain further insight into their respective roles in the maize plant, gene-specific expression profiles were established by semi-quantitative RT-PCR. While 7 CC genes were expressed in two or more tissues varying from gene to gene, CCI was preferentially expressed in immature tassels and CC8 and CC10 in developing kernels. As shown by in situ hybridisation of maize kernels, CC8 was specifically expressed in the basal region of the endosperm and CC10 both in the starchy endosperm and the scutellum of the embryo. The remaining, not kernel-specific genes, all had distinct expression kinetics during kernel development, generally with peaks during the early stages. In addition to developmental regulation, the effect of cold stress and water starvation were tested on cystatin expression. Two genes (CC8 and CC9) were induced by cold stress and 5 genes (CCII, CC3, CC4, CC5 and CC9) were down-regulated in response to water starvation. Taken together our data suggest distinct functions for CC genes in the maize plant.     

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.     

Proline-rich-protein promoters direct LacZ expression to the granular convoluted tubular cells of the submandibular gland in adult transgenic mice

The ability of two mouse PRP gene promoters to direct the expression of the bacterial lacZ reporter gene was tested in transgenic mice. Transgenes A1-lacZ and C1-lacZ consisted of 8.2 kb A1 and 7.8 kb C1 PRP promoters respectively fused to the lacZ coding sequence. A1 and C1 are two A-type PRP genes isolated from the inbred SWR mice, which show the same gene structure and similar sequence to the closely related MP2 and M14 PRP genes previously cloned from outbred CD-1 mice. We here show that both A1-lacZ and C1-lacZ transgenes have very similar expression patterns: (1) they expressed the lacZ gene in all 14 established transgenic lines under normal (non-stimulated) conditions; (2) the expression was restricted to the granular convoluted tubular cells of the submandibular glands; (3) the expression was developmentally regulated beginning at sexual maturation and lasting to at least 1.5 years of age; and (4) expression in some lines was probably influenced by sex hormones, since higher expression was found in males than in females. A1-lacZ and C1- lacZ are the first transgenes derived from the PRP/GRP (glutamine/glutamic acid-rich protein) gene superfamily to be expressed in the granular convoluted tubular cells (with known endocrine functions), rather than in the acinar cells (with mainly exocrine functions) of the submandibular glands     

Salivary cystatins influence ingestion of capsaicin-containing diets in the rat

Dietary capsaicin consumed by rats over several days induces cystatin-like substances in submandibular saliva. Yet the physiological role of these salivary proteins has not been thoroughly investigated. Salivary cystatins in the rat submandibular glands are known to be induced by chronic treatment with the sympathetic beta-agonist, isoproterenol. In the present study, the possible roles of the salivary proteins on food intake were examined by comparing consumption of a capsaicin-adulterated (0.05%) diet in rats with and without isoproterenol pretreatment (0.1 and 5.0 mg/kg, 5 days). Electrophoretic analysis performed prior to feeding trials revealed that the group pretreated with 5 mg/kg isoproterenol had large amounts of cystatin in the saliva compared with the group pretreated with 0.1 mg/kg isoproterenol and control group. The group treated with 5 mg/kg isoproterenol showed greater consumption of the capsaicin-adulterated diet than the other groups until the 3rd day of trials. Bilateral removal of the submandibular and sublingual glands neutralized the effects of isoproterenol. Induction of salivary cystatins by isoproterenol treatment was not mimicked by systemic and intragastric administration of capsaicin. These results suggest that cystatins are included in the salivary proteins induced by capsaicin and that they contribute to enhanced ingestion of the capsaicin diet. Induction of salivary cystatins may be triggered by irritation of the oral mucosa by capsaicin.     

Analysis of Blood Stem Cell Activity and Cystatin Gene Expression in a Mouse Model Presenting a Chromosomal Deletion Encompassing Csta and Stfa2l1

The cystatin protein superfamily is characterized by the presence of conserved sequences that display cysteine protease inhibitory activity (e.g., towards cathepsins). Type 1 and 2 cystatins are encoded by 25 genes of which 23 are grouped in 2 clusters localized on mouse chromosomes 16 and 2. The expression and essential roles of most of these genes in mouse development and hematopoiesis remain poorly characterized. In this study, we describe a set of quantitative real-time PCR assays and a global expression profile of cystatin genes in normal mouse tissues. Benefiting from our collection of DelES embryonic stem cell clones harboring large chromosomal deletions (to be reported elsewhere), we selected a clone in which a 95-kb region of chromosome 16 is missing (Del^16qB3Δ/+). In this particular clone, 2 cystatin genes, namely Csta and Stfa2l1 are absent along with 2 other genes (Fam162a, Ccdc58) and associated intergenic regions. From this line, we established a new homozygous mutant mouse model (Del^{16qB3Δ/16qB3Δ}) to assess the in vivo biological functions of the 2 deleted cystatins. Stfa2l1 gene expression is high in wild-type fetal liver, bone marrow, and spleen, while Csta is ubiquitously expressed. Homozygous Del^{16qB3Δ/16qB3Δ} animals are phenotypically normal, fertile, and not overtly susceptible to spontaneous or irradiation-induced tumor formation. The hematopoietic stem and progenitor cell activity in these mutant mice are also normal. Interestingly, quantitative real-time PCR expression profiling reveals a marked increase in the expression levels of Stfa2l1/Csta phylogenetically-related genes (Stfa1, Stfa2, and Stfa3) in Del^{16qB3Δ/16qB3Δ} hematopoietic tissues, suggesting that these candidate genes might be contributing to compensatory mechanisms. Overall, this study presents an optimized approach to globally monitor cystatin gene expression as well as a new mouse model deficient in Stfa2l1/Csta genes, expanding the available tools to dissect cystatin roles under normal and pathological conditions.     

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.     

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.