- Email: [email protected]
Mutation in a calpain-like protease affects the posttranslational mannosylation of phosphatases in Aspergillus nidulans
Fungal Genetics and Biology 38 (2003) 220–227 www.elsevier.com/locate/yfgbi
Mutation in a calpain-like protease affects the posttranslational mannosylation of phosphatases in Aspergillus nidulans S.R. Nozawa,a G.S. May,b,* N.M. Martinez-Rossi,a M.S. Ferreira-Nozawa,a J. Coutinho-Netto,c W. Maccheroni Jr.,a,1 and A. Rossic b
a Departamento de Gen etica, FMRP-USP, 14049-900 Ribeir~ ao Preto, SP, Brazil Division of Pathology and Laboratory Medicine, M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 54, The University of Texas, Houston, TX 77030, USA c Departamento de Bioquımica e Imunologia, FMRP-USP, 14049-900 Ribeir~ ao Preto, SP, Brazil
Received 11 March 2002; accepted 17 August 2002
Abstract In this communication, we show that the palB7 mutation drastically reduced the mannose and N-acetylgalactosamine content of the pacA-encoded acid phosphatase secreted by the fungus Aspergillus nidulans at pH 5.0, compared to a control strain. By using mRNA differential display reverse transcription and polymerase chain reaction, we isolated two cDNAs from the control pabaA1 strain that were not detected in the palB7 mutant strain that encode a mannosyl transferase and a NADH-ubiquinone oxidoreductase. Thus, a defect in the posttranslational mannosylation of proteins could be the consequence of mutations in the palB gene, which encodes for a nuclear calpain-like protease that may have specific functions in the processing of transcription factor(s) similar to its homolog, RIM13, in Saccharomyces cereviseae. Ó 2002 Elsevier Science (USA). All rights reserved.
1. Introduction Although it is well established that ambient pH affects growth, physiology, differentiation, and viability of all organisms, the molecular responses to environmental pH changes are only now being elucidated. In Aspergillus nidulans these responses are mediated by a conserved signal transduction pathway comprising at least seven genes that have been cloned and sequenced (Denison, 2000; Denison et al., 2001; Maccheroni et al., 1997). The pacC gene codes for a Zn-finger transcription factor that undergoes proteolysis at alkaline pH, yielding an active protein responsible for the induction of genes expressing products with optimal activity at alkaline pH (e.g., alkaline phosphatase) and repression of those with optimal activity at acid pH (e.g., acid phosphatase) (Caddick et al., 1986a,b; Nahas et al., 1982). * Corresponding author. Fax: 1-713-792-8460. E-mail address: [email protected] (G.S. May). 1 Present address: Departamento de Genetica, ESALQ-USP, 13400-970 Piracicaba, SP, Brazil.
The pacC transcription is induced under alkaline growth conditions. The six pal genes (palA, B, C, F, H, and I) are putative members of a signaling cascade involved in ambient alkaline pH sensing whose sole known function is to promote the proteolytic activation of PacC, because expression of C-terminal-truncated PacC derivatives suppresses pal mutant defects. Strains with mutations in the pal genes have reduced alkaline phosphatase activity but increased acid phosphatase activity, among other phenotypic effects that are consistent with pal mutants mimicking growth at acidic pH. The palB gene codes for a calpain-like protease that is not involved directly in PacC processing, and the other pal genes have revealed few functional features (Denison, 2000; Denison et al., 1995). An interesting finding, however, is that the transcription of the six pal genes, unlike pacC, do not appear to be pH-regulated (Maccheroni et al., 2000; Negrete-Urtasun et al., 1999). Furthermore, if the pal genes represent metabolic steps involved in the activation of a single regulatory protein, the properties of the secreted acid phosphatase should
1087-1845/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. doi:10.1016/S1087-1845(02)00521-2
S.R. Nozawa et al. / Fungal Genetics and Biology 38 (2003) 220–227
be identical in all pal mutants and wild-type strains. This is not the case, as indicated by molecular mass, electrophoretic mobility, and chromatographic behavior of the acid phosphatase secreted by the palB7 strain (Maccheroni et al., 1995). Also, the palB7 mutation altered the electrophoretic mobility of the constitutive acid phosphatase synthesized on high inorganic phosphate (Pi) medium. With this in mind, we purified the major acid phosphatase secreted by strains pabaA1, pabaA1 palB7, and pabaA1 palB7 pyrG89, and determined the neutral and aminosugar content of the purified enzyme secreted by these mutant strains. Our objective was to establish whether the palB7 mutation affected the properties of secreted enzymes by altering the pattern of posttranslational modification of this phosphatase. We show that strains carrying the palB7 mutation secreted acid phosphatase with low mannose and N-acetylgalactosamine content, an effect partially restored by the combination of palB7 and pyrG89 mutations. Furthermore, by using an mRNA differential display (DDRT-PCR) we isolated two cDNAs, not detected in the palB7 strain, that encode a mannosyl transferase and an NADH-ubiquinone oxidoreductase (chain 4).
2. Materials and methods 2.1. Strains and growth conditions The following strains of A. nidulans were used in the present study: pabaA1 (p-aminobenzoic acid requiring), pyrG89 (orotidine-50 -phosphate decarboxylase), palB7 (mutant for alkaline phosphatase production), pabaA1 pyrG89, pabaA1 palB7, palB7 pyrG89, and pabaA1 palB7 pyrG89. All of these strains are isogenic. The mutations carried by A. nidulans have been described previously (Clutterbuck, 1993 and references therein) and are available from the Fungal Genetic Stock Center (University of Kansas Medical Center, Kansas City, KS, USA). The solid complete and minimal liquid media of Cove (1966) were used. All strains were maintained on solid complete medium containing 55 mM glucose as the carbon source, 10 mM ammonium tartarate as the nitrogen source (final concentrations), and 11 mM Pi, pH 6.5. Unless otherwise stated, a growth temperature of 37 °C was used. The palB7 mutants grow at pH 5.0 following the addition of (final concentration) 50 mM Na citrate, pH 5.0 (Nozawa et al., 1995), and pyrG89 mutants grow on medium containing 5 mM uridine and 2.5 mM uracil. Low-Pi liquid medium was prepared by adding (final concentrations) 50 mM Na citrate, pH 5.0, 200 lM KH2 PO4 , and 10 mM KCl to Pi-free minimal medium. Conidia (109 ) from all strains obtained after growth on solid complete medium were inoculated into 500 ml Erlenmeyers containing 100 ml low-Pi liquid
221
minimal medium and incubated 14 h in an orbital shaker (160 rpm). 2.2. Purification of secreted acid phosphatases The procedure used for the purification of the acid phosphatase secreted by strains pabaA1, palB7 pabaA1 chaA1, and palB7 pyrG89 w A3 to apparent homogeneity as determined by SDS–PAGE was that previously described (Nozawa et al., 1998). 2.3. Assays and characterization of the purified enzymes Pi-repressible acid phosphatase activity was determined at pH 6.0 (100 mM Na maleate, 2 mM EDTA) using 6 mM p-nitrophenylphosphate (PNP-P) as substrate at 37 °C (Nozawa and Rossi, 2000). One unit of acid phosphatase activity was defined as 1 gmol substrate hydrolyzed min1 , at 37 °C. Protein was measured by the method of Lowry as modified by Hartree (1972), with bovine serum bovine fraction V as the standard. Mycelium-specific activities were expressed as units per mg dry weight mycelium. Staining for acid phosphatase activity in petri dishes was determined by flooding the colonies with 2 mg sodium a-naphthyl acid phosphate and 20 mg fast red TR salt dissolved in 4 ml of 0.6 M sodium maleate buffer, pH 6.0, at room temperature (Caddick et al., 1986a). Enzyme activity is indicated by a brown precipitate inside the mycelial mass. PAGE was carried out at pH 8.3 by the method of Davis as described by Nahas and Rossi (1984) using 7.5% (w/v) polyacrylamide slab gels (10 10 0:1 cm). The electrophoretic pattern of acid phosphatases was developed by the method of Dorn as described by Maccheroni et al. (1995) using Na a-naphthyl phosphate as substrate, pH 6.0 (as described above), without prior incubation of slab gels with any buffer. The ratio of the distance covered by the enzyme bands to distance covered by bromophenol blue (electrophoretic mobility, Rf ) was measured. The relative heat stability of the phosphatase was determined by incubating the purified enzyme in 10 mM Na maleate buffer, pH 6.0, at 60 °C. At intervals samples were removed to measure the remaining phophatase activities using the standard procedure. Limiting velocities (Vm ) and Michaelis constants (Km ) were determined at 37 °C, pH 6.0, as Lineweaver and Burk (1934) plots. The interaction constant for the substrate (n) was determined by the Hill procedure as described by Koshland (1970). The kinetic constants reported here were obtained by linear-square analysis calculated from data obtained in at least three independent experiments. Neutral sugars were measured as described by Dubois et al. (1965) with glucose as standard. The composition of monosaccharides present in purified acid phosphatases was analyzed as described by Fu and OÕNeill (1995). The
222
S.R. Nozawa et al. / Fungal Genetics and Biology 38 (2003) 220–227
1-phenyl-3-methyl-2-pyrazoline-5-one (PMP)-labeled monosaccharides were analyzed out on a Shimadzu model LC-10ADVP HPLC system equipped with a diode-array SPD-M10AVP UV/VIS detector and a SCL-10AVP controller. A CHO C-18 column, 220 2:1 mm (Perkin–Elmer, Applied Biosystems Division), optimized for the separation of PMP-labeled carbohydrates, was used. The flow rate was set to 200 ll/min, and the wavelength for UV detection was 245 nm. For neutral and aminosugar separations, buffers A and B were 100 mM ammonium acetate, pH 5.5, containing 10% and 25% (v/v) acetonitrile, respectively. A gradient of 45–100% buffer B in 80 min was used for separation. Data analysis was performed automatically by using the class VP (Shimadzu) program.
The level of Pi-repressible acid phosphatase secreted by A. nidulans was increased approximately 2-fold in strains carrying the palB7 mutation alone or in combination with other auxotrophic markers, as shown by colony staining or enzymatic hydrolysis of PNP-P (Table 1). Additionally, the palB7 mutation altered the electrophoretic mobility of the pacA-encoded acid phosphatase, as compared to those of the enzyme secreted by strains pabaA1 and pabaA1 palB7 pyrG89 (Fig. 1). Also as previously described, the elution pattern from DEAE-cellulose of the acid phosphatase secreted
2.4. RNA preparation and differential display RT-PCR
Table 1 Acid phosphatase secreted into the growth medium by various strains of A. nidulans grown on low Pi-medium at 37 °C, pH 6.5
Total RNA preparations and DDRT-PCR of mycelial mRNA from pabaA1 and pabaA1 palB7 strains were performed, as described previously (Liang and Pardee, 1992) by using arbitrary primers. Total RNA from each strain, extracted after the mycelium grown for 16 h in shaken liquid complete medium was transferred to and incubated for 2–3 h in shaken liquid low-Pi medium, reverse transcribed using random hexamers to prime the reaction (Kim et al., 2000; Pienta and Schwab, 1999). The amplified cDNAs were separated after electrophoresis on 14 11 cm plates of agarose gel at 2% (w/v) in 1 TAE buffer (Sambrook et al., 1989) (a 100-bp ladder was included as size reference) and visualized with ethidium bromide. Overexpressed bands in one of these strains were excised using the Concert Matrix (GIBCO) gel extraction kit and amplified by PCR. The cDNA was then ligated into the pGEM-T Easy vector (Promega) and used to transform bacteria. The insert was isolated, sequenced using the 377 automated DNA sequencer from Perkin–Elmer, and used as probes in Northern blot analysis.
3. Results and discussion
Strain
Colony staininga
Specific activityb
pyrG89 pabaA1 pyrG89 pabaA1 palB7 palB7 pyrG89 palB7 pabaA1 palB7 pyrG89 pabaA1
+ + + ++ ++ ++ ++
131 8 162 8 150 4 252 20 221 16 272 12 232 12
a (+) and (++) represent enzyme activity developed using a-naphthyl phosphate as the substrate and indicated by the color intensity of a brown precipitate formed inside the mycelium mass. b PNP-P hydrolysis expressed as units/mg dry weight mycelium. Data are reported as means SD. For details see Section 2.
2.5. Northern hybridization Total RNA (10 lg) was denatured, separated by electrophoresis, and blotted onto Amersham HybondN+ (Pharmacia Biotech) membranes following the procedure of Fourney et al. (1988). Hybridization was performed with cDNA probes random-prime labeled with ½a-33 PATP (Amersham-Pharmacia Biotech), and unincorporated nucleotides were eliminated from the probes by purification through Sephadex G-50 column. Hybridization was visualized by autoradiography after the membranes were washed twice (Sambrook et al., 1989). Ethidium bromide staining showed that in all experiments equal amounts of RNA were loaded. Actin mRNA level was also used as a loading control (Fidel et al., 1988).
Fig. 1. PAGE of Pi-repressible phosphatases secreted by A. nidulans at pH 5.0. Samples containing about 0.5 U enzyme were separated by electrophoresis, and the enzyme activity bands developed using anaphthyl phosphate as the substrate, pH 6.0, without prior incubation of the slab gels with any buffer. Enzymes of strains pabaA1 (lanes 1, 3, and 5), palB7 pabaA1 (lane 2), and palB7 pyrG89 pabaA1 (lane 4).
S.R. Nozawa et al. / Fungal Genetics and Biology 38 (2003) 220–227
Fig. 2. Stepwise fractionation on DEAE-cellulose of acid phosphatases secreted by the pabaA1 (A), pabaA1 palB7 (B), and palB7 pabaA1 pyrG89 (C) strains of A. nidulans grown in low-Pi medium at 37 °C, pH 5.0. After elution of nonabsorbed proteins with about 100 ml of equilibrating buffer, proteins were eluted by stepwise addition of 0.05, 0.10, 0.15, 0.30, 0.50, and 1 M NaCl dissolved in 10 mM MOPS, pH 7.2. 6 ml fractions were collected in tubes containing 1 ml of 0.10 M Na acetate buffer, pH 4.7, at flow rate of 120 ml/h.
223
by the pabaA1 palB7 strain was altered because only the form II-like activity was recovered (Fig. 2). In addition, the major acid phosphatase secreted by strain pabaA1 palB7 pyrG89 was recovered from DEAE-cellulose as a form IV-like activity (Fig. 2). We have shown that forms I–IV of acid phosphatase secreted by A. nidulans are encoded by the structural gene pacA and that these isozymes may be generated by different patterns of glycosylation and other posttranslational modifications of the protein (Justino et al., 2001; Maccheroni et al., 1995; Nozawa and Rossi, 2000; Nozawa et al., 1998). Thus, to further characterize the acid phosphatase secreted by A. nidulans, we purified to apparent homogeneity as determined by PAGE the form II-like and IV-like variants of this enzyme, respectively, secreted by pabaA1 palB7 and pabaA1 palB7 pyrG89 mutants (and both forms I and II of this enzyme secreted by pabaA1 strain), and determined their neutral and aminosugars content, molecular mass, and kinetic properties. The procedure summarized in Tables 2A and 2B provided optimal conditions for the purification of acid phosphatases II-like and IV-like secreted by the pabaA1 palB7 and pabaA1 palB7 pyrG89 mutants, respectively. All enzyme preparations (at least three independent preparations) appeared to be homogeneous as determined by 7.5% PAGE at pH 8.3, with the protein bands being superimposable on acid phosphatase activity (not shown), and they were purified 248- and 93-fold, respectively, with good yield (Tables 2A and 2B). The net neutral sugars content of the purified acid phosphatases II-like and IV-like was 13 2% and 24 3% (w/w), respectively, whereas the content of forms I and II secreted by strain pabaA1 was 40 5% and 31 3% (w/ w), respectively (Justino et al., 2001). In addition, HPLC analysis of neutral and aminosugars present in the acid phosphatase form II-like secreted by strain pabaA1 palB7 revealed a reduced content of mannose and N-
Table 2A Purification of the Pi-repressible acid phosphatase synthesized by the pabaA1 palB7 strain of A. nidulans grown in low-Pi medium at 37 °C, pH 5.0 Fraction
Volume (ml)
Total protein (mg)
Total activity (U)
Specific activity (U/mg)
Yield (%)
Purification (fold)
Culture medium ðNH4 Þ2 SO4 (65–95%) DEAE-cellulose (fraction II-like) Sephadex G-200 (fraction II-like)
1800 12 4.8 1.5
224 5.9 0.76 0.18
81.2 24 18.2 16
0.36 4.0 23.8 89.2
100 30 23 20
1 11.1 66.1 247.8
Table 2B Purification of the Pi-repressible acid phosphatase synthesized by the pabaA1 palB7 pyr G89 strain of A. nidulans grown in low-Pi medium at 37 °C, pH 5.0 Fraction
Volume (ml)
Total protein (mg)
Total activity (Units)
Specific activity (U/mg)
Yield (%)
Purification (fold)
Culture Medium ðNH4 Þ2 SO4 (65–95%) DEAE-cellulose (fraction IV-like) Sephadex G-200 (fraction IV-like)
1560 18 6 1.3
359 6.8 0.92 0.27
201 33.6 24.3 14
0.56 4.9 26.4 51.85
100 16.7 12.1 6.9
1 8.75 47.1 92.6
224
S.R. Nozawa et al. / Fungal Genetics and Biology 38 (2003) 220–227
acetylgalactosamine, an effect partially restored in the enzyme form IV-like secreted by strain pabaA1 palB7 pyrG89 (Fig. 3). The lower glycosylation level of these enzyme forms, as compared to those of the enzyme forms I and II secreted by the pabaA1 mutant, could account for the differences in electrophoretic mobility (Fig. 1), elution pattern from DEAE-cellulose (Fig. 2), and protective effect against heat inactivation when incubated at 60 °C, pH 6.0 (Fig. 4).
Fig. 4. Thermal inactivation of purified acid phosphatases at 60 °C, pH 6.0, secreted by A. nidulans grown in low-Pi-medium at 37 °C, pH 5.0. Open circle, and open triangle represent the enzyme forms I and II secreted by strain pabaA1. Open box, represent the enzyme form II-like secreted by strain pabaA1 palB7. Filled box, represent the enzyme form IV-like secreted by strain pabaA1 palB7 pyrG89.
Interestingly, although the glycosylation level was not fully restored in the form IV-like enzyme variant secreted by pabaA1 palB7 pyrG89 strain (Fig. 3), the protective effect against heat inactivation was comparable to that observed for the enzyme forms I and II secreted by pabaA1 strain (Fig. 4). Furthermore, no deviation was observed from Michaelis kinetics for the hydrolysis of both PNP-P and a-naphthyl phosphate by the acid phosphatases I and II secreted by strain pabaA1, and the acid phosphatases II-like and IV-like secreted by strains pabaA1 palB7 and pabaA1 palB7 pyrG89, respectively. The Km determined for both substrates showed quite similar values that ranged from 0:32 0:04 to 0:46 0:05 mM, except that forms II and II-like synthesized, respectively, by strains pabaA1 (Km ¼ 0:25 0:06 mM) and palB7 pabaA1 (Km ¼ 0:24 0:05 mM) had a higher affinity for the hydrolysis of a-naphthyl phosphate (low Km value). Also, the interaction constant for both substrates (n) showed values
Fig. 3. Composition analysis of monosaccharides present in the purified Pi-repressible acid phosphatases secreted by various strains of A. nidulans grown on low Pi-medium at 37 °C, pH 5.0. For details see Section 2. Forms I (A) and II (B) secreted by strain pabaA1. Form IIlike enzyme (C) secreted by strain pabaA1 palB7. Form IV-like enzyme (D) secreted by strain pabaA1 palB7 pyrG89 (key: Man, mannose; GlcN, glucosamine; GalN, galactosamine; Glc, glucose; Gal, galactose; Fuc, fucose).
Fig. 5. Differential display using total RNA from pabaA1 (lane 1) and pabaA1 palB7 (lane 2) strains of A. nidulans. Arrow heads indicate differential bands between pabaA1 and pabaA1 palB7 strains.
S.R. Nozawa et al. / Fungal Genetics and Biology 38 (2003) 220–227
225
Fig. 6. Comparison of the deduced amino acid sequences for the two cDNA probes DD-1 (mannosyl transferase) (A) and DD-2 (NADH-ubiquinone oxidoreductase) (B) from A. nidulans. For details see Fig. 5 and Section 2.
ranging from 0:96 0:06 to 1:10 0:05. In addition, no important variation was observed in their molecular masses, which showed values ranging from 110 to 113 kDa as determined by exclusion chromatography, and values ranging from 55 to 62 kDa, as determined by SDS–PAGE. Although these methods are not accurate for glycoproteins (Frank and Rodbard, 1975; Odds and Hierholzer, 1973) all enzyme forms are dimers. Also, determination of the pH activity profile showed an apparent optimum ranging from pH 6.0 to 6.2 for purified acid phosphatases II-like and IV-like enzyme. Two bands differentially expressed between the pabaA1 and pabaA1 palB7 strains of A. nidulans were identified as the amplified cDNAs, and these were designated DD-1 and DD-2 (Fig. 5). Comparison of the nucleotide sequences of these two cDNAs using sequences deposited in the NCBI data bank indicated that DD-1 was identical to a mannosyl transferase from A. niger (Accession No. AF396953) and that DD-2 was identical to the NADH-ubiquinone oxidoreductase previously identified in A. nidulans (Accession No. QXAS4M) (Fig. 6). We also confirmed the reduced expression profiles of the genes for both enzymes in the palB7 mutant by Northern blotting analysis using the cDNA probe for both enzymes labeled with 33 P (Fig. 7). In conclusion, the results described here show the involvement of the calpain-like protease encoded by
gene palB at least in the regulation of the enzymes NADH-ubiquinone oxidoreductase and mannosyl transferase. The involvement of mannosylations in posttranslational modifications of the pacA-encoded Pirepressible acid phosphatase secreted by A. nidulans grown at pH 5.0, clearly documented in this work (Figs. 3 and 7), is not compatible with the participation of this calpain-like protease only in signaling of ambient pH in A. nidulans (Maccheroni et al., 1995). If the palB gene (and all five other pal genes) represented a metabolic
Fig. 7. Northern blot hybridization of RNA prepared from strains pabaA1 (lanes 1 and 3) and pabaA1 palB7 (lanes 2 and 4) was carried out using cDNA probes labeled with 33 P-radiolabeled. Lanes 1 and 2 (B), DD-1 (Fig. 5); lanes 3 and 4 (E), DD-2 (Fig. 6). RNA was stained with ethidium bromide (A) and (D) and probed with actin (C) and (F). For details see Section 2.
226
S.R. Nozawa et al. / Fungal Genetics and Biology 38 (2003) 220–227
step involved only in the activation of PacC-mediated signal transduction of ambient alkaline pH, the properties of the acid phosphatase secreted by A. nidulans at acid pH by all pal mutants should be identical to that in wild-type strains, which is not the case (Maccheroni et al., 1995). Also interesting are the observations that the mammalian PalB homolog is enriched in the nucleus when expressed in COS cells (Futai et al., 2001; Sorimachi et al., 1993), suggesting that PalB has specific functions in the processing of transcription factor(s), as does its homolog Rim13p in Saccharomyces cerevisae (Lamb et al., 2001; Li and Mitchell, 1997). Further evidence in this direction comes from the demonstration that mutations in the mammalian calpain-like protease rather than a structural defect can cause limb–girdle muscular dystrophy type 2A (Richard et al., 1995), and that these mutations are pathogenic only in a specific mitochondrial context (Richard et al., 1995). We also observed reduced expression of the NADH-ubiquinone oxidoreductase in the palB7 mutant, an enzyme present in the mitochondrial complex I (Figs. 6 and 7). Thus, a defect in the posttranslational mannosylation of proteins could be the consequence of mutations in the proteolytic enzyme calpain 3 and could promote muscular dystrophy type 2A in humans. Acknowledgments This research was supported by grants from FAPESP and CNPq (Brazil) and by NIH (GM53027) and NSF (MCB-9513382) (G.S.M., USA). S.R.N and M.S.F.-N. were supported by postgraduate student fellowships from FAPESP and W.M. Jr. by a postdoctoral student fellowship from FAPESP. We thank Mercia V. Carlos (HPLC analysis, CAPES/CNPq agreement), Jose Seminate-Filho, and Newton R. Alves for their technical assistance.
References Caddick, M.X., Brownlee, A.G., Arst Jr., H.N., 1986a. Phosphatase regulation in Aspergillus nidulans: responses to nutritional starvation. Genet. Res. (Camb.) 47, 93–102. Caddick, M.X., Brownlee, A.G., Arst Jr., H.N., 1986b. Regulation of gene expression by pH of the growth medium in Aspergillus nidulans. Mol. Gen. Genet. 203, 346–353. Clutterbuck, A.J., 1993. In: OÕBrien, S.J. (Ed.), Genetic Maps, Locus Maps of Complex Genomes. Cold Spring Harbor Laboratory, New York, pp. 3.71–3.84. Cove, D.J., 1966. The induction and repression of nitrate reductase in the fungus Aspergillus nidulans. Biochim. Biophys. Acta 113, 51–56. Denison, S.H., 2000. pH regulation of gene expression in fungi. Fungal Genet. Biol. 29, 61–71. Denison, S.H., Negrete-Urtasun, S., Mingot, J.M., Tilburn, J., Mayer, W.A., Goel, A., Espeso, E.A., Penalva, M.A., Arst, H.N., 2001. Putative membrane components of signal transduction pathways for ambient pH regulation in Aspergillus and
meiosis in Saccharomyces are homologous (Addendum). Mol. Microbiol. 39, 211. Denison, S.H., Orejas, M., Arst Jr., H.N., 1995. Signaling of ambient pH in Aspergillus involves a cysteine protease. J. Biol. Chem. 270, 28519–28522. Dubois, B., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1965. Colorimetric method for determination of sugar and related substances. Analyt. Chem. 28, 350–356. Fidel, S., Doonan, J.H., Morris, N.R., 1988. Aspergillus nidulans contains a single actin gene which has unique intron locations and encodes a c-actin. Gene 70, 283–293. Fourney, R.M., Miakoshi, J., Day, R.S., Paterson, R.S., 1988. Northern blotting: efficient RNA staining and transfer. Focus 19, 5–6. Frank, R.N., Rodbard, D., 1975. Precision of sodium dodecyl sulfate– polyacrylamide-gel electrophoresis for molecular-weight estimation of a membrane glycoprotein—studies on bovine rhodopsin. Arch. Biochem. Biophys. 171, 1–13. Fu, D., OÕNeill, R.A., 1995. Monosaccharide composition analysis of oligosaccharides and glycoproteins by high-performance liquid chromatography. Anal. Biochem. 227, 377–384. Futai, E., Kubo, T., Sorimachi, H., Suzuki, K., Maeda, T., 2001. Molecular cloning of PalBH, a mammalian homologue of the Aspergillus atypical calpain PalB. Biochim. Biophys. Acta 1517, 316–319. Hartree, E.F., 1972. Determination of protein: a modification of Lowry method that gives a linear photometric response. Analyt. Biochem. 48, 422–427. Justino, A., Nozawa, S.R., Maccheroni Jr., W., May, G.S., MartinezRossi, N.M., Rossi, A., 2001. The Aspergillus nidulans pyrG89 mutation alters glycosylation of secreted acid phosphatase. Fungal Genet. Biol. 32, 113–120. Kim, Y.K., Liu, Z.M., Li, D., Kolattukudy, P.E., 2000. Two novel genes induced by hard-surface contact of Colletotrichum gloeosporioides conidia. J. Bacteriol. 182, 4688–4695. Koshland Jr., D.E., 1970. The molecular basis for enzyme regulation. In: Boyer, P.D. (Ed.), The Enzymes. Academic Press, New York, pp. 341–396. Lamb, T.M., Xu, W., Diamond, A., Mitchell, A.P., 2001. Alkaline response genes of Saccharomyces cerevisiae and their relationship to the RIM101 pathway. J. Biol. Chem. 276, 1850–1856. Li, W.S., Mitchell, A.P., 1997. Proteolytic activation of RIM1p, a positive regulator of yeast sporulation and invasive growth. Genetics 145, 63–73. Liang, P., Pardee, A.B., 1992. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257, 967–971. Lineweaver, H., Burk, D., 1934. The determinations of the enzyme dissociation constants. J. Am. Chem. Soc. 56, 658–666. Maccheroni Jr., W., May, G.S., Martinez-Rossi, N.M., Rossi, A., 2000. The levels of mRNA expressed by gene palF of A. nidulans do not appear to be pH regulated. Fungal Genet. Newsl. 47, 72–73. Maccheroni, W., Martinez-Rossi, N.M., Rossi, A., 1995. Does gene palB regulate the transcription or the post-translational modification of Pi-repressible phosphatases of Aspergillus nidulans. Braz. J. Med. Biol. Res. 28, 31–38. Maccheroni, W., May, G.S., Martinez-Rossi, N.M., Rossi, A., 1997. The sequence of palF, an environmental pH response gene in Aspergillus nidulans. Gene 194, 163–167. Nahas, E., Rossi, A., 1984. Properties of repressible alkaline phosphatase secreted by the wild-type strain 74A of Neurospora crassa. Phytochemistry 23, 507–510. Nahas, E., Terenzi, H.F., Rossi, A., 1982. Effect of carbon source and pH on the production and secretion of acid phosphatase (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1) in Neurospora crassa. J. Gen. Microbiol. 128, 2017–2021.
S.R. Nozawa et al. / Fungal Genetics and Biology 38 (2003) 220–227 Negrete-Urtasun, S., Reiter, W., Diez, E., Denison, S.H., Tilburn, J., Espeso, E.A., Penalva, M.A., Arst Jr., H.N., 1999. Ambient pH signal transduction in Aspergillus: completion of gene characterization. Mol. Microbiol. 33, 994–1003. Nozawa, S.R., Rossi, A., 2000. Gene pacAþ codes for the multiple active forms of Pi-repressible acid phosphatase in the Aspergillus nidulans. World J. Microbiol. Biotechnol. 16, 333–336. Nozawa, S.R., Maccheroni, W., Stabeli, R.G., Thedei Jr., G., Rossi, A., 1998. Purification and properties of Pi-repressible acid phosphatases from Aspergillus nidulans. Phytochemistry 49, 1517–1523. Nozawa, S.R., Rigoli, I.C., Thedei Jr., G., Rossi, A., 1995. Mind the buffering capacity of citric acid. Fungal Genet. Newsl. 42, 56. Odds, F.C., Hierholzer, J.C., 1973. Purification and properties of a glycoprotein acid phosphatase from Candida albicans. J. Bacteriol. 114, 257–266.
227
Pienta, K.J., Schwab, E.D., 1999. Modified differential display technique that eliminates radioactivity and decreases screening time. BioTechniques 28, 272–277. Richard, I., Broux, O., Allamand, V., Fougerousse, F., Chiannilkulchai, N., Brenguier, L., Devaud, C., Pasturaud, P., Roudaut, C., Hillaire, D., Passos-Bueno, M.R., Zatz, M., Tischfield, J.A., Fardeau, M., Jackson, C.E., Cohen, D., Beckmann, J.S., 1995. Mutations in the proteolytic-enzyme calpain 3 cause limb–girdle muscular-dystrophy type-2A. Cell 81, 27–40. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, New York. Sorimachi, H., Toyama-Sorimachi, N., Saido, T.C., Kawasaki, H., Sugita, H., Miyasaka, M., Arahata, K., Ishiura, S., Suzuki, K., 1993. Muscle-specific calpain, p94, is degraded by autolysis immediately after translation, resulting in disappearance from muscle. J. Biol. Chem. 268, 10593–10605.
Mutation in a calpain-like protease affects the posttranslational mannosylation of phosphatases in Aspergillus nidulans S.R. Nozawa,a G.S. May,b,* N.M. Martinez-Rossi,a M.S. Ferreira-Nozawa,a J. Coutinho-Netto,c W. Maccheroni Jr.,a,1 and A. Rossic b
a Departamento de Gen etica, FMRP-USP, 14049-900 Ribeir~ ao Preto, SP, Brazil Division of Pathology and Laboratory Medicine, M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 54, The University of Texas, Houston, TX 77030, USA c Departamento de Bioquımica e Imunologia, FMRP-USP, 14049-900 Ribeir~ ao Preto, SP, Brazil
Received 11 March 2002; accepted 17 August 2002
Abstract In this communication, we show that the palB7 mutation drastically reduced the mannose and N-acetylgalactosamine content of the pacA-encoded acid phosphatase secreted by the fungus Aspergillus nidulans at pH 5.0, compared to a control strain. By using mRNA differential display reverse transcription and polymerase chain reaction, we isolated two cDNAs from the control pabaA1 strain that were not detected in the palB7 mutant strain that encode a mannosyl transferase and a NADH-ubiquinone oxidoreductase. Thus, a defect in the posttranslational mannosylation of proteins could be the consequence of mutations in the palB gene, which encodes for a nuclear calpain-like protease that may have specific functions in the processing of transcription factor(s) similar to its homolog, RIM13, in Saccharomyces cereviseae. Ó 2002 Elsevier Science (USA). All rights reserved.
1. Introduction Although it is well established that ambient pH affects growth, physiology, differentiation, and viability of all organisms, the molecular responses to environmental pH changes are only now being elucidated. In Aspergillus nidulans these responses are mediated by a conserved signal transduction pathway comprising at least seven genes that have been cloned and sequenced (Denison, 2000; Denison et al., 2001; Maccheroni et al., 1997). The pacC gene codes for a Zn-finger transcription factor that undergoes proteolysis at alkaline pH, yielding an active protein responsible for the induction of genes expressing products with optimal activity at alkaline pH (e.g., alkaline phosphatase) and repression of those with optimal activity at acid pH (e.g., acid phosphatase) (Caddick et al., 1986a,b; Nahas et al., 1982). * Corresponding author. Fax: 1-713-792-8460. E-mail address: [email protected] (G.S. May). 1 Present address: Departamento de Genetica, ESALQ-USP, 13400-970 Piracicaba, SP, Brazil.
The pacC transcription is induced under alkaline growth conditions. The six pal genes (palA, B, C, F, H, and I) are putative members of a signaling cascade involved in ambient alkaline pH sensing whose sole known function is to promote the proteolytic activation of PacC, because expression of C-terminal-truncated PacC derivatives suppresses pal mutant defects. Strains with mutations in the pal genes have reduced alkaline phosphatase activity but increased acid phosphatase activity, among other phenotypic effects that are consistent with pal mutants mimicking growth at acidic pH. The palB gene codes for a calpain-like protease that is not involved directly in PacC processing, and the other pal genes have revealed few functional features (Denison, 2000; Denison et al., 1995). An interesting finding, however, is that the transcription of the six pal genes, unlike pacC, do not appear to be pH-regulated (Maccheroni et al., 2000; Negrete-Urtasun et al., 1999). Furthermore, if the pal genes represent metabolic steps involved in the activation of a single regulatory protein, the properties of the secreted acid phosphatase should
1087-1845/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. doi:10.1016/S1087-1845(02)00521-2
S.R. Nozawa et al. / Fungal Genetics and Biology 38 (2003) 220–227
be identical in all pal mutants and wild-type strains. This is not the case, as indicated by molecular mass, electrophoretic mobility, and chromatographic behavior of the acid phosphatase secreted by the palB7 strain (Maccheroni et al., 1995). Also, the palB7 mutation altered the electrophoretic mobility of the constitutive acid phosphatase synthesized on high inorganic phosphate (Pi) medium. With this in mind, we purified the major acid phosphatase secreted by strains pabaA1, pabaA1 palB7, and pabaA1 palB7 pyrG89, and determined the neutral and aminosugar content of the purified enzyme secreted by these mutant strains. Our objective was to establish whether the palB7 mutation affected the properties of secreted enzymes by altering the pattern of posttranslational modification of this phosphatase. We show that strains carrying the palB7 mutation secreted acid phosphatase with low mannose and N-acetylgalactosamine content, an effect partially restored by the combination of palB7 and pyrG89 mutations. Furthermore, by using an mRNA differential display (DDRT-PCR) we isolated two cDNAs, not detected in the palB7 strain, that encode a mannosyl transferase and an NADH-ubiquinone oxidoreductase (chain 4).
2. Materials and methods 2.1. Strains and growth conditions The following strains of A. nidulans were used in the present study: pabaA1 (p-aminobenzoic acid requiring), pyrG89 (orotidine-50 -phosphate decarboxylase), palB7 (mutant for alkaline phosphatase production), pabaA1 pyrG89, pabaA1 palB7, palB7 pyrG89, and pabaA1 palB7 pyrG89. All of these strains are isogenic. The mutations carried by A. nidulans have been described previously (Clutterbuck, 1993 and references therein) and are available from the Fungal Genetic Stock Center (University of Kansas Medical Center, Kansas City, KS, USA). The solid complete and minimal liquid media of Cove (1966) were used. All strains were maintained on solid complete medium containing 55 mM glucose as the carbon source, 10 mM ammonium tartarate as the nitrogen source (final concentrations), and 11 mM Pi, pH 6.5. Unless otherwise stated, a growth temperature of 37 °C was used. The palB7 mutants grow at pH 5.0 following the addition of (final concentration) 50 mM Na citrate, pH 5.0 (Nozawa et al., 1995), and pyrG89 mutants grow on medium containing 5 mM uridine and 2.5 mM uracil. Low-Pi liquid medium was prepared by adding (final concentrations) 50 mM Na citrate, pH 5.0, 200 lM KH2 PO4 , and 10 mM KCl to Pi-free minimal medium. Conidia (109 ) from all strains obtained after growth on solid complete medium were inoculated into 500 ml Erlenmeyers containing 100 ml low-Pi liquid
221
minimal medium and incubated 14 h in an orbital shaker (160 rpm). 2.2. Purification of secreted acid phosphatases The procedure used for the purification of the acid phosphatase secreted by strains pabaA1, palB7 pabaA1 chaA1, and palB7 pyrG89 w A3 to apparent homogeneity as determined by SDS–PAGE was that previously described (Nozawa et al., 1998). 2.3. Assays and characterization of the purified enzymes Pi-repressible acid phosphatase activity was determined at pH 6.0 (100 mM Na maleate, 2 mM EDTA) using 6 mM p-nitrophenylphosphate (PNP-P) as substrate at 37 °C (Nozawa and Rossi, 2000). One unit of acid phosphatase activity was defined as 1 gmol substrate hydrolyzed min1 , at 37 °C. Protein was measured by the method of Lowry as modified by Hartree (1972), with bovine serum bovine fraction V as the standard. Mycelium-specific activities were expressed as units per mg dry weight mycelium. Staining for acid phosphatase activity in petri dishes was determined by flooding the colonies with 2 mg sodium a-naphthyl acid phosphate and 20 mg fast red TR salt dissolved in 4 ml of 0.6 M sodium maleate buffer, pH 6.0, at room temperature (Caddick et al., 1986a). Enzyme activity is indicated by a brown precipitate inside the mycelial mass. PAGE was carried out at pH 8.3 by the method of Davis as described by Nahas and Rossi (1984) using 7.5% (w/v) polyacrylamide slab gels (10 10 0:1 cm). The electrophoretic pattern of acid phosphatases was developed by the method of Dorn as described by Maccheroni et al. (1995) using Na a-naphthyl phosphate as substrate, pH 6.0 (as described above), without prior incubation of slab gels with any buffer. The ratio of the distance covered by the enzyme bands to distance covered by bromophenol blue (electrophoretic mobility, Rf ) was measured. The relative heat stability of the phosphatase was determined by incubating the purified enzyme in 10 mM Na maleate buffer, pH 6.0, at 60 °C. At intervals samples were removed to measure the remaining phophatase activities using the standard procedure. Limiting velocities (Vm ) and Michaelis constants (Km ) were determined at 37 °C, pH 6.0, as Lineweaver and Burk (1934) plots. The interaction constant for the substrate (n) was determined by the Hill procedure as described by Koshland (1970). The kinetic constants reported here were obtained by linear-square analysis calculated from data obtained in at least three independent experiments. Neutral sugars were measured as described by Dubois et al. (1965) with glucose as standard. The composition of monosaccharides present in purified acid phosphatases was analyzed as described by Fu and OÕNeill (1995). The
222
S.R. Nozawa et al. / Fungal Genetics and Biology 38 (2003) 220–227
1-phenyl-3-methyl-2-pyrazoline-5-one (PMP)-labeled monosaccharides were analyzed out on a Shimadzu model LC-10ADVP HPLC system equipped with a diode-array SPD-M10AVP UV/VIS detector and a SCL-10AVP controller. A CHO C-18 column, 220 2:1 mm (Perkin–Elmer, Applied Biosystems Division), optimized for the separation of PMP-labeled carbohydrates, was used. The flow rate was set to 200 ll/min, and the wavelength for UV detection was 245 nm. For neutral and aminosugar separations, buffers A and B were 100 mM ammonium acetate, pH 5.5, containing 10% and 25% (v/v) acetonitrile, respectively. A gradient of 45–100% buffer B in 80 min was used for separation. Data analysis was performed automatically by using the class VP (Shimadzu) program.
The level of Pi-repressible acid phosphatase secreted by A. nidulans was increased approximately 2-fold in strains carrying the palB7 mutation alone or in combination with other auxotrophic markers, as shown by colony staining or enzymatic hydrolysis of PNP-P (Table 1). Additionally, the palB7 mutation altered the electrophoretic mobility of the pacA-encoded acid phosphatase, as compared to those of the enzyme secreted by strains pabaA1 and pabaA1 palB7 pyrG89 (Fig. 1). Also as previously described, the elution pattern from DEAE-cellulose of the acid phosphatase secreted
2.4. RNA preparation and differential display RT-PCR
Table 1 Acid phosphatase secreted into the growth medium by various strains of A. nidulans grown on low Pi-medium at 37 °C, pH 6.5
Total RNA preparations and DDRT-PCR of mycelial mRNA from pabaA1 and pabaA1 palB7 strains were performed, as described previously (Liang and Pardee, 1992) by using arbitrary primers. Total RNA from each strain, extracted after the mycelium grown for 16 h in shaken liquid complete medium was transferred to and incubated for 2–3 h in shaken liquid low-Pi medium, reverse transcribed using random hexamers to prime the reaction (Kim et al., 2000; Pienta and Schwab, 1999). The amplified cDNAs were separated after electrophoresis on 14 11 cm plates of agarose gel at 2% (w/v) in 1 TAE buffer (Sambrook et al., 1989) (a 100-bp ladder was included as size reference) and visualized with ethidium bromide. Overexpressed bands in one of these strains were excised using the Concert Matrix (GIBCO) gel extraction kit and amplified by PCR. The cDNA was then ligated into the pGEM-T Easy vector (Promega) and used to transform bacteria. The insert was isolated, sequenced using the 377 automated DNA sequencer from Perkin–Elmer, and used as probes in Northern blot analysis.
3. Results and discussion
Strain
Colony staininga
Specific activityb
pyrG89 pabaA1 pyrG89 pabaA1 palB7 palB7 pyrG89 palB7 pabaA1 palB7 pyrG89 pabaA1
+ + + ++ ++ ++ ++
131 8 162 8 150 4 252 20 221 16 272 12 232 12
a (+) and (++) represent enzyme activity developed using a-naphthyl phosphate as the substrate and indicated by the color intensity of a brown precipitate formed inside the mycelium mass. b PNP-P hydrolysis expressed as units/mg dry weight mycelium. Data are reported as means SD. For details see Section 2.
2.5. Northern hybridization Total RNA (10 lg) was denatured, separated by electrophoresis, and blotted onto Amersham HybondN+ (Pharmacia Biotech) membranes following the procedure of Fourney et al. (1988). Hybridization was performed with cDNA probes random-prime labeled with ½a-33 PATP (Amersham-Pharmacia Biotech), and unincorporated nucleotides were eliminated from the probes by purification through Sephadex G-50 column. Hybridization was visualized by autoradiography after the membranes were washed twice (Sambrook et al., 1989). Ethidium bromide staining showed that in all experiments equal amounts of RNA were loaded. Actin mRNA level was also used as a loading control (Fidel et al., 1988).
Fig. 1. PAGE of Pi-repressible phosphatases secreted by A. nidulans at pH 5.0. Samples containing about 0.5 U enzyme were separated by electrophoresis, and the enzyme activity bands developed using anaphthyl phosphate as the substrate, pH 6.0, without prior incubation of the slab gels with any buffer. Enzymes of strains pabaA1 (lanes 1, 3, and 5), palB7 pabaA1 (lane 2), and palB7 pyrG89 pabaA1 (lane 4).
S.R. Nozawa et al. / Fungal Genetics and Biology 38 (2003) 220–227
Fig. 2. Stepwise fractionation on DEAE-cellulose of acid phosphatases secreted by the pabaA1 (A), pabaA1 palB7 (B), and palB7 pabaA1 pyrG89 (C) strains of A. nidulans grown in low-Pi medium at 37 °C, pH 5.0. After elution of nonabsorbed proteins with about 100 ml of equilibrating buffer, proteins were eluted by stepwise addition of 0.05, 0.10, 0.15, 0.30, 0.50, and 1 M NaCl dissolved in 10 mM MOPS, pH 7.2. 6 ml fractions were collected in tubes containing 1 ml of 0.10 M Na acetate buffer, pH 4.7, at flow rate of 120 ml/h.
223
by the pabaA1 palB7 strain was altered because only the form II-like activity was recovered (Fig. 2). In addition, the major acid phosphatase secreted by strain pabaA1 palB7 pyrG89 was recovered from DEAE-cellulose as a form IV-like activity (Fig. 2). We have shown that forms I–IV of acid phosphatase secreted by A. nidulans are encoded by the structural gene pacA and that these isozymes may be generated by different patterns of glycosylation and other posttranslational modifications of the protein (Justino et al., 2001; Maccheroni et al., 1995; Nozawa and Rossi, 2000; Nozawa et al., 1998). Thus, to further characterize the acid phosphatase secreted by A. nidulans, we purified to apparent homogeneity as determined by PAGE the form II-like and IV-like variants of this enzyme, respectively, secreted by pabaA1 palB7 and pabaA1 palB7 pyrG89 mutants (and both forms I and II of this enzyme secreted by pabaA1 strain), and determined their neutral and aminosugars content, molecular mass, and kinetic properties. The procedure summarized in Tables 2A and 2B provided optimal conditions for the purification of acid phosphatases II-like and IV-like secreted by the pabaA1 palB7 and pabaA1 palB7 pyrG89 mutants, respectively. All enzyme preparations (at least three independent preparations) appeared to be homogeneous as determined by 7.5% PAGE at pH 8.3, with the protein bands being superimposable on acid phosphatase activity (not shown), and they were purified 248- and 93-fold, respectively, with good yield (Tables 2A and 2B). The net neutral sugars content of the purified acid phosphatases II-like and IV-like was 13 2% and 24 3% (w/w), respectively, whereas the content of forms I and II secreted by strain pabaA1 was 40 5% and 31 3% (w/ w), respectively (Justino et al., 2001). In addition, HPLC analysis of neutral and aminosugars present in the acid phosphatase form II-like secreted by strain pabaA1 palB7 revealed a reduced content of mannose and N-
Table 2A Purification of the Pi-repressible acid phosphatase synthesized by the pabaA1 palB7 strain of A. nidulans grown in low-Pi medium at 37 °C, pH 5.0 Fraction
Volume (ml)
Total protein (mg)
Total activity (U)
Specific activity (U/mg)
Yield (%)
Purification (fold)
Culture medium ðNH4 Þ2 SO4 (65–95%) DEAE-cellulose (fraction II-like) Sephadex G-200 (fraction II-like)
1800 12 4.8 1.5
224 5.9 0.76 0.18
81.2 24 18.2 16
0.36 4.0 23.8 89.2
100 30 23 20
1 11.1 66.1 247.8
Table 2B Purification of the Pi-repressible acid phosphatase synthesized by the pabaA1 palB7 pyr G89 strain of A. nidulans grown in low-Pi medium at 37 °C, pH 5.0 Fraction
Volume (ml)
Total protein (mg)
Total activity (Units)
Specific activity (U/mg)
Yield (%)
Purification (fold)
Culture Medium ðNH4 Þ2 SO4 (65–95%) DEAE-cellulose (fraction IV-like) Sephadex G-200 (fraction IV-like)
1560 18 6 1.3
359 6.8 0.92 0.27
201 33.6 24.3 14
0.56 4.9 26.4 51.85
100 16.7 12.1 6.9
1 8.75 47.1 92.6
224
S.R. Nozawa et al. / Fungal Genetics and Biology 38 (2003) 220–227
acetylgalactosamine, an effect partially restored in the enzyme form IV-like secreted by strain pabaA1 palB7 pyrG89 (Fig. 3). The lower glycosylation level of these enzyme forms, as compared to those of the enzyme forms I and II secreted by the pabaA1 mutant, could account for the differences in electrophoretic mobility (Fig. 1), elution pattern from DEAE-cellulose (Fig. 2), and protective effect against heat inactivation when incubated at 60 °C, pH 6.0 (Fig. 4).
Fig. 4. Thermal inactivation of purified acid phosphatases at 60 °C, pH 6.0, secreted by A. nidulans grown in low-Pi-medium at 37 °C, pH 5.0. Open circle, and open triangle represent the enzyme forms I and II secreted by strain pabaA1. Open box, represent the enzyme form II-like secreted by strain pabaA1 palB7. Filled box, represent the enzyme form IV-like secreted by strain pabaA1 palB7 pyrG89.
Interestingly, although the glycosylation level was not fully restored in the form IV-like enzyme variant secreted by pabaA1 palB7 pyrG89 strain (Fig. 3), the protective effect against heat inactivation was comparable to that observed for the enzyme forms I and II secreted by pabaA1 strain (Fig. 4). Furthermore, no deviation was observed from Michaelis kinetics for the hydrolysis of both PNP-P and a-naphthyl phosphate by the acid phosphatases I and II secreted by strain pabaA1, and the acid phosphatases II-like and IV-like secreted by strains pabaA1 palB7 and pabaA1 palB7 pyrG89, respectively. The Km determined for both substrates showed quite similar values that ranged from 0:32 0:04 to 0:46 0:05 mM, except that forms II and II-like synthesized, respectively, by strains pabaA1 (Km ¼ 0:25 0:06 mM) and palB7 pabaA1 (Km ¼ 0:24 0:05 mM) had a higher affinity for the hydrolysis of a-naphthyl phosphate (low Km value). Also, the interaction constant for both substrates (n) showed values
Fig. 3. Composition analysis of monosaccharides present in the purified Pi-repressible acid phosphatases secreted by various strains of A. nidulans grown on low Pi-medium at 37 °C, pH 5.0. For details see Section 2. Forms I (A) and II (B) secreted by strain pabaA1. Form IIlike enzyme (C) secreted by strain pabaA1 palB7. Form IV-like enzyme (D) secreted by strain pabaA1 palB7 pyrG89 (key: Man, mannose; GlcN, glucosamine; GalN, galactosamine; Glc, glucose; Gal, galactose; Fuc, fucose).
Fig. 5. Differential display using total RNA from pabaA1 (lane 1) and pabaA1 palB7 (lane 2) strains of A. nidulans. Arrow heads indicate differential bands between pabaA1 and pabaA1 palB7 strains.
S.R. Nozawa et al. / Fungal Genetics and Biology 38 (2003) 220–227
225
Fig. 6. Comparison of the deduced amino acid sequences for the two cDNA probes DD-1 (mannosyl transferase) (A) and DD-2 (NADH-ubiquinone oxidoreductase) (B) from A. nidulans. For details see Fig. 5 and Section 2.
ranging from 0:96 0:06 to 1:10 0:05. In addition, no important variation was observed in their molecular masses, which showed values ranging from 110 to 113 kDa as determined by exclusion chromatography, and values ranging from 55 to 62 kDa, as determined by SDS–PAGE. Although these methods are not accurate for glycoproteins (Frank and Rodbard, 1975; Odds and Hierholzer, 1973) all enzyme forms are dimers. Also, determination of the pH activity profile showed an apparent optimum ranging from pH 6.0 to 6.2 for purified acid phosphatases II-like and IV-like enzyme. Two bands differentially expressed between the pabaA1 and pabaA1 palB7 strains of A. nidulans were identified as the amplified cDNAs, and these were designated DD-1 and DD-2 (Fig. 5). Comparison of the nucleotide sequences of these two cDNAs using sequences deposited in the NCBI data bank indicated that DD-1 was identical to a mannosyl transferase from A. niger (Accession No. AF396953) and that DD-2 was identical to the NADH-ubiquinone oxidoreductase previously identified in A. nidulans (Accession No. QXAS4M) (Fig. 6). We also confirmed the reduced expression profiles of the genes for both enzymes in the palB7 mutant by Northern blotting analysis using the cDNA probe for both enzymes labeled with 33 P (Fig. 7). In conclusion, the results described here show the involvement of the calpain-like protease encoded by
gene palB at least in the regulation of the enzymes NADH-ubiquinone oxidoreductase and mannosyl transferase. The involvement of mannosylations in posttranslational modifications of the pacA-encoded Pirepressible acid phosphatase secreted by A. nidulans grown at pH 5.0, clearly documented in this work (Figs. 3 and 7), is not compatible with the participation of this calpain-like protease only in signaling of ambient pH in A. nidulans (Maccheroni et al., 1995). If the palB gene (and all five other pal genes) represented a metabolic
Fig. 7. Northern blot hybridization of RNA prepared from strains pabaA1 (lanes 1 and 3) and pabaA1 palB7 (lanes 2 and 4) was carried out using cDNA probes labeled with 33 P-radiolabeled. Lanes 1 and 2 (B), DD-1 (Fig. 5); lanes 3 and 4 (E), DD-2 (Fig. 6). RNA was stained with ethidium bromide (A) and (D) and probed with actin (C) and (F). For details see Section 2.
226
S.R. Nozawa et al. / Fungal Genetics and Biology 38 (2003) 220–227
step involved only in the activation of PacC-mediated signal transduction of ambient alkaline pH, the properties of the acid phosphatase secreted by A. nidulans at acid pH by all pal mutants should be identical to that in wild-type strains, which is not the case (Maccheroni et al., 1995). Also interesting are the observations that the mammalian PalB homolog is enriched in the nucleus when expressed in COS cells (Futai et al., 2001; Sorimachi et al., 1993), suggesting that PalB has specific functions in the processing of transcription factor(s), as does its homolog Rim13p in Saccharomyces cerevisae (Lamb et al., 2001; Li and Mitchell, 1997). Further evidence in this direction comes from the demonstration that mutations in the mammalian calpain-like protease rather than a structural defect can cause limb–girdle muscular dystrophy type 2A (Richard et al., 1995), and that these mutations are pathogenic only in a specific mitochondrial context (Richard et al., 1995). We also observed reduced expression of the NADH-ubiquinone oxidoreductase in the palB7 mutant, an enzyme present in the mitochondrial complex I (Figs. 6 and 7). Thus, a defect in the posttranslational mannosylation of proteins could be the consequence of mutations in the proteolytic enzyme calpain 3 and could promote muscular dystrophy type 2A in humans. Acknowledgments This research was supported by grants from FAPESP and CNPq (Brazil) and by NIH (GM53027) and NSF (MCB-9513382) (G.S.M., USA). S.R.N and M.S.F.-N. were supported by postgraduate student fellowships from FAPESP and W.M. Jr. by a postdoctoral student fellowship from FAPESP. We thank Mercia V. Carlos (HPLC analysis, CAPES/CNPq agreement), Jose Seminate-Filho, and Newton R. Alves for their technical assistance.
References Caddick, M.X., Brownlee, A.G., Arst Jr., H.N., 1986a. Phosphatase regulation in Aspergillus nidulans: responses to nutritional starvation. Genet. Res. (Camb.) 47, 93–102. Caddick, M.X., Brownlee, A.G., Arst Jr., H.N., 1986b. Regulation of gene expression by pH of the growth medium in Aspergillus nidulans. Mol. Gen. Genet. 203, 346–353. Clutterbuck, A.J., 1993. In: OÕBrien, S.J. (Ed.), Genetic Maps, Locus Maps of Complex Genomes. Cold Spring Harbor Laboratory, New York, pp. 3.71–3.84. Cove, D.J., 1966. The induction and repression of nitrate reductase in the fungus Aspergillus nidulans. Biochim. Biophys. Acta 113, 51–56. Denison, S.H., 2000. pH regulation of gene expression in fungi. Fungal Genet. Biol. 29, 61–71. Denison, S.H., Negrete-Urtasun, S., Mingot, J.M., Tilburn, J., Mayer, W.A., Goel, A., Espeso, E.A., Penalva, M.A., Arst, H.N., 2001. Putative membrane components of signal transduction pathways for ambient pH regulation in Aspergillus and
meiosis in Saccharomyces are homologous (Addendum). Mol. Microbiol. 39, 211. Denison, S.H., Orejas, M., Arst Jr., H.N., 1995. Signaling of ambient pH in Aspergillus involves a cysteine protease. J. Biol. Chem. 270, 28519–28522. Dubois, B., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1965. Colorimetric method for determination of sugar and related substances. Analyt. Chem. 28, 350–356. Fidel, S., Doonan, J.H., Morris, N.R., 1988. Aspergillus nidulans contains a single actin gene which has unique intron locations and encodes a c-actin. Gene 70, 283–293. Fourney, R.M., Miakoshi, J., Day, R.S., Paterson, R.S., 1988. Northern blotting: efficient RNA staining and transfer. Focus 19, 5–6. Frank, R.N., Rodbard, D., 1975. Precision of sodium dodecyl sulfate– polyacrylamide-gel electrophoresis for molecular-weight estimation of a membrane glycoprotein—studies on bovine rhodopsin. Arch. Biochem. Biophys. 171, 1–13. Fu, D., OÕNeill, R.A., 1995. Monosaccharide composition analysis of oligosaccharides and glycoproteins by high-performance liquid chromatography. Anal. Biochem. 227, 377–384. Futai, E., Kubo, T., Sorimachi, H., Suzuki, K., Maeda, T., 2001. Molecular cloning of PalBH, a mammalian homologue of the Aspergillus atypical calpain PalB. Biochim. Biophys. Acta 1517, 316–319. Hartree, E.F., 1972. Determination of protein: a modification of Lowry method that gives a linear photometric response. Analyt. Biochem. 48, 422–427. Justino, A., Nozawa, S.R., Maccheroni Jr., W., May, G.S., MartinezRossi, N.M., Rossi, A., 2001. The Aspergillus nidulans pyrG89 mutation alters glycosylation of secreted acid phosphatase. Fungal Genet. Biol. 32, 113–120. Kim, Y.K., Liu, Z.M., Li, D., Kolattukudy, P.E., 2000. Two novel genes induced by hard-surface contact of Colletotrichum gloeosporioides conidia. J. Bacteriol. 182, 4688–4695. Koshland Jr., D.E., 1970. The molecular basis for enzyme regulation. In: Boyer, P.D. (Ed.), The Enzymes. Academic Press, New York, pp. 341–396. Lamb, T.M., Xu, W., Diamond, A., Mitchell, A.P., 2001. Alkaline response genes of Saccharomyces cerevisiae and their relationship to the RIM101 pathway. J. Biol. Chem. 276, 1850–1856. Li, W.S., Mitchell, A.P., 1997. Proteolytic activation of RIM1p, a positive regulator of yeast sporulation and invasive growth. Genetics 145, 63–73. Liang, P., Pardee, A.B., 1992. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257, 967–971. Lineweaver, H., Burk, D., 1934. The determinations of the enzyme dissociation constants. J. Am. Chem. Soc. 56, 658–666. Maccheroni Jr., W., May, G.S., Martinez-Rossi, N.M., Rossi, A., 2000. The levels of mRNA expressed by gene palF of A. nidulans do not appear to be pH regulated. Fungal Genet. Newsl. 47, 72–73. Maccheroni, W., Martinez-Rossi, N.M., Rossi, A., 1995. Does gene palB regulate the transcription or the post-translational modification of Pi-repressible phosphatases of Aspergillus nidulans. Braz. J. Med. Biol. Res. 28, 31–38. Maccheroni, W., May, G.S., Martinez-Rossi, N.M., Rossi, A., 1997. The sequence of palF, an environmental pH response gene in Aspergillus nidulans. Gene 194, 163–167. Nahas, E., Rossi, A., 1984. Properties of repressible alkaline phosphatase secreted by the wild-type strain 74A of Neurospora crassa. Phytochemistry 23, 507–510. Nahas, E., Terenzi, H.F., Rossi, A., 1982. Effect of carbon source and pH on the production and secretion of acid phosphatase (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1) in Neurospora crassa. J. Gen. Microbiol. 128, 2017–2021.
S.R. Nozawa et al. / Fungal Genetics and Biology 38 (2003) 220–227 Negrete-Urtasun, S., Reiter, W., Diez, E., Denison, S.H., Tilburn, J., Espeso, E.A., Penalva, M.A., Arst Jr., H.N., 1999. Ambient pH signal transduction in Aspergillus: completion of gene characterization. Mol. Microbiol. 33, 994–1003. Nozawa, S.R., Rossi, A., 2000. Gene pacAþ codes for the multiple active forms of Pi-repressible acid phosphatase in the Aspergillus nidulans. World J. Microbiol. Biotechnol. 16, 333–336. Nozawa, S.R., Maccheroni, W., Stabeli, R.G., Thedei Jr., G., Rossi, A., 1998. Purification and properties of Pi-repressible acid phosphatases from Aspergillus nidulans. Phytochemistry 49, 1517–1523. Nozawa, S.R., Rigoli, I.C., Thedei Jr., G., Rossi, A., 1995. Mind the buffering capacity of citric acid. Fungal Genet. Newsl. 42, 56. Odds, F.C., Hierholzer, J.C., 1973. Purification and properties of a glycoprotein acid phosphatase from Candida albicans. J. Bacteriol. 114, 257–266.
227
Pienta, K.J., Schwab, E.D., 1999. Modified differential display technique that eliminates radioactivity and decreases screening time. BioTechniques 28, 272–277. Richard, I., Broux, O., Allamand, V., Fougerousse, F., Chiannilkulchai, N., Brenguier, L., Devaud, C., Pasturaud, P., Roudaut, C., Hillaire, D., Passos-Bueno, M.R., Zatz, M., Tischfield, J.A., Fardeau, M., Jackson, C.E., Cohen, D., Beckmann, J.S., 1995. Mutations in the proteolytic-enzyme calpain 3 cause limb–girdle muscular-dystrophy type-2A. Cell 81, 27–40. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, New York. Sorimachi, H., Toyama-Sorimachi, N., Saido, T.C., Kawasaki, H., Sugita, H., Miyasaka, M., Arahata, K., Ishiura, S., Suzuki, K., 1993. Muscle-specific calpain, p94, is degraded by autolysis immediately after translation, resulting in disappearance from muscle. J. Biol. Chem. 268, 10593–10605.