Archives of Medical Research 31 (2000) S160–S161
Entamoeba histolytica Contains a TER-ATPase Gloria León,* José Luis Sánchez-Salas,** María Isabel Salazar,* Miguel Angel Moreno* and María de Lourdes Muñoz* *Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del I.P.N. (Cinvestav), Mexico City, Mexico **Departamento de Quimica y Biología, Universidad de las Américas, Cholula, Puebla, Mexico
Key Words: Entamoeba histolytica, AAA family, TER-ATPase.
Introduction The AAA (ATPases associated with diverse cellular activities) protein family is a group of ATPases linked by a common ancestor that share a common sequence domain in one or two copies. The AAA domain is a conserved sequence of 220–250 amino acids that contains consensus motifs. These motifs include the Walker A and B boxes, which are important in ATP binding and hydrolysis, and can be found in many types of ATP-binding proteins. AAA proteins are involved in many cellular activities, including vesicle fusion, peroxisome biogenesis, cell cycle control, transcriptional regulation, intracellular proteolysis, mitotic and meiotic spindle interactions, and several mitochondrial functions. The first member of the AAA family identified was the porcine valosin-containing protein (VCP); it contains two ATP-binding modules. Two relatives—SEC 18 from yeast and NSF (N-ethyl-maleimide-Sensitive Fusion) protein from mammals—were described later. The SEC18 mutants are affected in the transport of proteins between the endoplasmic reticulum (ER) and Golgi. NSF and SEC18 do not have signal sequences or transmembrane segments, which indicates that they are not integral membrane proteins. Furthermore, they can be found in the cytoplasm, forming multisubunit complexes. This supports the idea that they are not permanently associated to the ER or the Golgi. The porcine VCP p97 and its orthologue from Xenopus laevis have also been involved in membrane fusion during vesicle transport from the ER to the Golgi. Additionally, the Cdc48p from Saccharonmyces cerevisiae and its rat orthologue TER ATPase are required for vesicle fusion. Little is known concerning the genes that codify vesicle fusion proteins in Entamoeba histolytica. In this paper, we re-
Address reprint requests to: Gloria León Avila, Departamento de Genética y Biología Molecular, Cinvestav, Av. Instituto Politécnico Nacional #2508, Col. San Pedro Zacatenco, 07300 México, D.F., México. Tel.: (⫹525) 747-3800, ext. 5352; FAX (⫹525) 747-7100; E-mail: [email protected]
cinvestav.mx Presenting author: Gloria León.
port the cloning and amino acid sequence characteristics of a TER-ATPase (transition endoplasmic reticulum ATPase).
Materials and Methods E. histolytica trophozoites strain HM-1:IMSS were cultured in TYI-S-33 medium and harvested at 48 h. The DNA was obtained using a Qiagen genomic tip. Synthetic deoxinucleotide design and PCR. The primer set used to amplify the TER-ATPase was: forward 5⬘-CCACGWTGTGGWGTWCCAGATGT and reverse 5⬘- GAATGWCCWAGTTCATGWGCWGC (W ⫽ A or T). The PCR conditions were denaturing at 94⬚C for 5 min, 40 cycles with annealing for 1 min at 42⬚C, extension at 72⬚C for 1 min, and a final extension at 72⬚C for 3 min. A 178-bp PCR product was precipitated with isopropanol, ligated into vector T pBluescript II KS⫹ (Stratagene, La Jolla, CA, USA), and designated as clone A27. The fragment was released by digesting the clone A27 with EcoRI-HindIII and purified. Cloning. The 178-bp insert was radiolabeled and used as a probe to screen an E. histolytica lambda zap II cDNA library. The pBluescript plasmids containing the inserts were excised from the positive recombinant lambda ZAPII phage and purified using Wizard Plus SV columns (Promega, Madison, WI, USA). Seven positive plates were obtained after the third screening. The longest cDNA (G14) clone was sequenced and digested with EcoRI. A 243-bp fragment was liberated and used as a probe to screen a genomic library; 4- and 5-kb clones were obtained, containing the 5⬘ end of the gene. DNA sequence. Plasmid sequencing was done by PCR using Big Dye Primer Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Branchburg, NJ, USA) using the M13 and specific primers. The sequencing reactions were purified by column, and were read in an ABI Prism 310 Sequence detection system (Perkin-Elmer).
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XIV Seminar on Amebiasis, Mexico City / Archives of Medical Research 31 (2000) S160–S161
Figure 1. Partial sequence alignment of the Entamoeba histolytica TER-ATPase with human orthologue. Bold letters show the Walker A box (ATP binding site), and italics show the Walker B box (hydrolysis site).
Northern blot analysis. E. histolytica mRNA was isolated using the Tripure reagent (Boehringer-Mannheim, Mannheim, Germany), separated on agarose-formaldehyde gel, transferred to a Hybond N⫹ membrane, and hybridized with the 243-bp radiolabeled fragment.
Results and Discussion We have identified an E. histolytica TER-ATPase that represents the first characterization of a member of the AAA family. Sequence analysis of the cloned 178 bp-PCR fragment indicated that it had an open reading frame with a 55% identity to TER-ATPase from mouse. Because this enzyme has an important role in vesicular transport from the ER to Golgi, we used it as a probe to screen a cDNA library from E. histolytica and obtained seven clones. The size of the inserts was determined by PCR analysis using the M13 primers and digestion with EcoRI-XhoI. The largest clone, G14, was sequenced in both strands and has an open reading frame of 2110 bp, truncated at the 5⬘ end. The missing 5⬘ end was obtained after screening a genomic library with a 243-bp fragment produced by G14 clone digestion with EcoRI. Two positive clones were selected and sequenced. A 2316-bp open reading frame was identified, and encodes a deduced protein composed of 772 amino acids. In silico analysis was done, and the results indicated that the predicted protein is a member of the AAA family. BLAST analysis shows a homology of 82%, and an overall identity of 67% with the human transitional endoplasmic reticulum ATPase (Figure 1). Similar results were obtained with TERATPases from other mammals such as rat, mouse, and pig.
Furthermore, the identity is greater with TER-ATPases from mammals than other members of the family, such as the VCP from African trypanosomes that, in theory, are evolutively more closely related. The E. histolytica TER-ATPase contains two copies of 176 and 172 amino acids of the AAA module. These modules contain the Walker A ATP-binding domain GPPGCGKT from 245–253 and 517–526, the Walker B hydrolysis motif DEID from 302–307, and DELD from 575–580. The results of the k-NN predictions show a probability of 82% of having a cytoplasmic localization as SEC18 and NSF. The sequence does not have hydrophobic signal sequences or transmembrane domains, nor a KDEL signal corresponding to ER retention. Northern blot analysis was done to determine the size of the mRNA, using the 243-bp fragment as a probe. Because only a single band of 2.34 kb was detected, we suggest that the sequence is not interrupted by introns, nor is there splicing (data not shown). We cloned the E. histolytica TER-ATPase gene. Further studies will be necessary to determine enzymatic activity, cytoplasmic localization, and the role of this protein in the transport of proteins between the ER and the Golgi. In conclusion, E. histolytica trophozoites contain a TER-ATPase that may be important in vesicle traffic and, consequently, in processes such as secretion. Acknowledgments The authors thank Dr. E. Orozco for the cDNA and genomic libraries. This work was supported by Conacyt (Mexico) Grant No. 211085-5-30581-M.