Beruflich Dokumente
Kultur Dokumente
Laboratoire de Biologie Cellulaire et Moleculaire, EA MESR 2413, UFR des Sciences Pharmaceutiques et Biologiques, Montpellier, France;
Centre de Biochimie Structurale, CNRS UMR C9955, INSERM U414, Universite Montpellier I, France; 3Department of Parasitology,
Intervet International BV, Boxmeer, the Netherlands
In the search for immunoprotective antigens of the intraerythrocytic Babesia canis rossi parasite, a new cDNA
was cloned and sequenced. Protein sequence database searches suggested that the 41-kDa protein belongs to the
phosphofructokinase B type family (PFK-B). However, because of the low level sequence identity (, 20%) of
the protein both with adenosine and sugar kinases from this family, its structural and functional features were
further investigated using molecular modelling and enzymatic assays. The sequence/structure comparison of the
protein with the crystal structure of a member of the PFK-B family, Escherichia coli ribokinase (EcRK),
suggested that it might also form a stable and active dimer and revealed conservation of the ATP-binding site.
However, residues specifically involved in the ribose-binding sites in the EcRK sequence (S and N) were
substituted in its sequence (by H and M, respectively), and were suspected of binding adenosine compounds
rather than sugar ones. Enzymatic assays using a purified glutathione S-transferase fusion protein revealed that
this protein exhibits rapid catalysis of the phosphorylation of adenosine with an apparent Km value of 70 nm,
whereas it was inactive on ribose or other carbohydrates. As enzymatic assays confirmed the results of the
structure/function analysis indicating a preferential specificity towards adenosine compounds, this new protein of
the PFK-B family corresponds to an adenosine kinase from B. canis rossi. It was named BcrAK.
Keywords: adenosine kinase; glutathione S-transferase fusion protein; homology modelling; intracellular parasite;
structure/function analysis.
M AT E R I A L S A N D M E T H O D S
Construction of the cDNA library
Total RNA and mRNA of B. canis rossi were isolated using,
respectively, the RNAgentsw Total RNA Isolation System and
the PolyATtract mRNA Isolation Systemw III as described by
the manufacturer (Promega). Poly(A) (5 mg) was converted
into double-stranded cDNA and used to construct the cDNA
library with the ZAP ExpressTM cDNA Gigapackw II Gold
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Molecular cloning of a B. canis rossi adenosine kinase (Eur. J. Biochem. 265) 1017
R E S U LT S A N D D I S C U S S I O N
Isolation and sequence of the BcrAK cDNA
Although effective chemotherapeutic treatment of infected
animals is available, the prevention of babesiosis by vaccination
seems to be a great opportunity especially using soluble
parasite antigens as vaccine [36]. Moreover, whatever the
species studied, soluble parasite antigens with molecular mass
around 40 kDa, seem to play an important role in the
development of efficient vaccine [37,38]. In an effort to
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Fig. 1. Sequence/structure alignment of BcrAK and three known PFK-B enzymes. The multiple sequence alignment including the fructokinase and
ribokinase from E. coli (ScrK, accession number P40713 and EcRK, accession number P05054, respectively), the BcrAK (accession number AJ223322), and
the human adenosine kinase (hAK, accession number P55263), was deduced from the structure/sequence comparison performed using the program tito.
Conserved motifs involved in the ATP binding are labelled ATP1, ATP2 and ATP3 as described in the text. Similarly the motifs lying in the substrate binding
pocket are labelled as SUB and the position interacting with the 2 0 -hydroxyl group is marked by a star. Identities are shown on a black background, and
similarities on a grey background. The putative signal peptide is marked by a hatched bar and the dimerization subdomain is underlined. The secondary
structures (a19 and b19) were predicted by similarity with those assigned in the known crystal structure of the ribokinase (EcRK) and they were labelled
to highlight the domain organization as well as the relationship of the catalytic domain with the Rossmann fold. The BcrAK sequence numbering is indicated
above the alignment.
derived from the sequence/structure comparison. The conservation of both motifs and the surrounding secondary structures
suggests that BcrAK and EcRK interact with their common
phosphorus donor, in the same way.
From this structural comparison, the main change in the
kinase structure corresponds to the region comprising the
substrate-binding site (designated SUB in Fig. 1). This change
includes particular residue substitutions as well as a 15 amino
acids long insertion in the BcrAK. In the current model this
extra sequence stretch points toward the active site of the
second monomer in the dimeric enzyme. This suggests that it
might be involved in adenine moiety recognition. Two specific
amino acid substitutions were highlited by the analysis of
ribose binding in the EcRK crystal structure. Residues H41 and
M43 in BcrAK (boldface type in Fig. 1) correspond in EcRK to
the S12 and N14 which interact with the sugar. In particular, the
larger and hydrophobic side chain of M43 would not allow the
ribose to bind in the same conformation in BcrAK as the N14
allows in EcRK. The particular conformation of ribose
complexed to EcRK remains unclear [16]. However, the
distinct ribose conformation observed in the adenosine
compounds in various protein complexes might explain the
discrimination between the ribose alone and the same sugar
attached to a purine. Further analysis revealed that the other
adenosine kinase as well as the fructokinases have in common a
valine on the position equivalent to that occupied by the
methionine M43 in BcrAK (indicated with a star in Fig. 1).
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Molecular cloning of a B. canis rossi adenosine kinase (Eur. J. Biochem. 265) 1019
CONCLUSIONS
The cloning, sequencing and expression of an immunogenic
antigen of the B. canis rossi parasite is presented here. The
molecule corresponds to a new adenosine kinase belonging to
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ACKNOWLEDGEMENTS
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The authors would like to thank S. Camillieri and V. Arnavielle for efficient
technical assistance and C. Rouveirol for secretarial assistance. The authors
are also grateful to J. P. Pin and A. M. Cathiard for valuable help. We thank
H. Vial for critical reading of the manuscript. This work was supported by a
grant from Intervet International (the Netherlands). C. C. is a PhD student
supported by a fellowship from the Ministere de l'Education Nationale, de
la Recherche et de la Technologie and Internet International BV
(Convention Cifre no. 679/95).
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