Prothymosin α Is Processed to Thymosin α1 and Thymosin α11 by a Lysosomal Asparaginyl Endopeptidase
2003; Elsevier BV; Volume: 278; Issue: 15 Linguagem: Inglês
10.1074/jbc.m213005200
ISSN1083-351X
AutoresConcepción S. Sarandeses, Guillermo Covelo, Cristina Dı́az-Jullien, Manuel Freire,
Tópico(s)Microtubule and mitosis dynamics
ResumoThymosin α1(Tα1) and thymosin Tα11(Tα11) are polypeptides with immunoregulatory properties first isolated from thymic extracts, corresponding to the first 28 and 35 amino acid residues, respectively, of prothymosin α (ProTα), a protein involved in chromatin remodeling. It has been widely supposed that these polypeptides are not natural products of the in vivo processing of ProTα, since neither was found in extracts in which proteolysis was prevented. Here we show that a lysosomal asparaginyl endopeptidase is able to process ProTα to generate Tα1 and Tα11. In view of its catalytic properties and structural and immunological analyses, this protease was identified as mammalian legumain. It selectively cleaves some of the asparaginyl-glycine residues in the ProTα sequence; specifically, Asn28-Gly29 and Asn35-Gly36 residues are cleaved with similar efficiency in vitro to generate Tα1 and Tα11, respectively. By contrast Tα1 is the main product detected in vivo, free in the cytosol, at concentrations similar to that of ProTα. The data here reported demonstrate that Tα1 is not an artifact but rather is naturally present in diverse mammalian tissues and raise the possibility that it has a functional role. Thymosin α1(Tα1) and thymosin Tα11(Tα11) are polypeptides with immunoregulatory properties first isolated from thymic extracts, corresponding to the first 28 and 35 amino acid residues, respectively, of prothymosin α (ProTα), a protein involved in chromatin remodeling. It has been widely supposed that these polypeptides are not natural products of the in vivo processing of ProTα, since neither was found in extracts in which proteolysis was prevented. Here we show that a lysosomal asparaginyl endopeptidase is able to process ProTα to generate Tα1 and Tα11. In view of its catalytic properties and structural and immunological analyses, this protease was identified as mammalian legumain. It selectively cleaves some of the asparaginyl-glycine residues in the ProTα sequence; specifically, Asn28-Gly29 and Asn35-Gly36 residues are cleaved with similar efficiency in vitro to generate Tα1 and Tα11, respectively. By contrast Tα1 is the main product detected in vivo, free in the cytosol, at concentrations similar to that of ProTα. The data here reported demonstrate that Tα1 is not an artifact but rather is naturally present in diverse mammalian tissues and raise the possibility that it has a functional role. thymosin fraction 5 thymosin α1 thymosin α11 prothymosin α high performance liquid chromatography reverse-phase HPLC phenylmethylsulfonyl fluoride fast protein liquid chromatography 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid isoelectric focusing polyacrylamide gel The α-thymosins are a group of acidic peptides (pI 3.5–4.5) present in a calf thymus extract denominated thymosin fraction 5 (TF5)1 (1Goldstein A.L. Guha A. Zatz M. Hardy M.A. White A. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 1800-1803Crossref PubMed Scopus (256) Google Scholar), which shows immunoregulatory properties in several in vitro and in vivo assay systems (2Low T.L.K. Thurman G.B. McAdoo M. McClure J. Rossio J.L. Naylor P.H. Goldstein A.L. J. Biol. Chem. 1979; 254: 981-986Abstract Full Text PDF PubMed Google Scholar). Thymosin α1(Tα1; 28 amino acids) is the most abundant α-thymosin in TF5 and was the first to be isolated and sequenced (3Goldstein A.L. Low T.L.K. McAdoo M. McClure J. Thurman G.B. Rossio J. Lai C-Y. Chang D. Wang S-S. Harvey C. Ramel A.H. Meienhofer J. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 725-729Crossref PubMed Scopus (317) Google Scholar). A less abundant TF5 component named thymosin α11(Tα11) has subsequently been characterized (4Caldarella J. Goodall G.J. Felix A.M. Heimer E.P. Salvin S.B. Horecker B.L. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 7424-7427Crossref PubMed Scopus (51) Google Scholar); this peptide comprises the Tα1 sequence plus an additional 7 residues at its C terminus (i.e. 35 residues in total). Both Tα1 and Tα11 show immunoregulatory properties similar to those of TF5 (5Low T.L.K. Goldstein A. Methods Enzymol. 1985; 116: 213-248Crossref PubMed Scopus (43) Google Scholar). A polypeptide including Tα1 in its structure has been detected among the translation products of calf thymus mRNAs (6Freire M. Crivellaro O. Isaacs C. Moschera S. Horecker B.L. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 6007-6011Crossref PubMed Scopus (24) Google Scholar,7Freire M. Hannappel E. Rey M. Freire J.M. Kibo H.K. Horecker B.L. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 192-195Crossref PubMed Scopus (17) Google Scholar). This apparent precursor of the α-thymosins was later isolated from thymus (8Haritos A.A. Goodall G.J. Horecker B.L. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1008-1011Crossref PubMed Scopus (220) Google Scholar) and other mammalian tissues (9Haritos A.A. Tsolas O. Horecker B.L. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1391-1393Crossref PubMed Scopus (119) Google Scholar, 10Franco F.J. Dı̀az C. Barcia M. Freire M. Biochim. Biophys. Acta. 1992; 1120: 43-48Crossref PubMed Scopus (35) Google Scholar). Sequencing indicated that it comprised 109–111 amino acids, including the sequence of Tα11 (and thus Tα1) at its N terminus (11Haritos A.A. Blacher R. Stein S. Caldarella J. Horecker B.L. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 343-346Crossref PubMed Scopus (95) Google Scholar, 12Pan L.X. Haritos A.A. Wideman J. Komiyama T. Chang M. Stein S. Salvin S.B. Horecker B.L. Arch. Biochem. Biophys. 1986; 250: 197-201Crossref PubMed Scopus (72) Google Scholar). This protein was denominated prothymosin α (ProTα) (8Haritos A.A. Goodall G.J. Horecker B.L. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1008-1011Crossref PubMed Scopus (220) Google Scholar). ProTα is a highly acidic protein (pI 3.55) with a highly conserved sequence (13Freire M. Dı́az-Jullien C. Covelo G. Pandalai S.G. Recent Research Developments in Proteins. 1. Transworld Research Network, Kerala, India2002: 257-276Google Scholar). Its wide distribution in mammalian tissues suggested that its function was probably not immunological, despite the apparent immunoregulatory properties of Tα1and Tα11. Subsequent studies indicated that ProTα has a generalized role in the proliferation of mammalian cells, by mechanisms involving migration to the nucleus (14Gómez-Márquez J. Segade F. FEBS Lett. 1988; 226: 217-219Crossref PubMed Scopus (78) Google Scholar, 15Manrow R.E. Sburlati A.R. Hanover J.A. Berger S.L. J. Biol. Chem. 1991; 266: 3916-3924Abstract Full Text PDF PubMed Google Scholar, 16Clinton M. Graeve L. el-Dorry H. Rodrı́guez-Boulan E. Horecker B.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6608-6612Crossref PubMed Scopus (64) Google Scholar) and cytosolic phosphorylation of ProTα (17Barcia M.G. Castro J.M. Jullien C.D. Freire M. J. Biol. Chem. 1993; 268: 4704-4708Abstract Full Text PDF PubMed Google Scholar, 18Pérez-Estévez A. Dı́az-Jullien C. Covelo G. Salgueiro M.T. Freire M. J. Biol. Chem. 1997; 272: 10506-10513Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Reports from our group and others over recent years have provided increasing evidence that the nuclear function of ProTα involves interactions with histones (19Papamarcaki T. Tsolas O. FEBS Lett. 1994; 345: 71-75Crossref PubMed Scopus (75) Google Scholar, 20Dı́az-Jullien C. Pérez-Estévez A. Covelo G. Freire M. Biochim. Biophys. Acta. 1996; 1296: 219-227Crossref PubMed Scopus (57) Google Scholar, 21Karetsou Z. Sandaltzopoulos R. Frangou-Lazaridis M. Lai C.Y. Tsolas O. Becker P.B. Papamarcaki T. Nucleic Acids Res. 1998; 26: 3111-3118Crossref PubMed Scopus (93) Google Scholar) and with other proteins related to DNA metabolism, including proliferating cell nuclear antigen, Cdk2, and cyclin A (22Freire J. Covelo G. Sarandeses C. Dı́az-Jullien C. Freire M. Biochem. Cell Biol. 2001; 79: 123-131Crossref PubMed Scopus (18) Google Scholar), arguing strongly for a role in chromatin remodeling. In further support of this view, ProTα enables nucleosome assembly (20Dı́az-Jullien C. Pérez-Estévez A. Covelo G. Freire M. Biochim. Biophys. Acta. 1996; 1296: 219-227Crossref PubMed Scopus (57) Google Scholar) and modulates the activity of histone acetyltransferase p300 (23Cotter II, M.A. Robertson E.S. Mol. Cell. Biol. 2000; 20: 5722-5735Crossref PubMed Scopus (104) Google Scholar). Independently of ProTα and its function, uncertainty remains about the status of Tα1 and Tα11. When ProTα was isolated in 1984 from thymus extracts prepared under conditions in which proteolytic activity was prevented, Tα1 and Tα11 were not detected in reverse-phase HPLC separation of the extracts (8Haritos A.A. Goodall G.J. Horecker B.L. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1008-1011Crossref PubMed Scopus (220) Google Scholar). This led to the suggestion that the presence of ProTα-derived α-thymosins in TF5 was an artifact resulting from uncontrolled proteolysis during preparation of TF5. In contrast, experiments performed in our laboratory, using isoelectric focusing rather than HPLC for α-thymosin detection, have indicated that Tα1 is indeed present in extracts of this type in diverse mammalian tissues (10Franco F.J. Dı̀az C. Barcia M. Freire M. Biochim. Biophys. Acta. 1992; 1120: 43-48Crossref PubMed Scopus (35) Google Scholar). To resolve this controversy, we have been performing experiments designed to detect a protease in mammalian cells that is capable of processing ProTα to generate the α-thymosins. In this paper, we report the characterization of a lysosomal asparaginyl endopeptidase with the required specificity. The characteristics of this enzyme match those of legumain, a cysteine endopeptidase initially known only from plants (24Hara-Nishimura I. Takeuchi Y. Nishimura M. Plant Cell. 1993; 5: 1651-1659Crossref PubMed Scopus (135) Google Scholar) and the trematode Schistosoma (25Klinkert M.Q. Felleisen R. Link G. Ruppel A. Beck E. Mol. Biochem. Parasitol. 1989; 33: 113-122Crossref PubMed Scopus (154) Google Scholar) but later found in mammals (26Chen J-M. Dando P.M. Rawlings N.D. Borwn M.A. Young N.E. Stevens R.A: Hewitt E. Watts C. Barret A.J. J. Biol. Chem. 1997; 272: 8090-8098Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). Experiments designed to assess the possible biological significance of the processing of ProTα by this enzyme are also presented. Cells used were transformed human B lymphocytes (NC-37). For subcellular fractionation, cells were washed and resuspended (about 2 × 108cells/ml) in 0.25 m sucrose, 10 mm acetic acid, 10 mm triethanolamine, and 1 mm EDTA, pH 7.4, and then homogenated (15 strokes) in a Potter Teflon glass blender and then centrifuged (2000 × g for 10 min) to yield the nuclear pellet. The supernatant was centrifuged at 20,000 ×g to obtain the cytoplasmic organelle fraction (mitochondria and lysosomes) as pellet, and the supernatant was centrifuged again at 100,000 × g to obtain the microsome fraction as pellet and cytosol fraction as supernatant. The nuclear pellet was resuspended (2 × 108nuclei/ml) in 10 mm Tris-HCl buffer, pH 8.0, containing 10 mm KCl, 1.5 mm MgCl2, 420 mm NaCl, 0.2 mm EDTA, 25% glycerol, 1 mm dithiothreitol, and 0.5 mm PMSF, and then homogenated (10 strokes) in a Potter Teflon glass blender. After centrifugation at 20,000 × g for 15 min at 4 °C, the supernatant was dialyzed against buffer A (50 mmTris-HCl buffer, pH 7.5, 5% (v/v) glycerol, 0.5 mm PMSF, 1 mm DTT) to yield the nucleoplasm fraction. The pellet was resuspended in 10 mm Tris-HCl buffer, pH 7.4, with 0.1 mm MgCl2, 420 mm NaCl, 1 mm DTT, and 0.5 mm PMSF containing 0.5m NaCl and then incubated for 5 min at 0 °C and centrifuged at 20,000 × g for 15 min; the supernatant was then dialyzed against buffer A to yield the nuclear membrane fraction. The lysosome and mitochondria fractions were purified from the cytoplasmic organelle fraction by density gradient centrifugation in two sequential Percoll/metrizamide density gradients (27Storrie B. Madden E.A. Methods Enzymol. 1990; 182: 203-221Crossref PubMed Scopus (497) Google Scholar). Lysosomes, mitochondria, microsomes, and nuclear membranes were extracted in citrate/phosphate buffer pH 4.5 or phosphate buffer pH 7.5, both containing 1% Triton X-114, for 30 min at 4 °C and then centrifuged at 13,000 × g for 5 min. Digitonin permeabilization was carried out as described (28Weigel P.H. Ray D.A. Oka J.A. Anal. Biochem. 1983; 133: 437-449Crossref PubMed Scopus (81) Google Scholar). Cells were resuspended in ice-cold PBS (30 volumes), and the suspension was brought to 0.02% (w/v) digitonin (diluted from a 14 mg/ml stock solution). Cells were allowed to permeabilize for 2 min on ice, and the released cytosolic components were recovered by centrifugation at 800 × g. A heat-stable acidic polypeptide fraction (including the α-thymosins) was obtained from whole cell extracts or from subcellular fractions by a modification of the procedure of Goldstein et al. (1Goldstein A.L. Guha A. Zatz M. Hardy M.A. White A. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 1800-1803Crossref PubMed Scopus (256) Google Scholar), as previously described (10Franco F.J. Dı̀az C. Barcia M. Freire M. Biochim. Biophys. Acta. 1992; 1120: 43-48Crossref PubMed Scopus (35) Google Scholar). Briefly, frozen cells or subcellular fractions were pulverized under liquid nitrogen with a chilled pestle and mortar, dispersed in 10 volumes of boiling 0.15 m NaCl, and homogenized in a Waring blender. The homogenates were centrifuged, the supernatants were brought to pH 2.5, and insoluble material was removed by centrifugation. The resulting supernatants were brought up to 70 mm NaCl, 50 mm Tris-HCl, pH 8, and then loaded into a DEAE-cellulose chromatography column, which was eluted with 0.4m NaCl to give the fraction of acidic polypeptides. ProTα was purified from the acidic polypeptides fraction by reverse-phase HPLC (RP-HPLC) on a Nucleosyl 300-C18 column (Sugelabor) (5 μm; 4.6 × 250 mm) in a Beckman HPLC System. Elution was done with a programmed gradient of n-propyl alcohol (0–50% v/v) in a 0.1% aqueous dilution of trifluoroacetic acid. The resulting purified ProTα was dephosphorylated by incubation for 30 min at room temperature with 1 unit of calf intestine alkaline phosphatase per μg of ProTα, in 20 mm Tris-HCl, pH 7.8, containing MgCl2 (1 mm) and ZnCl2(0.1 mm). Dephosphorylated ProTα was obtained from this reaction mixture by RP-HPLC as indicated above. Phosphorylation of ProTα with radioactive orthophosphate was done using ProTα protein kinase, purified as described (18Pérez-Estévez A. Dı́az-Jullien C. Covelo G. Salgueiro M.T. Freire M. J. Biol. Chem. 1997; 272: 10506-10513Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Briefly, a phosphorylation reaction mixture containing 50 μg of dephosphorylated ProTα, 50 mm Tris-HCl, pH 7.4, 150 mm KCl, 26 mm MgCl2, 1.6 mm EGTA, 3.3 mm DTT, 80 ng/ml protamine, 83 mm β-glycerol phosphate, and 100 mm [32P]ATP (3,000 Ci/mmol) in a total volume of 250 μl was incubated at 37 °C for 45 min. [32P]ProTα was then obtained by RP-HPLC as described above. Tα1 and Tα11 were a gift from Dr. Heimer (Hoffman-Roche). Isoelectrofocusing was carried out as described previously (10Franco F.J. Dı̀az C. Barcia M. Freire M. Biochim. Biophys. Acta. 1992; 1120: 43-48Crossref PubMed Scopus (35) Google Scholar). Briefly, samples were applied to PAG plates with a pH range of 4.0–6.5 (Amersham Biosciences) and electrofocused for 2.5 h (2000 V, 25 mA, 25 watts) in a LKB Multiphor isoelectric focusing cell thermostatted at 10 °C. The isoelectrofocused gel was fixed with trichloroacetic/sulfosalycilic acid and stained with Coomassie Brilliant Blue G. Slab gel electrophoresis was carried out on 18% polyacrylamide gels by the method of Laemmli (29Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207218) Google Scholar). The reaction mixture contained different concentrations of ProTα (15–30 μg) plus crude or purified cell extract (1–15 μg) in 45 μl of 0.1 mammonium acetate buffer, pH 4.5, plus 0.5 mm DTT or 0.1m phosphate buffer, pH 7.5, plus 0.5 mm DTT. In some experiments, protease inhibitors were added. The mixture was incubated at 37 °C for 8 h. The reaction products were then analyzed by SDS-PAGE, RP-HPLC, or isoelectrofocusing, as indicated under "Results." The structures of the processing products, obtained by RP-HPLC or from extracts of bands in SDS-PAGE, were established by analysis of the amino acid composition of their tryptic peptides, as previously described (10Franco F.J. Dı̀az C. Barcia M. Freire M. Biochim. Biophys. Acta. 1992; 1120: 43-48Crossref PubMed Scopus (35) Google Scholar). Lysosomes of NC-37 cells were extracted with 20 mm sodium acetate, pH 5.0, 1 mm EDTA containing 1% Triton X-114; the mixture was centrifuged at 13,000 ×g for 5 min, and the supernatant was passed through 0.22-μm nitrocellulose filters. The filtrate was then applied to anAmersham Biosciences Mono-S FPLC column (type HR 5/5) equilibrated with 20 mm sodium acetate, pH 5.2, 10 mm NaCl. Elution was with a NaCl gradient (10–1000 mm) in the same buffer. 1-ml fractions were collected and tested for asparaginyl endopeptidase activity by fluorimetric assays with the synthetic peptide benzyloxycarbonyl-Val-Ala-Asn-7-amido-4-methylcoumarin (Bachem) as substrate, as described (30Manoury B. Hewitt E.W. Morrice N. Dando P.M. Barrett A.J. Watts C. Nature. 1998; 396: 695-699Crossref PubMed Scopus (308) Google Scholar). ProTα processing activity was also assayed in the different fractions, as detailed above. Deglycosylation of the purified protease was carried out by incubation of aliquots of purified protease in 250 μl of buffer containing 0.1m citric acid, 0.2 m NaHPO4, 1 mm EDTA, 0.025% CHAPS, pH 7.2, at 100 °C for 5 min. After cooling, 0.53 milliunits of N-glucidase-F (Roche Molecular Biochemicals) was added, and the solution was incubated at 37 °C for 24 h. The protease was then precipitated with 10% trichloroacetic acid and analyzed by immuno-Western blotting as indicated below. Western blotting assays were performed by transferring the proteins separated by SDS-PAGE to polyvinylidene difluoride membranes. A polyclonal legumain antiserum was custom-produced by Neosystem Laboratories (Strasbourg, France) against the human legumain sequence fragment KGIGSGKVLKKSPQ, as previously used for antibody production (30Manoury B. Hewitt E.W. Morrice N. Dando P.M. Barrett A.J. Watts C. Nature. 1998; 396: 695-699Crossref PubMed Scopus (308) Google Scholar). Neosystem Laboratories also supplied preimmune serum from the same rabbit. The effect of the legumain antiserum antibodies on the ProTα-processing activity of the different cell fractions was investigated by previous incubation of the fraction of interest with different concentrations of immune or nonimmune serum, in the absence or presence of the peptide KGIGSGKVLKKSPQ in PBS, for 1 h at room temperature. ProTα processing was then assayed as described above. To investigate the intracellular processing of ProTα, we performed a systematic study of ProTα-proteolytic activities of various subcellular fractions of human lymphoma cells (NC-37), including nucleoplasm, cytosol, nuclear membranes, cytosolic organelles, and microsome fractions. The effect of pH on proteolytic activity was also investigated, on the basis of assays at physiological and acid pH. Analysis by SDS-PAGE of the various reaction mixtures (Fig.1A) indicated that proteolysis of ProTα occurred only in reaction mixtures containing the cytosolic organelle fraction. This proteolytic activity was dependent on pH, being detected only at pH 4.5. To further localize the proteolytic activity in the cytosolic organelle fraction, we separated mitochondrial and lysosomal subfractions from this fraction by density gradient centrifugation and assayed ProTα-proteolytic activity in extracts of these subfractions at different pH values. As indicated in Fig. 1B, proteolytic activity was localized only in the lysosomal subfraction and again required acid pH. Since the lysosomal extracts were originally prepared at pH 7.5, we investigated the possible influence of pH during extract preparation. To this end, lysosomes were extracted at pH 4.5, and proteolytic activity was then assayed at pH 4.5 or at higher pH. As shown in Fig. 1C, ProTα-proteolytic activity assayed at pH 4.5 was markedly higher in extracts prepared at pH 4.5 than in extracts prepared at pH 7.5 (see Fig. 1B) and markedly higher when assayed at pH 4.5 than when assayed at pH 6.0 or 8.0. Note that nucleoplasm, microsome, nuclear membranes, mitochondria, and cytosolic fractions prepared at pH 4.5 still did not show ProTα-proteolytic activity (data not shown). Moreover, ProTα-proteolytic activity was detected only when disrupted lysosomes were assayed, not in assays without previous lysosome disruption (data not shown). An important characteristic of the pattern of proteolysis of ProTα by the acidic lysosomal lysates is that two of the fragments of ProTα had the same electrophoretic mobility as synthetic Tα1 and Tα11 (Fig.1C). To further investigate processing of ProTα, we investigated the influence of various protease inhibitors on the pattern of fragments obtained with the lysosomal lysate. Analysis of the different reaction mixtures showed rather surprising results (Fig. 1D), since the ProTα fragmentation pattern was not modified by any of the broad spectrum protease inhibitors used (including serine, cysteine, and aspartic protease inhibitors) but was markedly modified by protease inhibitor iodoacetamide, which efficiently prevented the processing. This indicated that a particular cysteine protease activity is involved in the processing of ProTα. Since ProTα is phosphorylated by a cytosolic threonine protein kinase with unknown structure (18Pérez-Estévez A. Dı́az-Jullien C. Covelo G. Salgueiro M.T. Freire M. J. Biol. Chem. 1997; 272: 10506-10513Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), we next investigated the influence of ProTα phosphorylation on the proteolysis pattern. Specifically, we compared proteolysis of dephosphorylated ProTα and ProTα phosphorylated in vitro by the purified ProTα protein kinase with [32P]ATP as cosubstrate. Results of these experiments (data not shown) indicate that phosphorylation of ProTα did not affect its proteolysis by the lysosomal lysates. The above results suggest the existence of a specific lysosomal protease activity that is highly dependent on pH and that is able to cleave ProTα, even in the presence of broad spectrum protease inhibitors, yielding four fragments, two of which have the same electrophoretic mobility as Tα1 and Tα11. To characterize the different products derived from the processing of ProTα by the lysosomal protease activity, we used in the first instance RP-HPLC. The elution pattern for ProTα-processing products obtained with the lysosomal extract at pH 4.5 showed four peaks (Fig.2A). Analysis by SDS-PAGE of these four peaks (inset in Fig. 2A) showed that a polypeptide with the same electrophoretic mobility as Tα1is the main component in peak 1; peaks 2 and 3 are mixtures of this component and the other ProTα fragments, and peak 4 is whole ProTα. Similar elution patterns were obtained when chromatographic conditions were varied. Characterization of the component in peak 1 was accomplished by determining the amino acid composition of its tryptic peptides, as separated by RP-HPLC (Fig. 2B). The tryptic map and amino acid composition of the peak-1 polypeptide proved identical to that of Tα1 (Fig. 2B). Structural analysis of the other ProTα-processing products (those eluting in peaks 2 and 3) was carried out by tryptic digestion of the respective bands excised from the SDS-PAGE gels in which the components of the processing assay reaction mixtures had been separated. The amino acid compositions of the tryptic peptides derived from these products and separated by RP-HPLC (data not shown) indicate that the component with the same electrophoretic mobility as synthetic Tα11 is indeed Tα11, whereas the two fragments with higher electrophoretic mobility correspond to residues 29–109 and 36–109 of the ProTα sequence. In view of these structural analyses, summarized in Fig. 2C, we conclude that the lysosomal protease cleaves ProTα at Asn-Gly residues located at positions 28–29 to yield Tα1 and at positions 35–36 to yield Tα11, whereas the resulting C-terminal portions of ProTα, positions 29–109 and 36–109, remain undigested. To judge by the concentrations of the respective bands in the SDS-PAGE gel (Fig. 2C), both Tα1 and Tα11 are produced in similar proportions by the lysosomal protease, whereas the concentrations of fragments 29–109 and 36–109 appear to be lower. However, this discrepancy should probably be attributed to low efficiency in the Coomassie staining of the larger (highly acidic) fragments of ProTα, rather than to a lower concentration in the processing products, since extracts of the respective bands (from SDS-PAGE) separated by RP-HPLC prior to structural analysis showed similar spectrophotometrically determined concentrations to Tα1 and Tα11(data not shown). In view of its specificity, then, this protease can be considered as an asparaginyl-glycyl endopeptidase, and this specificity is consistent with its ability to generate α-thymosinsin vivo. The characteristics of the lysosomal protease processing ProTα in vitro to yield Tα1 and Tα11 (including its specificity, its response to pH variations, and its resistance to protease inhibitors) strikingly resemble those of the mammalian form of legumain, a protease originally described in plants and recently reported to be involved in major histocompatibility complex-restricted antigen presentation in mammalian cells (30Manoury B. Hewitt E.W. Morrice N. Dando P.M. Barrett A.J. Watts C. Nature. 1998; 396: 695-699Crossref PubMed Scopus (308) Google Scholar). We therefore performed experiments to investigate whether the lysosomal asparaginyl endopeptidase that processes ProTα might be legumain. These experiments included legumain activity assays, immunodetection assays, and structural characterization. Fractionation of the lysosomal extracts by ion exchange FPLC demonstrated that the fractions showing ProTα-processing activity likewise showed legumain activity, as determined by fluorimetric assay using the synthetic substrate benzyloxycarbonyl-Val-Ala-Asn-7-amido-4-methylcoumarin (Fig.3A). For immunodetection assays, we used antibodies raised against the peptide KGIGSKVCCKSCPQ (a fragment of the human legumain sequence that is coincident with that in other mammals) (30Manoury B. Hewitt E.W. Morrice N. Dando P.M. Barrett A.J. Watts C. Nature. 1998; 396: 695-699Crossref PubMed Scopus (308) Google Scholar). Western blotting analysis of the FPLC-purified lysosomal lysates, shown in Fig. 3B, detected a protein with the same size (46 kDa) as human legumain, at a concentration similar to that in crude extract. Since active human legumain is glycosylated (31Chen J-M. Fortunato M. Barrett A.J. Biochem. J. 2000; 352: 327-334Crossref PubMed Google Scholar), we investigated the effect of previous deglycosylation. In the FPLC-purified lysosomal lysate treated withN-glycosidase F, the legumain antiserum detected a protein with lower apparent molecular mass (41 kDa; Fig. 3B); this decrease is consistent with that reported after deglycosylation of other mammalian legumains (26Chen J-M. Dando P.M. Rawlings N.D. Borwn M.A. Young N.E. Stevens R.A: Hewitt E. Watts C. Barret A.J. J. Biol. Chem. 1997; 272: 8090-8098Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). To complete the characterization of the enzyme processing ProTα, we investigated whether the legumain antiserum blocks the proteolysis of ProTα by the lysosomal protease. As indicated in Fig. 3C, the antibodies efficiently blocked processing of ProTα, whereas the corresponding nonimmune serum had no effect. However, no blockade occurred when the synthetic peptide used to obtain the immune serum was included in the processing assay reaction mixture (Fig. 3C). Similar results were obtained using crude lysosomal extracts (data not shown). These results provide further support for the view that the asparaginyl endopeptidase responsible for the processing of ProTαin vitro to generate Tα1 and Tα11 is legumain. To further study the processing of ProTα, we decided to investigate relationships between the observed proteolysis of ProTα by the legumain in vitrowith the processing seen in vivo. To this end, we compared the polypeptide pattern observed after ProTα processing in vitro with the α-thymosins pattern observed in lymphocytes, in which both ProTα and legumain are known to be present. Whole cell extracts and subcellular fractions were obtained from lymphocytes (NC-37 cells), in all cases under conditions in which proteolytic activity was strictly prevented. α-Thymosins in the acidic polypeptide fraction obtained from the diverse extracts were detected by isoelectrofocusing (IEF), a technique that is highly effective for the separation and identification of these peptides in different cell extracts (10Franco F.J. Dı̀az C. Barcia M. Freire M. Biochim. Biophys. Acta. 1992; 1120: 43-48Crossref PubMed Scopus (35) Google Scholar). IEF analysis of the α-thymosin components from whole-cell extracts of NC-37 cells is shown in Fig.4A. This analysis indicates that NC-37 cells contain components with the same pI as ProTα and Tα1 but not components with the same pI as Tα11 or the acidic fragments 29–109 and 36–109 of ProTα detected after in vitro processing. Structural analysis confirmed that the more acidic components were ProTα and Tα1. This IEF pattern and the similar concentrations of ProTα and Tα1 determined by densitometry (Fig. 4A) are in agreement with our previous findings indicating that Tα1 is naturally present in various mammalian tissues (10Franco F.J. Dı̀az C. Barcia M. Freire M. Biochim. Biophys. Acta. 1992; 1120: 43-48Crossref PubMed Scopus (35) Google Scholar) and in line with the view that ProTα is processed in vivo by legumain or another protease with similar specificity to yield Tα1. We next investigated the intracellular location of Tα1 in NC-37 cells. IEF analysis of α-thymosins in the different subcellular fractions separated by differential centrifugation (Fig. 4B) and by digitonin permeabilization (Fig. 4C) indicated that Tα1 is located in the cytosolic fraction and is not associated with any cell organelle. The observed cytosolic location of ProTα (Fig. 4, B and C) is in agreement with the tendency of ProTα to leak out of isolated nuclei (15Manrow R.E. Sburlati A.R. Hanover J.A. Berger S.L. J. Biol. Chem. 1991; 266: 3916-3924Abstract Full Text PDF PubMed Google Scholar). It is worth pointing out that the α-thymosins obtained from both whole-cell extracts and from organelle fractions showed rather "messy" chromatographic behavior; thus, in the RP-HPLC analysis ProTα emerged as a clear peak, but Tα1 was retained for longer by the column and eluted as mixtures with other components in the α-thymosinic fraction (data not shown). Similar behavior was observed under diverse HPLC conditions, both reverse-phase and ion exchange. Data reported here strongly suggest that the presence of Tα1 in lymphocytes and other mammalian cells is due to the processing of ProTα by a lysosomal asparaginyl endopeptidase identified as legumain. This enzyme was recently discovered in mammals (26Chen J-M. Dando P.M. Rawlings N.D. Borwn M.A. Young N.E. Stevens R.A: Hewitt E. Watts C. Barret A.J. J. Biol. Chem. 1997; 272: 8090-8098Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar), having been known previously only from plants (24Hara-Nishimura I. Takeuchi Y. Nishimura M. Plant Cell. 1993; 5: 1651-1659Crossref PubMed Scopus (135) Google Scholar) and invertebrates (25Klinkert M.Q. Felleisen R. Link G. Ruppel A. Beck E. Mol. Biochem. Parasitol. 1989; 33: 113-122Crossref PubMed Scopus (154) Google Scholar). Mammalian legumain shows high specificity for a small subset of asparaginyl bonds; it appears to have no specific requirement for amino acids other than the asparagine, although it shows a preference for Ala or Pro at position P3 (i.e. the third residue in the N-terminal direction) (32Dando P.M. Fortunato M. Smith L. Knight C.G. McKendrick J.E. Barrett A.J. Biochem. J. 1999; 339: 743-749Crossref PubMed Scopus (64) Google Scholar). The specificity of legumain with ProTα as substrate is in line with these characteristics, but it also shows a special requirement for a Gly at P1′. Specifically, in vitro the enzyme only cleaves Asn–Gly bonds at positions 28–29 (with an Ala residue at P3) and 35–36 (with a Pro residue at P3) and appears not to cleave the Asn–Gly bond at positions 42–43 (which has a Glu at P3) or Asn bonds with other amino acids (Ala at position 38, Glu at positions 40 and 50). To judge from the analysis of the α-thymosins in cell extracts prepared under conditions in which proteolysis was absolutely prevented (Fig. 4), this protease shows even higher specificity in vivo, since only Tα1 (i.e. cleavage at positions 28–29) was detected at a concentration similar to ProTα, whereas Tα11 (i.e. cleavage at positions 35–36) was not detected, in line with previous studies of other mammalian tissues (10Franco F.J. Dı̀az C. Barcia M. Freire M. Biochim. Biophys. Acta. 1992; 1120: 43-48Crossref PubMed Scopus (35) Google Scholar). However, we cannot rule out the possibility that the apparent absence of Tα11 is due to rapid degradation of this peptide in vivo. The nondetection of the larger ProTα fragment 29–109 (or 36–109) in cell extracts is likewise presumably indicative of rapid degradation of this fragmentin vivo. The presence of Tα11 at low concentrations in the calf thymus fraction TF5 (4Caldarella J. Goodall G.J. Felix A.M. Heimer E.P. Salvin S.B. Horecker B.L. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 7424-7427Crossref PubMed Scopus (51) Google Scholar) might be due to the characteristics of the procedure used for its preparation. In fact, we did not detect this polypeptide in the α-thymosin fractions obtained (from calf thymus and other mammalian tissues) under the conditions used here (10Franco F.J. Dı̀az C. Barcia M. Freire M. Biochim. Biophys. Acta. 1992; 1120: 43-48Crossref PubMed Scopus (35) Google Scholar,33Freire M. Rey-Méndez M. Gómez-Márquez J. Arias P. Arch. Biochem. Biophys. 1985; 239: 480-485Crossref PubMed Scopus (15) Google Scholar). As noted, the characteristics of the in vivo processing of ProTα suggest that it is accomplished by legumain. It is worth noting that additional confirmation of this conclusion by in vivoenzyme blockade is difficult, since specific inhibitors of this enzyme are not currently available and since the use of Asn-containing peptides as competitive inhibitors is inefficient when incubations of over 1 h are necessary (30Manoury B. Hewitt E.W. Morrice N. Dando P.M. Barrett A.J. Watts C. Nature. 1998; 396: 695-699Crossref PubMed Scopus (308) Google Scholar). In light of the present results, previous failures to detect Tα1 in extracts prepared under drastic denaturing conditions (8Haritos A.A. Goodall G.J. Horecker B.L. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1008-1011Crossref PubMed Scopus (220) Google Scholar) may perhaps be attributable to the "messy" chromatographic behavior of α-thymosins prepared under these conditions, due to their tendency to aggregate, as evidenced in the present and previous reports (10Franco F.J. Dı̀az C. Barcia M. Freire M. Biochim. Biophys. Acta. 1992; 1120: 43-48Crossref PubMed Scopus (35) Google Scholar, 33Freire M. Rey-Méndez M. Gómez-Márquez J. Arias P. Arch. Biochem. Biophys. 1985; 239: 480-485Crossref PubMed Scopus (15) Google Scholar). In fact, original isolation of Tα1 included gel filtration in the presence of 6m guanidium chloride (3Goldstein A.L. Low T.L.K. McAdoo M. McClure J. Thurman G.B. Rossio J. Lai C-Y. Chang D. Wang S-S. Harvey C. Ramel A.H. Meienhofer J. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 725-729Crossref PubMed Scopus (317) Google Scholar). In the present and previous studies (10Franco F.J. Dı̀az C. Barcia M. Freire M. Biochim. Biophys. Acta. 1992; 1120: 43-48Crossref PubMed Scopus (35) Google Scholar, 33Freire M. Rey-Méndez M. Gómez-Márquez J. Arias P. Arch. Biochem. Biophys. 1985; 239: 480-485Crossref PubMed Scopus (15) Google Scholar), we have used isoelectrofocusing, which, unlike the HPLC procedures used by other authors (8Haritos A.A. Goodall G.J. Horecker B.L. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1008-1011Crossref PubMed Scopus (220) Google Scholar), is effective for separating and identifying the α-thymosins. In this connection, it is also worth noting that antibodies raised against fragments of the N-terminal region of ProTα (the most immunogenic region; such antibodies have been widely used for immunodetection of ProTα) do not differentiate between ProTα and its N-terminal derivatives Tα1 and Tα11. To judge by the wide distribution of both Tα1 (10Franco F.J. Dı̀az C. Barcia M. Freire M. Biochim. Biophys. Acta. 1992; 1120: 43-48Crossref PubMed Scopus (35) Google Scholar) and legumain (26Chen J-M. Dando P.M. Rawlings N.D. Borwn M.A. Young N.E. Stevens R.A: Hewitt E. Watts C. Barret A.J. J. Biol. Chem. 1997; 272: 8090-8098Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar) in different tissues, we would suggest that ProTα processing to yield Tα1 is a generalized process in mammalian tissues. Interestingly, tissues showing high legumain activity such as lymphoid tissues (26Chen J-M. Dando P.M. Rawlings N.D. Borwn M.A. Young N.E. Stevens R.A: Hewitt E. Watts C. Barret A.J. J. Biol. Chem. 1997; 272: 8090-8098Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 30Manoury B. Hewitt E.W. Morrice N. Dando P.M. Barrett A.J. Watts C. Nature. 1998; 396: 695-699Crossref PubMed Scopus (308) Google Scholar) also show high levels of Tα1 (10Franco F.J. Dı̀az C. Barcia M. Freire M. Biochim. Biophys. Acta. 1992; 1120: 43-48Crossref PubMed Scopus (35) Google Scholar). The highly conserved primary structure of ProTα, especially around the asparagine/glycine bonds cleaved by legumain (13Freire M. Dı́az-Jullien C. Covelo G. Pandalai S.G. Recent Research Developments in Proteins. 1. Transworld Research Network, Kerala, India2002: 257-276Google Scholar), provides further support for the view that ProTα processing to yield Tα1 is generalized. Moreover, the present results indicate that processing of ProTα in vitrois independent of its phosphorylation state (ProTα is phosphorylated at specific residues when cells are activated to proliferate) (17Barcia M.G. Castro J.M. Jullien C.D. Freire M. J. Biol. Chem. 1993; 268: 4704-4708Abstract Full Text PDF PubMed Google Scholar, 18Pérez-Estévez A. Dı́az-Jullien C. Covelo G. Salgueiro M.T. Freire M. J. Biol. Chem. 1997; 272: 10506-10513Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). The presence of phosphorylated Tα1 in proliferating cells and the inability of the ProTα protein kinase to phosphorylate Tα1 (34Pérez-Estévez A. Freire J. Sarandeses C. Covelo G. Dı́az-Jullien C. Freire M. Mol. Cell. Biochem. 2000; 208: 111-118Crossref PubMed Google Scholar) thus suggest that phospho-ProTα may be processed in vivo to yield phospho-Tα1. Proteolysis of ProTα at the Asp99 residue by caspase 3 has recently been reported to occur in HeLa cells in which apoptosis has been induced (35Evstafieva A.G. Belov G.A. Kalkum M. Chichkova N.V. Bogdanov A.A. Agol V.I. Vartapetian A.B. FEBS Lett. 2000; 467: 150-154Crossref PubMed Scopus (44) Google Scholar, 36Enkemann S.A. Wang R.H. Trumbore M.W. Berger S.L. J. Cell. Physiol. 2000; 182: 256-268Crossref PubMed Scopus (30) Google Scholar). This proteolysis has so far not been demonstrated to be a generalized process occurring in other cell types undergoing apoptosis and, in any case, occurs in an entirely different biological context (i.e. apoptosis) from that of the generation of Tα1 in proliferating or nonproliferating cells. Independently of this, the question remaining is why ProTα is processed by legumain to yield Tα1: simply as a step in the catabolism of ProTα, or in view of some biological function of Tα1? Our current knowledge about mammalian legumain may perhaps shed some light on this question. Legumain shows strict specificity for a restricted subset of asparaginyl bonds, which certainly suggests that it is unlikely to contribute to the gross catabolism of proteins but rather that it will tend to play more specific roles in protein processing (32Dando P.M. Fortunato M. Smith L. Knight C.G. McKendrick J.E. Barrett A.J. Biochem. J. 1999; 339: 743-749Crossref PubMed Scopus (64) Google Scholar). Of particular interest is the identification of legumain as a key protease in class-II MHC antigen processing (30Manoury B. Hewitt E.W. Morrice N. Dando P.M. Barrett A.J. Watts C. Nature. 1998; 396: 695-699Crossref PubMed Scopus (308) Google Scholar). In view of these considerations, the proteolysis of ProTα in lymphocytes and other mammalian cells is in our view likely to be selective processing rather than nonspecific degradation. The fact that Tα1 is present at high levels, similar to those of ProTα, suggests that Tα1 may have some biological function. The cytosolic location of Tα1, its demonstrated incapacity to migrate to the nucleus (15Manrow R.E. Sburlati A.R. Hanover J.A. Berger S.L. J. Biol. Chem. 1991; 266: 3916-3924Abstract Full Text PDF PubMed Google Scholar), and its lack of any known secretion signal (note that ProTα is synthesized in free polysomes) (37Eschenfeldt W.H. Manrow R.E. Krug M.S. Berger S.L. J. Biol. Chem. 1989; 264: 7546-7555Abstract Full Text PDF PubMed Google Scholar) argue for a nonnuclear intracellular function. This putative function may or may not be related to that of ProTα.
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