Biol. Chem. Hoppe-Seyler Vol. 373, pp. 1211-1215, December 1992
The Baculovirus Autographa californica Nuclear Polyhedrosis Virus Genome Includes a Papain-Like Sequence Neil D. RAWLiNGS 3 , Laurence H. PEARL b and David J. BUTTLE* a b
Biochemistry Department, Strangeways Research Laboratory, Cambridge, U.K. Biomolecular Structure and Modelling Unit, Department of Biochemistry, University College, London, U.K.
(Received 7 September 1992)
Summary: The published DNA sequence that includes the gene for the envelope glycoprotein gp67 of the baculovirus Autographa californica nuclear polyhedrosis virus also contains a segment that shows 32% amino acid identity to papain, a cysteine endopeptidase from the papaya plant (Carica papaya). The viral papain-like sequence is apparently affected by a frame-shift mutation, but otherwise appears capable of encoding a functional enzyme. Catalytically essential amino acids appear to be conserved, as do disulphide bridges. The overall structure of the putative protein is similar to that of papain, as judged by
hydropathy profile. Secondary structure prediction using a consensus of seven methods indicates that the putative viral enzyme retains the α/β domain structure of papain, including the long helix beginning with cysteine-25. Infecting SF9 cells with the virus did not lead to a detected increase in cysteine endopeptidase activity, and a cysteine endopeptidase inactivator appeared to have no effect on infectivity. Irrespective of whether the sequence encodes a function-ally active cysteine endopeptidase, this is the first example of a papain-related sequence in a viral genome.
Key terms: Cysteine endopeptidase, baculovirus, evolution, secondary structure, papain.
Enzymes that cleave internal peptide bonds of proteins and rely on a cysteine residue for catalysis are known as cysteine endopeptidases. Most of the cysteine endopeptidases so far sequenced are homologous to papain (EC 3.4.22.2) and comprise the papain superfamily (in the terminology of Dayhoff et al.[1]). At least 32 protein and cDNA sequences belong to this superfamily, and most have been shown to be functional cysteine endopeptidases. Examples exist in plants, e.g. papain^; vertebrates, e.g. human cathepsin H (EC 3.4.22.16)[3]; arthropods, e.g. house dust mite cysteine endopeptidase^; nematodes, e.g. Schistosoma mansoni^; protozoa, e.g. Plas; and slime moulds^. The cysteine endo-
peptidases identified in fungi, bacteria and viruses exhibit no significant sequence relationship to the papain superfamily. The sequence of an envelope glycoprotein (gp67) from AcMNPV, a double-stranded DNA baculovirus, has recently been reported^. The gp67 gene was mapped to the left end of the EcoRl fragment H in a right-to-left orientation on the consensus map of AcMNPV[9], between 80.1 and 81.5mu. The cDNA sequence continues beyond the four polyadenylation signals to the EcoRl H site. We now disclose the existence of a papain-like sequence within the EcoRl fragment H of this baculovirus genome.
Enzymes: Cathepsin H (EC 3.4.22.16); Papain (EC 3.4.22.2). Abbreviations: AcMNPV, Autographa californica nuclear polyhedrosis virus; E64;L-3-carboxy-2,3-iran.s-epoxypropionyl-leucylamido-(4-guanidino)butane; NHMec, 7-(4-methyl)coumarylamide; Ep453, mws-epoxypropionyl-leucylamido(3-methyl)butane ethyl ester; Z, benzyloxycarbonyl.
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N.D. Rawlings, L.H. Pearl and D.J. Buttle
Results
This has the effect of producing the sequence TGAGCTAG, which encodes a terminator codon in two reading frames. However, the deduced aminoacid sequence to the left of this extra adenine is 47% identical to papain (including the active site cysteine), and that to the right 28% identical (including the active site histidine), giving an over-all level of identity of 32%. The open reading frame ends with a stop codon at position 1817 (gp67 nucleotide numbering). Because the cDNA sequence commences with the EcoRl restriction site, the sequence equivalent to the N-terminus of papain is missing.
A papain-like sequence in AcMNPV A search of the GenBank database (release 63.0), using the FASTA program of Pearson and Lipman^, with the amino acid sequence of mature papain'11', revealed a relationship with the 3' end of the published sequence of the AcMNPV EcoRl H fragment. When read left to right, the AcMNPV cDNA sequence produced two "hits" against the papain sequence because of a frame shift resulting from an insertion of a single adenine base relative to papain.
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GAATTCACTGGCGTCGTCTCAACAAAGTCACTAGCGTAAAAAATCAGGGCATGTGTGGCGCCTGCTGGGCGTTT I H gKj R L N K [Hj S MJHHB M Hfl A Υ V D QE Q K G A QH P QHflDEH S HD S
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i + 2300-, a GCCACTCTGAGCTAGTTTGGAAAGTCAATTTGCAATCAAACATAACCAGTTGATTAATCTGTCGGAGCAGCAAATGATCGAT b A T L A S L S Q F A [ ] K H N Q | l l N L Β·ΠΚ! Q M I Q c S A V V T I Q G I I K y R T G N Q N E Y ^^B ! E L L g 30 40 50
a TGTGATTTTGTCGACGCTGGCTGTAACGGCGGCTTGTTGCACACAGCGTTCGAAGCCATCATTAAAATGGGCGGCGTACAGCTG
b PH F v D A nKMflQE L L H T Q F E A I I K M Q G V Q L c
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2000-I a - - ATTGTTAACTATAAACAGGGTATT - ATAAAATATTGTTTCAACAGCGGTCTAAACCATGCGGTTCTTTTAGTGGGT b - - I V N [ J K Q QH - I K Y g F f f l S G L N J^^H L L OQ c K D F Q L J j R G EH F V G P g G D K ~ V D ^DH A A D E
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Fig. 1. Alignment of the deduced amino-acid sequence of the baculovirus cysteine endopeptidase-like sequence with that of papain. Key to sequences: a) nucleotide sequence of the left hand end of baculovirus fragment EcoRl H; b) deduced amino-acid sequence from the baculovirus DNA, ignoring adenine 2361, marked by | ; c) the amino-acid sequence of mature papain.The nucleic acid s quence is numbered according toWhitford et al.[81. Amino-acid sequences are numbered according to the sequence of mature papain. Gaps are indicated by '-';'*' indicates the termination codon for the baculovirus sequence;' +' marks a potential glycosylation site for the baculovirus sequence. Identical amino-acid residues are shown in white-on-black.
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Vol. 373 (1992)
The deduced amino-acid sequence from the baculovirus DNAis compared with the amino acid sequence of papain in Fig. 1. We have assumed that the extra adenine at position 2361 is a sequencing error, and have presented the viral protein sequence as being continuous.
Hydrophobicity profiles and secondary structure prediction The level of identity (32%) between the two sequences is sufficient to suggest that the polypeptide backbones of the two proteins would be broadly similar. We tested this prediction by comparing the hydropathy profiles of the two sequences (Fig. 2). The profiles are very similar, with hydrophobic segments at positions 18-33, 65-75,122-132 and 155-162. The central core of each of the two domains of papain is hydrophobic, and the similar hydropathy profile for the AcMNPV sequence is therefore consistent with the maintenance of the same two-domain structure. This view is further supported by the conservation of all four buried charged residues (Lys17, Glu35, Glu50 and Lys174) that interact across the interface of the two domains in papain^. Similarity in over-all structure would also be reflected in conservation of secondary structure elements between papain and the baculovirus sequence. A consensus of seven methods^ was therefore used to predict the secondary structure of the baculovirus sequence. The prediction is compared with the known secondary structure of papain in Fig. 3.
Fig.2. Comparison of the hydropathy profiles for the baculovirus putative cysteine endopeptidase and papain. Hydropathy values have been calculated according to the method of Kyte & Doolittle'12', with a moving window of 8. Amino acid residue number is shown along the .x-axis, and relative hydropathy index (positive numbers being hydrophobic, and negative hydrophilic) along the y-axis. The solid line indicates the hydropathy profile of the baculovirus sequence, and the broken line that of papain. To align the profiles it has been assumed that the mature baculovirus endopeptidase N-terminus is four amino acids to the left of the deduced sequence.
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The L-domain in papain comprises residues 12-112 and 208-212, and is predominantly α-helical in structure. The R-domain (residues 1-11 and 113-207) is based upon an antiparallel -sheet. The secondary structure prediction for the baculovirus sequence indicates a similar over-all structure. In particular, the α-helices commencing with the active site Cys25, Ser49, Gly66 and Asn117 are all retained.Thus, both the hydropathy index and secondary structure prediction suggest that the baculovirus sequence could encode a protein with a similar backbone structure to that of papain. The conservation of all cysteine residues forming disulphide bridges in papain also supports this hypothesis (the baculovirus sequence also contains an extra cysteine at position 109). The active site The following amino acids have been implicated in the active site chemistry of papain[131 and are completely conserved in the viral sequence: Cys25 and His159 make up the catalytic ion pair; Asn175 is hydrogen bonded to His159 and orients the imidazolium ring of the histidine; this Η-bond is shielded from solvent by Trp177. The primary specificity determinant of the papain molecule is the hydrophobic pocket at S2 (in the terminology of Schechter and Berger^), binding both aromatic and aliphatic non-polar side-chains. The residues lining this pocket are different in the AcMNPV sequence with respect to papain: Tyr67^Leu, Pro68^Leu, Trp69-»His, Phe207^Ala, Val133-+Ala, Val157-*Leu and Ala160^Leu. The AcMNPV DNA sequence may thus code for a cys-
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N.D. Rawlings, L.H. Pearl and D.J. Buttle 16
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1 10 20 30 40 50 60 70 80 IPEYVDWRQKGAVTPVKNQGSCGSCWAFSAVVTIEGIIKIRTGNLNEYSEQELLDCDRRSYGCNGGYPWSALQLVAQYG-I IHWRRLNKVTSVKNQGMCGACWAFATLASLESQFAIKHNQLINLSEQQMIDCDFVDAGCNGGLLHTAFEAIIKMGGV
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Fig. 3. Comparison of the secondary structure predicted for the baculovirus sequence with the known secondary structure of papain. The consensus method of Eliopoulos'141 was used to predict the putative protein secondary structure .The structure shown was that predicted by at least five of the seven algorithms used. Key: pap, papain: bac. baculovirus putative endopeptidase: H, α-helix: B, /3-sheet; Τ,/3-turn.
teine endopeptidase with peptide bond specificity different to that of papain. The cysteine endopeptidase activity ofAcMNPV infected and uninfected SF9 cells SF9 cells (from Spodoptera fmgiperda), a gift from Dr. Tom Hassell, Celltech Limited, Slough, U.K., were grown in serum-free SF900 medium (GIBCO) at 28 °C. After 48h the conditioned medium and cells were separated by centrifugation.The cells were disrupted by freeze-thawing and the cell lysate and medium were assayed with a series of peptidylNHMec fluorescent substrates at pH 5.5, 40 °C in the presence of 0.02% Brij 35 and 4mM cysteine. The most sensitive substrate was found to be Z-Phe-ArgNHMec, hydrolysed by the cell lysate at a rate of 0.75pmol/s/106 cells (5μΜ substrate concentration). Even more activity was present in the conditioned medium: 19.5pmol/(s χ 106 cells); and the activity from both cells and medium was totally inhibited by the cysteine endopeptidase inactivator E64 (ΙΟμΜ) (Sigma). Sampling experiments demonstrated that the specific activity in the cell lysate remained roughly constant, whereas it increased with time in the medium, suggesting that SF9 cells secrete cysteine endopeptidase(s) with papain-like specificity. Cell ly-
sate and medium were run separately on the Mono S HR5/5 cation-exclicinge column, on a gradient of sodium acetate buffer, pH 5.O. Virtually all of the activity in the cell lysate eluted as a single peak at 0.18M Na®. In contrast, the activity in the medium displayed considerable charge heterogeneity, eluting between 0.2M and 0.7M counter-ion concentration (not shown). The simplest interpretation of these results is that SF9 cells synthesize several cysteine endopeptidases, all except the most acidic of which are secreted. SF9 cells were infected with wild-type AcMNPV(Invitrogen Corporation, San Diego, USA) (5p.f.u./ cell), and the cysteine endopeptidase activity of cell lysates and culture medium was monitored up to day 4. There was no detected increase in cysteine endopeptidase activity, assayed with a number of peptidyl-NHMec substrates, following viral infection. We used the membrane-permeant cysteine endopeptidase inactivator Ep453 (also known as EST)[16] to investigate whether the putative AcMNPV-encoded enzyme was required for the successful completion of the viral life-cycle. SF9 cell viability following infection with AcMNPV(10p.f.u./cell) was not improved by the presence of ΙΟΟμΜ of the cysteine endopeptidase inactivator in the culture medium. Brought to you by | Purdue University Libraries Authenticated Download Date | 5/31/15 12:33 AM
Vol. 373 (1992)
Baculovirus Papain Homologue
Discussion The search for cysteine endopeptidase activity associated with AcMNPV infection highlighted the large amount of such activity in the host SF9 cells and culture medium.The SF9-AcMNPVsystem is a popular choice for the expression of recombinant proteins, because of the presence of the efficient polyhedrin promoter. The addition of cysteine endopeptidase inactivators to the culture medium may be beneficial to the recovery of recombinant proteins in this system. After the completion of the work presented in this paper, a search of the NEWEMBL database release 28,2 June 1991, revealed an entry labelled "BANPAVCAT, Kuzio, J., Faulkner, P., Unpublished, 'Autographa California nuclear polyhedrosis virus cathepsin (v-cath) gene'".This includes the same nucleotide sequence as that shown in Fig. 1, except that the extra adenine base has been removed so that the papainlike sequence now continues in the same frame, and a guanine has been inserted immediately after the EcoRl H site, changing the first two amino acids in our sequence in Fig. 1 from Ile-His to Phe-Asp. The sequence also continues upstream of the EcoRI H site, to give the four N-terminal amino acids Gly-ProLeu-Glu, a propeptide, a probable leader peptide and initiating methionine. This new evidence, together with the work presented in this paper, makes the existence of a functionally active viral papain homologue seem very likely. That AcMNPV is economical in its use of DNA, often encoding overlapping genes on a DNA strand in different reading frames1171, sometimes transcribing one open reading frame in both directions^181, and using genes that do not contain introns^18'191, increases the likelihood still further that the papain-like sequence is converted into protein. In view of the presence of papain-like activity in host cells, it seems quite likely that the viral gene had a eukaryotic origin, having been captured from the insect host. It has already been noted that insect genes are probably incorporated into baculovirus genomes, and may possibly give certain viruses a selective advantage^201. However, the function of a cysteine endopeptidase in the viral life-cycle is unknown. Our inability to detect increased activity associated with in-
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fection certainly does not rule out the existence.of an enzyme with a different substrate specificity to that of papain. Similarly, the apparent lack of effect of a cysteine endopeptidase inactivator in a culture system does not preclude a role for the enzyme in some specific effect associated with infection of an insect host. Cloning and expression of this novel sequence will provide insights into structure-function relationships of enzymes of the papain superfamily.
References 1 2 3 4 5 6 7 8 9 10 11 12 13
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Dayhoff, M.O., Barker, W.C. & Hunt, L.T. (1983) Methods Enzymol. 91, 524-545. Cohen, L.W., Coghlan, V.M. & Dihel, L.C. (1986) Gene 48, 219-227. Ritonja, A., Popovic, T., Kotnik, M., Machleidt, W. & Turk, V. (1988) FEBS Lett. 228, 341-345. Chua, K.Y., Stewart, G.A., Thomas, W.R., Simpson, R.J., Dilworth, R.J., Plozza,T.M. &Turner, K.J. (1988) J. Exp. Med. 167, 175-182. Klinkert, M.-Q., Felleisen, R., Link, G., Ruppel, A. & Beck, E. (1989) Mol. Biochem. Parasitol. 33,113-122. Li,W.-B., Bzik, D.J., ΗΟΓΪΪ,Τ. & Inselburg, J. (1989) Mol. Biochem. Parasitol. 33, 13-26. Pears, C.J., Mahbubani, H.M. & Williams, J.G. (1985) Nucleic Acids Res. 13, 8853-8866. Whitford, M., Stewart, S., Kuzio, J. & Faulkner, P. (1989) J. Virol. 63, 1393-1399. Vlak, J.M. & Smith, G.E. (1982) /. Virol. 41,1118-1121. Pearson, W.R. & Lipman, D.J. (1988) Proc. Natl. Acad. Sei. USA 85, 2444-2448. Kamphuis, I.G., Drenth, J. & Baker, E.N. (1985) J. Mol. Biol. 182, 317-329. Kyte,J.&Doolittle,R.F.(1982)/. Mol. Biol. 157,105-132. Baker, E.N. & Drenth, J. (1987) in Biological Macromolecules and Assemblies. Vol. 3. Active Site of Enzymes (Jurnak, F.A. & McPherson, A., eds), pp. 314-368, John Wiley & Sons, Inc., New York. Eliopoulos, E., Geddes, A.J., Brett, M., Pappin, D.J.C. & Findlay, J.B.C. (1982) Int. J. Biol. Macromol. 4, 263268. Schechter, I. & Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162. Tamai, M., Matsumoto, K., Omura, S., Koyama, I., Ozawa, Y. & Hanada, K. (1986) J. Pharmacobio-Dyn. 9, 672-677. Oellig, C, Happ, B., M ller,T. & Doerfler, W. (1987) J. Virol. 61, 3048-3057. Miller, L.K. (1988) Anna. Rev. Microbiol. 42,177-199. Luckow, V. A. & Summers, M.D. (1988) Biol Technology 6, 47-55. Bussard, G.W. & Rohrmann, G.F. (1990) Anna. Rev. Entomol. 35,127-155.
N.D. Rawlings & D.J. Buttle*, Biochemistry Department, Strangeways Research Laboratory, Cambridge CB14RN, U.K. L.H. Pearl, Biomolecular Structure and Modelling Unit, Department of Biochemistry, University College, London WC1E 6BT, U.K. * To whom correspondence should be sent.
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The Baculovirus Autographa californica Nuclear Polyhedrosis Virus Genome Includes a Papain-Like Sequence Neil D. RAWLiNGS 3 , Laurence H. PEARL b and David J. BUTTLE* a b
Biochemistry Department, Strangeways Research Laboratory, Cambridge, U.K. Biomolecular Structure and Modelling Unit, Department of Biochemistry, University College, London, U.K.
(Received 7 September 1992)
Summary: The published DNA sequence that includes the gene for the envelope glycoprotein gp67 of the baculovirus Autographa californica nuclear polyhedrosis virus also contains a segment that shows 32% amino acid identity to papain, a cysteine endopeptidase from the papaya plant (Carica papaya). The viral papain-like sequence is apparently affected by a frame-shift mutation, but otherwise appears capable of encoding a functional enzyme. Catalytically essential amino acids appear to be conserved, as do disulphide bridges. The overall structure of the putative protein is similar to that of papain, as judged by
hydropathy profile. Secondary structure prediction using a consensus of seven methods indicates that the putative viral enzyme retains the α/β domain structure of papain, including the long helix beginning with cysteine-25. Infecting SF9 cells with the virus did not lead to a detected increase in cysteine endopeptidase activity, and a cysteine endopeptidase inactivator appeared to have no effect on infectivity. Irrespective of whether the sequence encodes a function-ally active cysteine endopeptidase, this is the first example of a papain-related sequence in a viral genome.
Key terms: Cysteine endopeptidase, baculovirus, evolution, secondary structure, papain.
Enzymes that cleave internal peptide bonds of proteins and rely on a cysteine residue for catalysis are known as cysteine endopeptidases. Most of the cysteine endopeptidases so far sequenced are homologous to papain (EC 3.4.22.2) and comprise the papain superfamily (in the terminology of Dayhoff et al.[1]). At least 32 protein and cDNA sequences belong to this superfamily, and most have been shown to be functional cysteine endopeptidases. Examples exist in plants, e.g. papain^; vertebrates, e.g. human cathepsin H (EC 3.4.22.16)[3]; arthropods, e.g. house dust mite cysteine endopeptidase^; nematodes, e.g. Schistosoma mansoni^; protozoa, e.g. Plas; and slime moulds^. The cysteine endo-
peptidases identified in fungi, bacteria and viruses exhibit no significant sequence relationship to the papain superfamily. The sequence of an envelope glycoprotein (gp67) from AcMNPV, a double-stranded DNA baculovirus, has recently been reported^. The gp67 gene was mapped to the left end of the EcoRl fragment H in a right-to-left orientation on the consensus map of AcMNPV[9], between 80.1 and 81.5mu. The cDNA sequence continues beyond the four polyadenylation signals to the EcoRl H site. We now disclose the existence of a papain-like sequence within the EcoRl fragment H of this baculovirus genome.
Enzymes: Cathepsin H (EC 3.4.22.16); Papain (EC 3.4.22.2). Abbreviations: AcMNPV, Autographa californica nuclear polyhedrosis virus; E64;L-3-carboxy-2,3-iran.s-epoxypropionyl-leucylamido-(4-guanidino)butane; NHMec, 7-(4-methyl)coumarylamide; Ep453, mws-epoxypropionyl-leucylamido(3-methyl)butane ethyl ester; Z, benzyloxycarbonyl.
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1212
N.D. Rawlings, L.H. Pearl and D.J. Buttle
Results
This has the effect of producing the sequence TGAGCTAG, which encodes a terminator codon in two reading frames. However, the deduced aminoacid sequence to the left of this extra adenine is 47% identical to papain (including the active site cysteine), and that to the right 28% identical (including the active site histidine), giving an over-all level of identity of 32%. The open reading frame ends with a stop codon at position 1817 (gp67 nucleotide numbering). Because the cDNA sequence commences with the EcoRl restriction site, the sequence equivalent to the N-terminus of papain is missing.
A papain-like sequence in AcMNPV A search of the GenBank database (release 63.0), using the FASTA program of Pearson and Lipman^, with the amino acid sequence of mature papain'11', revealed a relationship with the 3' end of the published sequence of the AcMNPV EcoRl H fragment. When read left to right, the AcMNPV cDNA sequence produced two "hits" against the papain sequence because of a frame shift resulting from an insertion of a single adenine base relative to papain.
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a TGTGATTTTGTCGACGCTGGCTGTAACGGCGGCTTGTTGCACACAGCGTTCGAAGCCATCATTAAAATGGGCGGCGTACAGCTG
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Fig. 1. Alignment of the deduced amino-acid sequence of the baculovirus cysteine endopeptidase-like sequence with that of papain. Key to sequences: a) nucleotide sequence of the left hand end of baculovirus fragment EcoRl H; b) deduced amino-acid sequence from the baculovirus DNA, ignoring adenine 2361, marked by | ; c) the amino-acid sequence of mature papain.The nucleic acid s quence is numbered according toWhitford et al.[81. Amino-acid sequences are numbered according to the sequence of mature papain. Gaps are indicated by '-';'*' indicates the termination codon for the baculovirus sequence;' +' marks a potential glycosylation site for the baculovirus sequence. Identical amino-acid residues are shown in white-on-black.
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Vol. 373 (1992)
The deduced amino-acid sequence from the baculovirus DNAis compared with the amino acid sequence of papain in Fig. 1. We have assumed that the extra adenine at position 2361 is a sequencing error, and have presented the viral protein sequence as being continuous.
Hydrophobicity profiles and secondary structure prediction The level of identity (32%) between the two sequences is sufficient to suggest that the polypeptide backbones of the two proteins would be broadly similar. We tested this prediction by comparing the hydropathy profiles of the two sequences (Fig. 2). The profiles are very similar, with hydrophobic segments at positions 18-33, 65-75,122-132 and 155-162. The central core of each of the two domains of papain is hydrophobic, and the similar hydropathy profile for the AcMNPV sequence is therefore consistent with the maintenance of the same two-domain structure. This view is further supported by the conservation of all four buried charged residues (Lys17, Glu35, Glu50 and Lys174) that interact across the interface of the two domains in papain^. Similarity in over-all structure would also be reflected in conservation of secondary structure elements between papain and the baculovirus sequence. A consensus of seven methods^ was therefore used to predict the secondary structure of the baculovirus sequence. The prediction is compared with the known secondary structure of papain in Fig. 3.
Fig.2. Comparison of the hydropathy profiles for the baculovirus putative cysteine endopeptidase and papain. Hydropathy values have been calculated according to the method of Kyte & Doolittle'12', with a moving window of 8. Amino acid residue number is shown along the .x-axis, and relative hydropathy index (positive numbers being hydrophobic, and negative hydrophilic) along the y-axis. The solid line indicates the hydropathy profile of the baculovirus sequence, and the broken line that of papain. To align the profiles it has been assumed that the mature baculovirus endopeptidase N-terminus is four amino acids to the left of the deduced sequence.
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Baculovirus Papain Homologue
The L-domain in papain comprises residues 12-112 and 208-212, and is predominantly α-helical in structure. The R-domain (residues 1-11 and 113-207) is based upon an antiparallel -sheet. The secondary structure prediction for the baculovirus sequence indicates a similar over-all structure. In particular, the α-helices commencing with the active site Cys25, Ser49, Gly66 and Asn117 are all retained.Thus, both the hydropathy index and secondary structure prediction suggest that the baculovirus sequence could encode a protein with a similar backbone structure to that of papain. The conservation of all cysteine residues forming disulphide bridges in papain also supports this hypothesis (the baculovirus sequence also contains an extra cysteine at position 109). The active site The following amino acids have been implicated in the active site chemistry of papain[131 and are completely conserved in the viral sequence: Cys25 and His159 make up the catalytic ion pair; Asn175 is hydrogen bonded to His159 and orients the imidazolium ring of the histidine; this Η-bond is shielded from solvent by Trp177. The primary specificity determinant of the papain molecule is the hydrophobic pocket at S2 (in the terminology of Schechter and Berger^), binding both aromatic and aliphatic non-polar side-chains. The residues lining this pocket are different in the AcMNPV sequence with respect to papain: Tyr67^Leu, Pro68^Leu, Trp69-»His, Phe207^Ala, Val133-+Ala, Val157-*Leu and Ala160^Leu. The AcMNPV DNA sequence may thus code for a cys-
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Vol. 373 (1992)
N.D. Rawlings, L.H. Pearl and D.J. Buttle 16
pap bac
1 10 20 30 40 50 60 70 80 IPEYVDWRQKGAVTPVKNQGSCGSCWAFSAVVTIEGIIKIRTGNLNEYSEQELLDCDRRSYGCNGGYPWSALQLVAQYG-I IHWRRLNKVTSVKNQGMCGACWAFATLASLESQFAIKHNQLINLSEQQMIDCDFVDAGCNGGLLHTAFEAIIKMGGV
pap bac
pap bac
pap bac
TT
HHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHH
HHHHHHHHH HHHH
TTTT HHHHHHHHHHHH TTT T HHHHHHHHH
90 100 110 120 130 140 150 160 HYRNTYPYEGVQRYCRSREKGPYAAKTDGVRQVQPYNEGALLYSIANQPVSVVLEAAGKDFQLYRGGIFVGPCGNK-VDHA QLESDYPYEADNNNCRMNSNKFLVQVKDCYRYITVYEEKLKDLLRLVGPIPMAIDAAD—IVNYKQGI-IKYCFNSGLNHA
TTTT
TTT T
BB
BB HHHHHHHHHHHH BB HHHHHHH BBBB HHHHHHHHHHH HHHHHH B
pap bac
170 180 190 200 210 VAAVGYGPNYIL IKNSWGTGWGENGYIRIKRGTGNSYGVCGLYTSSFYPVKN VLLVGYGVENNIPYWTFKNTWGTDWGEDGFFRV QQNINACGMRNELASTAVIY
pap bac
BBBBBBBB B BBBBBB
BBB BBB TTT
BBBBBBBBB
BBB-BBBBB TTT
HHHHHHHBBBB
Fig. 3. Comparison of the secondary structure predicted for the baculovirus sequence with the known secondary structure of papain. The consensus method of Eliopoulos'141 was used to predict the putative protein secondary structure .The structure shown was that predicted by at least five of the seven algorithms used. Key: pap, papain: bac. baculovirus putative endopeptidase: H, α-helix: B, /3-sheet; Τ,/3-turn.
teine endopeptidase with peptide bond specificity different to that of papain. The cysteine endopeptidase activity ofAcMNPV infected and uninfected SF9 cells SF9 cells (from Spodoptera fmgiperda), a gift from Dr. Tom Hassell, Celltech Limited, Slough, U.K., were grown in serum-free SF900 medium (GIBCO) at 28 °C. After 48h the conditioned medium and cells were separated by centrifugation.The cells were disrupted by freeze-thawing and the cell lysate and medium were assayed with a series of peptidylNHMec fluorescent substrates at pH 5.5, 40 °C in the presence of 0.02% Brij 35 and 4mM cysteine. The most sensitive substrate was found to be Z-Phe-ArgNHMec, hydrolysed by the cell lysate at a rate of 0.75pmol/s/106 cells (5μΜ substrate concentration). Even more activity was present in the conditioned medium: 19.5pmol/(s χ 106 cells); and the activity from both cells and medium was totally inhibited by the cysteine endopeptidase inactivator E64 (ΙΟμΜ) (Sigma). Sampling experiments demonstrated that the specific activity in the cell lysate remained roughly constant, whereas it increased with time in the medium, suggesting that SF9 cells secrete cysteine endopeptidase(s) with papain-like specificity. Cell ly-
sate and medium were run separately on the Mono S HR5/5 cation-exclicinge column, on a gradient of sodium acetate buffer, pH 5.O. Virtually all of the activity in the cell lysate eluted as a single peak at 0.18M Na®. In contrast, the activity in the medium displayed considerable charge heterogeneity, eluting between 0.2M and 0.7M counter-ion concentration (not shown). The simplest interpretation of these results is that SF9 cells synthesize several cysteine endopeptidases, all except the most acidic of which are secreted. SF9 cells were infected with wild-type AcMNPV(Invitrogen Corporation, San Diego, USA) (5p.f.u./ cell), and the cysteine endopeptidase activity of cell lysates and culture medium was monitored up to day 4. There was no detected increase in cysteine endopeptidase activity, assayed with a number of peptidyl-NHMec substrates, following viral infection. We used the membrane-permeant cysteine endopeptidase inactivator Ep453 (also known as EST)[16] to investigate whether the putative AcMNPV-encoded enzyme was required for the successful completion of the viral life-cycle. SF9 cell viability following infection with AcMNPV(10p.f.u./cell) was not improved by the presence of ΙΟΟμΜ of the cysteine endopeptidase inactivator in the culture medium. Brought to you by | Purdue University Libraries Authenticated Download Date | 5/31/15 12:33 AM
Vol. 373 (1992)
Baculovirus Papain Homologue
Discussion The search for cysteine endopeptidase activity associated with AcMNPV infection highlighted the large amount of such activity in the host SF9 cells and culture medium.The SF9-AcMNPVsystem is a popular choice for the expression of recombinant proteins, because of the presence of the efficient polyhedrin promoter. The addition of cysteine endopeptidase inactivators to the culture medium may be beneficial to the recovery of recombinant proteins in this system. After the completion of the work presented in this paper, a search of the NEWEMBL database release 28,2 June 1991, revealed an entry labelled "BANPAVCAT, Kuzio, J., Faulkner, P., Unpublished, 'Autographa California nuclear polyhedrosis virus cathepsin (v-cath) gene'".This includes the same nucleotide sequence as that shown in Fig. 1, except that the extra adenine base has been removed so that the papainlike sequence now continues in the same frame, and a guanine has been inserted immediately after the EcoRl H site, changing the first two amino acids in our sequence in Fig. 1 from Ile-His to Phe-Asp. The sequence also continues upstream of the EcoRI H site, to give the four N-terminal amino acids Gly-ProLeu-Glu, a propeptide, a probable leader peptide and initiating methionine. This new evidence, together with the work presented in this paper, makes the existence of a functionally active viral papain homologue seem very likely. That AcMNPV is economical in its use of DNA, often encoding overlapping genes on a DNA strand in different reading frames1171, sometimes transcribing one open reading frame in both directions^181, and using genes that do not contain introns^18'191, increases the likelihood still further that the papain-like sequence is converted into protein. In view of the presence of papain-like activity in host cells, it seems quite likely that the viral gene had a eukaryotic origin, having been captured from the insect host. It has already been noted that insect genes are probably incorporated into baculovirus genomes, and may possibly give certain viruses a selective advantage^201. However, the function of a cysteine endopeptidase in the viral life-cycle is unknown. Our inability to detect increased activity associated with in-
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fection certainly does not rule out the existence.of an enzyme with a different substrate specificity to that of papain. Similarly, the apparent lack of effect of a cysteine endopeptidase inactivator in a culture system does not preclude a role for the enzyme in some specific effect associated with infection of an insect host. Cloning and expression of this novel sequence will provide insights into structure-function relationships of enzymes of the papain superfamily.
References 1 2 3 4 5 6 7 8 9 10 11 12 13
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Dayhoff, M.O., Barker, W.C. & Hunt, L.T. (1983) Methods Enzymol. 91, 524-545. Cohen, L.W., Coghlan, V.M. & Dihel, L.C. (1986) Gene 48, 219-227. Ritonja, A., Popovic, T., Kotnik, M., Machleidt, W. & Turk, V. (1988) FEBS Lett. 228, 341-345. Chua, K.Y., Stewart, G.A., Thomas, W.R., Simpson, R.J., Dilworth, R.J., Plozza,T.M. &Turner, K.J. (1988) J. Exp. Med. 167, 175-182. Klinkert, M.-Q., Felleisen, R., Link, G., Ruppel, A. & Beck, E. (1989) Mol. Biochem. Parasitol. 33,113-122. Li,W.-B., Bzik, D.J., ΗΟΓΪΪ,Τ. & Inselburg, J. (1989) Mol. Biochem. Parasitol. 33, 13-26. Pears, C.J., Mahbubani, H.M. & Williams, J.G. (1985) Nucleic Acids Res. 13, 8853-8866. Whitford, M., Stewart, S., Kuzio, J. & Faulkner, P. (1989) J. Virol. 63, 1393-1399. Vlak, J.M. & Smith, G.E. (1982) /. Virol. 41,1118-1121. Pearson, W.R. & Lipman, D.J. (1988) Proc. Natl. Acad. Sei. USA 85, 2444-2448. Kamphuis, I.G., Drenth, J. & Baker, E.N. (1985) J. Mol. Biol. 182, 317-329. Kyte,J.&Doolittle,R.F.(1982)/. Mol. Biol. 157,105-132. Baker, E.N. & Drenth, J. (1987) in Biological Macromolecules and Assemblies. Vol. 3. Active Site of Enzymes (Jurnak, F.A. & McPherson, A., eds), pp. 314-368, John Wiley & Sons, Inc., New York. Eliopoulos, E., Geddes, A.J., Brett, M., Pappin, D.J.C. & Findlay, J.B.C. (1982) Int. J. Biol. Macromol. 4, 263268. Schechter, I. & Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162. Tamai, M., Matsumoto, K., Omura, S., Koyama, I., Ozawa, Y. & Hanada, K. (1986) J. Pharmacobio-Dyn. 9, 672-677. Oellig, C, Happ, B., M ller,T. & Doerfler, W. (1987) J. Virol. 61, 3048-3057. Miller, L.K. (1988) Anna. Rev. Microbiol. 42,177-199. Luckow, V. A. & Summers, M.D. (1988) Biol Technology 6, 47-55. Bussard, G.W. & Rohrmann, G.F. (1990) Anna. Rev. Entomol. 35,127-155.
N.D. Rawlings & D.J. Buttle*, Biochemistry Department, Strangeways Research Laboratory, Cambridge CB14RN, U.K. L.H. Pearl, Biomolecular Structure and Modelling Unit, Department of Biochemistry, University College, London WC1E 6BT, U.K. * To whom correspondence should be sent.
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