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Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center (G.A.C., D.M.H., N.C., N.H.), Lubbock, Texas 79430; Department of Biochemistry, University of Bristol (A.C.), Bristol, United Kingdom BS8 1TD; and Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center (I.L.), New Orleans, Louisiana 70112
Address all correspondence and requests for reprints to: Dr. Gail A. Cornwall, 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.
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Abstract |
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Introduction |
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The cystatin-related epididymal spermatogenic (CRES) protein defines a new subgroup within the family 2 cystatins by virtue of its low sequence identity (28%) with cystatin C but its conserved gene structure and cosegregation with the cystatin C gene to the distal region of mouse chromosome 2 (7). CRES also structurally resembles the cystatins by containing two putative intrachain disulfide bonds that dictate the tertiary structure of the proteins and by being a secreted protein. CRES, however, is not predicted to have the same biochemical activity as cystatins, because it possesses only the C-terminal PW site and lacks the two other consensus sites necessary for inhibition of C1 cysteine proteases (8). Furthermore, the N-terminal region of cystatin C responsible for its tight binding to cysteine proteases is poorly conserved in CRES, suggesting that the specificity of CRES differs from that of cystatin C. For these reasons, it has been difficult to predict whether CRES functions as a protease inhibitor or, alternatively, has acquired new noninhibitory functions as was shown for the cystatin-related protein fetuin (9).
CRES is also different from the cystatins in its reproductive- and neuroendocrine-specific expression. CRES is highly expressed in a region-dependent manner in the epididymis (8), postmeiotic germ cells in the testis (10), and anterior pituitary gonadotroph cells (11). In addition, we recently detected expression in the ovary (12). Furthermore, we have shown that CRES is localized within the mouse sperm acrosomal cap (13) and the human sperm equatorial segment (14) suggesting that CRES may function during fertilization. The recent identification of testatin (15), cystatin T (16), and other CRES-like genes (12) and their high levels of expression in reproductive tissues indicate that the CRES subgroup of proteins is diverse and may have evolved to perform tissue-specific functions. Interestingly, these subgroup members are similar to CRES in that they are expected to structurally resemble cystatins, but because they possess only the C-terminal PW site, their functions are likely to differ. Like CRES, however, the functions of testatin and cystatin T are not known.
The prohormone convertases (PC) are calcium-dependent serine proteases with catalytic domains related to bacterial subtilisins. The seven mammalian members of the convertase family function within the secretory pathway, where they cleave proproteins at mono- or dibasic sites to generate mature proteins (17, 18). Several of the family members are ubiquitously expressed, whereas PC1, PC2, and PC4 are primarily found within the neuroendocrine and reproductive systems, respectively. Like other proteases, endogenous inhibitors probably control the activities of prohormone convertases. However, to date only two convertase inhibitors have been identified within the secretory pathway: 7B2, which inhibits PC2 at nanomolar concentrations (19), and pro-SAAS, which inhibits PC1 at high nanomolar concentrations (20, 21, 22). Therefore, it is likely that other convertase inhibitors exist.
The purpose of the present study was to determine whether CRES functions as a protease inhibitor in in vitro assays. In this report we demonstrate that CRES, although structurally belonging to the cystatins, is not an inhibitor of papain or cathepsin B, but, rather, is a novel cross-class inhibitor of the serine protease PC2. Our results suggest that CRES and perhaps other CRES-like proteins may mediate proprotein-processing events within the reproductive and neuroendocrine systems.
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Materials and Methods |
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T3-1 and LßT2 gonadotroph cell lines were gifts from Pam Mellon (University of California-San Diego, La Jolla, CA). GH3 cells were obtained from American Type Culture Collection (Manassas, VA). The T3-1 and LßT2 cells were cultured in 100-mm tissue culture dishes in DMEM with 4.5 mg/ml glucose, 5% fetal calf serum, 5% calf serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin at 37 C in an atmosphere of 5% CO2. GH3 cells were maintained in DMEM with 10% fetal calf serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Media and antibiotics were obtained from Invitrogen (Grand Island, NY), and serum was purchased from HyClone Laboratories, Inc. (Logan, UT).
Enzymes and substrates
Papain, trypsin (bovine pancreas), human cathepsin B, subtilisin, and aprotinin were purchased from Sigma-Aldrich (St. Louis, MO). PC1, PC2, and the proprotein convertase furin were prepared as described previously (23, 24). The substrates Boc-Phe-Ser-Arg-MCA and pGlu-Arg-Thr-Lys-Arg-MCA were purchased from Peptides International (Louisville, KY), whereas Z-Phe-Arg-MCA was obtained from Peninsula Laboratories, Inc. (San Carlos, CA), and Suc-Ala-Ala-Pro-Phe-MCA was obtained from Sigma-Aldrich.
Preparation of histidine (His) fusion proteins
A 366-bp CRES cDNA and a 306-bp cystatin C cDNA containing the coding sequence minus the signal peptide sequence were generated by PCR from plasmids containing the mouse CRES or cystatin C cDNAs, respectively. The amplified products were cloned into the pGEM-T-Easy vector (Promega Corp., Madison, WI), followed by restriction digestion and ligation into the pQE9 expression plasmid (QIAGEN, Chatsworth, CA), resulting in CRES or cystatin C sequences that are downstream of a 6-His tag. An N-terminally truncated CRES protein lacking the first 16 amino acids of the mature protein (CRES
1–16) was prepared as previously described (10). cDNAs were sequenced by the Texas Tech University Biotechnology Core facility to verify in-frame cloning with the His tag.
The His fusion proteins were expressed in Escherichia coli M15[pREP4] and purified under denaturing conditions from inclusion bodies by nickel affinity chromatography following the manufacturer’s protocol (QIAGEN). To allow refolding of the proteins, the eluted fraction containing His-CRES or His-cystatin C fusion protein was dialyzed overnight at 4 C against 25 mM 2-(4-morpholino)-ethane sulfonic acid buffer (pH 5) containing 1 mM dithiothreitol, 5% glycerol, and 1 M guanidine, followed by changes into the same dialysis buffer containing 0.1 M guanidine, and then dialysis in buffer without guanidine. The protein amounts were quantified using the bicinchoninic acid assay (Pierce Chemical Co., Rockford, IL), concentrated by Centricon filters (cut-off, 3 kDa; Millipore Corp., Bedford, MA) if necessary, aliquoted, and stored at -20 C. A portion of the dialysis buffer from the final dialysis step was also aliquoted and stored at -20 C.
Enzyme assays
Trypsin assays were performed with 0.26 nM trypsin in 100 mM Tris buffer (pH 8), 10 mM CaCl2, and 1 mM Boc-Phe-Ser-Arg-MCA. Papain (0.18 nM) and cathepsin B (39 nM) were assayed in 100 mM sodium phosphate buffer (pH 6), 1 mM dithiothreitol, and 2 mM EDTA with 20 µM Z-Phe-Arg-MCA. Subtilisin (0.25 nM) was assayed in 100 mM HEPES (pH 7.4), 0.5% Triton X-100, 1 mM CaCl2, and 1 mM ß-mercaptoethanol with 25 µM Suc-Ala-Ala-Pro-Phe-MCA. PC2 (0.7 nM) was assayed in 100 mM sodium acetate (pH 5), 0.1% Brij, and 1 mM CaCl2 with 50 µM pGlu-Arg-Thr-Lys-Arg-MCA. Before use in the assays, pro-PC2 was diluted into 2x PC2 assay buffer and incubated at room temperature for 20 min to allow conversion to the active form (25). Reactions were initiated by the addition of substrate, and the enzymatically released MCA was measured using an Bowman fluorescence spectrophotometer (Aminco, Urbana, IL) with an excitation wavelength of 380 nm and an emission wavelength of 460 nm. Continuous rates were measured for 100–240 sec, during which linear progress curves were observed for each enzyme tested. All reactions were carried out in an 80-µl reaction volume. To test various inhibitors, inhibitor and enzyme were diluted into the appropriate enzyme buffer, and reactions were initiated by the addition of substrate. To control for the possible effects of the recombinant protein buffer on enzyme activity, a volume of dialysis buffer equivalent to the volume of the His-CRES or His-cystatin C protein was added to enzymes assayed in the absence of inhibitor. In some experiments CRES1–16 or CRES protein that had been heated at 100 C for 10 min was tested in inhibition assays of PC2.
To compare CRES inhibitory activities against the different prohormone convertase family members, increasing concentrations of His-CRES were combined with 7 nM PC2, 50 nM PC1, or 30 nM furin for 30 min at room temperature in a 96-well plate before the addition of 200 µM pGlu-Arg-Thr-Lys-Arg-MCA. PC1 was assayed in the same buffer as PC2. The assay for furin was performed using the same substrate as for PC1 and PC2, but in 100 mM HEPES (pH 7), 5 mM CaCl2, and 0.1% Brij (24). The Km values of PC1, PC2, and furin were 11, 42, and 8 µM, respectively as determined using a computerized least squares fitting technique with EnzFitter (Biosoft, Cambridge, UK). Unless stated otherwise, the data for all assays are reported as a percentage of the control, with control representing enzyme activity in the absence of inhibitor (100%). All assays were performed three to five times, and the mean ± SEM for each point are presented.
Kinetics of PC2 inhibition by CRES
A kinetic analysis of CRES inhibition of PC2 activity was performed by incubation of 0.7 nM preactivated PC2 with increasing concentrations of His-CRES protein for 30 min at room temperature, followed by the addition of 25–100 µM pGlu-Arg-Thr-Lys-Arg-MCA. Enzyme activity was determined by a continuous rate measurement of substrate hydrolysis. Kinetic constants were determined using nonlinear least squares regression analysis of the raw data using the software package DynaFit (Biokin Ltd., Pullman, WA).
RT-PCR
Total RNA was isolated from mouse tissues and cell lines using TRIzol reagent (Invitrogen, Grand Island, NY) following the manufacturer’s protocol. The RNA was quantitated by A260/A280 and visualized by gel electrophoresis in 1% agarose gel containing borate buffer (pH 8.2) and 0.66 M formaldehyde. For RT-PCR, 2.5 µg RNA were incubated in RT reaction buffer containing 5 mM MgCl2, 50 mM KCl, 10 mM Tris (pH 8.3), 0.5 mM deoxynucleotide triphosphates (dNTPs), 20 U RNasin (ribonuclease inhibitor, Promega Corp.), and 2.5 µM oligo(deoxythymidine) (Promega Corp.) for 30 min at 37 C in the presence of 2.5 U ribonuclease-free deoxyribonuclease I (Roche, Indianapolis, IN). After heat inactivation of deoxyribonuclease I at 75 C for 5 min, an aliquot was reserved for PCR amplification as a no RT control to ensure the removal of all DNA. Moloney murine leukemia virus reverse transcriptase (50 U; Roche) was added to the remainder, and the reactions were incubated at 42 C for 60 min, 99 C for 5 min, and 5 C for 5 min.
Two and a half microliters of each RT and no RT reaction were amplified by PCR in separate reactions using primers recognizing PC2, CRES, and S16 cDNAs. S16 RNA was amplified as a constitutive control to measure the relative efficiency of each RT reaction. PCR master mixes containing 10 mM Tris (pH 8.3), 50 mM KCl, 0.5 µM each of forward and reverse primers, and 1.25 U Taq DNA polymerase (Fisher Scientific, Pittsburgh, PA) were prepared for each set of PCR primers so that all compared PCRs were from the same master mix. MgCl2 and dNTP concentrations were optimized for each set of primers. PC2 PCRs were carried out in 1.5 mM MgCl2 and 0.14 mM dNTPs for 40 cycles. CRES reactions consisted of 2.2 mM MgCl2 and 0.19 mM dNTPs for 40 cycles, and S16 reactions were amplified using 2 mM MgCl2 and 0.14 mM dNTPs for 25 cycles. The cycling parameters consisted of 45 sec at 95 C for denaturing, 25 sec at annealing temperature for each primer set, and 1 min at 72 C for extension, after which the reactions were incubated at 72 C for 7 min using a minicycler (MJ Research, Inc., Watertown, MA). RT-PCR products were analyzed by electrophoresis on 1.5% agarose/1x Tris/acetate/EDTA gels. RT-PCR products generated with each primer pair were cloned into the pGEM-T-Easy vector (Promega Corp.) and sequenced to confirm identities.
Oligonucleotide primers
PCR primers (Invitrogen, La Jolla, CA) were designed from the known sequences for mouse PC2, CRES, and S16 cDNAs using PrimerSelect 5.0 (DNASTAR, Madison, WI): PC2 sense, 5'-GGC GGC CGG GTT CCT CTT CT-3'; antisense, 5'-GTT CCA TCG GCT TGC CCA GTG TT-3' (annealing temperature, 59 C); CRES sense, 5'-CAA GGA AAG TGA GGA CAA ATA TGT C-3'; antisense, 5'-GTG ACA GAC TTG AAC CAC AGG TT-3' (annealing temperature, 64 C); and S16 sense, 5'-CGC TGC AGT CCG TGC AGG TGT T-3'; antisense, 5'-TCC AAA CTT TTT GGA TTC GCA GCG-3' (annealing temperature, 57 C).
Western blot analysis
The T3-1 cells were grown to confluence and scraped into buffer containing 25 mM Tris (pH 7.4), 100 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM phenylmethylsulfonylfluoride, 0.5 mM p-chloromercuriphenylsulfonic acid, and 10 mM iodoacetamide. Cells were lysed by Polytron (Brinkmann Instruments, Inc., Westbury, NY), and lysates were centrifuged at 12,000 x g to remove insoluble material. Mouse testis and pituitary tissue were lysed by Polytron in buffer containing 20 mM Tris (pH 7.4), 50 mM NaCl, 0.5% Triton X-100, 0.5% deoxycholic acid, 0.5% sodium dodecyl sulfate, 0.5% aprotinin, 1 mM EDTA, and 8.6 mM iodoacetamide, followed by centrifugation at 12,000 x g. Protein concentrations were determined by the bicinchoninic acid reagent (Pierce Chemical Co.).
Ten to 40 µg protein were separated on 10% (PC2) or 15% (CRES) SDS-PAGE gels under reducing conditions, followed by transfer to polyvinylidene difluoride membrane (Millipore Corp.). Blots were incubated for 1 h with Tris-buffered saline [50 mM Tris-HCl (pH 7.4) and 200 mM NaCl] containing 0.2% Tween 20 (TBS-T) and 3% (wt/vol) nonfat dry milk at room temperature, followed by incubation with a polyclonal rabbit antimouse PC2 peptide antiserum (1:4,000) (26) or an affinity-purified polyclonal rabbit antimouse CRES antibody (0.2 µg antibody/cm2 membrane) at 4 C overnight. The blots were washed three times for 10 min each time in TBS-T and incubated with a goat antirabbit secondary antibody conjugated to horseradish peroxidase (Biosource Technologies, Inc., Camarillo, CA) at 1:20,000 for 2 h at room temperature. The blots were washed extensively in TBS-T, followed by 20 min in TBS, incubated with Supersignal reagent (Pierce Chemical Co.) for 5 min, and exposed to film.
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). Because CRES is expressed at sites of proprotein processing, and the CRES sequence contains several monobasic and dibasic sites, we next tested whether CRES could inhibit proprotein-processing enzymes, specifically prohormone convertases. Incubation of CRES with PC2 resulted in a dramatic decrease in the rate of substrate hydrolysis by PC2 compared with activity in the absence of CRES (Fig. 1). In contrast, cystatin C did not inhibit PC2 activity, demonstrating the specificity of the CRES/PC2 interaction. CRES did not inhibit the related serine protease subtilisin or trypsin, a serine protease with broad substrate specificities, supporting the idea that CRES is a specific inhibitor of PC2 and not a general inhibitor of serine proteases (Fig. 1). CRES also did not inhibit the convertase family members PC1 and furin, further indicating that CRES may be a specific inhibitor of PC2 (Fig. 2).
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. From this and the results of nonlinear least squares regression analysis of different inhibition types using the software package Dynafit (Biokin Ltd.), we concluded that CRES is a competitive inhibitor of PC2 with a Ki of 25 ± 3.1 nM.
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1–16. As shown in Fig. 4A, the truncated protein was less effective as a PC2 inhibitor than the full-length protein, implying that the N-terminal sequences are important for CRES inhibitory activity. At this time, however, we cannot rule out the possibility that the deletion of 16 amino acids could also induce a major structural change in the CRES protein that subsequently could affect its inhibitory activity. The heat stability of CRES was also assessed by heating CRES protein for 10 min at 95 C and then examining its inhibitory activity against PC2. Heat-treated CRES inhibited PC2 no differently than untreated CRES, suggesting that CRES is a stable protein and resistant to inactivation by heat (Fig. 4B).
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, both Cres and PC2 mRNAs were detected at varying levels in mouse testis, epididymis, ovary, and pituitary as well as in the T3-1 gonadotroph cells. PC2 was also detected in the somatotroph/lactotroph cell line GH3, but surprisingly was not detected in LßT2 cells, a gonadotroph cell line that expresses both the - and ß-subunits of LHß (27). Because LßT2 cells represent a more differentiated gonadotroph cell than T3–1 cells, which express only the -subunit (28), the lack of PC2 expression could reflect the absence of specific hormonal stimuli. Studies are currently in progress to determine whether the administration of steroid hormones and/or GnRH will induce PC2 mRNA expression in LßT2 cells. Alternatively, the lack of PC2 expression in these cells could be a result of the simian virus 40-mediated cell transformation used to generate the cell line (27).
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T3-1 cells, Western blot studies were next carried out to examine protein. Analysis of T3-1 cell extracts showed the presence of the 68-kDa PC2 protein as well as small amounts of the proform of PC2 (Fig. 5B
). Both PC2 proteins were blocked when the antibody was preincubated with immunizing peptide, indicating the specificity of the antibody (data not shown) (26). We have previously shown that there are several forms of CRES protein in the epididymis and testis, including a predominant 19-kDa N-linked glycosylated protein and a 14-kDa nonglycosylated protein as well as a 29-kDa protein that may represent a sodium dodecyl sulfate-stable CRES protein dimer (Fig. 5B) (10, 11, 13). As shown in Fig. 5B, CRES protein was also expressed in T3-1 cells. The majority of CRES was present as the 29-kDa protein, previously identified as the major CRES form in the LßT2 cells and pituitary gland (11). Longer exposure of the Western blot also revealed low levels of the 19-kDa monomeric CRES protein in the T3-1 cell extracts, whereas the 14-kDa CRES protein was not detected. The higher molecular mass protein of approximately 60 kDa present in the testis and at much lower levels in T3-1 cells may represent a cross-reacting epitope or a sodium dodecyl sulfate-stable CRES protein complex. All of the aforementioned CRES proteins disappeared or were dramatically reduced after incubation of similar blots with an antiserum depleted of CRES antibody, thus indicating that they represent specific interactions (data not shown). |
Discussion |
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An analysis of the kinetics of the CRES/PC2 interaction revealed competitive inhibition, indicating that CRES binds to the enzyme active site. Thus, CRES has a mechanism of action similar to that of 7B2 and pro-SAAS, which are competitive inhibitors of PC2 and PC1, respectively (20, 21, 29). PC2 inhibition by the 7B2 CT peptide has been previously shown to require a highly conserved VNPYLQG site in addition to a dibasic pair (30). The fact that a form of CRES lacking 16 amino acids from the N terminus inhibits PC2, albeit at greatly reduced levels, implies that the inhibitory region is located elsewhere. The C-terminal portion of CRES contains several pairs of basic residues (Fig. 6). Of these, the last pair (Lys115-Lys116, mouse, rat; Arg115-Lys116, human) is well conserved; in addition, the P4 aliphatic hydrophobic residue (Leu113), previously found to be important for inhibition of PC2 by the 7B2 CT peptide (31, 32), is present. Mutagenesis experiments will be required to define the actual sequences within CRES, including those within the N terminus, that participate in the inhibition of PC2; such studies are now in progress.
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Taken together, our studies suggest that CRES functions as a novel cross-class inhibitor with a structural relationship to the cystatins, but a functional relationship to serine protease inhibitors or serpins. Although there are several examples of cross-class inhibition between families of cysteine and serine proteases, most are of inhibition of cysteine proteases by serpins. For example, squamous cell carcinoma antigen 2 (SCCA2) is 92% identical to SCCA1, a well-characterized serpin; however, it does not inhibit chymotrypsin, a traditional serpin target, but, rather, inhibits the cysteine proteases cathepsins L and S (33). More recently, SQN-5, a serpin similar to SCCA1 and -2, was shown to exhibit dual mechanistic class inhibition by inhibiting both serine and cysteine proteases (34). Another example of cross-class inhibition has been identified within the apoptotic pathway. The viral cowpox protein crmA, a serpin, is a potent inhibitor of the IL-1B-converting enzyme, a cysteine protease (35). In addition, the serpin protease nexin2 has been shown to inhibit prohormone thiol protease, a cysteine protease (36). Finally, a synthetic peptide representing a domain of cystatin SA that contains the PW motif conserved in CRES and CRES-related proteins was shown to possess inhibitory activity against both cysteine and serine proteases (37), suggesting that, like serpins, some cystatins may also interact with other families of proteases. Indeed, our observation that CRES inhibits a serine protease rather than a cysteine protease, is one of the first examples of a cystatin that has acquired new protease inhibitory functions and exhibits cross-class inhibition of a serine protease. Most serpins inhibit their target proteases by forming a 1:1 stochiometric complex with the active site of the protease, resulting in sodium dodecyl sulfate-stable complexes (38). CRES does not appear to inhibit by the same mechanism as serpins, because preliminary analyses using recombinant proteins failed to reveal higher molecular mass complexes of CRES and PC2 by SDS-PAGE (Cornwall, G. A., et al., unpublished observations). Also, the kinetics of the CRES/PC2 interaction do not support the mechanism of irreversible protease inactivation exhibited by serpins. Finally, we cannot rule out that CRES may inhibit members of other families of cysteine proteases. However, preliminary studies indicate that CRES does not inhibit caspase-3, a member of the C14 family of cysteine proteases (Hsia, N., and G. A. Cornwall, unpublished observations), and it is also unlikely that CRES inhibits the C13 cysteine protease, legumain, because it lacks the Asn39 residue that is thought to be important for legumain binding by cystatin C (4). Mammalian homologs for most of the other families of cysteine proteases have yet to be identified.
Our data showing that CRES functions as a protease inhibitor, and in particular as a cross-class inhibitor of the serine protease PC2, represent a critical first step toward understanding the biological roles that CRES may perform in the neuroendocrine and reproductive systems. The Ki values we observed are low enough to suggest that CRES inhibition of PC2 is physiologically relevant. Indeed, our previous studies showing the colocalization of CRES with LHß in anterior pituitary gonadotroph cells in vivo and the presence of CRES in granular extracts from LßT2 cells (11), taken together with other reports showing that PC2 is expressed in gonadotroph cells (39, 40), suggest that within the pituitary gland CRES and PC2 may interact. In support of this, our RT-PCR and Western blot studies show coexpression of CRES and PC2 mRNA and protein in T3-1 gonadotroph cells. The role of CRES as a PC2 inhibitor in the testis and epididymis is less clear. PC2 has been shown previously to be expressed in the epididymis (41), and our RT-PCR studies confirm this. However, the epididymal cell population expressing PC2 has not been determined. In the testis the PC4 family member appears to be the predominant convertase and is present in round spermatids (42), where CRES is also localized, whereas in the ovary PC1/3 and PC4 are expressed in the corpus luteum (43, 44), where we have recently also detected Cres mRNA (12). Our RT-PCR analyses show, however, that PC2 mRNA is also present in the testis and ovary, and further studies are needed to identify the relevant cell populations. At this time it remains possible that CRES may perform distinct functions in different tissues and consequently may inhibit other convertase family members in these tissues. Alternatively, CRES may inhibit PC2-like proteases that have yet to be identified. Studies are currently in progress to examine CRES inhibition of other convertase family members as well as to assess PC2 expression in the epididymis, testis, and ovary. In addition, biochemical approaches are being used to examine CRES protein interactions in vivo. Taken together, the studies presented herein are significant in that they are the first to identify a function for a member of a growing family of cystatin-related proteins.
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Footnotes |
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Abbreviations: CRES, Cystatin-related epididymal spermatogenic; dNTP, deoxynucleotide triphosphate; His, histidine; MCA, methylcoumaryl amide; PC, prohormone convertase; SCCA, squamous cell carcinoma antigen; TBS-T, Tris-buffered saline containing 0.2% Tween 20.
Received October 25, 2002.
Accepted for publication November 6, 2002.
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-trace alkaline microprotein. The basic defect in hereditary cerebral hemorrhage with amyloidosis. N Engl J Med 311:1547–1549[Medline]
2-HS glycoprotein is a transforming growth factor-ß type II receptor mimic and cytokine antagonist. J Biol Chem 271:12755–12761This article has been cited by other articles:
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  H. H. von Horsten, S. S. Johnson, S. K. SanFrancisco, M. C. Hastert, S. M. Whelly, and G. A. Cornwall Oligomerization and Transglutaminase Cross-linking of the Cystatin CRES in the Mouse Epididymal Lumen: POTENTIAL MECHANISM OF EXTRACELLULAR QUALITY CONTROL J. Biol. Chem., November 9, 2007; 282(45): 32912 - 32923. [Abstract] [Full Text] [PDF]
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 V. Thimon, M. Belghazi, J.-L. Dacheux, and J.-L. Gatti Analysis of furin ectodomain shedding in epididymal fluid of mammals: demonstration that shedding of furin occurs in vivo. Reproduction, December 1, 2006; 132(6): 899 - 908. [Abstract] [Full Text] [PDF]
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 H. G. Sutton-Walsh, S. Whelly, and G. A. Cornwall Differential Effects of GnRH and Androgens on Cres mRNA and Protein in Male Mouse Anterior Pituitary Gonadotropes J Androl, November 1, 2006; 27(6): 802 - 815. [Abstract] [Full Text] [PDF]
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 Y. Li, C. A. Putnam-Lawson, H. Knapp-Hoch, P. J. Friel, D. Mitchell, R. Hively, and M. D. Griswold Immunolocalization and Regulation of Cystatin 12 in Mouse Testis and Epididymis Biol Reprod, November 1, 2005; 73(5): 872 - 880. [Abstract] [Full Text] [PDF]
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 V. Tohonen, J. Frygelius, M. Mohammadieh, U. Kvist, L. J. Pelliniemi, K. O'Brien, K. Nordqvist, and A. Wedell Normal Sexual Development and Fertility in testatin Knockout Mice Mol. Cell. Biol., June 15, 2005; 25(12): 4892 - 4902. [Abstract] [Full Text] [PDF]
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N. Hsia, J. P. Brousal, S. R. Hann, and G. A. Cornwall Recapitulation of Germ Cell- and Pituitary-Specific Expression With 1.6 kb of the Cystatin-Related Epididymal Spermatogenic (Cres) Gene Promoter in Transgenic Mice J Androl, March 1, 2005; 26(2): 249 - 257. [Abstract] [Full Text] [PDF]
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 M. J. Richer, C. A. Keays, J. Waterhouse, J. Minhas, C. Hashimoto, and F. Jean The Spn4 gene of Drosophila encodes a potent furin-directed secretory pathway serpin PNAS, July 20, 2004; 101(29): 10560 - 10565. [Abstract] [Full Text] [PDF]
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 N. Hsia and G. A. Cornwall Cres2 and Cres3: New Members of the Cystatin-Related Epididymal Spermatogenic Subgroup of Family 2 Cystatins Endocrinology, March 1, 2003; 144(3): 909 - 915. [Abstract] [Full Text] [PDF]
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, PC2 activity in the absence of CRES inhibitor (control); 
, 120 nM CRES; 
, 610 nM CRES.
