Colin Berry, John B. Damel, Ben M. Dunnzand. John Kay. Department of Biochemistry, University of Wales,. Cardiff, CF1 1ST and Depts of 1Infectious Diseases ...
Biochemical Society Transactions ( 1 994) 22
Aspartic proteinases from the human malarial parasite Plasmodium farcipamm Colin Berry, John B. Damel, Ben M. Dunnzand John Kay Department of Biochemistry, University of Wales, Cardiff, CF1 1ST and Depts of 1Infectious Diseases and 2Biochem & Mol Biol, University of Florida, Gainesville, FL 32610, U.S.A. During the intraerythrocytic stages of malarial infection, the parasite relies upon the degradation of host cell haemoglobin as the source of most of its nutrients. Aspartic proteinases play a key role in this degradation by catalysing the initial cleavage in the haemoglobin alpha chain prior to further cleavage [ 11. A gene encodmg an aspartic proteinase (PFAPD) has been characterised. The parasite enzyme shows approximately 30% identity to human aspartic proteinases ( cathepsins D and E, 36%, renin, 33%, pepsin, 31%). The PFAPD sequence was shown to be distinct from that of another aspartic proteinase (PFAPG) which was identified by Goldberg et al. in the food vacuoles of P. falciparum [I]. The gene encoding this proteinase has recently been sequenced [2]. An alignment of the mature PFAPD and PFAPG proteinase sequences showing 87% identity, is presented in Fig. 1.
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D:LNSGLTKTNYLGSSNDNIELVDFQNIMFYGDAEVGDNQQP G: AG S V T N VA V Y E Q I K K
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D:FTFILDTGSANLWVPSVKCTTAGCLTKHLYDSSKSRTYEK G: A F A Q N I K N N K
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0:DGTKVEMNYVSGTVSGFFSKDLVTVGNLSLPYKFIEVIDT G: I I A F T
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NGFEPTYTASTFDGILGLGWKDLSIGSVDPIVVELKNQNK
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LGQ
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0:IENALFTFYLPVHDKHTGFLTIGGIEERFYEGPLTYEKLN G: Q V FD K Y D Q
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0 : H D L Y W Q I T L DAHV G N I M L E KANC I V D S GT SA I T V P T D F L N G: VD L F LTV TA S A E
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D:KMLQNLDVIKVPFLPFYVTLCNNSKLPTFEFTSENGKYTL G: FFEG V I L I T P L R ATNV
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D:EPEYYLQHIEDVGPGLCMLNIIGLDFPVPTFILGDPFMRK G: Q F F I S VS PV LNKN 310
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D:YFTVFDYDNHSVGIALAKKNL G: T F K
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Fig. 1. Alignment of Plasmodium Aspartic proteinases D and G. The sequence of mature recombinant D enzyme is given in full. The mature G enzyme, as isolated from parasite food vacuoles [l]begins at residue -1; where no residue is shown, identity with D exists. Numbering is based on that for pig pepsin [3]. Typical features associated with aspartic proteinases can be identified in the sequences of the two Plasmodium enzymes. Two active site motifs, Hydrophobic-Hydrophobic-Asp-Thr/Ser-GI y sequences are present (around residues 32 and 215) in both enzymes. The substitution of Ser for Thr in both Plasmodium enzymes is somewhat unusual but is found also in the cathepsin D-like enzyme from Barley 141 and in some dimeric viral enzymes [5].A 3-dimensional model of PFAPD has been constructed using rule based procedures and shows that the primary sequence may be folded into a structure closely resembling those of other known aspartic proteinases. PFAPD has been expressed in E. coli in the pGEX2T expression vector. The expressed product was found to be insoluble. Insoluble material was solubilised in 6M urea and refolded by rapid 1:40 fold dilution [6]. Refolded enzyme was activated and partially purified by acidification by the method of Hill et al [7] and finally purified to homogeneity by affinity chromatography using pepstatin-agarose. Edman degradation of the purified product generated the N-terminal sequence depicted in Fig. 1. The cleavage site (Val-Asn-Phe*Leu) between the pro-segment and the mature recombinant enzyme was a typical aspartic proteinase auto-activation sequence. The recombinant proteinase was active against a range of synthetic peptide substrates and haemoglobin. Experiments using synthetic substrates have demonstrated that the specificity of PFAPD is quite broad and is not restricted to haemoglobin. The pH optimum of the proteinase is around 4.7 and its activity is totally inhibited by 100 nM pepstatin. 1. Goldberg, D.E., Stater, A.F.G., Beavis, R., Chait, B., Cerami, A. & Henderson, G.B. (1991) J. Exp. Med. 173,961-969. 2. Francis, S.E., Gluzman, I.Y., Oksman, A., Knickerbocker, A., Mueller, R., Bryant, M.L., Sherman, D.R., Russel, D.G., and Goldberg, G. (1994) EMBO J. 13,306-317. 3. Sogawa, K., Fujii-Kuriyama, Y ., Mizukami, Y ., Ichihara, Y. and Takahashi, K. (1983) J. Biol. Chem. 258,5306-53 11. 4. Runeberg-Roos, P., Tormakangas, K. & Ostman, A. (1991) Eur. J. Biochem. 202, 1021-1027. 5. Ohlendorf, D.H., Foundling, S.I., Wendoloski, J.J., Sedlacek, J., Strop, P. & Salemme, F.R. (1992) Proteins 14, 382-391. 6. Chen, Z., Koelsch, G., Han, H., Wang, X.J., Lin, X., Hartsuck, J.A. & Tang, J. (1991) J. Biol. Chem. 266, 11718-11725. 7. Hill, J, Montgomery, D.S. & Kay, J. (1993) FEBS Lett. 326, 101-104.
