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Cloning and characterization of a putative testis-specific pyruvate dehydrogenase β subunit from the parasitic nematode, Ascaris suum1
Molecular and Biochemical Parasitology 90 (1997) 391 – 394
Short communication
Cloning and characterization of a putative testis-specific pyruvate dehydrogenase b subunit from the parasitic nematode, Ascaris suum 1 Yue-Jin Huang, Richard Komuniecki * Department of Biology, Uni6ersity of Toledo, Toledo, OH 43606, USA Received 1 August 1997; received in revised form 26 September 1997; accepted 3 October 1997
Keywords: Ascaris suum; Pyruvate dehydrogenase complex; E1b subunit; Molecular cloning; Testis
The pyruvate dehydrogenase complex (PDC) plays a key role in energy metabolism during aerobic/anaerobic transitions in the development of the parasitic nematode, Ascaris suum [1,2]. PDC from early aerobic larval stages supplies acetyl CoA for TCA cycle oxidation [3]. In contrast, mitochondria in later larval stages and adult body wall muscle lack a functional TCA cycle and are anaerobic. Pyruvate is generated intramitochondrially and the PDC provides both the reducing power and thioester linkages needed to drive branched-chain fatty acid synthesis through a reversal of b-oxidation [2,4]. The PDC is composed Abbre6iations: E1b, b subunit of pyruvate dehydrogenase; PDC, pyruvate dehydrogenase complex. * Corresponding author. Tel.: + 1 419 5304595; fax: +1 419 5307737; e-mail: [email protected] 1 Note: Nucleotide sequence data reported in this paper is available in the GenBank™ data base under the accession number AF013755.
of three major catalytic components, pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2) and dihydrolipoyl dehydrogenase (E3) and its activity is regulated by the reversible phosphorylation/dephosphorylation of the a2b2 E1 tetramer [5,6]. To date, two a subunits and one b subunit have been cloned and sequenced from a cDNA library prepared from poly A + RNA isolated from adult A. suum muscle [3,7]. Interestingly, immunoblotting with antiserum against the E1b subunit isolated from adult A. suum muscle suggested that it was not present in early larval stages, even though the other subunits of the PDC were apparent [8]. Therefore, in an attempt to identify additional E1b subunits, we screened cDNA pools from a variety of other A. suum adult tissues and larval stages. In the present study, we report the identification of a putative testis-specific b-subunit that may play a role in the aerobic metabolism of early A. suum embryos.
0166-6851/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 6 - 6 8 5 1 ( 9 7 ) 0 0 1 7 5 - 8
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Y.-J. Huang, R. Komuniecki / Molecular and Biochemical Parasitology 90 (1997) 391–394
Fig. 1. Comparison of the predicted A. suum E1bII sequence with other known E1b sequences. Inserted gaps are spaced to optimize the alignment. Dots indicate identity with the A. suum E1bII sequence. c indicates residues that are identical and indicates residues that are homologous.
Total RNA was isolated from adult A. suum testis and cDNA pools were prepared using the Marathon cDNA synthesis kit from Clontech. A 480 nt product was identified by reverse transcriptase polymerase chain reaction using degenerate primers designed from conserved regions in the A. suum E1bI subunit (PICEFM109 and ECEVIN263). Full length sequence was then generated using the 3% and 5% rapid amplification of cDNA ends protocols. The full length 1195 bp cDNA (E1bII) had a number of structural features typical of eukaryotic mRNAs, including a short 5% untranslated region (reverse transcriptase polymerase chain reaction using the spliced leader sequence was negative), an open reading frame of 1071 nt, and a 3% untranslated region of 96 nt with a putative polyadenylation signal, AAUAAA, 14 nt upstream from a short poly(A) tail. The coding sequence predicted a mature protein of about 36 kDa with a putative 26 amino acid leader peptide [3]. Overall, the E1bII sequence was 60% identical to the previously identified A. suum E1b (E1bI)
[7]. In contrast, the two E1a subunits previously identified in A. suum were over 90% identical. The A. suum E1bII also exhibited significant identity to E1bs from a variety of other organisms, including the human and yeast E1bs (58%) (Fig. 1). Northern blotting with total RNA isolated from different A. suum larval stages and adult tissues and the E1bII cDNA clone as a probe indicated that E1bII was expressed only in testis (Fig. 2A). In contrast, mRNA for E1bI was present in both third-stage larva and adult muscle, in agreement with results reported previously [7]. The putative mature E1bII was expressed in E. coli as a fusion protein with a six his-tag attached to the N-terminus (pQE-30 from Qiagen) and purified to apparent homogeneity. A polyclonal antibody against recombinant E1bII was prepared and affinity purified as described previously for other A. suum proteins [8,9]. Immunoblotting of homogenates of different A. suum larval stages and adult tissues with the affinity-purified antisera against E1bII yielded a significant band in testis
Y.-J. Huang, R. Komuniecki / Molecular and Biochemical Parasitology 90 (1997) 391–394
393
Fig. 2. Stage and tissue specific expression of the A. suum E1bII. (A) Northern blot: Total RNA (20 mg) from different developmental stages was separated on a 1% formaldehyde agarose gel, transferred to a nylon membrane, and hybridized with the 32 P-labeled cDNAs coding for E1bI and E1bII. The transcript size of the E1bII mRNA was about 1.2 kb. As a control, the RNA filter was rehybridized with a cloned A. suum a-tubulin probe. UE, unembryonated eggs; L1, first-stage larvae; L2, second-stage larvae; L3, third-stage larvae; M, adult muscle; O, ovary; T, testis. (B) Immunoblot: Extracts of adult A. suum and unembryonated eggs were separated by 10% SDS-PAGE, transferred to nitrocellulose and reacted with an affinity-purified antiserum generated against recombinant E1bII. Lanes 1, unembryonated eggs (100 mg); lane 2, testis (10 mg). Molecular mass markers are shown at the right of the figure.
at about 36 kDa and a faint band in unembryonated eggs at the same apparent molecular mass (Fig. 2B). No band at 36 kDa was present in second- or third-stage larvae or adult muscle, ovary or intestine (data not shown). The expression of E1bII is unique. Its mRNA is only apparent in testis/sperm, but E1bII protein also appears to be present in unembryonated eggs, suggesting that it may have arisen from sperm mitochondria. Each A. suum sperm contains about 100 mitochondria. During fertilization, the sperm enters the oocyte and, once inside, the sperm cytoplasm, with its numerous mitochondria, concentrates around the nucleus [11,12]. In fact, intact sperm mitochondria are still visible in the oocyte after fertilization [13]. The subsequent fate of the sperm mitochondria is unclear but recent experiments suggest that mitochondrial inheritance in A. suum is maternal as in other organisms [14].
The anaerobic energy metabolism of adult body wall muscle has been well characterized [1,2,10]. In addition, it appears that mitochondria isolated from A. suum testis/sperm are also anaerobic and cytochrome c oxidase activity is not detectable [10]. Interestingly, the activities of many of the TCA cycle enzymes are dramatically elevated in testis/sperm mitochondria relative to their activities in body wall muscle [10], even though testis/ sperm mitochondria do not appear to contain a functional TCA cycle. The lumen of the testis and seminal vesicle are densely packed with sperm. Since sperm mitochondria are larger and much more numerous than those of the testis, most of the mitochondria isolated from A. suum testis probably come from sperm [10]. The function of the TCA cycle enzymes present in testis is not clear, but it has been suggested that they might be used in the aerobic metabolism of the developing embryo. The presence of the testis/sperm specific E1bII in unembryonated eggs in the apparent
Y.-J. Huang, R. Komuniecki / Molecular and Biochemical Parasitology 90 (1997) 391–394
394
absence of its mRNA provides additional data suggesting that the sperm mitochondria may contribute to aerobic energy generation in early A. suum embryos.
[5]
[6]
Acknowledgements The authors would like to thank the personnel at Routh Packing in Sandusky, OH for their permission to collect ascarids at their facility. This work was supported by National Institutes of Health Grant AI 19427 to RWK.
References [1] Komuniecki R, Harris B. Carbohydrate and energy metabolism in helminths. In: Marr J, Mueller M, editors. Parasite Biochemistry and Molecular Biology. New York: Academic Press, 1995:49–66. [2] Komuniecki R, Komuniecki PR. Aerobic–anaerobic transitions in energy metabolism during the development of the parasitic nematode, Ascaris suum. In: Boothroyd JC, Komuniecki R, editors. Molecular Approaches to Parasitology. New York: Wiley, 1995:109–121. [3] Johnson KR, Komuniecki R, Sun Y, Wheelock MJ. Characterization of cDNA clones for the a subunit of pyruvate dehydrogenase from Ascaris suum. Mol Biochem Parasitol 1992;51:37–47. [4] Komuniecki R, Klingbeil MM, Arnette R, Walker DJ, Diaz F. Regulation of the pyruvate dehydrogenase complex in anaerobic mitochondria of parasitic nematodes.
.
[7]
[8]
[9]
[10]
[11] [12] [13]
[14]
In: Roche T, Patel M, Harris RA, editors. a-Keto Acid Dehydrogenase Complexes. Basel: Birkhauser, 199:96 – 99. Patel MS, Roche T. Molecular biology and biochemistry of pyruvate dehydrogenase complexes. FASEB J 1990;4:3224 – 33. Roche TS, Cox DJ. Multifunctional 2-oxoacid dehydrogenase complexes. In: Agius L, Sherrat HS, editors. Channeling in Intermediary Metabolism. London: Portland Press, 1996:115 – 132. Wheelock MJ, Komuniecki R, Duran E, Johnson KR. Characterization of cDNA clones for the b subunit of pyruvate dehydrogenase from Ascaris suum. Mol Biochem Parasitol 1991;45:9 – 18. Klingbeil M, Walker D, Arnette R, Sidawy E, Hayton K, Komuniecki PR, Komuniecki R. Identification of a novel dihydrolipoyl dehydrogenase binding protein in the pyruvate dehydrogenase complex of the anaerobic parasitic nematode Ascaris suum. J Biol Chem 1996;271:5451 – 7. Wawrzynczak EJ, Cumber AJ. Immunoaffinity purification and quantification of antibody-toxin conjugates. In: Manson M, editor. Methods in Molecular Biology, vol. 10, Immunochemical Protocols. Totowa (NJ): Humana, 1992:295 – 305. Komuniecki PR, Johnson J, Kamhawi M, Komuniecki R. Mitochondrial heterogeneity in the parasitic nematode Ascaris suum. Exp Parasitol 1993;76:424 – 37. Foor WE. Zygote formation in Ascaris lumbricoides (Nematoda). J Cell Biol 1968;39:119 – 34. Foor WE. Spermatozoan morphology and zygote formation in nematodes. Biol Reprod Suppl 1970;2:177 – 202. Foor WE. Spermatogenesis and sperm function. In: Adiyodi KG, Adiyodi RG, editors. Reproductive Biology of Invertebrates. New York: Wiley, 1983:221 – 256. Anderson TJC, Komuniecki R, Komuniecki PR, Jaenike J. Are mitochondria inherited paternally in Ascaris? Int J Parasitol 1995;25:1001 – 4.
Short communication
Cloning and characterization of a putative testis-specific pyruvate dehydrogenase b subunit from the parasitic nematode, Ascaris suum 1 Yue-Jin Huang, Richard Komuniecki * Department of Biology, Uni6ersity of Toledo, Toledo, OH 43606, USA Received 1 August 1997; received in revised form 26 September 1997; accepted 3 October 1997
Keywords: Ascaris suum; Pyruvate dehydrogenase complex; E1b subunit; Molecular cloning; Testis
The pyruvate dehydrogenase complex (PDC) plays a key role in energy metabolism during aerobic/anaerobic transitions in the development of the parasitic nematode, Ascaris suum [1,2]. PDC from early aerobic larval stages supplies acetyl CoA for TCA cycle oxidation [3]. In contrast, mitochondria in later larval stages and adult body wall muscle lack a functional TCA cycle and are anaerobic. Pyruvate is generated intramitochondrially and the PDC provides both the reducing power and thioester linkages needed to drive branched-chain fatty acid synthesis through a reversal of b-oxidation [2,4]. The PDC is composed Abbre6iations: E1b, b subunit of pyruvate dehydrogenase; PDC, pyruvate dehydrogenase complex. * Corresponding author. Tel.: + 1 419 5304595; fax: +1 419 5307737; e-mail: [email protected] 1 Note: Nucleotide sequence data reported in this paper is available in the GenBank™ data base under the accession number AF013755.
of three major catalytic components, pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2) and dihydrolipoyl dehydrogenase (E3) and its activity is regulated by the reversible phosphorylation/dephosphorylation of the a2b2 E1 tetramer [5,6]. To date, two a subunits and one b subunit have been cloned and sequenced from a cDNA library prepared from poly A + RNA isolated from adult A. suum muscle [3,7]. Interestingly, immunoblotting with antiserum against the E1b subunit isolated from adult A. suum muscle suggested that it was not present in early larval stages, even though the other subunits of the PDC were apparent [8]. Therefore, in an attempt to identify additional E1b subunits, we screened cDNA pools from a variety of other A. suum adult tissues and larval stages. In the present study, we report the identification of a putative testis-specific b-subunit that may play a role in the aerobic metabolism of early A. suum embryos.
0166-6851/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 6 - 6 8 5 1 ( 9 7 ) 0 0 1 7 5 - 8
392
Y.-J. Huang, R. Komuniecki / Molecular and Biochemical Parasitology 90 (1997) 391–394
Fig. 1. Comparison of the predicted A. suum E1bII sequence with other known E1b sequences. Inserted gaps are spaced to optimize the alignment. Dots indicate identity with the A. suum E1bII sequence. c indicates residues that are identical and indicates residues that are homologous.
Total RNA was isolated from adult A. suum testis and cDNA pools were prepared using the Marathon cDNA synthesis kit from Clontech. A 480 nt product was identified by reverse transcriptase polymerase chain reaction using degenerate primers designed from conserved regions in the A. suum E1bI subunit (PICEFM109 and ECEVIN263). Full length sequence was then generated using the 3% and 5% rapid amplification of cDNA ends protocols. The full length 1195 bp cDNA (E1bII) had a number of structural features typical of eukaryotic mRNAs, including a short 5% untranslated region (reverse transcriptase polymerase chain reaction using the spliced leader sequence was negative), an open reading frame of 1071 nt, and a 3% untranslated region of 96 nt with a putative polyadenylation signal, AAUAAA, 14 nt upstream from a short poly(A) tail. The coding sequence predicted a mature protein of about 36 kDa with a putative 26 amino acid leader peptide [3]. Overall, the E1bII sequence was 60% identical to the previously identified A. suum E1b (E1bI)
[7]. In contrast, the two E1a subunits previously identified in A. suum were over 90% identical. The A. suum E1bII also exhibited significant identity to E1bs from a variety of other organisms, including the human and yeast E1bs (58%) (Fig. 1). Northern blotting with total RNA isolated from different A. suum larval stages and adult tissues and the E1bII cDNA clone as a probe indicated that E1bII was expressed only in testis (Fig. 2A). In contrast, mRNA for E1bI was present in both third-stage larva and adult muscle, in agreement with results reported previously [7]. The putative mature E1bII was expressed in E. coli as a fusion protein with a six his-tag attached to the N-terminus (pQE-30 from Qiagen) and purified to apparent homogeneity. A polyclonal antibody against recombinant E1bII was prepared and affinity purified as described previously for other A. suum proteins [8,9]. Immunoblotting of homogenates of different A. suum larval stages and adult tissues with the affinity-purified antisera against E1bII yielded a significant band in testis
Y.-J. Huang, R. Komuniecki / Molecular and Biochemical Parasitology 90 (1997) 391–394
393
Fig. 2. Stage and tissue specific expression of the A. suum E1bII. (A) Northern blot: Total RNA (20 mg) from different developmental stages was separated on a 1% formaldehyde agarose gel, transferred to a nylon membrane, and hybridized with the 32 P-labeled cDNAs coding for E1bI and E1bII. The transcript size of the E1bII mRNA was about 1.2 kb. As a control, the RNA filter was rehybridized with a cloned A. suum a-tubulin probe. UE, unembryonated eggs; L1, first-stage larvae; L2, second-stage larvae; L3, third-stage larvae; M, adult muscle; O, ovary; T, testis. (B) Immunoblot: Extracts of adult A. suum and unembryonated eggs were separated by 10% SDS-PAGE, transferred to nitrocellulose and reacted with an affinity-purified antiserum generated against recombinant E1bII. Lanes 1, unembryonated eggs (100 mg); lane 2, testis (10 mg). Molecular mass markers are shown at the right of the figure.
at about 36 kDa and a faint band in unembryonated eggs at the same apparent molecular mass (Fig. 2B). No band at 36 kDa was present in second- or third-stage larvae or adult muscle, ovary or intestine (data not shown). The expression of E1bII is unique. Its mRNA is only apparent in testis/sperm, but E1bII protein also appears to be present in unembryonated eggs, suggesting that it may have arisen from sperm mitochondria. Each A. suum sperm contains about 100 mitochondria. During fertilization, the sperm enters the oocyte and, once inside, the sperm cytoplasm, with its numerous mitochondria, concentrates around the nucleus [11,12]. In fact, intact sperm mitochondria are still visible in the oocyte after fertilization [13]. The subsequent fate of the sperm mitochondria is unclear but recent experiments suggest that mitochondrial inheritance in A. suum is maternal as in other organisms [14].
The anaerobic energy metabolism of adult body wall muscle has been well characterized [1,2,10]. In addition, it appears that mitochondria isolated from A. suum testis/sperm are also anaerobic and cytochrome c oxidase activity is not detectable [10]. Interestingly, the activities of many of the TCA cycle enzymes are dramatically elevated in testis/sperm mitochondria relative to their activities in body wall muscle [10], even though testis/ sperm mitochondria do not appear to contain a functional TCA cycle. The lumen of the testis and seminal vesicle are densely packed with sperm. Since sperm mitochondria are larger and much more numerous than those of the testis, most of the mitochondria isolated from A. suum testis probably come from sperm [10]. The function of the TCA cycle enzymes present in testis is not clear, but it has been suggested that they might be used in the aerobic metabolism of the developing embryo. The presence of the testis/sperm specific E1bII in unembryonated eggs in the apparent
Y.-J. Huang, R. Komuniecki / Molecular and Biochemical Parasitology 90 (1997) 391–394
394
absence of its mRNA provides additional data suggesting that the sperm mitochondria may contribute to aerobic energy generation in early A. suum embryos.
[5]
[6]
Acknowledgements The authors would like to thank the personnel at Routh Packing in Sandusky, OH for their permission to collect ascarids at their facility. This work was supported by National Institutes of Health Grant AI 19427 to RWK.
References [1] Komuniecki R, Harris B. Carbohydrate and energy metabolism in helminths. In: Marr J, Mueller M, editors. Parasite Biochemistry and Molecular Biology. New York: Academic Press, 1995:49–66. [2] Komuniecki R, Komuniecki PR. Aerobic–anaerobic transitions in energy metabolism during the development of the parasitic nematode, Ascaris suum. In: Boothroyd JC, Komuniecki R, editors. Molecular Approaches to Parasitology. New York: Wiley, 1995:109–121. [3] Johnson KR, Komuniecki R, Sun Y, Wheelock MJ. Characterization of cDNA clones for the a subunit of pyruvate dehydrogenase from Ascaris suum. Mol Biochem Parasitol 1992;51:37–47. [4] Komuniecki R, Klingbeil MM, Arnette R, Walker DJ, Diaz F. Regulation of the pyruvate dehydrogenase complex in anaerobic mitochondria of parasitic nematodes.
.
[7]
[8]
[9]
[10]
[11] [12] [13]
[14]
In: Roche T, Patel M, Harris RA, editors. a-Keto Acid Dehydrogenase Complexes. Basel: Birkhauser, 199:96 – 99. Patel MS, Roche T. Molecular biology and biochemistry of pyruvate dehydrogenase complexes. FASEB J 1990;4:3224 – 33. Roche TS, Cox DJ. Multifunctional 2-oxoacid dehydrogenase complexes. In: Agius L, Sherrat HS, editors. Channeling in Intermediary Metabolism. London: Portland Press, 1996:115 – 132. Wheelock MJ, Komuniecki R, Duran E, Johnson KR. Characterization of cDNA clones for the b subunit of pyruvate dehydrogenase from Ascaris suum. Mol Biochem Parasitol 1991;45:9 – 18. Klingbeil M, Walker D, Arnette R, Sidawy E, Hayton K, Komuniecki PR, Komuniecki R. Identification of a novel dihydrolipoyl dehydrogenase binding protein in the pyruvate dehydrogenase complex of the anaerobic parasitic nematode Ascaris suum. J Biol Chem 1996;271:5451 – 7. Wawrzynczak EJ, Cumber AJ. Immunoaffinity purification and quantification of antibody-toxin conjugates. In: Manson M, editor. Methods in Molecular Biology, vol. 10, Immunochemical Protocols. Totowa (NJ): Humana, 1992:295 – 305. Komuniecki PR, Johnson J, Kamhawi M, Komuniecki R. Mitochondrial heterogeneity in the parasitic nematode Ascaris suum. Exp Parasitol 1993;76:424 – 37. Foor WE. Zygote formation in Ascaris lumbricoides (Nematoda). J Cell Biol 1968;39:119 – 34. Foor WE. Spermatozoan morphology and zygote formation in nematodes. Biol Reprod Suppl 1970;2:177 – 202. Foor WE. Spermatogenesis and sperm function. In: Adiyodi KG, Adiyodi RG, editors. Reproductive Biology of Invertebrates. New York: Wiley, 1983:221 – 256. Anderson TJC, Komuniecki R, Komuniecki PR, Jaenike J. Are mitochondria inherited paternally in Ascaris? Int J Parasitol 1995;25:1001 – 4.