JOSEPHINE GRIMA, LI-JI ZHU, SHU D. ZONG, JAMES F. CAlTERALL, C. WAYNE BARDIN, and C. YAN CHENG 2. The Population Council, New York, New York 10021

Transcription

1 BIOLOGY OF REPRODUCTION 52, (1995) Rat Testin Is a Newly Identified Component of the Junctional Complexes in Various Tissues Whose mrna Is Predominantly Expressed in the Testis and Ovary' JOSEPHINE GRIMA, LI-JI ZHU, SHU D. ZONG, JAMES F. CAlTERALL, C. WAYNE BARDIN, and C. YAN CHENG 2 The Population Council, New York, New York ABSTRACT Testin I and testin II are the two molecular variants of testin that are synthesized and secreted by Sertoli cells in vitro. N- Terminal and partial internal amino acid sequence analysis of testin I and testin II reveals that these molecules are identical with the exception that testin II has three extra N-terminal amino acids of TAP compared to testin I. Studies using immunohistochemistry suggested that testin is a component of the specialized junctional complexes in the seminiferous epithelium and other tissues. Immunoreactive testin is localized not only at Sertoli-Sertoli and Sertoli-germ cell junctions, but also at sites of similar junctions in the liver, epididymis, kidney, and intestine. Other physiological studies have shown that the secretion of testin is tightly coupled to the presence of germ cells. In view of its possible role in germ cell development and its unique localization in the cell junction, the purpose of the present study was to determine the structure of testin by sequencing its full-length cdna. Two synthetic degenerate oligonucleotides based on the N-terminal and an internal amino acid sequence were used for polymerase chain reaction (PCR) to obtain a 289-bp cdna fragment. This PCR product was subsequently used to isolate a 1371-bp cdna from a cdna expression library constructed from Sertoli cell poly(a) RNA. This cdna coded for a 333 amino acid peptide that starts with an ATG initiation codon from the 5' end and ends with a TGA termination codon located 245 nucleotides before the polyadenylation site. The deduced amino acid sequence indicates that testin contains a 16 amino acid signal peptide with two possible cleavage sites that yield 314 and 317 amino acids for testin I and testin II with calculated molecular weights of and , respectively. Comparison of the entire coding region of testin with existing sequences at Genbank, EMBL, and Protein Identification Resource indicates that testin shares 58%, 5%, and 61% identity with rat, mouse, and human cathepsin L at the amino acid level, respectively. The positions of all of the 7 Cys residues and 8 of the 10 Trp residues in testin are conserved with respect to those present in cathepsin L. It is noted that Cys-122 in the predicted active site of cathepsin L was replaced with Ser-122 in testin. In view of the striking primary sequence homology between testin and cathepsin L, we assayed the proteolytic activity of testin using conditions known to activate cathepsin L. Results of these studies showed that no cathepsin L-like protease activity was detected. Northern blot analysis revealed that the testin mrna was detectable in Sertoli cells, testis, and ovary, but not in brain, liver, kidney, spleen, intestine, epididymis, or uterus from either male or female adult rats. However, hot-nested reverse transcriptase (RT)-PCR yielded a distinctive band with the expected size not only from the RNA of Sertoli cells, testis, and ovary, but also from epididymis, kidney, and intestine. However, the expression of the testin mrna transcript in the gonads is several orders of magnitude higher than in other tissues, suggesting that its expression is correlated with the rapid tissue remodeling in the gonads during germ cell and follicle development. In conclusion, testin is a newly identified component of cell junction whose mrna is predominantly expressed in the gonads. INTRODUCTION In the testis, the tight junctions between adjacent Sertoli cells create a unique microenvironment in the adluminal compartment behind the blood-testis barrier that segregates the developing germ cells from the systemic circulation [1-3]. Since the developing germ cells must traverse through the tight junctions between Sertoli cells during different stages of the spermatogenic cycle, there are extensive degradation and continual renewal of the components in these cellular junctions. It is known that Sertoli cells produce several proteases and protease inhibitors, such as cathepsin L [4, 5], plasminogen activator [6], cystatin c [7], tissue inhibitor of metalloproteases [8], and ac 2 -macroglobulin [9-11], that are involved in the tissue remodeling of the Accepted September 19, Received June 27, 'This work was supported in part by NIH grants (HD and DK 07313). The nucleotide sequence reported in this paper has been submitted to the Genbank Data Bank with accession number U Correspondence: Dr. C. Yan Cheng, Senior Scientist, The Population Council, 1230 York Avenue, New York, NY FAX: (212) seminiferous epithelium. It is therefore proposed that the Sertoli cell would also secrete proteins that are involved in the formation of the junctional complexes. Previous studies from our laboratory have shown that Sertoli cells synthesize and secrete two proteins, designated testin I and testin II, of Mr and , respectively [12-14]. N-Terminal amino acid sequence of these proteins reveals that they are identical except that testin II has three extra amino acids of TAP at its N-terminus. Thus, testin I and II appear to be the molecular variants of the same protein. However, the physiological significance of testin is not known. Three separate studies suggested that the secretion of testin by Sertoli cells is tightly coupled to the presence of germ cells. First, the secretion of testin by seminiferous tubules cultured in vitro decreases steadily with maturation of the tubular epithelium as germ cells appear [12, 13]. Secondly, there is a dramatic increase in the testicular content of testin after a single dose of busulfan treatment, which selectively depletes germ cells from the seminiferous epithelium without interfering with androgen 340

2 TESTIN IS A NEW COMPONENT OF CELL JUNCTION 341 production by Leydig cells; as the testes recover and germ cells reappear in the tubule, the testicular testin content declines to normal levels [13, 15]. Thirdly, when adult rats receive an acute dose of X-irradiation sufficient to destroy spermatogonia and some preleptotene spermatocytes, there is a significant increase in the level of testicular testin on Days following irradiation that is associated with a predominant depletion of late spermatids [16]. These results support the postulate that there is an inverse correlation between the concentrations of testicular testin and germ cells. Another interpretation is that when germ cells are removed by chemical or irradiation treatments, Sertoli cells are induced to secrete testin to be used to replenish the Sertoli-germ cell junctions as it is an integral component of the junctional complex. In this study we report the primary sequence of testin and show that this protein is widely distributed in junctional complexes in multiple tissues as evidenced by immunohistochemistry. Northern blots, reverse transcriptasepolymerase chain reaction (RT-PCR), and immunoblots suggest that there is greater expression of testin in the gonads than in other organs. MATERIALS AND METHODS Purification of Testin from Sertoli Cell-Enriched Culture Medium and Amino Acid Sequence Analysis Testin I and testin II were purified from primary Sertoli cell-enriched culture medium by sequential HPLCs consisting of anion exchange, chromatofocusing, and high-performance electrophoresis chromatography (HPEC) [13,15]. N-Terminal amino acid sequence analysis was performed through use of about 0.1 nmol of purified testin I or testin II [17-19]. To generate peptide fragments of testin for internal amino acid sequencing, testin was cleaved with either Staphylococcus aureus protease V8 (a Glu-C endopeptidase; Calbiochem, San Diego, CA), Arg C endopeptidase (from mouse submaxillary gland; Calbiochem), N-tosyl-Lphenylalanyl-chloromethyl ketone-treated trypsin (TPCKtrypsin, protein sequencing grade; Pierce, Rockford, IL), or low ph; the peptide fragments were fractionated onto a 15% T SDS-polyacrylamide gel, transferred onto polyvinylidene difluoride (PVDF) membrane, and sequenced via an Applied Biosystems 473A (Foster City, CA) pulsed-liquid phase protein sequencer as previously described [18, 20, 21]. Immunohistochemical Staining Adult rats between 250 and 280 g in body weight were killed by cervical dislocation following anesthesia with Metofane (methoxyflurane) from Pitman-Moore, Inc. (Mundelein, IL). Testis, epididymis, kidney, liver, and small intestine were removed and immediately frozen in liquid nitrogen. The unfixed tissues were sectioned to a 5-zm thickness in a cryostat microtome at -22 C. Sections were placed directly on poly-l-lysine-coated slides, air-dried at room temperature for 30 sec, and fixed in 4% formaldehyde (v/v) in PBS buffer (10 mm sodium phosphate, 0.15 M NaCl, ph, at 22 0 C) or modified Bouin's solution containing 4% formaldehyde in saturated picric acid for 3 min at room temperature. They were subsequently washed with PBS three times (5 min each). The endogenous peroxidase activity was blocked by treatment with 0.03% hydrogen peroxide (v/v) for 3 min. Nonspecific sites were blocked by treatment with 10% BSA (w/v) for 10 min and the sections were dried at 4 C. Immunostaining with streptavidin-biotin complex was performed according to procedures specified by the manufacturer (Zymed Laboratories, Inc., South San Francisco, CA). Briefly, tissue sections were incubated with the rabbit anti-rat testin II antibody at a dilution of 1:300 at 37 C for 1 h, washed three times with PBS (5 min each), incubated with biotinylated second antibody (goat anti-rabbit IgG) for 30 min, washed three times with PBS (5 min each), and incubated with streptavidin-peroxidase complex for 10 min. The sections were washed thoroughly with PBS and developed in an AEC-H staining system (Zymed) for 3-15 min. The slides were then washed in water for at least 15 min. to stop the reaction, counterstained with Mayer's hematoxylin, mounted, and examined microscopically. Controls were performed by substituting the primary antibody in adjacent sections with 1) antibody preabsorbed with purified testin II, the absorbed antibody having been prepared by incubating anti-testin II antibody at 1:300 dilution (1 ml) with 10 Iig of purified rat testin II overnight at 4 C; 2) substituting the primary antibody with preimmune serum; 3) substituting the primary antibody with normal rabbit serum; 4) substituting the second antibody with PBS buffer; 5) substituting the primary antibody with antitestin II antibody preabsorbed by protein A-Sepharose (antiserum:resin, 0.1 ml:10 ml). PCR Amplification of Testin Fragment from Its N- Terminus We had initially attempted to isolate the testin full-length cdna using several batches of polyclonal antibodies prepared against purified testin I or testin II, as well as several rat testis expression libraries, without success, primarily because of its low abundancy. To overcome this difficulty, we used PCR to prepare a partial cdna clone for testin that was then used to isolate its full-length cdna from a Sertoli cell cdna expression library. Briefly, a cdna clone for testin was prepared through use of two degenerate oligonucleotides synthesized based on the partial N-terminal amino acid sequence for testin (5'-TCTAGAGAYGTNGARTGGAAY- GARTGG-3') and a downstream internal amino acid sequence (5'-TCTAGANACYCANGCYCANAYRTG-3') as primers. Xba I (TCTAGA, underlined) sites were incorporated into

3 342 GRIMA ET AL. these primers to allow subsequent cloning of the PCR product into a plasmid (N, any base; Y, T, or C; R, A, or G). For RT-PCR, total RNA isolated from Sertoli cells through the use of 20-day-old rat testes was first reverse transcribed to DNA and used as a template. Cycling parameters for PCR were denaturation at 94 C for 1 min, annealing at 57 0 C for 2 min, extension at 72 0 C for 1 min; a total of 40 cycles were performed. The cycles were followed by a 15-min extension period at 72 C. No testin-specific PCR product was obtained during the first PCR reaction. Consequently, one tenth of the first PCR reaction was used for reamplification and a 289-bp PCR product was obtained that coded for about one third of the protein from the N-terminus. It was then cloned into pgem 4Z (Promega, Madison, WI), sequenced, and used to screen a rat Sertoli cell cdna library to isolate the full-length cdna clone. cdna Library Construction, Screening, and Sequencing The use of a rat Sertoli cell cdna library was critical to isolate the full-length testin cdna. We had tried to screen more than a million plaques with three different rat testis cdna expression libraries using the 289-bp PCR product, without success. We subsequently constructed a rat Sertoli cell cdna expression library with the Uni-ZAP XR unidirectional vector from Stratagene (LaJolla, CA) using poly(a) RNA isolated from primary cultures of Sertoli cells prepared from 20-day-old rat testis. Approximately 1 x 106 clones were screened by plaque hybridization with the a-[ 32 P]-labeled 289-bp testin PCR product. Nitrocellulose filters were prehybridized for 4 h in 50% deionized formamide, 5-strength SSPE (0.75 M sodium chloride, 0.5 M sodium phosphate, and 5 mm EDTA, ph, at 22 0 C), 5-strength Denhardt's buffer (0.2% Ficoll 70 [w/v], 0.2% polyvinylpyrrolidone [w/v], and 0.2% BSA [w/v]), 0.5% SDS (w/v), and 100 ig/ml denatured salmon sperm DNA at 42 C. Filters were hybridized by adding 1 x 106 cpm/ml of the nick-translated probe and were incubated overnight at 42 C. Filters were washed three times for 30 min each in doublestrength SSC buffer (0.3 M sodium chloride and 30 mm sodium citrate, ph 7.0)/0.2% SDS (w/v) at room temperature, one time in single-strength SSC/0.1% SDS (w/v) for 30 min at 65 C, and one time in 0.1-strength SSC/0.1% SDS (w/v) for 30 min at room temperature. More than 70 positive clones were isolated. In vivo excision of the pbluescript phagemid was performed on two of the isolated clones according to the protocols supplied by the manufacturer, and these clones were designated T44 and T51. Nucleotide sequence analysis from both orientations was performed by dideoxynucleotide chain termination method with Sequenase (Version 2.0; U.S. Biochemicals, Cleveland, OH), according to procedures supplied by the manufacturer, for both clones as previously described [22]. Protease Assays Protease activity was assayed by using various proteases and methyl-a-[1 4 C]-casein, and [ 3 H]-collagen as substrate [9,15]. Briefly, protease activity was assayed by incubating increasing quantities of enzyme in 200 l1 of a protease buffer. Tubes were incubated with [1 4 C]casein (10 pl, 0.5 RCi/ml, 1 about cpm) for 30 min at 37 C. Thereafter, 200 l of ice-cold trichloroacetic acid (20%, w/v) and 300,ug of BSA (10 Il of a 30 mg/ml stock solution, w/v) were added to each tube. After sedimentation at x g for 2 min, hydrolyzed fragments in the supernatants were quantified by spectrometry using 5 ml of Ready Safe scintillation fluid (Beckman Instr., Palo Alto, CA) in a 3-counter (Model 1209 Rackbeta Liquid Scintillation Counter; LKB, Rockville, MD) at 28% counting efficiency. Trypsin, a serine protease, was assayed using M Tris, ph, as a buffer containing 3% NaCl. Cathepsin L, a cysteine protease, was assayed using a buffer of 0.4 mm sodium acetate, 4 mm EDTA, and 1 mm dithiothreitol (DTT), ph ; and cathepsin D, an aspartic protease, was assayed using 40 mm sodium citrate, ph, as a buffer. Human cathepsin L and bovine cathepsin D were obtained from Calbiochem (San Diego, CA) and Sigma (St. Louis, MO), respectively. These two proteases were used in all experiments performed in this study except that a limited amount of rat testicular cathepsin L was purified from Sertoli cell-enriched culture medium as described below in order to study its protease activity and possible cross-reactivity with testin. Preincubation of testin for activation by trypsin and cathepsin D was performed in 50 Il of the appropriate buffer (described above) for the specified incubation time at 37 0 C. Protease activity of testin was then assayed at either ph,, or to monitor for either aspartic, cysteine, or serine protease activity by 1 addition of 140 Il1 of the specified buffer and 10 of [ 14 C]casein for 30 min at 37 0 C. Hydrolyzed fragments were quantified using a 13-counter. To visualize the processing of testin into smaller fragments, testin was incubated with either trypsin, cathepsin L, or cathepsin D, with appropriate buffers as described above at specific ph or at ph in 40 mm sodium citrate buffer for the specified time; the samples were resolved on a 12.5% T SDS-polyacrylamide gel, transferred to nitrocellulose, and stained with anti-testin I antibody. Similar experiments were performed using [125I]- testin II labeled with Bolton-Hunter reagent [13]. RNA Extraction and Northern Blot Analysis Total RNA was isolated from primary cultures of Sertoli cells from 20-day-old rats or adult tissues by RNAzol B solution (Tel-Test "B," Inc., Friendswood, TX) according to the manufacturer's protocols. Total RNA was denatured in 6% formaldehyde/50% formamide in single-strength MOPS buffer (40 mm 3-[N-morpholine]propane sulfonic acid [MOPS], 10 mm sodium acetate, 1 mm EDTA, ph 7.0, at 22 0 C) at 65 C for 15 min. RNA was resolved onto 1.1% agarose-formaldehyde gel in 0.5-strength MOPS buffer and electrophoretically transferred to Nytran nylon filters (Schleicher & Schuell, Keene, NH). RNA was immobilized

4 TESTIN IS A NEW COMPONENT OF CELL JUNCTION 343 by UV cross-linking by exposing the membrane to 254 nm UV for 120 mj/cm 2. Prehybridization was performed in 50% deionized formamide/5-strength Denhardt's buffer/3% dextran sulfate (w/v)/5-strength SSPE/1% SDS (w/v) and 100 RIg/ml denatured salmon sperm DNA for 4 h at 60 C. By means of an [a- 32 P]-labeled antisense RNA probe transcribed from the 289-bp testin PCR product, approximately 1 x 106 cpm/ml was added and hybridized overnight at 60 C. The blots were washed three times in single-strength SSPE/0.5% SDS (w/v) for 15 min each at 65 C and then washed once in 0.1-strength SSPE/0.5% SDS (w/v) for 15 min at 60 C. The filters were subjected to autoradiography with a double intensifying screen for 4 days. Amplification of RNA by Hot-Nested RT-PCR Since testin mrna was not readily detectable in many of the tissue extracts by Northern blots using as many as 30 Rg total RNA or 10 pxg poly(a) RNA, except in the gonads, hot-nested PCR was performed to amplify the testin mrna. About 4 Rig of total RNA for each tissue was reversed transcribed to DNA at 42 0 C for 60 min via an AMV reverse transcriptase kit (Promega) as suggested by the manufacturer. Briefly, 4 units of reverse transcriptase, 10 units of RNasin, 2.5 l of dntps (10 mm each nucleotide), 2.5 li of DTT (100 mm), 5 l of 5-strength RT buffer, 0.5 RIg of an antisense primer (5' -ACCATGGGTAACATTAGATCCCATG-3', complementary to the nucleotide sequence between nucleotides 459 and 483), and sterilized double-distilled water were incubated with the total RNA in a final reaction volume of 25 Il at 42 C for 60 min. The whole reaction product served as a template for PCR using 0.5 g of the antisense primer and 1 g of the sense primer 5'-GGACAA- AACATGGGAAAACATACAA-3' (nucleotide sequence 50-74), 10 Il of 10-strength PCR buffer, 16 pl of dntps (200 p.m each nucleotide), 5 units of Taq DNA polymerase (Stratagene), and sterilized double-distilled water in a final reaction volume of 100 l. The cycling parameters for the PCR were denaturation at 94 C for.1 min, annealing at 56 C for 2 min, and extension at 72 0 C for 3 min; a total of 40 cycles were performed. The cycles were followed by a 15- min extension period at 72 C. Specific amplification of the testin mrna with these primers yielded a 434-bp product. An aliquot of 5 l1 of this PCR reaction product served as a template for hot-nested PCR [23] using a nested primer that was prepared from an internal nucleotide sequence (5'- TGTGAAAATGATGACAGGCTTTCAAAGG-3') corresponding to nucleotides This internal primer was end-labeled by T4 polynucleotide kinase with [y- 32 PATP (specific activity, 7000 Ci/mmol; ICN Biochemicals, Costa Mesa, CA), and about 42 x 106 cpm was used per reaction tube. About 1 g of the antisense primer (nucleotides ) as described above was used as another primer in this second PCR reamplification reaction, together with 10 pil 10-strength PCR buffer, 16 1 l dntps (200 M each nucleotide), 5 units Taq DNA polymerase, and sterilized double-distilled water in a final reaction volume of 100 l. The cycling parameters for the PCR were 94C for 1 min, annealing at 58 C for 2 min, and extension at 72 0 C for 3 min; a total of 40 cycles were performed. The cycles were followed by a 15-min extension period at 72 C. A 10-pLl aliquot was resolved onto a 5% T polyacrylamide gel in 0.5-strength buffer (45 mm Tris, 45 mm boric acid, 1 mm EDTA, ph 8.0 at 22 0 C) and exposed to X-Omat AR film (Eastman Kodak, Rochester, NY) to detect the 268-bp PCR product. RIA of Testin Since cathepsin L shares extensive sequence identity with testin, we assessed whether or not rat cathepsin L, human cathepsin L, or bovine cathepsin D can cross-react with antirat testin antibody using an RIA developed specifically for rat testin [12,13]. Serial doses of cathepsins between 0.01 and 50 Rg were added onto the assay tube to determine whether they competed with the binding of [ 125 I]-testin to its antibody. RIA was performed as previously described [13]. Purified testin II was radiolabeled with Bolton-Hunter reagent, and a monospecific anti-testin II antiserum (CYCMB 22-B-2) at a final dilution of 1: was used [12, 13]. The minimal detectable dose and the 50% displacement were 0.06 ng and 1.32 ng/assay tube, respectively. The intraassay and interassay coefficients were 7 and 11%, respectively. Rat cathepsin L was purified from Sertoli cell-enriched culture medium by sequential anion exchange, gel permeation, and C8 reversed-phase HPLC; its identity was confirmed by direct N-terminal amino acid sequencing, which yielded a partial sequence of NH 2 -TPKFDQTFNAQXH. RESULTS Purification of Testin and Determination of Partial Internal Amino Acid Sequences Testin I (Mr ) and testin II (Mr ) were purified from primary Sertoli cell-enriched culture medium by sequential HPLC and HPEC [12, 13,15]; the purified testin I and testin II are shown in Figure 1. Protein sequence data from the N-terminus of testin I and testin II as well as from internal fragments were obtained after specific cleavage of the purified testin I and testin II with either a Glu- C or Arg-C endopeptidase; these data are shown in Figure 3B. N-Terminal amino acid sequence and partial internal sequence analysis revealed that testin I and testin II were identical, with the exception that testin II had three extra N-terminal amino acids of TAP that were absent in testin I. It was noted that in one batch of purified testin II, the N- terminus amino acid was Ala instead of Thr, suggesting the existence of polymorphism. Immunohistochemical Localization of Testin The localization of testin in the testis and in other tissues was determined by immunohistochemistry. Figure 2, A and

5 344 GRIMA ET AL. 2G) and of the liver (Fig. 21). Figure 2, D, F, H, and J, show the controls for the corresponding tissues using preimmune serum. These results clearly demonstrate that testin is present in the junctional complexes in multiple tissues. FIG. 1. Characterization of testin I and testin II (testins). Testin I and testin II were isolated from primary Sertoli cell-enriched culture medium by HPEC using a 10% T SDS-polyacrylamide gel (3.5 x 50 mm, i.d.). Lanes 3-5 and lanes 6-7 represent the purified testin I and testin II eluted from the polyacrylamide gel in the HPEC step. This is a silver-stained 12.5% T SDSpolyacrylamide gel that shows the purified proteins. Lane 1 contains high molecular weight standards consisting of 0.2 FLg protein each of myosin, Mr ; -galactosidase, Mr ; phosphorylase b, Mr ; BSA, M r ; ovalbumin, Mr Lanes 2 and 8 are low molecular weight standards consisting of 0.2 g protein each of phosphorylase b, Mr ; BSA, Mr ; ovalbumin, Mr ; carbonic anhydrase, Mr ; and lysozyme, Mr D, dye front. B, show a cross section of a seminiferous tubule of the testis at stage VIII. This stage of the spermatogenic cycle is characterized by the presence of elongated spermatids (es) on the border of the epithelium and a homogenous zone of round spermatids (rs). Immunoreactive testin was observed as dark red-brown particles localized in the adult rat testis at the sites of Sertoli-Sertoli junctions below the pachytene spermatocytes (p) and above the spermatogonia (sg, arrowheads). Testin was also observed in the concave side of the elongated spermatids (es, arrowhead with an asterisk). Figure 2B is the preabsorbed control, in which the antibody was preincubated with excessive antigen as described in Materials and Methods. Other controls described in Materials and Methods also yielded similar results (shown in Figure 2B) without any apparent testin immunostaining, indicating the specificity of the immunostaining shown in Figure 2A. In addition to the testis, several other organs were used to determine the localization of testins. In the cauda epididymis (Fig. 2C), testin was localized at the border of the epithelial cell junctions. Testin was localized in the cell junctions of the cortical collecting ducts of the kidney (Fig. 2E). In addition, testin was localized in the cell-cell junctions of the small intestine (Fig. Cloning, Nucleotide Sequence Analysis of the Testin cdna Clone, and Its Primary Structure Determination The strategy for sequencing the full-length testin cdna clone and a partial restriction map are shown in Figure 3A. In order to obtain clones for testin, two degenerate oligonucleotide pools, based on the most frequent codon usage in the rat species [24], were synthesized on the basis of the N-terminal amino acid sequence for testin and a downstream internal amino acid sequence as described in Materials and Methods. These oligonucleotides were used for PCR to obtain a 289-bp cdna fragment coding for part of testin near its N-terminus corresponding to nucleotides of the testin gene shown in Figure 3B. This PCR product was then used to screen a cdna expression library constructed from Sertoli cell poly(a) RNA to isolate the fulllength cdna. Of the 70 positive clones obtained, two clones designated T44 (1371 bp) and T51 (1335 bp), containing the entire testin I and testin II coding regions, were sequenced. The nucleotide sequence and the deduced amino acid sequence are shown in Figure 3B. The two clones, T44 and T51, were identical except that T44 had an additional 36-bp sequence of 5'-GAAACAGATCTGTAGAACCCAA- GATTCTCTGAAACT-3' in the 5' noncoding region as shown in Figure 3B. The open reading frame of this cdna coded for a 333 amino acid protein that started with an ATG initiation codon from the 5' end and ended with a TGA termination codon located 245 nucleotides before the polyadenylation site. The deduced amino acid sequence indicated that testin contained a 16 amino acid signal peptide with FIG. 2. A-J) Immunohistochemical localization of testin in the rat testis (A,B), epididymis (C,D), kidney (E,F), small intestine (G,H), and liver (I,J). Cryostat sections were immunostained by Zymed Histostain-SP with Mayer's hematoxylin as a counterstain as described in Materials and Methods. A) This is a stage VIII tubule of the spermatogenic cycle, characterized by the presence of elongated spermatids (es) on the border of the epithelium and a homogenous zone of round spermatids (rs). Immunoreactive testin was observed as dark red-brown deposits (arrowheads) localized in the adult rat testis at the sites of Sertoli-Sertoli junctions below the pachytene spermatocytes (p) and above the spermatogonia (sg). Testin was also observed in the concave side (arrowhead with an asterisk) of the elongated spermatids (es). Bar = 10 bm. B) Same stage as shown in (A) but stained with anti-testin II antibody preabsorbed with purified testin II. The absence of the red-brown deposits indicates that the staining shown in (A) was specific. C) In this cross section of the cauda epididymis, immunoreactive testin was detected in the luminal border (arrowhead) of epithelium consistent with localization in the epithelial junctional complexes. E) In the rat kidney cortex, testin was localized in the junctions of cortical collecting ducts (arrowhead). (G) A section of the rat small intestine where testin was localized in the columnar epithelium of the intestine villi coincides with the zona occlusions in the intestine epithelium (arrowhead). (I) A section of the adult rat liver showing that testin was localized in junctions between hepatocytes (arrowhead). Figures D, F, H, and J are the corresponding controls using preimmune testin serum. Magnification x285.

6 TESTIN IS A NEW COMPONENT OF CELL JUNCTION 345

7 346 GRIMA ET AL. two possible cleavage sites that yield 314 and 317 amino acids for testin I and testin II with calculated molecular weights of and , respectively. There is only one potential N-glycosylation site at amino acid 157 from the N-terminus. Comparison of the entire coding region of testin with existing sequences in the database in Genbank, EMBL, or PIR revealed that testin shares 58%, 5%, and 61% identity with rat [25, 26], mouse [27, 28], and human [28-30] cathepsin L, respectively, at the amino acid level (Fig. 3C). The testin cdna shares 68% identity with rat cathepsin L at the nucleotide level. The positions of all seven cysteine residues and of eight of the ten tryptophan residues in testin are conserved with respect to those present in cathepsin L. It is noted that of the two predicted active sites (Cys-122 and His-260) of cathepsin L [31, 32], Cys-122 was replaced with Ser-122 in testin while His-260 remained unchanged (Fig. 3C). In addition, the N-terminal region of testin from amino acids 1-98, which is analogous to the activation peptide of cathepsin L, displays a 48% identity with rat cathepsin L, whereas the latter half of the testin sequence shows a 64% identity to the active enzyme of cathepsin L. Testin also displayed 40% sequence identity with mouse CTLA-2a and CTLA-2, two novel molecules whose mrnas are expressed only in activated T lymphocytes and in mast cells [33]. Figure 3D shows the hydrophobicity analysis of testin using the amino acid sequence deduced from its nucleotide sequence based on a program of Kyte and FIG. 3. A-D) Sequence analysis of the testin full-length cdna and comparison of its deduced amino acid sequence with cathepsin L. A) Restriction map of T44 and the sequencing strategy. Closed box indicates predicted open reading frame of the clone and orientation of the PCR product. Selected restriction sites are shown above the diagram. Arrows represent the strand and the extent of DNA sequenced. B) The nucleotide sequence and the predicted amino acid sequence of testin. The sequences are derived from two cdna clones, designated T44 and T51. The two clones are identical except that the T44 has 36 extra nucleotides, marked by broken underline, that are absent from the T51 clone. The lower numbers on the left margin show the nucleotide positions. Negative values indicate nucleotide sequence of the 5' noncoding region. The upper numbers on the left margin designate the deduced amino acid positions of the secreted protein. The signal peptide is in a shaded box and starts with the ATG codon for Met. The single arrow is the predicted cleavage site of testin II, and the double arrow is the predicted cleavage site of testin. N-Terminal and internal (after Glu-C or Arg-C cleavage) amino acid sequences confirmed by direct protein sequencing are underlined. The polyadenylation consensus sequence is boxed. Solid circle indicates the potential N-linked glycosylation site. C) Comparison of the deduced amino acid sequence of testin to rat [25, 261, mouse 127, 281, and human ] cathepsin L and mouse CTLA- 2a and CTLA-2) [331-. Amino acids that are identical to testin are boxed and shaded. It was noted that testin displays 58%, 5%, and 61% similarity to rat, mouse, and human cathepsin L, respectively, at the amino acid level. The positions of eight of the ten tryptophan residues in the testin are conserved with respect to those present in cathepsin L, as are all of the cysteine residues. The Cys-122 in the active site of all the cathepsins was replaced with Ser-122 in testin. D) Hydropathy plot of testin. This plot was generated with the DNASIS program from Hitachi America, Ltd. (version 7.0) using a window of 6 residues. The average hydropathic index of the entire sequence was -5.7, indicated by the horizontal line, suggesting that the overall testin sequence is hydrophilic. Full scale on the y axis extends from -40 for the most hydrophilic to +40 for the most hydrophobic sequence. A PCR Product p, 5 p 4-4 _r~~~~(r 4- Sequencing 04 Strategy B -144 CAC GAG CTg AAA gag ATC TT AGA ACC AA GAT TCT CTG AACTC AAG TCT -15 Met I1e -93 GAG GAG ATC ATC TGA GGA GCA GCA GAC ATC CCA GGT GTA TGA GAC ATG ATT 1 3 [Ala Val Leu Ph. Leu Ala Ile Leu Cys Leu Glu Val Asp SerThr Ale Pro -42 GCT GTT CTC TTC CTA GCG ATT CTC TGC TTG GAA GTT GAC TCA ACT GCT CCA Vl 20 Thr Pro Asp Pro Ser Leu Asp Val Glu Trp Asn Glu Trp Arg Thr Lys His 10 ACA CCA GAT CCC AGT TT GAT GTT GAG TGG AAT GAA TGG AGG ACA AAA CAT 37 Gly Lys Thr Tyr Asn Met Asen Glu Glu Arg Leu Lys Ara Ala Val Trp Glu 61 GGG AAA ACA TAC AAC ATG AAT GAA GAA AGA C AAG AGA GCT GTG TG GAA 54 Lys Asn Phe Lys Met Ile Glu Leu Nis Asn Trp Glu Tyr Leu Glu Gly Arg 112 AAG AAT TTT AAA ATG ATT GAA CTG CAT AAT TGG GAA TAC CTT GAG 000 AGG 71 His Asp Phe Thr Met Ala Met Aen Ala Phe Gly Asp Leu Thr Asn Ile Glu 163 CAT GAC TTC ACC AMT GCA ATG AAT GCC m T GGT GAC TTG ACC AAT ATA GAA 88 Phe Val Lys Met Met Thr Gly Phe Gln Arg Gln Lys Ile Lys Lys Thr His 214 TT GTG AAA ATG ATG A A CA GGC TTT CAA AGG CAG AAO ATC AAG AAG ACG CAC 105 Ile Phe Gin Asp His Gln Phe Leu Tyr Val Pro Lye Ar g Val Asp Trp Arg 265 ATA TTC CAG GAT CAT CAG TTT CTT TAT GTC CCA AAA CGT GT GAT TGG AGA 122 Gln Leu Gly Tyr Val Thr Pro Val Lys Asn Gin Gly His Cys Al. Ser Ser 316 CAA CTT GGC TAT GTG ACT CCT GTG AAG AAT CAG GGT CAT TGT GCC TCT AGT 139 Trp Ala Phe Ser Ala Thr Gly Ser Leu Glu Gly Gln Met Phe Arg Lys Thr 367 TGG GCT TTT AGT GCA ACT GGG TCC CTA GAA GGA CAA ATG TTC AGG AAA ACA 156 Glu Arg Leu Ile Pro Leu Ser Glu Gin Asn Le Leu Asp Cy Met Gly Sr 418 GAA AGA CTG ATC CCT CTG AGT GAG CAG AAC CTA CTG GAC TGC ATG GGA TCT 173 Asn Val Thr His Gly Cys Ser Gly Gly Phe Met Gln Tyr Ale Phe Gin Tyr 469 AAT GTT ACC CAT GGT TGC AGC GGT GGC TTC ATG CAG TAT GCC TTC CAA TAC 190 Val Lys Asp Asn Gly Gly Leu Ale Thr Glu Glu Sr Tyr Pro Tyr Arg Gly 520 GTO AAA GAC AAT GGC GGC CTT GCA ACT GAG GAA TCC TAT CCT TAC AGA GGA 207 Gin Gly Arg Glu Cy Arg Tyr His Al Glu Asen Ser Ala Ale Asn Val Arg 571 CAA GGT AGA GAG TGC AGG TAC CAC GCT GAG AAT TCT GCG GCT AAT GTC AGA 224 Asp Phe Val Gin Ile Pro Gly Ser Glu Glu Ala Leu Met Lys Ale Val Ale 622 GAT TTT GG CAA ATC CCA GGA AGC GAG GAA GCC CTT ATG AAG GCA GTG GCT 241 Lys Val Gly Pro I1e Ser Val Ale Val Asp Ale Ser His Gly Ser Phe Gln 673 AAA GTG GG CCC ATC TCT GTT GCA GTT GAT GT GCC AGC CAT GG GT TCC TTC TCC CAG 258 Phe Tyr Gly Ser Gly Ile Tyr Tyr Glu Pro Gln Cys Lys Arg Veal His Leu 724 TTC TAC GGT AGT GGC ATC TAT TAT GAG CCA CAG TGT AAA AGG GTT CAC CTA 275 An His Ale Val Leu Val Val Gly Tyr Gly Phe Glu Gly Glu Glu Ser Asp 775 AAT CAT GCT GTT CTA GTG GTC GGC TAT GGC T GAA GGA GAA GAA TCA GAT 292 Gly Asn Ser Phe Trp Leu Val Lys Asn Ser Trp Gly Glu Glu Trp Gly Met 826 GGC AAC AGT TTT TG CTC GTC AAG AAC AGC TOG GOT GAG GAA TGG GGC ATG 309 Lys Gly Tyr Met Lys Leu Ale Lys Asp Trp Ser An His Cys Gly Ile Ale 877 AAG GGC TAC AT AAG CTT GCC AAA GAC TG AGC AAC CAC TGC GGG ATT GCT Thr Tyr Ser Thr Tyr Pro Ile Val 928 ACA TAT TCC ACA TAC CCC ATT GTA TG A GCT ACT GCA CAG TGA CAA GGA AGG 979 AGT GGA CTG GAA TGT ACT GAG CAG TTT TCT CCT TCC ACA GAC CAG CCT CTG 1030 CTG TGG GGC AAA GGG GAC AGG GGT AGT GAC AGC TGA GTC ACT GAG GAT CCA 1081 CA CTG ATG AGA ATT AAA CTC ATA AAC ATT AGT GCC ATT AAC AGC mtt GTA 1132 GGA GAG ACT CTC ATA TAA AAA AAA AAT GAA T TCA GTT TAA AAT GCT GTA 1183 CAA ATT TAC ATA TC A CTT AAT TTC ACA ACG GTA AAA

8 TESTIN IS A NEW COMPONENT OF CELL JUNCTION 347

9 348 GRIMA ET AL. FIG. 4. A-C) Processing of testin II by cathepsin D, cathepsin L, and trypsin and at ph. A) Immunoblot of testin II after incubation with cathepsin D, cathepsin L, and trypsin and at low ph for 36 h or 6 h at 37 0 C. Lane 1, testin II alone without treatment. Lanes 2 and 6, testin II incubated with 0.1 Ig of cathepsin D. Lanes 3 and 7 contain testin II incubated with 0.01 pg cathepsin L, and lanes 4 and 8 contain testin II incubated with 0.1 g cathepsin L. Lanes 5 and 9, testin II incubated with 0.05 g trypsin; lanes 10 and 11, testin II incubated at 37 C at low ph for 36 h and 6 h, respectively. Testin II was incubated in the optimal assay conditions for each reaction (see Materials and Methods) for 36 h (lanes 2-5) or 6 h (lanes 6-9) at 37 C. The buffers used for cathepsin D, cathepsin L, and trypsin incubation are described in Materials and Methods. Proteins were then resolved on a 12.5% T SDS-PAGE reducing gel, transferred to nitrocellulose, and subsequently stained with anti-testin II. Each lane contains about 0.25 ILg of purified testin II. Lanes 12 and 13 are identical to lanes 10 and 11, except that [' 25 1]-testin (about cpm/lane) was used and the processing of testin was visualized by autoradiography instead of the anti-testin antibody. Arrowhead indicates the testin 31-kDa fragment. B) The 31-kDa fragment cleaved from testin II in the presence of trypsin was purified by HPEC and sequenced; this indicated that the cleavage site was at Gln-94 from the N-terminus. The sequence was obtained by direct protein sequencing using protein electroblotted onto a PVDF member as previously described [19, 21]. C) Radioautograph of [ 1 ' 25 1]-testin II (about cpm/lane) incubated with increasing amounts of trypsin. Lane 1 contains [' 25 1]-testin II alone. Lanes 2 and 3 contain [ 125 1]-testin II incubated with 0.1 RIg and 0.5 ig of trypsin, respectively. [12 5 l]-testin II was incubated with increasing amounts of trypsin for 1 h at room temperature in the trypsin buffer at ph and then resolved onto a 12.5% T SDS-polyacrylamide gel under reducing conditions, fixed with 50% methanol for 1 h, dried, and exposed to x-ray film for 8 h. Two fragments of M, and Mr were noted. Doolittle [34]. There are five major hydrophobic domains in testin between amino acids 216 and 317 close to the C- terminus (Fig. 3D). The apparent pi for the entire sequence is 6.69 as determined by the PROSIS program (version 7.0) from Hitachi America, Ltd. (Brisbane, CA), whereas the observed pi for testin is as determined by isoelectric focusing [12]. Such differences may be attributed to the carbohydrate moiety, since testin is a concanavalin A (Con A)- and wheat germ agglutinin (WGA)-reactive protein [12]. Processing of Testin by Cathepsin D, Cathepsin L, Trypsin, and at Low ph In view of the striking similarity of the primary sequences of testin and cathepsin L, we investigated whether or not testin would possess the proteolytic activity of cathepsin L. It is known that the activation of cathepsin L involves specific cleavage at low ph at Gln-97 and Asp 273 (Fig. 3C) to release the active enzymatic peptide, amino acids , from the pro-region (amino acid 1-97), designated activation peptide. The mature form of the active protease is a dimer containing two peptides of amino acids and that are associated by disulfide bonds [25]. Other studies have shown that cathepsin D can also activate cathepsin L by releasing its activated peptide from the proregion [35-37]. In light of these observations, we investigated whether or not testin could be processed under conditions known to cleave the activation peptide of cathepsin L to release the enzymatically active peptide. Purified testin II was incubated with various concentrations of cathepsin D, cathepsin L, and trypsin at ph,, and, respectively, in buffers that are optimal for these proteases and also incubated at low ph alone for 6 and 36 h. Incubation of testin with cathepsin D resulted in complete hydrolysis at 36 h (Fig. 4A, lane 2) and partial hydrolysis at 6 h (Fig. 4A, lane 6). The fragments generated by cathepsin D were no longer immunoreactive. Since Sertoli cells are known to produce cathepsin L, which can undergo auto-activation at an acidic ph [5], we sought to examine whether this enzyme would activate testin. Testin II incubated with increasing amounts of cathepsin L for 36 h (Fig. 4A, lanes 3-4)

10 TESTIN IS A NEW COMPONENT OF CELL JUNCTION 349 TABLE 1. Study on the hydrolysis of [ 4 C]casein by trypsin, cathepsin D, cathepsin L, and testin under various conditions.* Protein used** ph*** Hydrolysis products (cpm) Testin II: intact protein without preincubation 0 pg 1 jg 3 jig 1 jg 3 ig 1 Ag 3,Jg Testin II: preincubated at ph for 15 h 1 g 3 plg Testin II: preincubated at ph for 6 h 1 jg 3 jg Trypsin: 0 g 0.01 jlg 0.05 jig 0.1 pg 0.01 ig 0.05 jg 0.1 jg 0.01 g 0.05 g 0.1 jg Testin II: preincubated with 0.05 jig trypsin for 6 h at ph 1.0 jg g 1.0 ig jig 1.0 ig ig Cathepsin D: 0.1 jg 0.5 jg 1.0 g Testin II: preincubated with 0.1 pg of cathepsin D for 1 h at ph 0.5 jig 1.0 jg jg 0.5 jg 1.0 jg jg Human cathepsin L: 0 ig 0.01 jig 0.1 jg 0.5 g 1088 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 37 TABLE 1. Study on the hydrolysis of [ 4 C]casein by trypsin, cathepsin D, cathepsin L, and testin under various conditions. (continued)* Protein used** ph*** Hydrolysis products (cpm) Rat testicular cathepsin L: preincubated at ph for 3 h 0 pg jig jig pig without pre-incubation at ph for 3 h Testin II: preincubated with 0.1 jig of activated rat testicular cathepsin L for 3 h at ph 1 tpg 3826 ± jig 3759 ±+ 158 Testin II: fragment isolated by HPEC as shown in Fig. 4B: 0.1 g 1178 ± jg jig 937 ± pg 1028 ± 58 *The protease assays were performed as described in Materials and Methods using various purified proteins. The total count was about 10,000 cpm containing pci [ 14 C]casein in each assay tube. Each number represents the mean ± SD of triplicate determinations of a given experiment. These experiments were repeated twice using two separate batches of purified testin II and similar results were obtained in each set of experiments. Another set of experiments was also performed using purified testin I and identical results were obtained. **Human cathepsin L (Calbiochem) and bovine cathepsin D (Sigma) used for this study were obtained commercially without further purification. Rat testicular cathepsin L and testin II were isolated from Sertoli cellenriched culture medium as described in Materials and Methods and their purity was confirmed by SDS-PAGE and direct protein sequencing. ***The buffers used for the protease assays were as follows: trypsin (a serine protease), 25 mm Tris, ph containing 3% NaCI; cathepsin L (a cysteine protease), 0.4 mm sodium acetate, 4 mm EDTA, 1 mm DTT, ph ; cathepsin D (an aspartic protease), 40 mm sodium citrate, ph. Preincubations of testin with different proteases was performed at the ph optimal for the specified protease in a reaction volume of Thereafter, the sample was suspended in a final reaction volume of 200 il using corresponding buffers described above at specified ph. and 6 h (lanes 7-8) also showed no specific proteolytic cleavage, suggesting that cathepsin L, in contrast to cathepsin D, did not hydrolyze testin II. Since other studies have shown that collagenase, a metalloprotease, can be activated by trypsin [38], we also examined the effect of trypsin on testin II. Trypsin induced specific cleavage of testin II to yield a 31-kDa immunoreactive fragment (Fig. 4A, lanes 5 and 9). Direct amino acid sequencing of this fragment using protein electrophoretically blotted onto PVDF membrane indicated that testin II was cleaved at Gln-94 from the N-terminus to generate this 31-kDa fragment vs. Gln-97 in cathepsin L. This site is not a site specifically cleaved by trypsin, since trypsin is a Lys-C and Arg-C protease. Thus, the mechanism by which trypsin induced cleavage of testin at Gln-94 is not known. We have subsequently purified the 31-kDa fragment by HPEC and confirmed its cleavage site by direct protein sequencing (Fig. 4B). Also, it was dem-

11 350 GRIMA ET AL.

12 TESTIN IS A NEW COMPONENT OF CELL JUNCTION 351 onstrated that the purified 31-kDa fragment did not possess any proteolytic activity (Table 1). Since cathepsin L can be activated by exposure to acidic ph alone, testin was incubated at low ph () for 36 h and 6 h (Fig. 4A, lanes 10 and 11). Short exposure time (6 h) to acidic conditions could also produce a 31-kDa fragment but not with the efficiency of trypsin (Fig. 4A, lane 11 vs. lanes 5 and 9), whereas longer exposure time (36 h) (Fig. 4A, lane 10) produced a more extensive hydrolysis compared to the 6-h exposure (Fig. 4A, lane 11 vs. lane 10); experiments using [1 25 I]-testin confirmed this observation (Fig. 4A, lanes 12, 13 vs. lanes 10, 11). In contrast, exposure of testin to ph, the assay condition for cathepsin L, did not induce cleavage (Fig. 4A, lanes 3, 4, 7, 8 vs. lanes 10-13). It was also noted that the 31-kDa fragment generated by exposure of testin to ph had the identical cleavage site as that generated by trypsin treatment as verified by direct protein sequencing. Further studies were also performed on the enzymatic cleavage of radiolabeled testin by trypsin to determine the proteolytic processing of testin. When [ 125 I]-testin II was incubated with increasing amounts of trypsin, only two fragments, with apparent molecular weights of and , were produced (Fig. 4C, lanes 2-3 vs. lane 1). The presence of the Mr fragment was not noted in the immunoblots described above (Fig. 4A), suggesting that this fragment was no longer immunoreactive. Hydrolysis of [ 14 C]Casein by Trypsin, Cathepsin D, and Cathepsin L, and Their Comparison to Testin When intact and native testin was assayed alone at serial doses in buffers known to activate serine, cysteine, or aspartic proteases, testin did not possess any protease activity whereas cathepsin L isolated from Sertoli cell-enriched culture medium cleaved [1 4 C]casein dose-dependently (Table 1). Casein was used as the substrate for these assays because it is a universal protease substrate [39]. It was noted FIG. 5. A-D) Tissue distribution of testin mrna examined by Northern blot (A,B), hot-nested PCR (C), and immunoblot (D). A) Total RNA from various male and female rat tissues (approximately 30 g/lane) was resolved onto a 1.1% agarose-formaldehyde gel, transferred to Nytran nylon filter, and hybridized with an [-32P]-labeled antisense RNA probe synthesized from the 289-bp PCR product as described in Materials and Methods. SC, Sertoli cells prepared from 20-day-old rat testes; Ti, 20-day-old testis; T, adult testis from 60-day-old rats; E, epididymis; K, kidney; B, brain; L, liver; SP, spleen; I, intestine; 0, ovary. All organs were obtained from 60-day-old male or female rats except where noted. B) The same blot as shown in (A) but stained with ethidium bromide showing the 28S and 18S rrna subunits. C) Hot-nested PCR from 4 g of total RNA from various male and female rat tissues. SC, Sertoli cell; T. testis; E, epididymis; K, kidney; B, brain; L, liver; SP, spleen; I, intestine; 0, ovary; U, uterus. (D) Immunoblot of rat testin in various tissues of adult rats. Sertoli cell-enriched culture media and cytosols from various tissues were fractionated by SDS-PAGE on a 10% T polyacrylamide gel, transferred to nitrocellulose, and stained with anti-testin II antibody. Approximately 2 and 10 jig of Sertoli cell-enriched culture medium (SCCM) were loaded. All other lanes contain approximately 100 ig of total protein from T, testis; SP, spleen; P, prostate; L, liver; B. brain; K, kidney; E, epididymis; 0. ovary; U, uterus; S. serum. v m 100o O-OSCCCM A--Ca Ithepsin D \ O-1- OCc ithepsin L,,,, ,,=U DOSE OF COMPETITOR (LI or g/assoy tube) 0 *.. I FIG. 6. Competition on the displacement of [ 25 1]-testin onto the testin antibody by Sertoli cell-enriched culture medium (SCCM) in microliters per assay tube and bovine cathepsin D and rat testicular cathepsin L in micrograms per assay tube. The abscissa is the log dose of competitor. The ordinate is expressed as percentage of B/Bo, where B and Bo are the counts bound in the presence (B) and absence (Bo) of unlabeled competitor. that rat testicular cathepsin L failed to cleave [ 14 C]casein without a preincubation period to activate the enzyme. When the testin fragment isolated by HPEC (as shown in Figure 4B) was assayed for protease activity, it did not possess any protease activity in contrast to human cathepsin L, bovine cathepsin D, and rat testicular cathepsin L (Table 1). Also, the activated rat testicular cathepsin L failed to induce protease activity in testin (Table 1). Proteolytic activity of testin was also assayed at various phs and in several buffer systems to check for the activity of serine, aspartic, and cysteine proteases since it was noted that of the two predicted active sites, Cys-122 and His-260, of cathepsin L, Cys-122 was replaced with Ser-122 in testin while His-260 remained unchanged. Since low ph, trypsin, and possibly cathepsin D may release the activated peptide of testin, we assayed the proteolytic activity of testin preincubated at low ph or with one of the specified proteases on the hydrolysis of [ 14 C]casein, as shown in Table 1. Neither low ph, known to activate cathepsin L, nor preincubation with other proteases could induce protease activity in testin (Table 1). When increasing doses of testin were preincubated at ph for 15 h and tested for their ability to proteolytically cleave [' 4 C]casein, no hydrolysis products were found in testing for a cysteine protease (Table 1). Thus, under all the conditions that are known to activate serine, aspartic, and cysteine proteases, testin did not display any proteolytic activity (Table 1). Tissue Distribution of Testin mrna Northern blot analysis revealed a 1.6-kb testin transcript in cultured Sertoli cells (Fig. 5A), immature and adult rat testis, and ovary of the rat (Fig. 5A), but not in the epididymis, kidney, brain, liver, or spleen from either male or female adult rats. Figure 5B is an ethidium bromide staining of the gel shown in Figure 5A and demonstrates that

13 352 GRIMA ET AL. similar amounts of total RNA, about 30 ltg/lane, were used for each tissue. Through the use of a ribonuclease protection assay with an [c- 32 P]-labeled antisense RNA probe based on the 289-bp testin PCR product, testin mrna was detectable only in the gonads (results not shown). It was postulated that if testin is indeed an integral component of the junctional complexes in multiple tissues, its mrna expression would be low in tissues undergoing minimal restructuring such as the epididymis and kidney vs. the gonads. To test this hypothesis, we amplified the RNA detection sensitivity by hot-nested RT-PCR using about 4 g of total RNA from the various tissues. As shown in Figure 5C, a distinctive band of 268 bp was obtained not only from Sertoli cells, testis, and ovary RNA but also from the epididymis, kidney, and intestine (Fig. 5C). Immunoblot analysis on the distribution of testin in these tissues showed positive immunoreactive testin only in the Sertoli cell-enriched culture medium and ovary, yielding both testin I and testin II (Fig. 5D). However, in the testis, spleen, prostate, liver, brain, kidney, epididymis, uterus, and serum of male or female adult rats, testin was not detectable by immunoblot (Fig. 5D). The apparent absence of testin in the testis and other tissues might simply reflect the limited sensitivity of this technique, which required ng of protein for visualization. This study, however, indicated that testin was highly expressed in the Sertoli cell and ovary-an observation consistent with the postulate that the expression of testin is correlated with rapid tissue restructuring in the gonads. Cross-Reactivity between Testin and Cathepsin L Since testin shares extensive primary sequence identity with cathepsin L, we examined whether cathepsin L crossreacted with the testin antibody. Serial doses of rat cathepsin L isolated from Sertoli cell-enriched culture medium and bovine cathepsin D, between 0.1 and 50 g/assay tube, were compared with Sertoli cell-enriched culture medium for their ability to displace bound radioactivity (Fig. 6). It was noted that rat testicular cathepsin L and cathepsin D showed no cross-reactivity with testin. Similarly, human cathepsin L at jig/assay tube did not cross-react with testin antibody (data not shown). DISCUSSION In the present study we have reported the primary amino acid sequence of testin deduced from its nucleotide sequence. Comparison of the testin amino acid sequence with existing sequences at Genbank and EMBL revealed that testin is related to cathepsin L and to mrnas of mouse CTLA- 2a and CTLA-2[3. The physiological function of CTLA-2a and CTLA-23 is not known; it has been shown that their mrnas are detectable in activated T lymphocytes and in mast cells but not in fibroblasts, thymocytes, and liver cells [33]. Interestingly, their nucleotide sequences display striking homology to the mouse cysteine proteinase precursor, but only to the pro-region [32,33] instead of the enzymatic moiety. Thus, it is unlikely that CTLA-2a and CTLA-2f are proteases. Since the protein molecules encoded by these cdnas have not been isolated for protease assay, whether or not they are proteases is uncertain. Cathepsin L is a lysosomal cysteine protease initially identified in the liver and kidney [40-42]. Subsequent studies have shown that it is also secreted by Sertoli cells in the testis [4,5]. Cathepsin L is an Mr protein that consists of an active peptide (amino acids disulfide-bonded with amino acids ) and a proregion of amino acids 1-97 from the N-terminus [25, 26]. This pro-region peptide must be removed either by autoactivation at low ph or by another enzyme such as cathepsin D [43] to activate the enzyme [36,44]. When the precursor form of cathepsin L is treated at ph 3-5, specific cleavage occurs at Gln-97 to yield an intermediate peptide of Ile 98-Asn 318; this then forms the mature protease consisting of two subunits of Mr between Ile-98 and Asp- 273 and Mr 5000 between Ser-274 and Asn-318 where the two subunits are disulfide-bonded [25,44]. It is noteworthy that incubation of testin at ph or with trypsin at ph yields a 31-kDa fragment formed by specific cleavage at Gln- 94, similar to what is observed for cathepsin L, as examined by immunoblot analysis. Through use of [' 25 I]-testin II incubated with increasing doses of trypsin, an additional peptide with an apparent molecular weight of was also noted; this might represent the N-terminal region of amino acids 1-94 in addition to the Mr fragment. Direct protein sequencing of this Mr fragment after ph incubation or trypsin treatment failed to determine its identity. The N-terminus was blocked, possibly because of modification upon releasing the fragment between Phe-95 and Val-317. Nonetheless, the processing of testin is similar to only one aspect of that of cathepsin L, in that testin can undergo site-specific cleavage at Gln-94, whereas the generation of the 5-kDa fragment from the 31-kDa fragment was not noted. The action of trypsin on testin does not appear to be the result of enzymatic cleavage, since trypsin is a serine protease that cleaves specifically at Lys and Arg from the C-terminus [45] and since the purity of the trypsin used in this study was confirmed by reverse-phase HPLC that yielded a single peak as monitored by a diode array detector at nm. It is possible that the exposure of testin to ph or trypsin might somehow destabilize its secondary or tertiary structure to allow this specific cleavage to occur. During the purification of testin from Sertoli cell-enriched culture medium, we did not observe the existence of the 31-kDa fragment that cross-reacted with the testin antibody. Also, pulse-chase labeling analysis using [ 3 5S]methionine with primary cultures of Sertoli cells did not yield a 31-kDa fragment either in the cytosol or in the medium [14]. These studies indicate that under normal culture conditions, Sertoli cells do not process testin to the 31-kDa fragment. It is not known, however, whether such cleavage can occur in vivo.

14 TESTIN IS A NEW COMPONENT OF CELL JUNCTION 353 Under the conditions that are known to activate serine, aspartic, and cysteine proteases, testin did not exhibit any proteolytic activity. This is not entirely unexpected as it could be attributable to a single amino acid substitution at the predicted active site of cathepsin L from Cys-122 to Ser-122 in testin. Casein is a universal protease substrate [39] that is cleaved by trypsin, rat testicular cathepsin L, human cathepsin L, and bovine cathepsin L. By contrast, testin had no apparent effect on casein, thus excluding the possibility that it is a cathepsin L-like protease. Other experiments performed during the course of this investigation, in which a quantitative [ 3 H]collagen film assay was used [46], also showed that trypsin, bovine cathepsin D, and rat testicular cathepsin L cleaved [ 3 H]collagen dose-dependently under conditions known to activate the serine, aspartic, and cysteine protease, respectively; testin or its Mr fragment, however, failed to hydrolyze this other substrate. However, it is possible that testin is a unique protease such as cathepsin H, which is a sulfhydryl aminopeptidase [47] that has limited substrate specificity. For instance, cathepsin H cleaves N-terminal amino acid residues with large hydrophobics such as Phe, Trp, Leu, and Tyr or basic side chain (Arg and Lys) and cannot hydrolyze amino acids containing small or polar side chains such as Gly, His, or Pro [47]. However, in view of its unique and widespread localization in the junctional complexes of multiple tissues, testin appears to be a junctional complex-associated molecule that is structurally related to the cysteine protease superfamily but devoid of any protease activity. Such an observation and conclusion are not without precedence. For instance, corticosteroidbinding protein (CBG), an extracellular steroid-binding protein that binds corticosteroid with high affinity [48,49], is a member of the serine protease inhibitor superfamily that displays 43, 44, and 31% identity to cxi-antitrypsin, alantichymotrypsin, and antithrombin III, respectively; however, CBG does not exhibit any protease or protease inhibitor activity. Also, E-cadherin (ovomorulin; a Ca 2 +-dependent cell adhesion molecule) is synthesized as a 135-kDa precursor polypeptide that must be specifically cleaved to release a 129 amino acid peptide to generate the 120-kDa mature protein to yield the uvomorulin-mediated adhesion function: it was found that in vitro-expressed uvomorulin, containing a mutated site that could no longer be cleaved to release the 129 amino acid peptide, could not function as a cell adhesion molecule [50]. The two cdna clones, T44 and T51, that contain the complete coding sequence for testin are identical in sequence except that T44 has an extra 36 bp in the 5' noncoding region located 52 nucleotides before the initiation codon. The significance of these 36 bp is not known. On the basis of the primary amino acid sequence deduced from testin cdna, we postulate that there are two possible cleavage sites that yield two virtually identical proteins in a manner similar to that observed for the rat epididymal retinoic acid-binding protein (ERABP). ERABP is an androgen-dependent secretory protein in the epididymis that has two molecular variants, of Mr and Mr , the latter form having three extra amino acids of TEG from the N- terminus; both molecular forms also appear to be coded by the same gene with two different cleavage sites [51-53]. This postulate is strengthened by the fact that direct amino acid sequencing of five different fragments of testin I and testin II (derived from both an Arg-C and a Glu-C endopeptidase) yields identical sequences for both proteins with the exception of the three amino acid difference at the N- terminus. It is therefore possible that the signal peptide is 16 amino acids for testin II and 19 amino acids for testin I to give rise to these two proteins from the same gene. However, from the work of von Heijne [54], who examined a sample of 78 eukaryotic signal sequences, it should be noted that the last residue of the signal peptide can be either Ala, Cys, Gin, Gly, Ser, or Thr, but not Pro. It therefore seems improbable for testin I to cleave at Pro, which is its -1 amino acid from the N-terminus. Thus, testin I may arise from testin II as a result of post-translational processing. Alternatively, the two molecular variants of testin that arise from a single gene may be the result of alternative splicing, analogous to the situation with the activin receptor [55], vinculin [56], and cathepsin L [57], which give rise to several molecular variants by alternative splicing of the same gene. To provide a definite answer to this question, we must await the cloning and structural determination of the testin genomic DNA. Analysis of the hydropathy of the testin primary structure that defines the relative hydrophobicity and hydrophilicity according to Kyte and Doolittle [34] has shown that testin is a hydrophilic protein with an average hydropathic index of 5.7, having five major hydrophobic domains between 216 and 317 amino acids near its C-terminus whereas the sequence around its N-terminus is mainly hydrophilic. Other studies using pulse-chase labeling analysis have shown that testin is rapidly secreted into the medium upon its synthesis [14]. In order for this protein to become an integral component of the cell junction, we postulate that the secreted protein is either activated or internalized so that the hydrophobic domain near its C-terminus attaches to the cell membrane that anchors the protein molecule to become a component of the cell junction. In this study we noted that testin I and testin II differed by three amino acids at the N-terminus, but the differences in their apparent molecular weights on SDS-PAGE cannot be accounted for by these amino acids. Therefore, it is possible that differential glycosylation might explain the discrepancy, since both testin I and testin II are shown to be Con A- and WGA-reactive [12]. The localization of testin in the cellular junctions of many tissues examined reveals that this protein is a novel component of these junctions. We have also examined the immunohistochemical localization of testin in the adult rat testis between Sertoli cells and elongated spermatids at stages

15 354 GRIMA ET AL. VII and VIII of the cycle prior to release of the germ cells into the tubular lumen. Immunoreactive testin was localized on the concave surface of the elongated spermatids but not in the round spermatids. Since testin appears to be a component of the cell junction and is localized predominantly near the heads of the elongated spermatids before their release into the seminiferous tubular lumen, it may function as a cell junctional molecule that anchors the elongated spermatids in the seminiferous epithelium until their maturation is completed. The expression of testin mrna in gonadal tissues is several orders of magnitude higher than in other tissues, suggesting that its expression is related to the constant turnover of the junctions during germ cell and follicle development. The low abundance of testin mrna expression in the non-gonadal tissues is correlated with the low rate of junctional complex turnover. Such a postulate can also be used to interpret the drastic increase in the amount of testicular testin in X-irradiated or busulfan-treated rats [13,15,16], during which treatments the germ cells were selectively depleted. Such a dramatic loss in germ cells may induce Sertoli cells to secrete testin to reconstruct the Sertoli-germ cell junctions in the testis. There have been many junctional proteins isolated and studied to date that give a junctional immunocytochemical staining pattern similar to that of testin, such as ZO-1 [58], vinculin [59], cadherins [60], cingulin [61], -, 13-, and y-catenins [62], uvomorulin [63], and a-actinin [64]. These proteins interact with each other and can become tightly associated with actin to induce cell proliferation and cell migration during embryonic development, differentiation, and tissue morphogenesis. These proteins are located at sites where the intracellular microfilament network establishes a morphologically defined junction with the extracellular matrix. For instance, cadherins are a family of transmembrane cell-cell adhesion molecules that are localized in specialized cellular junctions such as the intercellular adherens junction, the intermediate junction, or zonular adherens [65] and in the desmosome [66]. It is known that cadherins interact with each other via their extracellular domain in the presence of calcium [67]. The cytoplasmic domain of cadherins has been shown to interact with a variety of adhesion molecules such as actin, oa-, -, and y-catenins, talins, vinculin, and t-actinin [68]. Recent immunogold studies of vinculin in the rat testis indicated its localization in the ectoplasmic specializations both at sites of attachment of spermatids and adjacent to basal Sertoli cell junctions [69], similar to the localization of testin. All these molecules have been isolated directly from partially purified junctional complexes or identified with antibodies against such complexes or their components. Several of these tight junction-associated molecules, such as vinculin, has been shown to be synthesized by fibroblasts [70]. In contrast, testin has been shown to be synthesized and secreted by Sertoli cells [13,14]. In view of its unusual distribution in the cell junctions of various tissues, we postulate that testin is a new component of the cell junction whose expression is correlated with the rate of junctional complex turnover. In summary, we have determined the primary sequence structure of testin and shown that it is related to members of the cysteine protease superfamily structurally. However, testin is not a cathepsin L-like protease, but is a component of the junctional complexes in the testis and other organs. ACKNOWLEDGMENT We wish to thank Ms. Jean Schweis for her assistance in the preparation of this manuscript. REFERENCES 1. Fawcett DW. The Sertoli cell. In: Handbook of Physiology, Vol. 5. Washington DC: American Physiological Society; 1975: Bardin CW, Cheng CY, Musto NA, Gunsalus GL. The Sertoli cell. In: Knobil E, Neill JD, Ewing LL, Greenwald CS, Markert CL, Pfaff DW (eds.), The Physiology of Reproduction. New York: Raven Press; 1988: 1: Pelletier RM, Byers SW. The blood-testis barrier and Sertoli cell junctions: structural considerations. Microsc Res Tech 1992; 20: Cheng CY, Zwain I, Grima J, Bardin CW. Structure and functions of Setoli cell proteins. In: Baccetti B (ed.), Comparative Spermatology, 20 Years After. New York: Raven Press; 1991: Erickson-Lawrence M, Zabludoff SD, Wright WW. Cyclic protein-2, a secretary product of rat Sertoli cells, is the proenzyme form of cathepsin L. Mol Endocrinol 1991; 5: Lacroix M, Smith FE, Fritz lb. Secretion of plasminogen activator by Sertoli cellenriched cultures. Mol Cell Endocrinol 1977; 9: TsurutaJK, O'Brien DA, Griswold MD. Sertoli cell and germ cell cystatin c: stagedependent expression of two distinct messenger ribonucleic acid transcripts in rat testes. Biol Reprod 1993; 49: Cheng C, Calcagno K, Tio S, Silvestrini B. Identification and purification of a tissues inhibitor of metalloproteinases-2 (TIMP-2) from Sertoli cell-enriched culture medium. Mol Biol Cell 1993; 4(suppl):218a (abstract 1266). 9. Cheng CY, Grima J, Stahler MS, Guglielmotti A, Silvestrini B, Bardin CW. Sertoli cell synthesizes and secretes a protease inhibitor, 2 -macroglobulin. Biochemistry 1990; 29: Stabler MS, Schlegel P, Bardin CW, Silvestrini B, Cheng CY. a,-macroglobulin is not an acute-phase protein in the rat testis. Endocrinology 1991; 128: Cheng CY, GrimaJ, Pineau C, Stahler MS,Jegou B, Bardin CW. 2 -Macroglobulin protects the seminiferous epithelium during stress. In: Sheppard KE, BoublikJH, Funder JW (eds.), Stress and Reproduction. New York: Raven Press; 1992: 86: Cheng CY, Bardin CW. Identification of two testosterone-responsive testicular proteins in Sertoli cell-enriched culture medium whose secretion is suppressed by cells of the intact seminiferous tubule. J Biol Chem 1987; 262: Cheng CY, Grima J, Stahler MS, Lockshin RA, Bardin CW. Testins are two structurally related Sertoli cell secretory proteins whose secretion is tightly coupled to the presence of germ cells. J Biol Chem 1989; 264: Grima J, Pineau C, Bardin CW, Cheng CY. Rat Sertoli cell clusterin, a 2 -macroglobulin, and testins: biosynthesis and differential regulation by germ cells. Mol Cell Endocrinol 1992; 89: Cheng CY, Morris I, Bardin CW. Testins are structurally related to the mouse cysteine proteinase precursor proregion but devoid of any protease/anti-protease activity. Biochem Biophys Res Commun 1993; 191: Jegou B, Pineau C, Velez de la Calle JF, Touzalin A-M, Bardin CW, Cheng CY. Germ cell control of testin production is inverse to that of other Sertoli cell products. Endocrinology 1993; 132: Cheng CY, Mathur PP, Grima J. Structural analysis of clusterin and its subunits in ram rete testis fluid. Biochemistry 1988; 27: Cheng CY. Purification of a calcium binding protein (rat SPARC) from primary Sertoli cell-enriched culture medium. Biochem Biophys Res Commun 1990; 167: Saso L, Silvestrini B, Cheng CY. The use of high performance electrophoresis chromatography (HPEC) for the micropurification of cerebrospinal fluid proteins in the rat. Anal Biochem 1993; 212:

16 TESTIN IS A NEW COMPONENT OF CELL JUNCTION Saso L, Silvestrini B, Zwain, Guglielmotti A, Luparini MR, Cioli V, Cheng CY. Abnormal glycosylation of hemopexin in arthritic rats can be blocked by bindarit, 2-[(l-benzyl-indazol-3-y)methoxyl-2-methyl propionic acid. J Rheumatol 1992; 19: Leone MG, Saso L, Del Vecchio A, Mo MY, Silvestrini B, Cheng CY. Micropurification of cerebrospinal fluid proteins by high performance electrophoresis chromatography (HPEC). J Neurochem 1993; 61: Cheng CY, Chen C-LC, Feng ZM, Marshall A, Bardin CW. Rat clusterin isolated from primary Sertoli cell-enriched culture medium is sulfated glycoprotein-2 (SGP- 2). Biochem Biophys Res Commun 1988; 155: Simmonds P, Balfe P, Peutherer JF, Ludlam CA, Bishop JO, Brown AJL. Human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheral mononuclear cells at low copy numbers. J Virol 1990; 64: Wada K, Aota S, Tsuchiya R, Ishibashi F, Gojobori T, Ikemura T. Codon usage tabulated from the GenBank genetic sequence data. Nucleic Acids Res 1990; 18: Ishidoh K, Towatari T, Imajoh S, Kawasaki H, Kominami E, Katunuma N, Suzuki K. Molecular cloning and sequencing of cdna for rat cathepsin L. FEBS Lett 1987; 223: Ishidoh K, Kominami E, Suzuki K, Katunuma N. Gene structure and 5'-upstream sequence of rat cathepsin L. FEBS Lett 1989; 259: Troen BR, Gal S, Gottesman MM. Sequence and expression of the cdna for MEP (major excreted protein), a transformation-regulated secreted cathepsin. Biochem J 1987; 246: Joseph LJ, Chang LC, Stamenkovich D, Sukhatme VP. Complete nucleotide and deduced amino acid sequences of human and murine preprocathepsin L. J Clin Invest 1988; 81: Gal S, Gottesman MM. Isolation and sequence of a cdna for human pro-(cathepsin L). Biochem J 1988; 253: Ritonja A, Popovic T, Kotnik M, Machleidt W, Turk V. Amino acid sequences of the human kidney cathepsins H and L. FEBS Lett 1988; 228: Kamphuis IG, Drenth J, Baker EN. Comparative studies based on the high-resolution structures of papain and actinidin, and on amino acid sequence information for cathepsins B and H, and stem bromelain. J Mol Biol 1985; 182: Portnoy DA, Krickson AH, Kochan J, Ravetch JV, Unkeless JC. Cloning and characterization of a mouse cysteine proteinase. J Biol Chem 1986; 261: Denizot F, Brunet JF, Roustan P, Harper K, Suzan M, Luciani MF, Mattei MG, Golstein P. Novel structures CTLA-2a and CTLA-2p expressed in mouse activated T cells and mast cells and homologous to cysteine proteinase proregions. Eur J Immunol 1989; 19: Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein. J Mol Biol 1982; 157: Nishimura Y, Kawabata T, Kato K. Identification of latent procathepsins B and L in microsomal lumen: characterization of enzymatic activation and proteolytic processing in vitro. Arch Biochem Biophys 1988; 261: Wiederanders B, Kirschke H. The processing of a cathepsin L precursor in vitro. Arch Biochem Biophys 1989; 272: Nishimura Y, Kawabata T, Furuno K, Kato K. Evidence that aspartic protease is involved in the proteolytic processing event of procathepsin L in lysosomes. Arch Biochem Biophys 1989; 271: Liotta AL, Tryggvason K, Garbisa S, Gehron Robey P, Abe S. Partial purification and characterization of a neutral protease which cleaves type IV collagen. Biochemistry 1981; 20: Twining SS. Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes. Anal Biochem 1984; 143: Cleveland DW, Fischer SG, Kirschner MW, Laemmli UK. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem 1977; 252: McDonald PN, Ong DE. A lecithin:retinol acyltransferase activity inhuman and rat liver. Biochem Biophys Res Commun 1988; 827: Baricos WH, Zhou Y, Mason RW, Barrett AJ. Human kidney cathepsins B and L. Characterization and potential role in degradation of glomerular basement membrane. J Biochem 1988; 252: Erickson A. Biosynthesis of lysosomal endopeptidases. J Cell Biochem 1989; 40: Bando Y, Kominami E, Katunuma N. Purification and tissue distribution of rat cathepsin L. J Biochem 1986; 100: Gonias SL, Pizzo SY. Conformation and protease binding activity of binary and ternary human ci 2 -macroglobulin-protease complexes. J Biol Chem 1983; 258: Johnson-Wint B. A quantitative collagen film collagenase assay for large numbers of samples. Anal Biochem 1980; 104: Takahashi T, Dehdarani AH, Tang J. Porcine spleen cathepsin H hydrolyses oligopeptides solely by aminopeptidase activity. J Biol Chem 1988; 263: Hammond GL, Smith CL, Goping IS, Underhill DA, Harley MJ, Reventos J, Musto NA, Gunsalus GL, Bardin CW. Primary structure of human corticosteroid binding globulin deduced from hepatic and pulmonary cdnas exhibits homology with serine protease inhibitors. Proc Natl Acad Sci USA 1987; 84: Bardin CW, Gunsalus GL, Musto NA, Cheng CY, Reventos J, Smith C, Underhill DA, Hammond G. Corticosteroid binding globulin, testosterone-estradiol binding globulin, and androgen binding protein belong to protein families distinct from steroid receptors. J Steroid Biochem 1988; 30: Ozawa M, Kemler R. Correct proteolytic cleavage is required for the cell adhesive function of uvomorulin. J Cell Biol 1990; 111: Brooks DE, Means AR, Wright EJ, Singh SP, Tiver KT. Molecular cloning of the cdna for two major androgen-dependent secretory proteins of 18.5 kilodaltons synthesized by the rat epididymis. J Biol Chem 1986; 246: Newcomer ME, Ong DE. Purification and crystallization of a retinoic acid-binding protein from rat epididymis. J Biol Chem 1990; 265: Zwain I, Grima J, Cheng CY. Rat epididymal retinoic acid-binding protein (EP- RABP): Development of a radioimmunoassay, its tissue distribution, and its changes in selected androgen-dependent organs following orchiectomy. Endocrinology 1992; 131: von Heijne G. Patterns of amino and near signal-sequence cleavage sites. Eur J Biochem 1983; 133: Attisano L, WranaJL, Cheifetz S, MassagueJ. Novel activin receptors: distinct genes and alternative mrna splicing generates a repertoire of serine threonine kinase receptors. Cell 1992; 68: Moiseyeva EP, Weller PA, Zhidkova NI, Corben EB, Patel B, Jasinska I, Koteliansky VE, Critchley DR. J Biol Chem 1993; 268: Chauhan SS, Popescu NC, Ray D, Fleischmann R, Gottesman MM, Troen BR. Cloning, genomic organization, and chromosomal localization of human cathepsin L. J Biol Chem 1993; 268: Stevenson BR, Siliciano JD, Mooseker MS, Goodenough DA. Identification of ZO- 1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J Cell Biol 1986; 103: Grove BD, Pfeiffer DC, Allen S, Vogl AW. Immunofluorescence localization of vinculin in ectoplasmic ("junctional") specializations of rat Sertoli cells. Am J Anat 1990; 188: Takeichi M. Cadherins: a molecular family important in selective cell-cell adhesion. Annu Rev Biochem 1990; 59: Citi S, Sabanay H, Jakes R, Geiger B, Kendrick-Jones J. Cingulin, a new peripheral component of tight junctions. Nature 1988; 333: Ozawa M, Baribault H, Kemler R. The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different specific species. EMBO J 1989; 8: Ozawa M, Kemler R. Molecular organization of the uvomorulin-catenin complex. J Cell Biol 1992; 116: Geiger B. Membrane cytoskeleton interaction. Biochim Biophys Acta 1983; 737: Geiger B, Ginsberg D. The cytoplasmic domain of adherens-type junctions. Cell Motil Cytoskeleton 1991; 20: Buxton RS, Magee AI. Structure and interactions of desomosomal and other cadherins. Semin Cell Biol 1992; 3: Takeichi M. Cadherin cell adhesion receptors as a morphogenetic regulator. Science 1991; 251: Burridge K, Fath K, Kelly T, Nuckolls G, Turner C. Focal adhesions: Transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu Rev Cell Biol 1988; 4: Pfeiffer DC, Vogl AW. Evidence that vinculin is co-distributed with actin bundles in ectoplasmic ("junctional") specializations of mammalian Sertoli cells. Anat Rec 1991; 231: Ungar F, Geiger B, Ben-Ze'ev A. Cell contact- and shape-dependent regulation of vinculin synthesis in cultured fibroblasts. Nature 1986; 319: