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Endocrinology Vol. 144, No. 3 909-915
Copyright © 2003 by The Endocrine Society

ARTICLE

Cres2 and Cres3: New Members of the Cystatin-Related Epididymal Spermatogenic Subgroup of Family 2 Cystatins

Nelson Hsia and Gail A. Cornwall

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430

Address all correspondence and requests for reprints to: Gail A. Cornwall, Ph.D., Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, Texas 79430. E-mail: gail.cornwall{at}ttuhsc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cystatin-related epididymal spermatogenic (CRES) and recently identified testatin and cystatin T proteins define a new subgroup within the family 2 cystatins of cysteine protease inhibitors. Members of the CRES subgroup are predominantly expressed in reproductive tissues and lack critical cystatin active-site sequences implying divergent functions. To determine whether there are additional members of the subgroup, we searched nucleotide databases and identified two novel genes that we designated Cres2 and Cres3. These genes, like other subgroup members, encode proteins with four conserved cysteine residues and predicted molecular weights characteristic of family 2 cystatins but have divergent cystatin inhibitory sequences. Furthermore, the genes exhibited reproductive-specific expression with Cres2 exclusively expressed in the epithelial cells of the proximal and midcaput epididymal regions and Cres3 expressed in the proximal caput epididymal epithelium, Sertoli cells of the testis, and early follicles and corpora lutea in the ovary. Additional studies showed that, like Cres, both Cres2 and Cres3 genes are dependent on testicular factors for epididymal expression. Taken together, CRES2 and CRES3 represent new members of a subgroup of cystatin family 2 proteins that likely carry out tissue-specific functions distinct from that of typical cystatins.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE FAMILY 2 CYSTATINS (cystatin C, D, SA, E, SN, S) of the cystatin superfamily of cysteine protease inhibitors are secreted proteins of 12–13 kDa that are broadly expressed and contain two characteristic intrachain disulfide bonds. Mutation analyses and x-ray crystallography have revealed three conserved regions in the cystatins that are thought to be critical for cysteine protease inhibition including an N-terminal glycine, a glutamine-valine-glycine (QXVXG) motif, and a C-terminal proline-tryptophan (PW) sequence (1). Although in vitro studies have established cystatins as inhibitors of papain-like cysteine proteases, the in vivo functions of these proteins have not been well characterized. However, putative roles in tumor invasion, inflammation, and prohormone processing have been proposed (2, 3, 4), and several neurological diseases have been attributed to cystatin gene mutations including familial epilepsy and amyloid angiopathy (5, 6).

The identification of divergent cystatin proteins such as cystatin-related epididymal spermatogenic (CRES; Ref. 7), testatin (8), and cystatin T (9), which are specifically expressed in the reproductive and neuroendocrine systems, led to the hypothesis that a subgroup of family 2 cystatins has evolved to perform tissue-specific functions distinct from the general housekeeping functions of the classic cystatins (10). Specifically, CRES, testatin, and cystatin T are secretory proteins of a similar molecular weight as the archetypal cystatin C and contain the four conserved cysteine residues that direct tertiary structure, thus predicting the proteins conformationally resemble the family 2 cystatins. However, these proteins have divergent N termini and lack the conserved QXVXG sequences suggesting distinct functions. In support, our recent in vitro studies showed that CRES did not inhibit the cysteine proteases papain or cathepsin B but rather inhibited the calcium-dependent serine protease prohormone convertase 2, an enzyme involved in the proteolytic maturation of prohormones in the neuroendocrine system (11).

In the present report, we describe the identification of two new members of the CRES subgroup of family 2 cystatins. Cres2 and Cres3 share the features of other subgroup members including reproductive expression and divergent cystatin inhibitory sequences.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
Mature male and female CD-1 mice were obtained from Charles River Laboratories, Inc. (Wilmington, MA) and were maintained under a constant 12-h light, 12-h dark cycle, with food and water ad libitum. Orchiectomies and efferent duct ligations were done by the scrotal route under ketamine/xylazine anesthesia. Sham and castrate mice were killed 14 d after castration, and efferent duct ligated mice were killed 7 d post ligation. Hormonal maintenance began at the time of surgery and included daily injections (sc) of vehicle, 25 µg testosterone propionate (12), 25 µg 5{alpha}-androstan-17ß-ol-3-one (DHT; Ref. 13), or 300 ng 17ß-estradiol (E2; Ref. 13) in 100 µl sesame oil for 2 wk. Animals were killed the day following the final injections. All animal studies were conducted in accordance with the principles and procedures outlined in the NIH Guidelines for the Care and Use of Experimental Animals.

RNA isolation and Northern blot analysis
Total RNA was isolated from mouse tissues using Trizol reagent (Invitrogen, Carlsbad, CA) in accordance with the manufacturer’s protocol. Northern blot analysis was carried out as previously described (7) with the following modifications. Membranes were hybridized for 1.5 h at 65 C in Church buffer containing 0.5 M NaPO4 buffer, pH 7.4; 7% sodium dodecyl sulfate; and 1 mM EDTA, followed by hybridization overnight at 65 C in the presence of 3 x 106 cpm probe/ml hybridization buffer. Cres2 and Cres3 cDNA probes were prepared using a random prime labeling method (Prime-It II, Stratagene, La Jolla, CA). After hybridization, the blots were washed twice in 1x saline sodium citrate, 0.1% sodium dodecyl sulfate at room temperature for 15 min, and then twice at 65 C for 15 min before exposure to film.

In situ hybridization
Antisense and sense [35S]-UTP-labeled Cres, Cres2, and Cres3 probes were generated from linearized plasmids using the Riboprobe Gemini system (Promega Corp., Madison, WI). In situ hybridization was carried out as previously described (7, 14). The slides were dipped in NTB-2 emulsion (Kodak, New Haven, CT) and exposed at 4 C for 1 wk for epididymal and testis sections and 2 wk for ovarian sections. Slides were developed with D19 Developer (Kodak), counterstained with toluidine blue, and viewed with a BX50 microscope (Olympus Corp., Leeds Instruments, Inc., Irving, TX) under light and dark field optics.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of Cres2 and Cres3 cDNAs
To identify new members of the CRES subgroup, we searched GenBank databases using nucleotide and amino acid sequences corresponding to the mouse CRES amino acids 76–142 (see Fig. 2CGo). This region represents the C-terminal half of CRES protein and includes the divergent QXVXG motif, four conserved cysteine residues, and the PW sequence. Sequences selected for further study were chosen based on the presence of the cysteine residues but nonconsensus QXVXG sites, thus predicting family 2-related proteins with potentially divergent functions.



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  		Figure 2. Relationship of CRES2 and CRES3 with the CRES subgroup of family 2 cystatins. A, Dendrogram showing the evolutionary relationship of mouse CRES2 and -3 proteins with the cystatin superfamily. The phylogenetic tree was constructed with MegAlign using the ClustalW method with the Gonnet series (DNASTAR Inc., Madison, WI). Signal sequences were removed before alignment, and only the cystatin domains in the kininogens were included. B, Percent sequence identity among CRES subgroup members. C, Alignment based on nearest neighbor of CRES2 and CRES3 amino acid sequences with other CRES subgroup members and mouse cystatin C. The arrowhead denotes the putative signal peptide cleavage site. Dashes indicate gaps introduced to optimize alignment. Bold lines indicate residues that are involved in cysteine protease inhibitory activities of family 2 cystatins. The four cysteine residues conserved in all family 2 cystatins are boxed.

 
Of the several sequences fulfilling these criteria, two showed reproductive tissue origins and were characterized further. Cres2 (GenBank accession no. AF454373) was an uncharacterized RIKEN cDNA (GenBank accession no. AK020300) isolated from the adult mouse epididymis. The 610-bp Cres2 cDNA included an open reading frame encoding a 139-residue proprotein of which the first 28 residues are predicted to represent a signal sequence (Fig. 1Go). Cres3 was an uncharacterized cDNA identified in mouse spermatocytes (GenBank accession no. BF019223). Recently, a database entry labeled this sequence cystatin TE-1 (GenBank accession no. AF440737). A comparison of the two Cres3 cDNA sequences showed a 2-bp insertion in the 5' end of the BF019223 clone that may be a result of cloning or sequencing errors because the frame shift would result in translation of only a short signal peptide sequence. The 580-bp Cres3 cDNA lacking the insertion encodes a 128-residue proprotein with a putative 21-residue signal sequence (Fig. 1Go).



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  		Figure 1. Nucleotide and predicted amino acid sequences of mouse Cres2 and Cres3 cDNAs. The predicted translation initiation codons are underlined (32 ), and the stop codons are indicated by asterisks. The predicted signal sequence cleavage sites are designated by arrowheads (33 ), and the polyadenylation signals are double underlined.

 
As shown in Fig. 2AGo, a dendrogram examining the evolutionary relationship of these new cystatin-related proteins with cystatin superfamily members demonstrated that they were related to the family 2 cystatins and, in particular, clustered within the CRES subgroup. CRES2 protein is most similar to CRES (33.6%) and cystatin T (32.9%), whereas CRES3 is more closely related to cystatin SC (50.3%), another recently identified subgroup member (AF440375) (15) and testatin (30.5%) (Fig. 2BGo). Both CRES2 and CRES3 are expected to be of a similar molecular weight as other subgroup members and contain the conserved cysteine residues; however, these proteins are not predicted to function as classic cystatins because they lack the N-terminal glycine and have divergent QXVXG sequences (Fig. 2CGo). Although the PW site is conserved in CRES2, CRES3 has an AW sequence. In contrast to CRES, which is present in tissues as N-linked glycosylated as well as nonglycosylated forms (16), CRES2 and CRES3 lack consensus sites for N-linked glycosylation. Another common feature among CRES2, CRES3, and other subgroup members is that they exhibit different N termini. Because the N terminus has been shown to be important for the specificity of the interaction between cystatin C and its target protease papain (17, 18), the divergence in N termini of CRES2 and CRES3 as well as in other subgroup members suggests that this region could impart some specificity in their functions.

Cres2 and Cres3 genes are expressed in the reproductive tract
To determine the tissue expression of Cres2 and Cres3 mRNAs, Northern blot analysis was performed on RNA from a variety of mouse tissues. Both genes showed tissue-specific expression with the Cres2 mRNA detected in the mouse epididymis and Cres3 present in the epididymis, testis, and ovary (Fig. 3AGo). The fact that the Cres2 and Cres3 mRNAs detected by Northern blot were slightly larger than the predicted sizes suggests some 5' untranslated sequences may be missing from the cDNAs. The Cres mRNA also has been shown to contain large 5' untranslated regions (10). Interestingly, the Cres3 mRNA detected in the epididymis and ovary was approximately 660 bp in size, compared with a larger 820-bp mRNA in the testis. In addition, a minor 2.2-kb mRNA was also detected in the testis. Although the mechanism(s) causing these mRNAs is not known, the smaller Cres3 mRNA may represent an alternatively spliced variant of the 820-bp transcript in the testis. This is not unlike Cres, which is present in tissues as a 700-bp mRNA as well as a 520-bp splice variant. Further studies need to be carried out to determine whether a similar splicing event is occurring in the Cres3 gene. Finally, it is possible that the different-size transcripts observed with the Cres3 cDNA may represent cross-reactivity with another CRES subgroup member. However, this is unlikely because the sequence identity among all subgroup members is low, and Southern blot analysis showed no cross-hybridization among Cres, Cres2, and Cres3 sequences (data not shown).



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  		Figure 3. Tissue expression of Cres2 and Cres3 genes. A, Northern blot analysis of 5 µg total RNA from various mouse tissues. B, Different regions of the mouse epididymis probed with Cres2 and Cres3 cDNAs. The blots were stripped and reprobed with 18S cDNA to confirm equal loading of RNA. Representative blots are shown. TE, Testis; EP, epididymis; VS, vas deferens; SV, seminal vesicle; BR, brain; PT, pituitary gland; SM, submaxillary gland; TH, thymus; PR, prostate; OV, ovary; OD, oviduct; UT, uterus; ST, stomach; SI, small intestine; LI, large intestine; SK, skeletal muscle; BL, bladder; HT, heart; LU, lung; LV, liver; SP, spleen; PN, pancreas; AD, adrenal; KD, kidney. Epididymal regions are 1, proximal caput; 2, midcaput; 3, distal caput; 4, corpus; and 5, cauda epididymidis.

 
Within the epididymis, both Cres2 and Cres3 exhibited regionalized expression and were highly restricted to the proximal caput region (Fig. 3BGo). In situ hybridization showed that within the proximal caput, Cres2 and -3 mRNAs were expressed by the principal cells, the columnar epithelial cells that actively synthesize and secrete proteins into the epididymal lumen (Fig. 4Go, A1–A6 and B1–B6, respectively) and that have been previously shown to also express Cres mRNA (7). The expression profiles for Cres2 and Cres3 were slightly different as Cres2 mRNA was also detected in a part of the midcaput epididymidis (Fig. 4A3Go), whereas Cres3 was not (Fig. 4B3Go).



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  		Figure 4. In situ hybridization of antisense and sense Cres, Cres2, and Cres3 RNA probes to the mouse epididymis and ovary. Mouse epididymal sections were hybridized with 35S-labeled Cres2 (A1–A4) and Cres3 (B1–B4) antisense probes and with corresponding sense probes (A5–A6, B5–B6). Ovarian sections were hybridized with Cres (C1–C4) and Cres3 (D1–D4, E1–E2) antisense probes and corresponding sense probes (C5–C6, D5–D6, E3). Sections were photographed under bright field (A1–A2, B1–B2, C1–C2, D1–D2, E1) and dark field (A3–A6, B3–B6, C3–C6, D3–D6, E2–E3) illumination. Bar, 225 µm (A1, A3, A5, B1, B3, B5, C1, C3, C5, D1, D3, D5); 45 µm (A2, A4, A6, B2, B4, B6, C2, C4, C6, D2, D4, D6); 20 µm (E1–E3). ED, Efferent ducts; 1, proximal caput; 2, midcaput epididymidis; CL, corpus luteum; F, follicle. To differentiate follicles from corpora lutea, histological analyses of hematoxylin- and eosin-stained ovarian sections were carried out. In addition to differences in cell morphology, corpora lutea were typically acidophilic, compared with the basophilic follicles (data not shown).

 
Northern blot analysis also detected Cres3 mRNA in the testis and ovary (Fig. 3AGo), and previous studies had also detected Cres mRNA in the ovary (Cornwall, G. A., unpublished observations). In situ hybridization revealed that, in the testis, Cres3 mRNA was present in all stages of the seminiferous tubules and associated with cells exhibiting finger-like projections toward the lumen, a distinguishing feature of Sertoli cells (Fig. 5Go). Although we cannot rule out that other cell types express Cres3, these studies demonstrate that the predominant cell type expressing Cres3 in the testis is the Sertoli cell. In further studies, in situ hybridization revealed overlapping expression of Cres and Cres3 mRNAs in the ovary. Cres mRNA was detected in corpora lutea but not in any stages of developing follicles (Fig. 4Go, C1–C6), whereas Cres3 was present in the corpora lutea as well as early follicles (Fig. 4Go, D1–D6 and E1–E3). Preliminary examination of several ovarian sections suggested that both Cres and Cres3 mRNAs were primarily associated with luteal cells of early corpora lutea rather than in cells undergoing luteolysis (data not shown). Furthermore, Cres3 mRNA also exhibited temporal expression in the developing follicles with mRNA detected in granulosa cells of primary and secondary follicles and little RNA present in the mature follicles (Fig. 4Go, D1–D6 and E1–E3).



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  		Figure 5. In situ hybridization of antisense and sense Cres3 RNA probes to the mouse testis. Mouse testis sections were hybridized with an 35S-labeled antisense Cres3 (A–D) or control, sense probe (E, F). Sections of seminiferous tubules were photographed under brightfield (A, C, E) and dark-field (B, D, F) illumination. Bar, 190 µm (A, B, E, F), 45 µm (C, D).

 
Regulation of Cres2 and Cres3 expression in the mouse epididymis
Because the epididymis is a highly androgen-dependent organ, studies were next carried out to investigate whether Cres2 and Cres3 mRNA expression was regulated by hormones. Cres, which has been previously shown to be regulated by testis factors rather than steroid hormones, was included for comparison (7). As shown in Fig. 6Go, similar to Cres, 2-wk castration resulted in a dramatic decrease in the levels of Cres2 and Cres3 mRNA, implying that androgens might be important for expression. However, the administration of testosterone (T), DHT, or E2 during the 2-wk castration period did not prevent the decrease in mRNA levels, suggesting that steroid hormones are not directly involved in the regulation of Cres2 and Cres3. Indeed, the loss of the estrogen receptor {alpha} gene also did not affect Cres, Cres2, or Cres3 mRNA levels, supporting that E2 is likely not directly involved in the regulation of these subgroup members (Fig. 6Go). Surprisingly, the administration of T and DHT to castrated mice resulted in a further decrease in Cres, Cres2, and Cres3 mRNA levels, compared with that in castrated mice that did not receive exogenous hormone (C) (Fig. 6Go). This response, however, likely reflects the recovery of androgen-dependent epididymal tubule contractions and a subsequent clearing of remaining testis factors from the proximal caput epididymal lumen rather than a direct effect of hormone (19).



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  		Figure 6. Regulation of Cres, Cres2, and Cres3 gene expression in the epididymis. Left panel, Northern blot analysis of 5 µg total RNA isolated from the epididymides of mice that were intact, (I); 2 wk bilaterally castrated, (C); and 2 wk bilaterally castrated with testosterone (+T), dihydrotestosterone (+DHT), or estradiol (+E2) maintenance. Right panel, Northern blot analysis of 5 µg total RNA isolated from the epididymides of mice 2 wk unilaterally castrated, intact side (I) and castrate side (C); and bilaterally efferent duct ligated (edl); heterozygous (+/-) and homozygous (-/-) c-kit mutant mice; and C/EBPß and estrogen receptor {alpha} gene knockout mice. Blots were stripped and reprobed with 18S cDNA to confirm equal loading of RNA. Representative blots are shown. T and DHT levels as determined by RIA of serum pooled from four to six males were within normal range for intact (0.81 ng/ml, 1.66 ng/ml, respectively), hormone-replaced mice (13.07 ng/ml, 9.5 ng/ml, respectively), and unilaterally castrate (0.8 ng/ml, 1.33 ng/ml, respectively) mice, whereas levels in castrate mice were substantially decreased (.02 ng/ml and 0.31 ng/ml, respectively).

 
To test whether factors from the testis may regulate Cres2 and Cres3, unilateral castration and efferent duct ligation experiments were performed. In the epididymis in which the connection between the testis and epididymis was maintained (unilateral castration, intact side, I), both genes were expressed (Fig. 6Go). However, in the contralateral epididymis (unilateral castrate, castrate side C) that did not receive input from the testis, the mRNAs were no longer detected. Similarly, mice in which the connecting tubules between the testis and epididymis were ligated, thereby preventing testicular fluid from entering the epididymis (efferent duct ligation), also showed a loss of Cres2 and Cres3 expression (Fig. 6Go). These data suggest that, like Cres, Cres2 and Cres3 are regulated by factors from the testis. Furthermore, the loss of Cres2 and Cres3 expression from the epididymides of c-kit mutant mice that are germ cell deficient (20) suggests that the testis factors that regulate these genes are associated with spermatogenesis (Fig. 6Go).

We have previously demonstrated that the transcription factor CCAAT/enhancer-binding protein (C/EBP)ß binds to the Cres gene promoter, is important for high levels of Cres gene expression in the epididymis and that the loss of the C/EBPß gene profoundly decreased epididymal Cres mRNA levels (21). Interestingly, in contrast to Cres, Cres2 and Cres3 mRNAs were unaffected by the loss of the C/EBPß gene, suggesting that, although these three CRES subgroup members exhibit overlapping patterns of expression in the proximal caput region, the mechanisms controlling their expression appear to differ (Fig. 6Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cres2 and Cres3: new members of the CRES subgroup
The studies presented herein describe Cres2 and Cres3, two new members of the CRES subgroup within the family 2 cystatins. With the identification of these two genes, the CRES subgroup now has six members all sharing the unique features of divergent cystatin inhibitory sequences and predominant expression in the reproductive tract. Furthermore, there is a remarkable overlap yet distinctive expression pattern for each CRES subgroup member. Cres, Cres2, and Cres3 are all expressed by the proximal caput epithelium. Cres is also expressed in round spermatids (22) as is cystatin T (23), whereas testatin (8), cystatin SC (15), and Cres3 are present in Sertoli cells. Finally, Cres and Cres3 overlap in their expression in the corpus luteum, whereas Cres3 is also in the follicle. Cres is also expressed by the anterior pituitary gland (16), and it is possible that other subgroup members may be expressed there as well. Studies are currently ongoing to examine Cres2 and Cres3 expression in other tissues using real-time RT-PCR.

Cres2 and Cres3 are also distinct from the typical cystatins by their highly regulated mRNA expression. Cres2 and Cres3 are like Cres as well as several other proximal caput epididymal-expressed genes in that they are dependent on unknown testicular factors for expression. In addition, we have previously determined that CRES protein levels in the gonadotroph cells are altered by the hormonal state of the animal (16) and more recently that Cres mRNA levels are negatively regulated by GnRH (Sutton-Walsh, H. G., and G. A. Cornwall, manuscript in preparation). Although studies have yet to determine whether Cres3 is regulated by hormones or other factors in the Sertoli cells and ovary, together the data suggest that subgroup members carry out highly regulated cell- and tissue-specific functions.

Potential functions of CRES2 and CRES3
The overlapping tissue- and cell-specific expression of all CRES subgroup members implies potential redundancies in function. However, the relatively low level of sequence identity among the proteins argues either divergent functions or alternatively, related but substrate-specific functions. Within the archetypal family 2 cystatins, there are also relatively low levels of sequence identity (30–40%) among family members, yet all inhibit C1 cysteine proteases (1). The inhibitory specificities of the cystatins for C1 cysteine proteases vary, however, with cystatin C and E/M inhibiting cathepsin B, whereas cystatin D and F do not (24). The structural element postulated to mediate this specificity is the N-terminal region (24). Our recent observation that CRES inhibited the serine protease prohormone convertase PC2 but did not inhibit the convertase family members PC1/3 or furin (11) raises the possibility that CRES2 and CRES3 may also function as protease inhibitors. Considering the differences in the N termini between CRES2 and -3 as well as in other regions of the proteins, it is possible that whereas there may be some overlap in inhibitory activities, these proteins may specifically inhibit other convertase family members or convertase-like proteases.

In support, like Cres, Cres2 and Cres3 localize to cellular sites that express proprotein convertases and are active in proprotein processing. For example, proprotein convertase family members PC1/3, PC4, and furin are expressed in the corpus luteum and Sertoli cells, respectively (25, 26, 27), whereas PC5, PACE4, and furin are expressed in the epididymis (28). Unlike the neuroendocrine system, little is known about proprotein processing in the reproductive tract. However, a number of proteins present in the testis are produced from precursors including growth factors TGFß and inhibin (29). Furthermore, several sperm-associated antigens including fertilinß and {alpha}-D-mannosidase are proteolytically processed during epididymal transit and convertase-like proteases have been implicated (30, 31) Therefore, it is possible that CRES2 and CRES3 may mediate proteolytic processing events that are an integral part of sperm development and maturation. Alternatively, CRES2 and CRES3 may have evolved to acquire new functions unrelated to protease inhibition. Studies are currently ongoing to determine whether CRES2 and CRES3 function as protease inhibitors and, in particular, as inhibitors of proprotein convertases.


   Footnotes
 
This work was supported by NIH Grants HD-33903 (to G.A.C.) and T32-HD-07271 (to N.H.).

Abbreviations: C/EBP, CCAAT/enhancer-binding protein; CRES, cystatin-related epididymal spermatogenic; DHT, 5{alpha}-androstan-17ß-ol-3-one; E2, 17ß-estradiol; PW, proline-tryptophan; T, testosterone.

Received August 26, 2002.

Accepted for publication November 21, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Turk V, Bode W 1991 The cystatins: protein inhibitors of cysteine proteinases. FEBS Lett 285:213–219[CrossRef][Medline]
  2. Calkins CC, Sloane BF 1995 Mammalian cysteine protease inhibitors: biochemical properties and possible roles in tumor progression. Biol Chem Hoppe Seyler 376:71–80[Medline]
  3. Leung-Tack J, Tavera C, Martinez J, Colle A 1990 Neutrophil chemotactic activity is modulated by human cystatin C, an inhibitor of cysteine proteases. Inflammation 14:247–258[CrossRef][Medline]
  4. Orlowski M 1983 Pituitary endopeptidases. Mol Cell Biochem 52:49–74[Medline]
  5. Pennacchio LA, Lehesjoki AE, Stone NE, Willour VL, Virtaneva K, Miao J, D’Amato E, Ramirez L, Faham M, Koskiniemi M, Warrington JA, Norio R, de la Chapelle A, Cox DR, Myers RM 1996 Mutations in the gene encoding cystatin B in progressive myoclonus epilepsy (EPM1). Science 271:1731–1734[Abstract]
  6. Grubb A, Jensson O, Gudmundsson G, Arnason A, Lofberg H, Malm J 1984 Abnormal metabolism of gamma-trace alkaline microprotein. The basic defect in hereditary cerebral hemorrhage with amyloidosis. N Engl J Med 311:1547–1549[Medline]
  7. Cornwall GA, Orgebin-Crist MC, Hann SR 1992 The CRES gene: a unique testis-regulated gene related to the cystatin family is highly restricted in its expression to the proximal region of the mouse epididymis. Mol Endocrinol 6:1653–1664[Abstract/Free Full Text]
  8. Tohonen V, Osterlund C, Nordqvist K 1998 Testatin: a cystatin-related gene expressed during early testis development. Proc Natl Acad Sci USA 95:14208–14213[Abstract/Free Full Text]
  9. Shoemaker K, Holloway JL, Whitmore TE, Maurer M, Feldhaus AL 2000 Molecular cloning, chromosome mapping and characterization of a testis-specific cystatin-like cDNA, cystatin T. Gene 245:103–108[CrossRef][Medline]
  10. Cornwall GA, Hsia N, Sutton HG 1999 Structure, alternative splicing and chromosomal localization of the cystatin-related epididymal spermatogenic gene. Biochem J 340(Part 1):85–93
  11. Cornwall GA, Cameron A, Lindberg I, Hardy DM, Cormier N, Hsia N 2003 The cystatin-related epididymal spermatogenic protein inhibits the serine protease prohormone convertase 2. Endocrinology 144:901–908[Abstract/Free Full Text]
  12. Naik SI, Young LS, Charlton HM, Clayton RN 1984 Pituitary gonadotropin-releasing hormone receptor regulation in mice. I: Males. Endocrinology 115:106–113[Abstract/Free Full Text]
  13. Fallest PC, Trader GL, Darrow JM, Shupnik MA 1995 Regulation of rat luteinizing hormone ß gene expression in transgenic mice by steroids and a gonadotropin-releasing hormone antagonist. Biol Reprod 53:103–109[Abstract]
  14. Cornwall GA, Collis R, Xiao Q, Hsia N, Hann SR 2001 B-Myc, a proximal caput epididymal protein, is dependent on androgens and testicular factors for expression. Biol Reprod 64:1600–1607[Abstract/Free Full Text]
  15. Li Y, Griswold MD Isolation, molecular cloning and characterization of a Sertoli cell-specific cystatin-related gene, cystatin SC. In: Regulatory mechanisms of testicular cell differentiation. Proc XVIth Testis Workshop 62, Newport Beach, CA, 2001, p 62 (Abstract I-47)
  16. Sutton HG, Fusco A, Cornwall GA 1999 Cystatin-related epididymal spermatogenic protein colocalizes with luteinizing hormone-ß protein in mouse anterior pituitary gonadotropes. Endocrinology 140:2721–2732[Abstract/Free Full Text]
  17. Abrahamson M, Mason RW, Hansson H, Buttle DJ, Grubb A, Ohlsson K 1991 Human cystatin C. Role of the N-terminal segment in the inhibition of human cysteine proteinases and in its inactivation by leucocyte elastase. Biochem J 273(Part 3):621–626
  18. Lindahl P, Nycander M, Ylinenjarvi K, Pol E, Bjork I 1992 Characterization by rapid-kinetic and equilibrium methods of the interaction between N-terminally truncated forms of chicken cystatin and the cysteine proteinases papain and actinidin. Biochem J 286(Part 1):165–171
  19. Hib J, Ponzio RO 1977 Effect of efferent duct ligation, gonadectomy and testosterone replacement on epididymal contractility in the rat. J Reprod Fertil 50:327–329[Abstract/Free Full Text]
  20. De Franca LR, Bartke A, Borg KE, Cecim M, Fadden CT, Yagi A, Russell LD 1994 Sertoli cells in testes containing or lacking germ cells: a comparative study of paracrine effects using the W (c-kit) gene mutant mouse model. Anat Rec 240:225–232[CrossRef][Medline]
  21. Hsia N, Cornwall GA 2001 CCAAT/enhancer binding protein ß regulates expression of the cystatin-related epididymal spermatogenic (Cres) gene. Biol Reprod 65:1452–1461[Abstract/Free Full Text]
  22. Cornwall GA, Hann SR 1995 Transient appearance of CRES protein during spermatogenesis and caput epididymal sperm maturation. Mol Reprod Dev 41:37–46[CrossRef][Medline]
  23. Friel PJ, Johnston DS, Wright WW, Shoemaker K, Holloway JL, Whitmore TE, Maurer M, Feldhaus AL, Griswold MDMolecular cloning and characterization of cystatin T, a novel testis-specific member of the cystatin family. Proc SSR, Madison, WI, 2000, p 335 (Abstract 584)
  24. Ni J, Fernandez MA, Danielsson L, Chillakuru RA, Zhang J, Grubb A, Su J, Gentz R, Abrahamson M 1998 Cystatin F is a glycosylated human low molecular weight cysteine proteinase inhibitor. J Biol Chem 273:24797–24804[Abstract/Free Full Text]
  25. Renegar RH, Owens CR, Dai G, McMurtry JP, Kwok SC 2000 Expression and localization of prohormone convertase 1/3 (SPC3) in porcine ovary. Mol Reprod Dev 57:361–365[CrossRef][Medline]
  26. Tadros H, Chretien M, Mbikay M 2001 The testicular germ-cell protease PC4 is also expressed in macrophage-like cells of the ovary. J Reprod Immunol 49:133–152[CrossRef][Medline]
  27. Torii S, Yamagishi T, Murakami K, Nakayama K 1993 Localization of Kex2-like processing endoproteases, furin and PC4, within mouse testis by in situ hybridization. FEBS Lett 316:12–16[CrossRef][Medline]
  28. Seidah NG, Chretien M, Day R 1994 The family of subtilisin/kexin like pro-protein and pro-hormone convertases: divergent or shared functions. Biochimie (Paris) 76:197–209[Medline]
  29. Skinner MK 1991 Cell-cell interactions in the testis. Endocr Rev 12:45–77[Abstract/Free Full Text]
  30. Lum L, Blobel CP 1997 Evidence for distinct serine protease activities with a potential role in processing the sperm protein fertilin. Dev Biol 191:131–145[CrossRef][Medline]
  31. Tulsiani DR, NagDas SK, Skudlarek MD, Orgebin-Crist MC 1995 Rat sperm plasma membrane mannosidase: localization and evidence for proteolytic processing during epididymal maturation. Dev Biol 167:584–595[CrossRef][Medline]
  32. Kozak M 1981 Possible role of flanking nucleotides in recognition of the AUG initiator codon by eukaryotic ribosomes. Nucleic Acids Res 9:5233–5262[Abstract/Free Full Text]
  33. Nielsen H, Engelbrecht J, Brunak S, von Heijne G 1997 Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6[Abstract/Free Full Text]



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