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Endocrinology Vol. 143, No. 7 2787-2796
Copyright © 2002 by The Endocrine Society

REPRODUCTION-DEVELOPMENT

Cystatin 11: A New Member of the Cystatin Type 2 Family

Katherine G. Hamil, Qiang Liu, P. Sivashanmugam, Suresh Yenugu, Rama Soundararajan1, Gail Grossman, Richard T. Richardson, Yong-Lian Zhang, Michael G. O’Rand, Peter Petrusz, Frank S. French and Susan H. Hall

Departments of Pediatrics (K.G.H., Q.L., S.Y., F.S.F., S.H.H.) and Cell and Developmental Biology (P.S., G.G., R.T.R., M.G.O., P.P.) and the Laboratories for Reproductive Biology (K.G.H., Q.L., P.S., G.G., R.S., R.T.R., M.G.O., P.P., F.S.F., S.H.H.), University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599; and the State Key Laboratory of Molecular Biology (Q.L., Y.-L.Z.), Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

Address all correspondence and requests for reprints to: Susan H. Hall, Laboratories for Reproductive Biology, CB#7500, University of North Carolina, Chapel Hill, North Carolina 27599. E-mail: . shh{at}med.unc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cystatin (CST)11, a novel member of the CST type 2 family of cysteine protease inhibitors, was identified in Macaca mulatta epididymis by subtractive hybridization cloning. The human CST11 gene on chromosome 20p11.2 is located near three other CST genes expressed predominantly in the male reproductive tract. The CST11 gene spans three exons, a structure similar to that of other CST family 2 genes. An exon 2-deleted alternative transcript (CST11{Delta}2) was also identified. CST11 mRNA is expressed only in the epididymis as judged by Northern blot hybridization and is androgen regulated. The protein is most abundant in the initial segment, but is detected throughout the epididymis and on ejaculated human sperm. The calculated tertiary structure of CST11 reveals that the three regions corresponding to the protease inhibitory wedge of CST3 are similarly juxtaposed in CST11, consistent with protease inhibitor function. Intact and exon 2-deleted CST11 recombinant proteins were tested for antibacterial activity. After a 2-h incubation of Escherichia coli with 50 µg/ml recombinant CST11 or CST11{Delta}2, bacterial colony-forming units were reduced to 30% of control, indicating that both forms have antimicrobial activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CYSTATIN TYPE 2 family of competitive reversible cysteine protease inhibitors (1, 2) contains at least 10 members in addition to CST11, 3 of which are expressed predominantly in the male reproductive tract. CST8 [or CST-related epididymal spermatogenic protein (CRES)] is expressed in epididymis, testis, and male and female pituitary (3, 4). Testatin, or CST9 is expressed in spermatogonia (5) and in Sertoli cells, where it may be involved in testis development (6). CST T is expressed exclusively in the testis (7). Human homologs of these three CST genes found originally in mouse are clustered near the CST11 gene on chromosome 20p11.2, suggesting close evolutionary relationships.

CST C or CST3 and its avian homolog chicken CST are the most widely expressed and investigated members of CST type 2 family (1, 8). CST3 is a potent inhibitor of C1 family cysteine peptidases, which include the plant enzyme papain and the related mammalian cathepsins B, L, and H (9). CST3 is produced in testis, epididymis, prostate, seminal vesicles, and many other tissues (10, 11). Cathepsin L, a major secretory product of Sertoli cells (12), is also expressed in the caput epididymis (13) and prostate (14), and is present in sperm (15). Germ cell adherence to Sertoli cells, the formation of intercellular junctions (16), and germ cell migration from basal to luminal regions all involve CST3 and cathepsin L (17). Furthermore, CST3 and cathepsin L are thought to promote sperm maturation through modification of sperm surface proteins and soluble proteins in the surrounding fluid (18).

In addition to modulating cathepsin activities specific to sperm development and maturation, evidence suggests more general roles for CSTs in the maintenance of reproductive function. CST8 may regulate proprotein processing (4). CSTs protect against tissue damage by released lysosomal cathepsins (19, 20). Lower expression levels of cysteine protease inhibitors relative to cathepsins in cancerous tissues (21, 22) may permit accelerated extracellular matrix destruction and tumor invasion. CSTs may also target proteolytic enzymes that are essential virulence factors for prokaryotic and eukaryotic parasites during all stages of the infectious process (23). Peptides and derivatives that mimic the CST3 sequence that binds cysteine proteases can block the growth of group A Streptococcus apparently by inhibiting a cysteine proteinase specific to these bacteria (24). Such peptide fragments, as well as the intact CST3 protein, are also active against other bacteria (25, 26) and viruses (27, 28). In addition, the structurally similar plant CSTs inhibit cysteine proteases required for viral infection (29) and insect feeding (30).

Studies elucidating the three dimensional structure of chicken CST (31), human CST3 (hCST3) (32), and other CSTs (33, 34, 35) established the conserved structure of the CST superfamily. Prominent features include an {alpha}-helix arranged across an upward curving and somewhat twisted five-stranded antiparallel ß-sheet. At one end of the ß-sheet, the N-terminal peptide and two loops connecting ß-strands form a wedge that blocks the V-shaped active sites of papain and related cysteine proteinases (36). The steric fit of this inhibitor wedge into the enzyme active-site cleft determines the strength of the interaction. Binding strength depends primarily on nonspecific hydrophobic interactions, rather than on specific amino acids (36). Thus, differences in amino acid sequences in the inhibitor-wedge regions of CST family members may indicate inhibition of different proteases.

Here we report the sequence analysis of the CST11 cDNA. The encoded protein, its predicted structure, and relationship to the closest CSTs are described. The CST11 gene location and structure, the sites of expression of the mRNA and protein, androgen regulation of the mRNA, and the antibacterial function of CST11 protein are presented.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
DNA sequencing
Plasmid DNA was prepared by standard alkaline lysis procedures (37), and the sequence was determined at the University of North Carolina-Chapel Hill Automated DNA Sequencing Facility. Equipment includes an ABI 3100 Genetic Analyzer (PE Applied Biosystems, Foster City, CA). Reactions were performed using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase FS (PE Applied Biosystems). Primers for sequencing and PCR were synthesized on an automated PE Applied Biosystems DNA synthesizer Model 394 using standard cyanoethyl phosphoramidite chemistry.

DNA and protein sequence analysis
Consensus transcription factor binding sites were identified by the Genetics Computer Group (Madison, WI) program FindPatterns using the patterns in the Transcription Factor Database (release 7.5; Wisconsin Package Version 10.2, Genetics Computer Group). Amino acid sequences were deduced from the cDNAs using the Genetics Computer Group program Translate and the web site http://ca.expasy.org/tools/dna.html and compared using Pileup. The signal peptide cleavage sites were predicted using the web site http://www.cbs.dtu.dk/services/SignalP/ (38). N-Glycosylation sites and phosphorylation sites were predicted using the ProfileScan website http://hits.isb-sib.ch/cgi-bin/PFSCAN? (39).

Molecular modeling
The bioinbgu server (http://www.cs.bgu.ac.il/~bioinbgu/) (40) was used to thread the sequence of hCST11 onto the structure of chicken CST in the Protein Data Bank (1CEW.pdb) (41). A model of hCST11 was built using the Modeler module of the Insight II molecular modeling system from Accelrys Inc. (San Diego, CA). The figure was created using SPOCK (42) in the Structural BioInformatics Core Facility, University of North Carolina at Chapel Hill under the direction of Dr. Brenda Temple.

Peptide synthesis
A hCST11 N-terminal peptide (cKTFLSVHEVMAVENY) was synthesized using a Rainin Symphony multiple peptide synthesizer (Rainin Instrument, Woburn, MA) using fluoroenylmethyloxycarbonyl chemistry in the University of North Carolina Program in Molecular Biology Protein Chemistry Facility. The peptide was purified by HPLC and conjugated to keyhole limpet hemocyanin using maleimidobenzoyl-N-hydroxysuccinimide ester. The amino-terminal cysteine was added to mediate coupling. Antibodies were raised in rabbits no. 6344 and no. 6345 at Bethyl Laboratories, Inc. (Montgomery, TX). An affinity column was prepared by attaching 2 mg of the antigen peptide to SulfoLink gel (Pierce Chemical Co., Rockford, IL). First preimmune and later immune no. 6344 antiserum were passed over the column, and bound antibody was eluted in 0.1M glycine (pH 2.6) and neutralized with 1M Tris (pH 9.5), according to the column manufacturer’s recommendations.

Tissue sources and Northern hybridization
Tissues for Northern analysis and for immunostaining were obtained from Rhesus macaques (Macaca mulatta), 10–12 yr of age with proven breeding history (Covance Research Products Inc., Alice, TX; and Dr. Catherine VandeVoort, CA Regional Primate Center, Davis, California). Surplus human testes and epididymides from prostate cancer patients ranging in age from 58 to 83 yr were made available by Dr. James L. Mohler (Department of Urology Surgery, University of North Carolina at Chapel Hill). Other human tissues were obtained from the Tissue Procurement Core Facility of the Lineberger Comprehensive Cancer Center (University of North Carolina at Chapel Hill). Human tissues are not accompanied by identifying information and cannot be traced to the donor. Tissues for RNA isolation were frozen in liquid nitrogen and total RNA was purified as previously described (43). Briefly, tissues were homogenized in guanidine thiocyanate (Fluka, Milwaukee, WI) and RNA was pelleted through a 5.7M CsCl cushion (Gallard and Schlesinger, Carle Place, NY). Northern hybridizations were performed using glyoxalated RNA as previously described (43). Human and monkey cDNAs (93% identical) were labeled using 32P-dCTP and random priming.

For analysis of androgen regulation, male Rhesus macaques of similar age, weight, and testicular size underwent subcapsular orchiectomy or sham operation as previously described (43). One orchiectomized monkey was immediately injected im with testosterone enanthate, 30 mg/kg body weight (400 mg total), and the other with vehicle. Epididymides and remaining testes were removed 6 d later and frozen in liquid nitrogen. Serum samples for testosterone RIA were taken just before surgery on d 0 and 6. All animals used in these studies were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The protocol follows accepted veterinary medical practice and was approved by the University of North Carolina Animal Care and Use Committee. The animals were given analgesics and were monitored closely following surgery.

Immunohistochemical staining
Surplus swim-up human sperm were provided by Dr. Stan Beyler (Assisted Reproductive Technology Clinic, Department of Obstetrics and Gynecology, University of North Carolina at Chapel Hill). Swim-up sperm were prepared by standard methods (44). Briefly, semen was diluted 1:4 with Ham’s F10 medium or Human Tubal Fluid (Irvine Sci, Irvine, CA) and centrifuged 350 x g for 10 min. The pellet was resuspended in 0.5 ml medium and layered under two tubes containing 1.5 ml medium. Sperm that swam up within 1 h were harvested from the supernatant and maintained in medium for several hours at 37 C. Sperm used for double staining were cryopreserved. Sperm were washed in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate, 1.8 mM potassium phosphate, pH 7.4) and fixed in 4% paraformaldehyde for 30 min. Sperm were washed in PBS containing 50 mM glycine and were smeared on glass slides and stored at -20 C. On the day of staining, sperm were rehydrated in PBS for 10 min, blocked in 3% BSA, 5% normal goat serum in PBS for 1 h. To determine acrosomal status, sperm were incubated with rodamine-conjugated Pisum sativum agglutinin (45, 46) (Vector Laboratories, Inc., Burlingame, CA) diluted 1:1000 in PBS. This lectin binds {alpha}-mannosyl units in the carbohydrates of acrosomal proteins. After a 1-h incubation, sperm were washed twice in PBS and mounted in Vectashield (Vector Laboratories, Inc.). For immunostaining, sperm were incubated with affinity-purified preimmune or specific antibody no. 6344 or the same antibody preincubated overnight with 100 µg/ml antigen peptide. These antisera were used without dilution. Sperm slides were washed twice in PBS and incubated for 1 h with biotinylated goat antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1:250 in 3% BSA, 5% normal goat serum. After washing twice in PBS, sperm were incubated with Texas Avidin Red (Vector Laboratories, Inc.) diluted 1:1000 in PBS for 1 h at room temperature. Where sperm were double stained for CST11 and acrosome, the CST11 antibodies were detected using fluorescein-conjugated goat antirabbit IgG (ICN-Cappel, Costa Mesa, CA). Sperm were washed twice in PBS and mounted in Vectashield. Sperm images were taken using a Carl Zeiss (Thornwood, NY) Axiophot microscope with a Carl Zeiss Axiocam digital camera and were arranged and labeled using Adobe Photoshop 5.0 (Adobe Systems, Mountain View, CA).

Tissues were fixed by immersion in Bouin’s fluid (75 ml saturated picric acid, 5 ml glacial acetic acid, 25 ml 37% formaldehyde) promptly after excision and embedded in paraffin according to standard protocols as described (47). The double peroxidase-antiperoxidase method was used for immunohistochemical staining (47). Digital photos were taken using a Nikon (Melville, NY) Eclipse E600 microscope equipped with a Spot Digital Camera and Spot Advanced software (Diagnostic Instruments, Inc., Sterling Heights, MI).

Recombinant protein preparation
The cDNAs for CST11 and CST11{Delta}2 full-length coding regions were amplified by PCR using primers O86FL: GCG GAATTC ATG ATG GCT GAG CCC and O86R: GCG GGTACC GAATTC CTA GTC ACT GC and a lambda human caput library as template (47). The gel-purified PCR products were cloned into pBluescriptIISK- (Stratagene, La Jolla, CA) and sequenced. Using these plasmids as templates, the cDNAs for CST11 and CST11{Delta}2 without signal peptide regions were amplified by PCR using primers O86TEV: GC GGATCC GAG AAC TTG TAC TTC CAA GGT GCA AGG AGG AAA ACC and O86R: GCG GGTACC GAATTC CTA GTC ACT GC. The forward primer includes a tobacco etch virus (TEV) protease recognition site for removal of the His-tag. The cDNAs were cloned into the pQE30 expression vector (QIAGEN, Valencia, CA) as described (47). The recombinant proteins bearing N-terminal 6-histidine tags and TEV site were affinity purified using nickel-nitrilotriacetic acid-agarose. Proteins were eluted in low pH buffer (pH 4.5), and dialyzed against 10 mM sodium phosphate (pH 7.4). The calculated molecular weight added to recombinant proteins by the His-tag, TEV site, and associated amino acids is 2339.

Antibacterial assays
Human recombinant CST11, CST11{Delta}2 and, lipocalin (LCN)6 (unpublished lipocalin) proteins were tested for antibacterial activity using the colony-forming unit (CFU) assay as described (48). Escherichia coli XL1-Blue (Stratagene) were grown to mid-log phase (OD600 = 0.4). Bacteria were diluted to working concentration in 10 mM sodium phosphate buffer, pH 7.4. Aliquots of bacteria were incubated for 2 h at 37 C with 10–100 µg/ml 6-His-tagged recombinant mature protein that had been dialyzed against the same buffer. The reaction mixtures were diluted 1:1000 in prewarmed 10 mM sodium phosphate buffer. Aliquots of the diluted bacteria were plated on Luria Broth agar plates and incubated overnight at 37 C. The colonies were hand-counted.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To identify novel proteins involved in sperm maturation and other aspects of reproductive health, a M. mulatta epididymis-specific cDNA library was constructed and analyzed (43). In this library, epididymal subtracted clone 13 was found to encode a novel protein related to the CST type 2 family of cysteine protease inhibitors and was given the name CST11, approved by the Human Genome Organisation, accessible at the home page http://www.hugo-international.org/hugo/. The human CST11 cDNA corresponds to UniGene cluster Hs.128100, Loc164378, and to Sanger gene CST8L (49) located on human chromosome 20p11.21 (Fig. 1Go). The CST11 gene is located approximately 50 kb distal to the CST8 gene. Beyond CST4, outside the range of Fig. 1Go on 20p are the CST5 and CST7 genes. Human expressed sequence tags (ESTs) in GenBank derived from the CST8, CST9, CST T-like, and CST11 genes were isolated primarily from male reproductive tract tissues. The CST11 gene contains three exons and two introns (Figs. 1Go and 2AGo), similar to other family 2 CST genes. The intron/exon junctions are exactly conserved in CST3 (50, 51), CST5 (52), CST8 (53), and CST11. A CST11 cDNA missing exon 2 (CST11{Delta}2), a nonframe-shifting deletion was amplified by PCR using a human caput/corpus library as template indicating the presence of an alternatively spliced mRNA. CST11{Delta}2 is also represented in the GenBank EST database (accession nos. AI200857 and AI025187). Alternative splicing of mouse CST8 mRNA was also reported, and the resulting shorter mRNA would encode a protein lacking the signal peptide and additional amino acids present in mature CST8 N terminus (53).



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  		Figure 1. hCST11 gene location on chromosome 20p11.2. A, Ideogram of chromosome 20. Filled triangle indicates location of the CST family 2 gene cluster. B, Organization of the CST gene cluster from 23.38 megabases (Mb) to 23.63 Mb from the short-arm telomere. CSTT-like corresponds to Loc164380; CST9 corresponds to Unigene Hs.121554 and Sanger gene CST9L (49 ). Arrows indicate direction of transcription. Boxes beneath arrows diagram intron-exon structures (not to scale). C, Expanded view of CST11 gene structure. Narrow portions of black boxes represent untranslated regions and wide portions represent translated regions of the mRNA.

 


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  		Figure 2. hCST11 gene sequence aligned with the mRNA and amino acid sequences. A, The gene sequence was extracted from GenBank accession no. AL096677. The cDNA nucleotide sequence is indicated in capital letters (accession no. AF335480). Intron sequence is in lowercase. The predicted poly(A)+ addition site is double underlined. The amino acid sequence is indicated in single- and triple-letter abbreviations. The signal peptide is italicized. The conserved cysteines and amino acids corresponding to those in CST3 involved in protease binding are bold. B, The predicted promoter region. The predicted transcription start site is indicated ({wedge}). The predicted TATA box is bold and underlined (-21 to -26). Potential PEA3, androgen receptor, Sp1, NF-{kappa}B, and CCAAT transcription factor binding sites are underlined or overlined.

 
The presumed promoter region (Fig. 2BGo) immediately upstream from the cDNA contains a TATA-box-like pyrimidine sequence (TTAAAT) 21 bp upstream from a predicted transcription start site and 53 bp upstream from the longest cDNA obtained. A consensus CCAAT enhancer binding site is present as well as sites for binding polyomavirus enhancer activator 3 (PEA3), nuclear factor {kappa}B (NF-{kappa}B), Sp1, and androgen receptor.

CST11 protein sequence is 30–40% identical and 50–67% similar to the most closely related CSTs (Fig. 3Go, A and B). In Fig. 3Go, A and B, CST proteins are arranged in order of their similarity to CST11. Human CST11 is most closely related to rhesus CST11 and hCST8 and most distantly related to hCST9. The mouse CST T sequence is included in this comparison because transcripts in GenBank (ESTs BG772612, BI463136, BI827901) of the human CST T-like gene do not encode a full-length CST. Macaque and hCST11 contain a predicted C-terminal N-glycosylation site at N106 also conserved in CST9. T4 is a predicted cAMP-dependent protein kinase site, and S7 a predicted casein kinase II phosphorylation site. Both highly conserved tyrosines, Y37 and Y57, are predicted phosphorylation sites in macaque and hCST11. Although Y37, located in the ß3 strand, is conserved in all the CSTs in Fig. 3Go, the neighboring amino acids vary, and only the corresponding tyrosine in CST8 and the adjacent tyrosine in CST4 are conserved phosphorylation sites. Tyrosine 57, conserved in all the known human CST type 2 family except CST9, is a predicted phosphorylation site only in CST11. Thus, tyrosines may be conserved in these positions for a reason unrelated to phosphorylation; for example, structural stabilization. Tyrosine 34 in human CST3 {alpha}1 corresponding to Y29 in CST11, also a conserved tyrosine in family 2, was shown to make van der Waals contacts with L68 in ß3 (32). The L68 position in CST3 is replace by a methionine in CST11. Figure 3BGo shows the clustering relationships within the CST family. Horizontal branch lengths in the dendrogram indicate proportional differences between sequences.



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  		Figure 3. CST11 and related CSTs. A, Alignment of amino acid sequences of CST11 and closely related CSTs. hCST11, hCST11{Delta}2 (accession no. AF335481), and the M. mulatta CST11 (accession no. AF218043) are compared with hCST3 (accession no. X5607), hCST4 (accession no. X54667), chicken CST (accession no. J05077), hCST8 (accession no. AF059244), hCST9 (protein accession no. CAC05421), and mouse CST T (EST accession no. AK005665). Amino acid numbers refer to the CST11 mature protein. Signal peptides are not shown. Amino acids forming the {alpha}-helices are indicated in green and ß-strands in blue. Amino acids forming the inhibitory wedge in hCST3 and chicken CST, and the corresponding amino acids in the other CSTs are red and double underlined. The hCST11 N-terminal peptide used to raise the antibody for immunostaining is indicated in bold and is underlined. The four cysteines are bold, and brackets indicate disulfide linkages established in hCST3 and chicken CST. Predicted phosphorylation sites are in bold italics with dotted underlining. The predicted N-glycosylation site at 106 is single underlined. B, Dendrogram diagramming pairwise comparisons of the amino acid sequences in A. Panels A and B were constructed using Pileup in the GCG Wisconsin Package. The ß-strands and {alpha}-helices in hCST3 and chicken CST were determined by x-ray crystallography (31 32 ) and in CST11 were predicted as described for Fig. 4Go.

 
To understand the three-dimensional arrangement of key amino acids in the CST11, the model was built (Fig. 4Go) using chicken CST as the template (31). Chicken CST is the most closely related protein for which x-ray crystallography data are available. The model is oriented to place at the top of the figure the three-part inhibitory wedge that includes N-terminal L6, loop 1 Q50-H54, and loop 2 P97W98. The amino acid alignment (Fig. 3AGo) shows that, except for positions corresponding to Q55 and P105W106 in hCST3, the amino acids forming the inhibitory wedge are not well conserved in CSTs 8, 9, T, and 11. The diversity of predicted and known reactive site sequences in the CST type 2 family is consistent with inhibition of a range different proteases. These could be proteases secreted by the epithelium of the male tract, the female tract, or proteases produced by invading microbes.



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  		Figure 4. hCST11 protein model. {alpha}-Helices are indicated in green, ß-strands in blue and black, and unstructured regions in red. Key amino acids indicated in Fig. 3Go are shown in red single-letter abbreviations. The side chain of Y57 is shown extending outward from {alpha}1. The N terminus is truncated to begin with leucine 6 and the C terminus to end at S110.

 
To investigate the expression of CST11, RNA purified from different M. mulatta tissues was analyzed by Northern hybridization. CST11 mRNA is abundant in all three regions of the epididymis (Fig. 5Go) and migrates with an apparent size of about 0.8 kb. The lower abundance 2-kb transcripts may represent incompletely processed mRNAs retaining one intron. Transcripts migrating at 3.0–4.0 kb may represent CST11 mRNAs that include regions beyond those we identified as components of the CST11 gene, possibly including an upstream fourth exon as described for CST8 (53) or an additional 3' exon. Faint bands present in the seminal vesicle and testis may indicate spillover from the epididymal lanes, or low levels of expression in these tissues. In other experiments, hybridization was not detected with RNA isolated from M. mulatta hypothalamus, pituitary, colon, lung, retina, tonsil, cervix, and from CV-1 cells, LNCaP cells, PC3 cells, and DU145 cells (data not shown). Immunohistochemical staining of human epididymis revealed CST11 protein in epithelial cell cytoplasm, nuclei, and in the lumen throughout the epididymis (Fig. 6Go, A and C). Cytoplasmic immunoreactive protein is especially prominent in the initial segment. CST11 is expressed in some epithelial cells of the efferent ducts but not others (Fig. 6AGo, right and upper right), a pattern seen with other proteins in the epididymis (43). Both cytoplasmic and nuclear immunoreactivity were abolished by preincubation of the antibody with the peptide antigen (Fig. 6Go, B and D).



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  		Figure 5. Expression of CST11 mRNA in different M. mulatta tissues. Total RNA (10 µg/lane) was isolated from cerebrum, bladder, kidney, spleen, liver, stomach, small intestine, pancreas, heart, adrenal, salivary gland, testis, caput, corpus, cauda, seminal vesicle, prostate, ovary, oviduct, and uterus. The Northern blot was hybridized to CST11 cDNA encoding the mature protein. Film was overexposed to attempt to detect hybridization to nonepididymal RNAs.

 


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  		Figure 6. Immunolocalization of CST11 protein in human epididymis. Panels A and B, Caput and efferent ducts (x10 objective). A, CST11 appears brown against toluidine blue counterstain. B, Serial section using antibody preadsorbed with peptide antigen. C, CST11 in cauda (x10 objective). D, Serial section using preadsorbed antibody.

 
The presence of CST11 in the epididymal lumen indicated that it is available to interact with maturing sperm. CST11 location on sperm was confirmed by immunofluorescent staining (Fig. 7Go). Because CST11 mRNA was abundant in epididymis, but little if any was detected in testis (Fig. 5Go), the sperm-associated CST11 was most likely of epididymal origin and thus located on the outer surface. The sperm in Fig. 7Go, A–D, were predominantly acrosome-reacted based on staining of duplicate slides (not shown). The three sperm in Fig. 7Go, E–G, were acrosome-intact as indicated by the red color in the acrosomal regions (Fig. 7FGo) that resulted from interaction with rhodamine-conjugated Pisum sativum agglutinin. The results suggest that CST11 is localized in the postacrosomal and tail regions.



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  		Figure 7. Immunolocalization of CST11 on human sperm. A, Phase contrast view of the sperm in B. B, Texas red immunofluorescent staining of CST11. C, Phase contrast view of the sperm in D. D, Texas red immunofluorescent staining using anti-CST11 preincubated with peptide antigen. E, Phase contrast of human sperm in F and G. F, Red staining on sperm heads indicates intact acrosomes. G, Fluorescein immunofluorescent green staining indicates CST11. Magnification, x500.

 
To determine whether CST11 expression is regulated by testosterone, a key hormone in sperm maturation, RNA was obtained from rhesus macaques that were sham operated, castrated 6 d, and castrated 6 d but given a single injection of 400 mg testosterone enanthate immediately following testis removal (Fig. 8Go). By six d after castration, CST11 mRNA levels in caput had declined to 20% of normal levels. In corpus, RNA levels were unchanged by castration. The CST11 mRNA detected in sham-operated corpus was not seen in the castrated or testosterone-replaced animals. Expression in caput was maintained by exogenous testosterone, but was undetectable in corpus of the androgen-replaced animal. Serum testosterone levels before testis removal in shamoperated, castrated, and castrated/androgen-replaced macaques were 3.0, 3.8, and 1.4 ng/ml, respectively. Six days after injection, at the time the epididymides were removed, serum testosterone levels were 2.0 ng/ml in the sham, 0.15 ng/ml in the castrated, and 64.9 ng/ml in the androgen replaced. Previously reported serum testosterone levels in rhesus macaques ranged from 5 ng/ml in the morning to 18 ng/ml during the nocturnal surge (54).



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  		Figure 8. Androgen regulation of CST11 mRNA M. mulatta were sham operated, castrated, or castrated and androgen replaced with immediate injection of testosterone enanthate. Epididymides were removed 6 d postcastration. Upper panel, Total RNAs (10 µg/lane) from the indicated epididymal regions were analyzed by Northern hybridization to 32P-labeled CST11 full-length cDNA. Lower panel, The same blot was hybridized to 32P-labeled 18S ribosomal RNA cDNA.

 
Antimicrobial activities of CST3, CST3-derivative peptides and plant CSTs prompted us to determine whether CST11 also can kill bacteria. After a 2-h incubation of E. coli with 25–100 µg/ml CST11 or CST11{Delta}2 recombinant protein, a substantial loss of CFU was observed (Fig. 9Go). Incubation of E. coli with 100 µg/ml CST11{Delta}2 resulted in 100% loss of CFU. In similar experiments, incubation of E. coli with 25 µg/ml human ß-defensin-1 (American Peptide Co., Inc., Sunnyvale, CA) resulted in 50% loss and recombinant His-tagged CST3 resulted in 80% loss of CFU in 2 h (data not shown). Consistent with antimicrobial activity, recombinant CST11 and CST11{Delta}2 were purified from bacteria in relatively low yields (0.7 mg recombinant protein per gram of bacteria for CST11, 0.3 mg/g for CST11{Delta}2), whereas with LCN6 yields were approximately 4.0 mg/g.



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  		Figure 9. Antibacterial activity of CST11. Mid-log phase E. coli were incubated 2 h with 0–100 µg/ml recombinant LCN6 ({blacktriangledown}, CST11 ({blacksquare}), or CST11{Delta}2 ({blacktriangleup}). Aliquots (1/10,000th) of each reaction mixture were spread on duplicate agar plates and CFU counted. Similar results were obtained in two other experiments.

 
Each assay tube received 100 x 104/ml CFU, which tripled in the control tubes receiving no test protein (0 µg/ml initial point on the y-axis) during the 2-h incubation. In the presence of LCN6, by contrast, the bacteria doubled in concentration and were not further affected by increasing doses of this recombinant His-tagged protein prepared by the same protocol as the CST11 proteins. Thus, it is unlikely that the 6-histidine tag or copurifying E. coli proteins were responsible for the antimicrobial activity of CST11 or CST11{Delta}2.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report here for the first time, CST11, a gene in the CST 2 family located in a cluster of male reproductive tract-expressed CST genes on chromosome 20q11.2. The family gene structure is well conserved; two introns are positioned identically in the coding regions of CSTs 3, 5, 8, and 11. Conserved intron/exon structure and low amino acid sequence homology characterizes the CST type 2 family and many vertebrate protein families involved in extracellular and often immune-related activities (55). A proposed role for conserved intron position is to preserve sites for alternative splicing, a mechanism widespread in mammals that can result in the expression of multiple RNAs and proteins from a single gene (47, 56). Presumably, the alternative splice product, CST11{Delta}2, exists because either the mRNA or the translated protein has an important function. CST11 exon 2 encodes the loop 1 reactive site beginning with the second amino acid in the Q50-H54 motif and includes the entire third ß-strand as well as the loop joining the third and fourth ß-strands. Thus, in the predicted CST11{Delta}2 protein, the second and fourth ß-strands would join directly. The resulting structure is unknown. Methods used to generate the CST11 model failed to provide a probable structure for the exon 2-deleted protein.

Consistent with androgen regulation of CST11 mRNA (Fig. 8Go), the proposed CST11 promoter region contains a potential androgen response element and at least two half sites. The region also contains predicted binding sites for CCAAT/enhancer-binding proteins (C/EBP). C/EBP binding sites are present in the promoter of mouse CST8, and the ß-form, the most abundant C/EBP in the epididymis, regulates CST8 expression (57). The site for PEA3 (58), a testis factor-regulated protein in epididymis, may also mediate regulated CST11 expression. PEA3 and androgen receptor binding sites are also present in the promoter of Eppin, a male-specific sperm-binding protein containing protease inhibitor consensus sequences (59). The binding site for NF-{kappa}B, an important cellular factor mediating responses to microbial pathogens (60), is consistent with involvement of CST11 in host defense. The functions of these potential sites and the alternating purine/pyrimidine repeat sequences in the CST11 promoter remain to be analyzed.

The abundance of CST11 mRNA in the corpus of the sham-operated and castrated epididymides contrasts with undetectable levels in the corpus of the testosterone-treated macaque. In this same experiment in the testosterone-replaced animal, similar abolition of corpus expression of epididymal subtracted clone 42 was seen (43) but not human epididymal clone 2 (47). Region-specific regulation of gene expression by androgens in the epididymis is complex and was discussed in a recent review (61). The testosterone administered to the castrated/replaced macaque raised circulating total serum testosterone to levels that were similar to levels in caput fluid in the rat epididymis (62). CST11 transcription likely requires multiple proteins in addition to androgens. The presence or activities of certain critical proteins may be dependent on testis factors. Further experiments would be required to explain the regional differences in the response to testosterone.

The location of immunoreactive CST11 in the nuclear as well as cytoplasmic compartments was unexpected because the predicted protein contains a signal peptide and is found in the epididymal lumen. A splice variant of CST11 missing part or all of exon 1 that would encode a truncated protein lacking the signal peptide is possible, but was not found. However, such a variant of CST8 was reported (53). Translation of CST11 from the internal methionine located within the antigen peptide could result in a truncated, nonsecreted form recognizable by the antibody and is a possible explanation for the nuclear protein.

Several proteins in addition to CSTs defend the male reproductive tract against invading pathogens including the cationic peptides, ß-defensin-1 (63), the defensin-like Bin1b (64), and the cathelicidin hCAP18. The protease inhibitors {alpha}2-macroglobulin and secretory leukocyte protease inhibitor also defend the male tract in addition to tear LCN, lactoferrin, lysozyme, and lung surfactant protein D (65). Of particular interest are antimicrobial proteins attached to sperm, including lactoferrin, hCAP18 (66), and now CST11. On the sperm surface, these proteins have the potential to defend sperm in the female, as well as in the male, reproductive tract against pathogens. They may promote fertilization through interactions with other sperm proteins or with proteins of the egg or its vestments.

The abundance of four different members of the CST type 2 family abundant in the male reproductive tract is consistent with specialized requirements for regulated proteolysis in these tissues. The diversity in the inhibitory wedge sequences would allow for inhibition of a range of proteases involved in male tract function that could include maturation of proteins on sperm or in luminal fluid (67), as well as protection against invading pathogens by inhibiting microbial proteases or other mechanisms. Location of these CSTs on sperm surface or within the acrosome would permit a role in regulating enzymatic dispersal of cumulus cells or degradation of zona pellucida. It will be important to determine which enzymes can be inhibited by the CSTs in the male reproductive tract. It will also be important to understand whether protease inhibitor and antimicrobial activities reside in the same regions of the protein and whether both activities are involved in sperm maturation, sperm survival, or fertilization mechanisms.


   Acknowledgments
 
We thank Dr. James L. Mohler for performing surgical castrations of monkeys and Zang De-Ying for expert technical assistance. Special thanks to Betty F. Horton and Richard L. Pippin for expert preparation of the figures.


   Footnotes
 
This work was supported by the Consortium for Industrial Collaboration in Contraceptive Research Program of the Contraceptive Research and Development Program, Eastern Virginia Medical School. The views expressed by the authors do no necessarily reflect the views of Contraceptive Research and Development or Consortium for Industrial Collaboration in Contraceptive Research. This work is also supported by NIH Grants R37-HD04466, by National Institute of Child Health and Human Development/NIH through cooperative agreement U54-HD35041 as part of the Specialized Cooperative Centers Program in Reproduction Research, and by the Fogarty International Center Training and Research in Population and Health Grant D43TW/HD00627.

1 Present address: Department of Biochemistry, Indian Institute of Science, Bangalore, India 560-012. Back

Abbreviations: C/EBP, CCAAT/enhancer-binding protein; CFU, colony-forming unit; CST, cystatin; EST, expressed sequence tag; h, human; LCN, lipocalin; NF-{kappa}B, nuclear factor {kappa}B; PEA, polyomavirus enhancer activator 3; TEV, tobacco etch virus.

Received January 16, 2002.

Accepted for publication April 2, 2002.


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 Results
 Discussion
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