WO1993006125A1 - ISOLEMENT ET CARACTERISATION DE L'ADNc DE LA DESHYDROGENASE DE GLUCOSE-6-PHOSPHATE DU $i(PLASMODIUM FALCIPARUM) - Google Patents

ISOLEMENT ET CARACTERISATION DE L'ADNc DE LA DESHYDROGENASE DE GLUCOSE-6-PHOSPHATE DU $i(PLASMODIUM FALCIPARUM) Download PDF

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Publication number
WO1993006125A1
WO1993006125A1 PCT/US1992/007807 US9207807W WO9306125A1 WO 1993006125 A1 WO1993006125 A1 WO 1993006125A1 US 9207807 W US9207807 W US 9207807W WO 9306125 A1 WO9306125 A1 WO 9306125A1
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WIPO (PCT)
Prior art keywords
protein
glucose
asn
lys
falciparum
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Application number
PCT/US1992/007807
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English (en)
Inventor
David C. Kaslow
Mohammed Shahabuddin
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The United States Of America, Represented By The Secretary, Department Of Health And Human Services
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Publication date
Application filed by The United States Of America, Represented By The Secretary, Department Of Health And Human Services filed Critical The United States Of America, Represented By The Secretary, Department Of Health And Human Services
Publication of WO1993006125A1 publication Critical patent/WO1993006125A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • 6-phosphate dehydrogenase from Plasmodium falciparum and to the DNA segment which encodes it.
  • Glucose-6-phosphate dehydrogenase is a key enzyme in the pentose phosphate pathway. In most organisms the pathway has two main functions: production of pentose (ribose) for biosynthesis of nucleic acids and several coenzymes, and reduction of NADP for a variety of detoxification and reductive biosynthetic reactions. Recently, Vander Jagt et al. reported that isocitrate dehydrogenase may be responsible for providing much of the NADPH required for reductive biosynthesis within the Plasmodium falciparum parasite (D.L. Vander Jagt, L.A. Hunsaker, M. Kibirige, N.M. Campos, Blood.
  • HEET parasites initially have a reduced growth rate, but following an adaptation period, the growth again approximates in vivo rates (Usanga et al. (1985) ; I.T. Ling, R.J.M. Wilson, Mol. & Biochem. Parasit. , 31, 47-56 (1988)); E.F. Roth, C. Raventos-Suarez, A. Rinaldi, R.L. Nagel, PNAS. 80, 298-299 (1983)); and E.F. Roth, S. Schulman, Brit. J. Hema.. 70, 363-367 (1988) .
  • the present invention relates to DNA segments encoding glucose-6-phosphate dehydrogenase in Plasmodium falciparum .
  • the present invention additionally relates to the amino acid sequence of Plasmodium falciparum glucose-6-phosphate dehydrogenase.
  • FIGURES Figure 1 shows the nucleotide sequence (SEQ ID N0:1) of the cDNA encoding Plasmodium falciparum glucose-6-phosphate dehydrogenase protein.
  • SUBSTITUTE SHEET Figure 2 shows the deduced amino acid sequence (SEQ ID NO:2) of the protein encoded by the cDNA of Figure 1.
  • the present invention relates to a cDNA clone isolated by polymerase chain reaction techniques which encodes the glucose-6-phosphate dehydrogenase protein from Plasmodium falciparum.
  • the isolated cDNA clone can be obtained in a substantially pure form by using conventional methods used by those of ordinary skill in the art.
  • the present invention also relates to the glucose-6-phosphate dehydrogenase protein from Plasmodium falciparum encoded by the cDNA.
  • the protein has a novel structure as compared to all other (human, rat, fruit fly, yeast, and E. coli) G6PD deduced amino acid sequences.
  • the P. falciparum enzyme Although the predicted NADP binding site and glucose-6-phosphate binding site is conserved, the P. falciparum enzyme apparently has a secretory signal sequence, a membrane spanning segment, and a transmembrane helix, none of which are found in other G6PD deduced amino acid sequences.
  • the present invention further relates to a recombinantly produced P. falciparum G6PD protein with the amino acid sequence given in Figure 1, plus any allelic and/or biologically functioning variants of this sequence, or any unique portion of this sequence.
  • the recombinant protein can be expressed in a number of expression systems, including both bacterial and eukaryotic.
  • the present invention relates to a synthetic P. falciparum G6PD protein.
  • the present invention relates to a recombinant DNA molecule comprising a vector and a DNA segment encoding the P. falciparum G6PD protein.
  • recombinant DNA molecules of the present invention can be constructed. Possible vectors for use in the present invention include, but are not limited to pUC 13, pUC 19, pcDNAII, pBluescriptll.
  • the DNA segment can be present in the vector operably linked to regulatory elements, including, for example, a promoter.
  • the invention further relates to host cells comprising the above-described recombinant DNA molecule.
  • the recombinant DNA molecule may be stably transformed, stably transfected, transiently transfected into the host cell or in alive attenuated virus.
  • the host cell expresses a functionally active form of the protein encoded by the recombinant DNA molecule.
  • the host cells used can be either bacterial or eukaryotic. Some non-limiting examples of bacterial host cells are Escherichia coli and Staphylococcus aureus . Non-limiting examples of eukaryotic host cells are Saccharomyces cerevi ⁇ iae , CHO cells, COS cells, and Sf9 cells. Transformation with the recombinant molecules can be effected using methods well known in the art.
  • the present invention further relates to a method of screening drugs for anti-malarial activity by contacting a drug to the recombinant P. falciparum G6PD protein under conditions such that inhibition of said P. falciparum G6PD activity can be effected.
  • a drug to the recombinant P. falciparum G6PD protein under conditions such that inhibition of said P. falciparum G6PD activity can be effected.
  • SUBSTITUTE SHEET features of the amino acid sequence of the protein can be exploited in the design of a chemotherapeutic intervention for malaria.
  • the strong genetic and epidemiological evidence that human G6PD deficiency affords protection against malaria further suggests that malaria parasite G6PD may be a rational target for drug therapy.
  • Comparative assays were conducted to determine G6PD activity in the transfected cells which had been contacted with a drug versus G6PD activity in uncontacted transfected cells. After being contacted with the drug, the cells were placed in an environment where labeled glucose was the only source of carbon. Comparative assays were also conducted with untransfected cells as a control.
  • the present invention further relates to antibodies specific for the P. falciparum G6PD protein of the present invention.
  • antibodies such as monoclonal, polyclonal, anti- idotypic and monoclonal catalytic [Sastry et al.
  • such antibodies can be used in assays to detect the presence of P. falciparum G6PD protein in serum from a patient suspected of being infected with P. falciparum.
  • Antibodies specific for the P. falciparum G6PD protein or a unique portion thereof can be coated on to a solid surface such as a plastic and contacted with the serum sample. After- washing, the presence or absence of the protein from the serum bound to the fixed antibodies is deteted by addition of a labeled (e.g. fluorescently labeled) antibody specific for the P. falciparum G6PD protein.
  • a labeled antibody e.g. fluorescently labeled
  • the present invention also relates to a vaccine for use in humans against malaria.
  • the P. falciparum G6PD protein or a unique portion thereof, can be delivered to a human in a pharmacologically acceptable vehicle.
  • a pharmacologically acceptable vehicle for example, a synthetic polypeptide corresponding to the P. falciparum G6PD protein
  • Pharmacologically acceptable carriers commonly used in vaccines can be used to deliver the protein or peptide.
  • Such carriers include MTP, tetanus toxoid or liposomes.
  • Vaccines of the present invention can include effective amounts of immunological adjuvants known to enhance an immune response.
  • Such adjuvants include IL-2 and alum.
  • the protein or polypeptide is present in the vaccine in an amount sufficient to induce an immune response against the antigenic protein and thus to protect against Plasmodium infection thereby protecting the human against malaria.
  • Protective antibodies are usually best elicited by a series of 2-3 doses given about 1 to 6 months apart. The series can be repeated when concentrations of circulating antibodies in the human drops.
  • the vaccine can be used to immunize a human against other forms of malaria (that is, heterologous immunization) .
  • RNA Total cellular RNA, purified from stage III to IV 3D7 gametocytes and from HB3 asexual parasites, was used to construct oligo dT primed, size-selected, BstXI linkered cDNA libraries in plasmid pcDNA II (Invitrogen) .
  • the libraries were screened (J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual. 2d Ed. (1989)) with radiolabelled, random primed DNA probes (A.P. Feinberg, B. Vogelstein, Anal. Biochem. 137. 266-267 (1984)).
  • Pulsed field gel electrophoresis was performed as described by Welle s et al. (T.E. Wellems, D. Walliker, C.L. Smith, V.E. Do Rosario,
  • PCR polymerase chain reaction
  • highly degenerate oligonucleotides I. Nogae, M. Johnston, Gene. 96, 161-169 (1990).
  • 6 sense and 11 antisense primers were used in PCR, only a single pair of primers was found to yield a fragment of the yeast gene.
  • this latter pair of primers was used in PCR with genomic yeast DNA or genomic P. falciparum DNA, a product was observed only in the reaction containing yeast DNA template.
  • a further 13 permutations with 9 primers were examined by PCR using P. falciparum DNA as the template.
  • One pair of primers FOG.
  • G6PD P. falciparum gametocytes express parasite encoded G6PD at a high level. Therefore, to clone G6PD cDNA, a gametocyte specific cDNA library constructed in pcDNAII (Invitrogen) was screened with the 193bp PCR product. pPfg6pd2 (wpMS2) was selected for further characterization, and was found to have a 1750 bp insert, but did not contain the full length coding sequence (FIG. 1) . An asexual stage cDNA library was also screened from which several additional clones were isolated. pPfg6pd6 (MS6) contained the most 5 1 sequence.
  • pcDNAII Invitrogen
  • the insert from pPfg6pd2 hybridized to chromosome 14 by Southern blot analysis of size-fractionated P. falciparum chromosomes, confirming that the cDNA originated from P. falciparum and not human RNA or other potential contaminants.
  • SUBSTITUTE SHEET differences of the malaria parasite to other G6PD can be easily identified.
  • the reactive lysyl residue in the predicted binding site for glucose-6-phosphate were identical in mammalian (human and rat) , fruit fly, yeast, bacterial and parasite G6PD.
  • the NADP binding site proposed by Beutler and colleagues based on convincing genetic evidence (A. Hirono, W. Kuhl, T. Gelbart, L. Forman, V.F. Fairbanks, E. Beutler, PNAS. 86, 10015-10017 (1989)) is not present in falciparum G6PD; however, the region proposed by Persson et al.
  • Pfg6pd as compared to all of the other G6PD genes except E . coli that have been analyzed so far, has the least number of identical residues, and has a large insertion (residues 1-147) between the N-terminus and the putative NADP binding site and another large insertion (268-354) of 61 amino acids between that binding site and the G6P binding sites. These insertions make the predicted molecular mass of the monomer at least 82kDa rather than the 50-55kDa predicted for the other known G6PD enzymes.
  • the N-terminal insertion contains two potentially important structures: a secretory signal sequence (residues 63-76) and a hydrophilic region (residues 123-135) .
  • the other insertion contains a potential transmembrane helical structure (residues 349-364) that the other G6PD proteins lack, despite the identity of a number of residues in this region.
  • Another membrane associated structure, a membrane spanning segment, is predicted toward the C-terminus (residues 614-630) .
  • the remarkably slow migration of P. falciparum G6PD in native PAGE may be explained by its predicted higher molecular mass. Whether the unique features of P.
  • falciparum G6PD target the enzyme to the endoplasmic reticulum for transport to the parasitophorous vacuole, or even to the RBC cytoplasm, or to another compartment within the parasite itself remain to be determined. Wherever the enzyme resides, the striking differences in the structure of G6PD between parasite and other organisms may potentially be exploited in the design of new chemotherapeutic agents against malaria.
  • AAAATAAGAC AAATGAATAT TTTCAAATGT GTACTCCAAA AAATTGCCCT GAT ATGTAT 1020
  • AAAAATATTA CAATTATGGT AAAAATTATA CGCACAGACC TGAGTTTGTT AGAAAATCCT 2220
  • TAAAATTC A GTATAATTAA ATAAAAGAAA ATATTTGGAA CAATTTGCAT TTTTTATGTA 2520
  • Tyr lie Glu Val Asn Tyr Asn Leu Tyr Pro Ala Thr Tyr Leu lie Asp 100 105 110
  • Lys lie Tyr Pro Ala Leu Phe Lys Leu Phe Cys Asn Asn Ser Leu Pro 195 200 205
  • Lys Asp Leu Leu lie lie Gly Phe Ala Arg Thr Val Gin Asp Phe Asp 210 215 220
  • Lys lie lie Lys Lys Asn Cys Leu Asn Ser Lys Gly Thr Asp Lys lie 370 375 380
  • Lys Leu Glu Asp Thr lie Gly Gin Tyr Glu Lys Ala 515 520 525

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Des segments d'ADN codant la protéine de déshydrogénase de glucose-6-phosphate de Plasmodium falciparum sont obtenus par des techniques de réaction de chaîne de polymérase.
PCT/US1992/007807 1991-09-20 1992-09-21 ISOLEMENT ET CARACTERISATION DE L'ADNc DE LA DESHYDROGENASE DE GLUCOSE-6-PHOSPHATE DU $i(PLASMODIUM FALCIPARUM) WO1993006125A1 (fr)

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US76213791A 1991-09-20 1991-09-20
US762,137 1991-09-20

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7153666B2 (en) 2003-07-17 2006-12-26 General Atomics Methods and compositions for determination of glycated proteins
US7855079B2 (en) 2006-07-25 2010-12-21 General Atomics Methods for assaying percentage of glycated hemoglobin
US7943385B2 (en) 2006-07-25 2011-05-17 General Atomics Methods for assaying percentage of glycated hemoglobin
US8673646B2 (en) 2008-05-13 2014-03-18 General Atomics Electrochemical biosensor for direct determination of percentage of glycated hemoglobin

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"Current Protocols in Molecular Biology", Published 1987, by WILEY AND SONS, see pages 11.3-11.11.4. *
MOLECULAR AND BIOCHEMICAL PARASITOLOGY, Volume 31, issued 1988, I.R. LING et al., "Glucose-6-Phosphate Dehydrogenase Activity of the Malaria Parasite Plasmodium Falciparum", pages 47-51. *
NATURE, Volume 304, issued 07 July 1983, F.E.G. COX., "Cloning Genes for Antigens of Plasmodium Falciparum", pages 13-14. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7153666B2 (en) 2003-07-17 2006-12-26 General Atomics Methods and compositions for determination of glycated proteins
US7855079B2 (en) 2006-07-25 2010-12-21 General Atomics Methods for assaying percentage of glycated hemoglobin
US7943385B2 (en) 2006-07-25 2011-05-17 General Atomics Methods for assaying percentage of glycated hemoglobin
US8318501B2 (en) 2006-07-25 2012-11-27 General Atomics Methods for assaying percentage of glycated hemoglobin
US8338184B2 (en) 2006-07-25 2012-12-25 General Atomics Methods for assaying percentage of glycated hemoglobin
US8557591B2 (en) 2006-07-25 2013-10-15 General Atomics Methods for assaying percentage of glycated hemoglobin
US8673646B2 (en) 2008-05-13 2014-03-18 General Atomics Electrochemical biosensor for direct determination of percentage of glycated hemoglobin

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