Prothymosin alpha  Is Processed to Thymosin alpha 1 and Thymosin alpha 11 by a Lysosomal Asparaginyl Endopeptidase*

Concepción S. Sarandeses, Guillermo Covelo, Cristina Díaz-Jullien, and Manuel FreireDagger

From the Departamento de Bioquímica y Biología Molecular, Facultad de Biología, Universidad de Santiago de Compostela, Santiago de Compostela 15782, Spain

Received for publication, December 20, 2002, and in revised form, January 24, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Thymosin alpha 1 (Talpha 1) and thymosin Talpha 11 (Talpha 11) are polypeptides with immunoregulatory properties first isolated from thymic extracts, corresponding to the first 28 and 35 amino acid residues, respectively, of prothymosin alpha  (ProTalpha ), a protein involved in chromatin remodeling. It has been widely supposed that these polypeptides are not natural products of the in vivo processing of ProTalpha , since neither was found in extracts in which proteolysis was prevented. Here we show that a lysosomal asparaginyl endopeptidase is able to process ProTalpha to generate Talpha 1 and Talpha 11. 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 ProTalpha sequence; specifically, Asn28-Gly29 and Asn35-Gly36 residues are cleaved with similar efficiency in vitro to generate Talpha 1 and Talpha 11, respectively. By contrast Talpha 1 is the main product detected in vivo, free in the cytosol, at concentrations similar to that of ProTalpha . The data here reported demonstrate that Talpha 1 is not an artifact but rather is naturally present in diverse mammalian tissues and raise the possibility that it has a functional role.

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The alpha -thymosins are a group of acidic peptides (pI 3.5-4.5) present in a calf thymus extract denominated thymosin fraction 5 (TF5)1 (1), which shows immunoregulatory properties in several in vitro and in vivo assay systems (2). Thymosin alpha 1 (Talpha 1; 28 amino acids) is the most abundant alpha -thymosin in TF5 and was the first to be isolated and sequenced (3). A less abundant TF5 component named thymosin alpha 11 (Talpha 11) has subsequently been characterized (4); this peptide comprises the Talpha 1 sequence plus an additional 7 residues at its C terminus (i.e. 35 residues in total). Both Talpha 1 and Talpha 11 show immunoregulatory properties similar to those of TF5 (5).

A polypeptide including Talpha 1 in its structure has been detected among the translation products of calf thymus mRNAs (6, 7). This apparent precursor of the alpha -thymosins was later isolated from thymus (8) and other mammalian tissues (9, 10). Sequencing indicated that it comprised 109-111 amino acids, including the sequence of Talpha 11 (and thus Talpha 1) at its N terminus (11, 12). This protein was denominated prothymosin alpha  (ProTalpha ) (8). ProTalpha is a highly acidic protein (pI 3.55) with a highly conserved sequence (13). Its wide distribution in mammalian tissues suggested that its function was probably not immunological, despite the apparent immunoregulatory properties of Talpha 1 and Talpha 11. Subsequent studies indicated that ProTalpha has a generalized role in the proliferation of mammalian cells, by mechanisms involving migration to the nucleus (14-16) and cytosolic phosphorylation of ProTalpha (17, 18). Reports from our group and others over recent years have provided increasing evidence that the nuclear function of ProTalpha involves interactions with histones (19-21) and with other proteins related to DNA metabolism, including proliferating cell nuclear antigen, Cdk2, and cyclin A (22), arguing strongly for a role in chromatin remodeling. In further support of this view, ProTalpha enables nucleosome assembly (20) and modulates the activity of histone acetyltransferase p300 (23).

Independently of ProTalpha and its function, uncertainty remains about the status of Talpha 1 and Talpha 11. When ProTalpha was isolated in 1984 from thymus extracts prepared under conditions in which proteolytic activity was prevented, Talpha 1 and Talpha 11 were not detected in reverse-phase HPLC separation of the extracts (8). This led to the suggestion that the presence of ProTalpha -derived alpha -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 alpha -thymosin detection, have indicated that Talpha 1 is indeed present in extracts of this type in diverse mammalian tissues (10).

To resolve this controversy, we have been performing experiments designed to detect a protease in mammalian cells that is capable of processing ProTalpha to generate the alpha -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 (24) and the trematode Schistosoma (25) but later found in mammals (26). Experiments designed to assess the possible biological significance of the processing of ProTalpha by this enzyme are also presented.

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Cells and Subcellular Fractionation-- Cells used were transformed human B lymphocytes (NC-37). For subcellular fractionation, cells were washed and resuspended (about 2 × 108 cells/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 × 108 nuclei/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 mM Tris-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 0.5 M 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 (27). 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 (28). 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.

Obtention of alpha -Thymosins-- A heat-stable acidic polypeptide fraction (including the alpha -thymosins) was obtained from whole cell extracts or from subcellular fractions by a modification of the procedure of Goldstein et al. (1), as previously described (10). 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 0.4 M NaCl to give the fraction of acidic polypeptides.

ProTalpha 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 ProTalpha was dephosphorylated by incubation for 30 min at room temperature with 1 unit of calf intestine alkaline phosphatase per µg of ProTalpha , in 20 mM Tris-HCl, pH 7.8, containing MgCl2 (1 mM) and ZnCl2 (0.1 mM). Dephosphorylated ProTalpha was obtained from this reaction mixture by RP-HPLC as indicated above. Phosphorylation of ProTalpha with radioactive orthophosphate was done using ProTalpha protein kinase, purified as described (18). Briefly, a phosphorylation reaction mixture containing 50 µg of dephosphorylated ProTalpha , 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 beta -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]ProTalpha was then obtained by RP-HPLC as described above. Talpha 1 and Talpha 11 were a gift from Dr. Heimer (Hoffman-Roche).

Isoelectrofocusing and SDS-PAGE-- Isoelectrofocusing was carried out as described previously (10). 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 (29).

ProTalpha -processing Assays-- The reaction mixture contained different concentrations of ProTalpha (15-30 µg) plus crude or purified cell extract (1-15 µg) in 45 µl of 0.1 M ammonium acetate buffer, pH 4.5, plus 0.5 mM DTT or 0.1 M 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 (10).

Purification of the Lysosomal Asparaginyl Endopeptidase-- 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 an Amersham 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 (30). ProTalpha 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 0.1 M 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.

Immunoassays-- 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 (30). Neosystem Laboratories also supplied preimmune serum from the same rabbit.

The effect of the legumain antiserum antibodies on the ProTalpha -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. ProTalpha processing was then assayed as described above.

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ProTalpha -processing Activity in Subcellular Fractions-- To investigate the intracellular processing of ProTalpha , we performed a systematic study of ProTalpha -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 ProTalpha 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 ProTalpha -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, ProTalpha -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 ProTalpha -proteolytic activity (data not shown). Moreover, ProTalpha -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 ProTalpha by the acidic lysosomal lysates is that two of the fragments of ProTalpha had the same electrophoretic mobility as synthetic Talpha 1 and Talpha 11 (Fig. 1C).


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Fig. 1.   SDS-PAGE analysis of ProTalpha proteolysis by different subcellular fractions of human lymphocytes. Aliquots of ProTalpha (about 20 µg) were incubated with different NC-37 cell fractions, and each reaction mixture was then analyzed by SDS-PAGE under the conditions indicated under "Experimental Procedures." A, analysis of ProTalpha proteolysis by the nucleoplasm, cytosol, nuclear membrane, cytosolic organelle, and microsome fractions. Aliquots of about 10 µg of the nucleoplasmic and cytosolic fractions and that of nuclear membranes, microsomes, and cytosolic organelles extracted at pH 7.5 were used to assay the proteolytic activity at pH 7.5 or 4.5. The relative mobility of ProTalpha is indicated. B, analysis of ProTalpha proteolysis by the mitochondria and lysosome fractions. Extracts of these organelles were prepared in phosphate buffer, pH 7.5, and assayed (aliquots of about 5 µg of extract) at pH 7.5 or 4.5. The Mr of ProTalpha is indicated. C, effect of pH on the proteolysis of ProTalpha by the lysosome fraction. The lysosomes were extracted in citrate buffer, pH 4.5, and assayed (aliquots of about 5 µg of extract) at pH 4.5, 6.0, or 8.0. The relative mobilities of ProTalpha and synthetic Talpha 1 and Talpha 11 are shown in the right lane. D, effect of different protease inhibitors on ProTalpha proteolysis by the lysosome fraction. Lysosomal extracts were prepared in citrate buffer, pH 4.5, and assayed (aliquots of about 5 µg of extract) at pH 4.5 in reaction mixtures containing (a) pepstatin (10 µg/ml) plus E-64 (0.1 mg/ml), (b) PMSF (1 mM) plus leupeptin (10 µg/ml), (c) pepstatin plus E-64 plus PMSF plus leupeptin at these concentrations, or (d) iodoacetamide (1 mM).

To further investigate processing of ProTalpha , 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 ProTalpha 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 ProTalpha .

Since ProTalpha is phosphorylated by a cytosolic threonine protein kinase with unknown structure (18), we next investigated the influence of ProTalpha phosphorylation on the proteolysis pattern. Specifically, we compared proteolysis of dephosphorylated ProTalpha and ProTalpha phosphorylated in vitro by the purified ProTalpha protein kinase with [32P]ATP as cosubstrate. Results of these experiments (data not shown) indicate that phosphorylation of ProTalpha 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 ProTalpha , even in the presence of broad spectrum protease inhibitors, yielding four fragments, two of which have the same electrophoretic mobility as Talpha 1 and Talpha 11.

Characterization of the Products of Processing of ProTalpha -- To characterize the different products derived from the processing of ProTalpha by the lysosomal protease activity, we used in the first instance RP-HPLC. The elution pattern for ProTalpha -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 Talpha 1 is the main component in peak 1; peaks 2 and 3 are mixtures of this component and the other ProTalpha fragments, and peak 4 is whole ProTalpha . 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 Talpha 1 (Fig. 2B). Structural analysis of the other ProTalpha -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 Talpha 11 is indeed Talpha 11, whereas the two fragments with higher electrophoretic mobility correspond to residues 29-109 and 36-109 of the ProTalpha sequence. In view of these structural analyses, summarized in Fig. 2C, we conclude that the lysosomal protease cleaves ProTalpha at Asn-Gly residues located at positions 28-29 to yield Talpha 1 and at positions 35-36 to yield Talpha 11, whereas the resulting C-terminal portions of ProTalpha , 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 Talpha 1 and Talpha 11 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 ProTalpha , 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 Talpha 1 and Talpha 11 (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 alpha -thymosins in vivo.


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Fig. 2.   A, separation of the ProTalpha -processing products by reverse-phase HPLC. The products of processing of ProTalpha by the lysosome fraction were analyzed by RP-HPLC on a Vydac column (5 µm, 300 Å, 4.6 × 150 mm) with a programmed acetonitrile gradient (0-30%) in 0.1% trifluoroacetic acid in water (flow 0.7 ml/min). Peaks 1-4 were concentrated, and aliquots were analyzed by SDS-PAGE (inset). B, tryptic mapping of peak 1. 5 µg of the peak 1 concentrate was digested with trypsin, and the resulting peptides were separated by RP-HPLC. The structures of the tryptic peptides deduced from their amino acid composition are shown, together with the Talpha 1 sequence. C, summary of the structural analysis of the ProTalpha processing products. Bottom, arrows indicate sites in the ProTalpha sequence that are cleaved in vitro by the lysosomal asparaginyl endopeptidase. Top, concentrations of the different fragments observed in assays in vitro, as determined by densitometric analysis of SDS-PAGE bands (means of three assays).

Identification of the Lysosomal Protease That Processes ProTalpha -- The characteristics of the lysosomal protease processing ProTalpha in vitro to yield Talpha 1 and Talpha 11 (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 (30). We therefore performed experiments to investigate whether the lysosomal asparaginyl endopeptidase that processes ProTalpha 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 ProTalpha -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) (30). 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 (31), we investigated the effect of previous deglycosylation. In the FPLC-purified lysosomal lysate treated with N-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 (26).


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Fig. 3.   Characterization of the lysosomal asparaginyl endopeptidase. A, fractionation of the asparaginyl endopeptidase activity by FPLC. The lysosomal extract at pH 4.5 (5 mg) was chromatographed on a Mono-S FPLC column, and proteolytic activity with ProTalpha (red) or benzyloxycarbonyl-Val-Ala-Asn-7-amido-4-methylcoumarin (Z-Val-Ala-Asn-AMC) (blue) as substrate was assayed in 20-µl aliquots of the different fractions collected, as detailed under "Experimental Procedures." Fractions with the higher proteolytic activity were combined (purified protease activity). B, immunoblotting analysis of the lysosomal asparaginyl endopeptidase. A legumain antiserum (1:400) and a nonimmune serum at the same dilution were used to probe the lysosomal extracts (15 µg) (Crude), the purified protease activity (25 µl) (FPLC-Purified), or the same purified protease activity following treatment with N-glucidase-F (0.2 milliunits), as described under "Experimental Procedures." C, effect of the legumain antiserum on the ProTalpha -processing activity. ProTalpha processing was assayed with aliquots of the purified protease activity (25 µl) previously incubated with legumain antiserum (1:2), alone or in the presence of 20 µg of the peptide KGIGSGKVLKSGPQ used to raise the serum (see "Experimental Procedures"), with nonimmune serum (1:2) or with an equivalent volume of reaction mixture buffer (control). The different reaction mixtures were separated by SDS-PAGE, and the relative concentrations of ProTalpha and its derived peptides Talpha 1 and Talpha 11 were determined by densitometric analysis (means of three experiments).

To complete the characterization of the enzyme processing ProTalpha , we investigated whether the legumain antiserum blocks the proteolysis of ProTalpha by the lysosomal protease. As indicated in Fig. 3C, the antibodies efficiently blocked processing of ProTalpha , 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 ProTalpha in vitro to generate Talpha 1 and Talpha 11 is legumain.

Processing of ProTalpha in Vivo-- To further study the processing of ProTalpha , we decided to investigate relationships between the observed proteolysis of ProTalpha by the legumain in vitro with the processing seen in vivo. To this end, we compared the polypeptide pattern observed after ProTalpha processing in vitro with the alpha -thymosins pattern observed in lymphocytes, in which both ProTalpha 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. alpha -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 (10). IEF analysis of the alpha -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 ProTalpha and Talpha 1 but not components with the same pI as Talpha 11 or the acidic fragments 29-109 and 36-109 of ProTalpha detected after in vitro processing. Structural analysis confirmed that the more acidic components were ProTalpha and Talpha 1.


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Fig. 4.   alpha -Thymosin presence and subcellular distribution in vivo. The heat-stable acidic polypeptide fraction containing the alpha -thymosins was obtained and separated by IEF as detailed under "Experimental Procedures." A, alpha -thymosins in whole-cell NC-37 extracts. An aliquot of the acidic polypeptide fraction (150 µg) from whole-cell extract (2 g of cells) was separated by IEF, and the Coomassie-stained bands of the more acidic components were quantified by densitometry, extracted, and structurally analyzed as detailed under "Experimental Procedures." Histograms indicate the concentration of ProTalpha and Talpha 1 identified by the structural analysis. Mobilities of synthetic Talpha 11 and the ProTalpha fragments 29-109 and 36-109 (previously determined by IEF analysis of the respective bands excised from SDS-PAGE gels in which the components of the processing assay were separated) are also indicated in the gel. Similar results were obtained in IEF analyses of heat-stable acidic polypeptide fractions obtained from five different whole cell extracts. B, subcellular distribution of alpha -thymosins (fractions obtained by differential centrifugation). NC-37 cell homogenates (2 g of cells) were fractionated by differential centrifugation; the heat-stable acidic polypeptide fraction was obtained in each case, and aliquots of 150 µg were separated by IEF. The more acidic components were analyzed, as for A. Histograms indicate the concentration of products identified as ProTalpha and Talpha 1 (means of three experiments). C, subcellular distribution of alpha -thymosins (fractions obtained by digitonin permeabilization). NC-37 cell homogenates (2 g of cells) were fractionated by digitonin permeabilization as detailed under "Experimental Procedures," giving the cytosolic (digitonin-released) and the noncytosolic (digitonin-retained) fractions; the heat-stable acidic polypeptide fraction was obtained in each case, and aliquots of 150 µg were separated by IEF. The more acidic components were analyzed, as for A. Histograms indicate the concentration of products identified as ProTalpha and Talpha 1 (means of three experiments).

This IEF pattern and the similar concentrations of ProTalpha and Talpha 1 determined by densitometry (Fig. 4A) are in agreement with our previous findings indicating that Talpha 1 is naturally present in various mammalian tissues (10) and in line with the view that ProTalpha is processed in vivo by legumain or another protease with similar specificity to yield Talpha 1.

We next investigated the intracellular location of Talpha 1 in NC-37 cells. IEF analysis of alpha -thymosins in the different subcellular fractions separated by differential centrifugation (Fig. 4B) and by digitonin permeabilization (Fig. 4C) indicated that Talpha 1 is located in the cytosolic fraction and is not associated with any cell organelle. The observed cytosolic location of ProTalpha (Fig. 4, B and C) is in agreement with the tendency of ProTalpha to leak out of isolated nuclei (15).

It is worth pointing out that the alpha -thymosins obtained from both whole-cell extracts and from organelle fractions showed rather "messy" chromatographic behavior; thus, in the RP-HPLC analysis ProTalpha emerged as a clear peak, but Talpha 1 was retained for longer by the column and eluted as mixtures with other components in the alpha -thymosinic fraction (data not shown). Similar behavior was observed under diverse HPLC conditions, both reverse-phase and ion exchange.

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Data reported here strongly suggest that the presence of Talpha 1 in lymphocytes and other mammalian cells is due to the processing of ProTalpha by a lysosomal asparaginyl endopeptidase identified as legumain. This enzyme was recently discovered in mammals (26), having been known previously only from plants (24) and invertebrates (25). 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) (32). The specificity of legumain with ProTalpha 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 alpha -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 Talpha 1 (i.e. cleavage at positions 28-29) was detected at a concentration similar to ProTalpha , whereas Talpha 11 (i.e. cleavage at positions 35-36) was not detected, in line with previous studies of other mammalian tissues (10). However, we cannot rule out the possibility that the apparent absence of Talpha 11 is due to rapid degradation of this peptide in vivo. The nondetection of the larger ProTalpha fragment 29-109 (or 36-109) in cell extracts is likewise presumably indicative of rapid degradation of this fragment in vivo.

The presence of Talpha 11 at low concentrations in the calf thymus fraction TF5 (4) might be due to the characteristics of the procedure used for its preparation. In fact, we did not detect this polypeptide in the alpha -thymosin fractions obtained (from calf thymus and other mammalian tissues) under the conditions used here (10, 33).

As noted, the characteristics of the in vivo processing of ProTalpha suggest that it is accomplished by legumain. It is worth noting that additional confirmation of this conclusion by in vivo enzyme 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 (30).

In light of the present results, previous failures to detect Talpha 1 in extracts prepared under drastic denaturing conditions (8) may perhaps be attributable to the "messy" chromatographic behavior of alpha -thymosins prepared under these conditions, due to their tendency to aggregate, as evidenced in the present and previous reports (10, 33). In fact, original isolation of Talpha 1 included gel filtration in the presence of 6 M guanidium chloride (3). In the present and previous studies (10, 33), we have used isoelectrofocusing, which, unlike the HPLC procedures used by other authors (8), is effective for separating and identifying the alpha -thymosins. In this connection, it is also worth noting that antibodies raised against fragments of the N-terminal region of ProTalpha (the most immunogenic region; such antibodies have been widely used for immunodetection of ProTalpha ) do not differentiate between ProTalpha and its N-terminal derivatives Talpha 1 and Talpha 11.

To judge by the wide distribution of both Talpha 1 (10) and legumain (26) in different tissues, we would suggest that ProTalpha processing to yield Talpha 1 is a generalized process in mammalian tissues. Interestingly, tissues showing high legumain activity such as lymphoid tissues (26, 30) also show high levels of Talpha 1 (10). The highly conserved primary structure of ProTalpha , especially around the asparagine/glycine bonds cleaved by legumain (13), provides further support for the view that ProTalpha processing to yield Talpha 1 is generalized. Moreover, the present results indicate that processing of ProTalpha in vitro is independent of its phosphorylation state (ProTalpha is phosphorylated at specific residues when cells are activated to proliferate) (17, 18). The presence of phosphorylated Talpha 1 in proliferating cells and the inability of the ProTalpha protein kinase to phosphorylate Talpha 1 (34) thus suggest that phospho-ProTalpha may be processed in vivo to yield phospho-Talpha 1.

Proteolysis of ProTalpha at the Asp99 residue by caspase 3 has recently been reported to occur in HeLa cells in which apoptosis has been induced (35, 36). 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 Talpha 1 in proliferating or nonproliferating cells.

Independently of this, the question remaining is why ProTalpha is processed by legumain to yield Talpha 1: simply as a step in the catabolism of ProTalpha , or in view of some biological function of Talpha 1? 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 (32). Of particular interest is the identification of legumain as a key protease in class-II MHC antigen processing (30). In view of these considerations, the proteolysis of ProTalpha in lymphocytes and other mammalian cells is in our view likely to be selective processing rather than nonspecific degradation. The fact that Talpha 1 is present at high levels, similar to those of ProTalpha , suggests that Talpha 1 may have some biological function. The cytosolic location of Talpha 1, its demonstrated incapacity to migrate to the nucleus (15), and its lack of any known secretion signal (note that ProTalpha is synthesized in free polysomes) (37) argue for a nonnuclear intracellular function. This putative function may or may not be related to that of ProTalpha .

    FOOTNOTES

* This work was supported by Dirección General de Investigación Científica y Técnica Grant PB96-0965 and Consellería de Ordenación Universitaria Grant XUGA PGIDT00PXX20001PR and PGIDT00BIO20001PR from the Spanish Ministerio de Educación y Ciencia and Xunta de Galicia, respectively.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. de Bioquímica y Biología Molecular, Facultad de Biología, 15782 Santiago de Compostela, Spain. Tel.: 34-981-563100 (ext. 13316); Fax: 34-981-596904; E-mail: bnfreire@usc.es.

Published, JBC Papers in Press, January 28, 2003, DOI 10.1074/jbc.M213005200

    ABBREVIATIONS

The abbreviations used are: TF5, thymosin fraction 5; Talpha 1, thymosin alpha 1; Talpha 11, thymosin alpha 11; ProTalpha , prothymosin alpha ; HPLC, high performance liquid chromatography; RP-HPLC, reverse-phase HPLC; PMSF, phenylmethylsulfonyl fluoride; FPLC, fast protein liquid chromatography; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; IEF, isoelectric focusing; PAG, polyacrylamide gel.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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