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.     

Transient appearance of CRES protein during spermatogenesis and caput epididymal sperm maturation

In previous studies we identified an epididymal gene that exhibits homology to the cystatin family of cysteine protease inhibitors. The expression of this gene, termed CRES (cystatin-related epididymal and spermatogenic), was shown to be highly restricted to the proximal caput epididymal epithelium with less expression in the testis and no expression in the 24 other tissues examined. In this report, studies were carried out to examine CRES gene expression in the testis as well as to characterize the CRES protein in the testis and epididymis. In situ hybridization experiments revealed that within the testis CRES gene expression is stage-specific during spermatogenesis and is exclusively expressed by the round spermatids of Stages VII-VIII and the early elongating spermatids of Stages IX and X. Immunohistochemical studies demonstrated that CRES protein was transiently expressed in both the testis and epididymis. Within the testis the protein was localized to the elongating spermatids, whereas within the epididymis CRES protein was exclusively synthesized by the proximal caput epithelium and then secreted into the lumen. Surprisingly, the secreted CRES protein had completely disappeared from the epididymal lumen by the distal caput epididymidis. Western blot analysis of testicular and epididymal proteins showed that the CRES antibody specifically recognized a predominant 19 kDa CRES protein and a less abundant 14 kDa form. These observations suggest that the CRES protein performs a specialized role during sperm development and maturation. © 1995 Wiley-Liss, Inc.     

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.     

Antimicrobial Activity and Molecular Mechanism of the CRES Protein

Cystatin-related epididymal spermatogenic (CRES) protein, a member of the cystatin superfamily of cysteine protease inhibitors (also known as CST8), exhibits highly specific, age-dependent expression in mouse testis and epididymis. The CRES protein possesses four highly conserved cysteine residues which govern the overall conformation of the cystatins through the formation of two disulfide bonds. Previous studies have revealed that other cystatin family members, such as cystatin 3 and cystatin 11, show antibacterial activity in vitro. This prompted us to investigate the potential antimicrobial activity of the CRES protein. Colony forming assays and spectrophotometry were used to investigate the effects of recombinant CRES protein on Escherichia coli (E. coli) and Ureaplasma urealyticum (Uu), respectively, in vitro. After incubation of E. coli with CRES recombinant protein fused with glutathione-S-transferase (GST), a substantial decrease in colony forming units was observed, and the effect was dose and time dependent. Furthermore, it took longer for Uu to grow to plateau stage when incubated with GST-CRES recombinant protein compared with the control GST. The antibacterial and Anti-Uu activities were not impaired when the cysteine residues of CRES protein were mutated, indicating that the antimicrobial effect was not dependent on its disulfide bonds. Functional analysis of three CRES polypeptides showed that the N-terminal 30 residues (N30) had no antimicrobial activity while N60 showed similar activity as full-length CRES protein. These results suggest that the active center of CRES protein resides between amino acid residues 31 and 60 of its N-terminus. Mechanistically, E. coli membrane permeabilization was increased in a dose-dependent manner, and macromolecular synthesis was inhibited on treatment with GST-CRES. Together, our data on the antimicrobial activities of CRES protein suggest that it is a novel and innate antimicrobial protein which protecting the male reproductive tract against invading pathogens.     

Cloning,Characterization and Primary Function Study of a Novel Gene,Cymg1,Related to Family 2 Cystatins

Cystatins are cysteine proteinase inhibitors,We found two expression sequence tags (ESTs),CA463109 and AV042522,from a mouse testis library using Digital differential display (DDD).By electricalhybridization,a novel gene,Cymgl(GenBank accession No.AY600990),which has a full length of 0.78 kb,and contains four exons and three introns,was cloned from a mouse testis eDNA library.The gene is locatedin the 2G3 area of chromosome 2.The full eDNA encompasses the entire open reading frame,encoding 141amino acid residues.The protein has a cysteine protease inhibitor domain that is related to the family 2cystatins but lacks critical consensus sites important for cysteine protease inhibition.These characteristicsare seen in the CRES subfamily,which are related to the family 2 cystatins and are expressed specifically inthe male reproductive tract.CYMG1 has a 44%(48/108)identity with mouse CRES and 30%(42/140)identity with mouse cystatin C.Northern blot analysis showed that the Cymgl is specifically expressed inadult mouse testes.Cell location studies showed that the GFP-tagged CYMG 1 protein was localized in thecytoplasm of HeLa cells,lmmunohistochemistry revealed that the CYMG1 protein was expressed in mousetestes spermatogonium,spermatocytes,round spermatids,elongating spermatids and spermatozoa.RT-PCRresults also showed that Cymgl was expressed in mouse testes and spermatogonium.The Cymgl expressionlevel varied in different developmental stages:it was low 1 week postpartum,steadily increased 2 to 5 weekspostpartum,and was highest 7 weeks postpartum.The expression level at 5 weeks postpartum was main-tained during 13 to 57 weeks postpartum.The Cymgl expression level in the testes over different develop-mental stages correlates with the mouse spermatogenesis and sexual maturation process.All these indicatethat Cymgl might play an important role in mouse spermatogenesis and sexual maturation. Cystatins are cysteine proteinase inhibitors,We found two expression sequence tags(ESTs),CA463109 and AV042522,from a mouse testis library using Digital differential display (DDD).By electricalhybridization,a novel gene,Cymgl(GenBank accession No.AY600990),which has a full length of 0.78 kb,and contains four exons and three introns,was cloned from a mouse testis eDNA library.The gene is locatedin the 2G3 area of chromosome 2.The full eDNA encompasses the entire open reading frame,encoding 141amino acid residues.The protein has a cysteine protease inhibitor domain that is related to the family 2cystatins but lacks critical consensus sites important for cysteine protease inhibition.These characteristicsare seen in the CRES subfamily,which are related to the family 2 cystatins and are expressed specifically inthe male reproductive tract.CYMG1 has a 44%(48/108)identity with mouse CRES and 30%(42/140)identity with mouse cystatin C.Northern blot analysis showed that the Cymgl is specifically expressed inadult mouse testes.Cell location studies showed that the GFP-tagged CYMG 1 protein was localized in thecytoplasm of HeLa cells,lmmunohistochemistry revealed that the CYMG1 protein was expressed in mousetestes spermatogonium,spermatocytes,round spermatids,elongating spermatids and spermatozoa.RT-PCRresults also showed that Cymgl was expressed in mouse testes and spermatogonium.The Cymgl expressionlevel varied in different developmental stages:it was low 1 week postpartum,steadily increased 2 to 5 weekspostpartum,and was highest 7 weeks postpartum.The expression level at 5 weeks postpartum was main-tained during 13 to 57 weeks postpartum.The Cymgl expression level in the testes over different develop-mental stages correlates with the mouse spermatogenesis and sexual maturation process.All these indicatethat Cymgl might play an important role in mouse spermatogenesis and sexual maturation.     

Cloning, characterization and primary function study of a novel gene, Cymg1, related to family 2 cystatins

Cystatins are cysteine proteinase inhibitors. We found two expression sequence tags (ESTs), CA463109 and AV042522, from a mouse testis library using Digital differential display (DDD). By electrical hybridization, a novel gene, Cymg1 (GenBank accession No. AY600990), which has a full length of 0.78kb, and contains four exons and three introns, was cloned from a mouse testis cDNA library. The gene is located in the 2G3 area of chromosome 2. The full cDNA encompasses the entire open reading frame, encoding 141 amino acid residues. The protein has a cysteine protease inhibitor domain that is related to the family 2 cystatins but lacks critical consensus sites important for cysteine protease inhibition. These characteristics are seen in the CRES subfamily, which are related to the family 2 cystatins and are expressed specifically in the male reproductive tract. CYMG1 has a 44% (48/108) identity with mouse CRES and 30% (42/140) identity with mouse cystatin C. Northern blot analysis showed that the Cymg1 is specifically expressed in adult mouse testes. Cell location studies showed that the GFP-tagged CYMG1 protein was localized in the cytoplasm of HeLa cells. Immunohistochemistry revealed that the CYMG1 protein was expressed in mouse testes spermatogonium, spermatocytes, round spermatids, elongating spermatids and spermatozoa. RT-PCR results also showed that Cymg1 was expressed in mouse testes and spermatogonium. The Cymg1 expression level varied in different developmental stages: it was low 1 week postpartum, steadily increased 2 to 5 weeks postpartum, and was highest 7 weeks postpartum. The expression level at 5 weeks postpartum was maintained during 13 to 57 weeks postpartum. The Cymg1 expression level in the testes over different developmental stages correlates with the mouse spermatogenesis and sexual maturation process. All these indicate that Cymg1 might play an important role in mouse spermatogenesis and sexual maturation.     

Molecular cloning and sequence analysis of a cDNA encoding a cysteine proteinase inhibitor fromSorghum bicolor seedlings

A 711-bp cDNA encoding a cysteine proteinase inhibitor (cystatin) was isolated from a cDNA library prepared from 7–10 cmSorghum bicolor seedlings. The nearly full-length cDNA clone encodes 130 amino acid residues, which include the Gln-Val-Val-Ala-Gly motif, conserved among most of the known cystatins as a probable binding site for cysteine proteinases. The amino acid sequence of sorghum cystatin deduced from the cDNA clone shows significantly homology to those of other plant cystatins. The sorghum cystatin expressed inE. coli showed a strong papain-inhibitory activity.     

Molecular cloning and sequence analysis of a cDNA encoding a cysteine proteinase inhibitor fromSorghum bicolor seedlings

A 711-bp cDNA encoding a cysteine proteinase inhibitor (cystatin) was isolated from a cDNA library prepared from 7–10 cmSorghum bicolor seedlings. The nearly full-length cDNA clone encodes 130 amino acid residues, which include the Gln-Val-Val-Ala-Gly motif, conserved among most of the known cystatins as a probable binding site for cysteine proteinases. The amino acid sequence of sorghum cystatin deduced from the cDNA clone shows significantly homology to those of other plant cystatins. The sorghum cystatin expressed inE. coli showed a strong papain-inhibitory activity.     

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     

Molecular cloning of cDNA for sarcocystatin A and analysis of the expression of the sarcocystatin A gene during development of Sarcophaga peregrina

Sarcocystatin A is a cysteine proteinase inhibitor purified from the hemolymph of Sarcophaga peregrina larvae [Suzuki, T., & Natori, S. (1985) J. Biol. Chem. 260, 5115-5120]. We isolated a cDNA clone for sarcocystatin A and analyzed the structure and expression of the sarcocystatin A gene. Sarcocystatin A consists of 102 amino acid residues. Significant homology was found between amino acid sequences of sarcocystatin A and other mammalian cystatins, and highly conserved sequences among mammalian cystatins were also found in sarcocystatin A. Using cloned cDNA as a probe, we investigated expression of the sarcocystatin A gene during the development of Sarcophaga. Results showed that this gene was transiently activated in the very early embryonic stage and in the pupal stage, suggesting that sarcocystatin A participates in morphogenesis of larval and adult structures of Sarcophaga.     

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

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

Comparative 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     

Modulating the proteinase inhibitory profile of a plant cystatin by single mutations at positively selected amino acid sites

Cysteine proteinase inhibitors of the cystatin superfamily have several important functions in plants, including the inhibition of exogenous cysteine proteinases during herbivory or infection. Here we used a maximum-likelihood approach to assess whether plant cystatins, like other proteins implicated in host-pest interactions, have been subject to positive selection during the course of their evolution. Several amino acid sites were identified as being positively selected in cystatins from either Poaceae (monocots) and Solanaceae (dicots). These hypervariable sites were located at strategic positions on the protein: on each side of the conserved glycine residues in the N-terminal trunk, within the first and second inhibitory loops entering the active site of target enzymes, and surrounding the larfav motif, a sequence of unknown function conserved among plant cystatins. Supporting the assumption that positively selected, hypervariable sites are indicative of amino acid sites implicated in functional diversity, mutants of the 8th cystatin unit of tomato multicystatin including alternative residues at positively selected sites in the N-terminal trunk exhibited highly variable affinities for the cysteine proteases papain, cathepsin B and cathepsin H. Overall, these observations support the hypothesis that plant cystatins have been under selective pressure to evolve in response to predatory challenges by herbivorous enemies. They also indicate the potential of site-directed mutagenesis at positively selected sites for the generation of cystatins with improved binding properties.     

Primary Structure of a Cysteine Proteinase Inhibitor from the Fruit of Avocado (Persea americana Mill)

The complete amino acid sequence of a proteinaceous cysteine proteinase inhibitor from the fruit of avocado (avocado cystatin) is presented. The protein consists of 100 amino acid residues and has a molecular mass of 11,300 Da. Comparison of this sequence with sequences of plant cysteine proteinase inhibitors (phytocystatins), including oryzacystatins I and II from rice seeds, cowpea cystatin, and corn cystatin, showed that the avocado cystatin molecule has 60% and 54% residues identical with the two forms of the rice seed proteins, oryzacystatins I and II, respectively, and 64% and 63% with the cowpea and corn proteins, respectively. The totally conserved sequence, Gln-Val-Val-Ala-Gly, among several of the animal cystatins as well as phytocystatins, is at positions 47-51 in the avocado cystatin molecule.     

Molecular cloning of a cysteine proteinase inhibitor of rice (oryzacystatin). Homology with animal cystatins and transient expression in the ripening process of rice seeds

A cDNA clone for a cysteine proteinase inhibitor of rice (oryzacystatin) was isolated from a lambda gt10 cDNA library of rice immature seeds by screening with synthesized oligonucleotide probes based on partial amino acid sequences of oryzacystatin. A nearly full-length cDNA clone was obtained which encoded 102-amino acid residues. The amino acid sequence of oryzacystatin deduced from the cDNA sequence was significantly homologous to those of mammalian cystatins, especially family 2 cystatins. Oryzacystatin contained the sequence Gln-Val-Val-Ala-Gly conserved among most members of the cystatin superfamily. The gene for oryzacystatin was transcribed into a single mRNA species of about 700 nucleotides. The content of mRNA reached its highest level 2 weeks after flowering and then gradually decreased to undetectable levels at 10 weeks. This feature of transient expression is coordinate with that of glutelin (a major storage protein), although the expression of oryzacystatin precedes that of glutelin by about 1 week.     

Immunolocalization of CRES (Cystatin-related epididymal spermatogenic) protein in the acrosomes of mouse spermatozoa.

The CRES (cystatin-related epididymal spermatogenic) protein is a member of the cystatin superfamily of cysteine protease inhibitors and exhibits highly restricted expression in the reproductive tract. We have previously shown that CRES protein is present in elongating spermatids in the testis and is synthesized and secreted by the proximal caput epididymal epithelium. The presence of CRES protein in developing germ cells and in the luminal fluid surrounding maturing spermatozoa prompted us to examine whether CRES protein is associated with spermatozoa. In the studies presented, indirect immunofluorescence, immunogold electron microscopy, and Western blot analysis demonstrated that CRES protein is localized in sperm acrosomes and is released during the acrosome reaction. Interestingly, while the 19- and 14-kDa CRES proteins were present in testicular and proximal caput epididymal spermatozoa, the 14-kDa CRES protein was the predominant form present in mid-caput to cauda epididymal spermatozoa. Furthermore, following the ionophore-induced acrosome reaction, CRES protein localization was similar to that of proacrosin/acrosin in that it was detected in the soluble fraction as well as associated with the acrosome-reacted spermatozoa. The presence of CRES protein in the sperm acrosome, a site of high hydrolytic and proteolytic activity, suggests that CRES may play a role in the regulation of intraacrosomal protein processing or may be involved in fertilization.     

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.