An Asparaginyl Endopeptidase Mediates in Vivo Protein Backbone Cyclization

Proteases can catalyze both peptide bond cleavage and formation, yet the hydrolysis reaction dominates in nature. This presents an interesting challenge for the biosynthesis of backbone cyclized (circular) proteins, which are encoded as part of precursor proteins and require post-translational peptide bond formation to reach their mature form. The largest family of circular proteins are the plant-produced cyclotides; extremely stable proteins with applications as bioengineering scaffolds. Little is known about the mechanism by which they are cyclized in vivo but a highly conserved Asn (occasionally Asp) residue at the C terminus of the cyclotide domain suggests that an enzyme with specificity for Asn (asparaginyl endopeptidase; AEP) is involved in the process. Nicotiana benthamiana does not endogenously produce circular proteins but when cDNA encoding the precursor of the cyclotide kalata B1 was transiently expressed in the plants they produced the cyclotide, together with linear forms not commonly observed in cyclotide-containing plants. Observation of these species over time showed that in vivo asparaginyl bond hydrolysis is necessary for cyclization. When AEP activity was suppressed, either by decreasing AEP gene expression or using a specific inhibitor, the amount of cyclic cyclotide in the plants was reduced compared with controls and was accompanied by the accumulation of extended linear species. These results suggest that an AEP is responsible for catalyzing both peptide bond cleavage and ligation of cyclotides in a single processing event. Proteases can catalyze both peptide bond cleavage and formation, yet the hydrolysis reaction dominates in nature. This presents an interesting challenge for the biosynthesis of backbone cyclized (circular) proteins, which are encoded as part of precursor proteins and require post-translational peptide bond formation to reach their mature form. The largest family of circular proteins are the plant-produced cyclotides; extremely stable proteins with applications as bioengineering scaffolds. Little is known about the mechanism by which they are cyclized in vivo but a highly conserved Asn (occasionally Asp) residue at the C terminus of the cyclotide domain suggests that an enzyme with specificity for Asn (asparaginyl endopeptidase; AEP) is involved in the process. Nicotiana benthamiana does not endogenously produce circular proteins but when cDNA encoding the precursor of the cyclotide kalata B1 was transiently expressed in the plants they produced the cyclotide, together with linear forms not commonly observed in cyclotide-containing plants. Observation of these species over time showed that in vivo asparaginyl bond hydrolysis is necessary for cyclization. When AEP activity was suppressed, either by decreasing AEP gene expression or using a specific inhibitor, the amount of cyclic cyclotide in the plants was reduced compared with controls and was accompanied by the accumulation of extended linear species. These results suggest that an AEP is responsible for catalyzing both peptide bond cleavage and ligation of cyclotides in a single processing event. Backbone-cyclized (circular) proteins have been identified in bacteria, plants, and mammals (1Craik D.J. Science. 2006; 311: 1563-1564Crossref PubMed Scopus (244) Google Scholar, 2Trabi M. Craik D.J. Trends Biochem. Sci. 2002; 27: 132-138Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). The largest family of circular proteins are the cyclotides, which combine a cyclic backbone with a cystine knot arrangement of three disulfide bonds. The cyclic cystine knot (CCK) 2The abbreviations used are:CCKcyclic cystine knotNTRN-terminal repeatMES4-morpholineethanesulfonic acidAEPasparaginyl endopeptidaseVPEvacuolar processing enzymedpidays postinfiltrationERendoplasmic reticulumMALDI-TOFmatrix-assisted laser desorption/ionization-time of flight. framework endows cyclotides with a number of advantages over conventional (i.e. acyclic) proteins. Cyclotides are resistant to thermal and biochemical extremes, treatment with endoproteases and, because of the absence of termini, exoproteases (3Colgrave M.L. Craik D.J. Biochemistry. 2004; 43: 5965-5975Crossref PubMed Scopus (462) Google Scholar). These features have motivated the development of cyclotides as stable scaffolds in drug design (4Craik D.J. Cěmažar M. Daly N.L. Curr. Opin. Drug Discov. Devel. 2006; 9: 251-260PubMed Google Scholar). Fig. 1 shows the compact structure of the prototypic cyclotide kalata B1 (5Saether O. Craik D.J. Campbell I.D. Sletten K. Juul J. Norman D.G. Biochemistry. 1995; 34: 4147-4158Crossref PubMed Scopus (376) Google Scholar). Although cyclotides display a broad range of bioactivities (6Daly N.L. Love S. Alewood P.F. Craik D.J. Biochemistry. 1999; 38: 10606-10614Crossref PubMed Scopus (196) Google Scholar, 7Gran L. Medd. Nor. Farm. Selsk. 1970; 12: 173-180Google Scholar, 8Gustafson K.R. Sowder R.C.I. Henderson L.E. Parsons I.C. Kashman Y. Cardellina J.H.I. McMahon J.B. Buckheit R.W.J. Pannell L.K. Boyd M.R. J. Am. Chem. Soc. 1994; 116: 9337-9338Crossref Scopus (263) Google Scholar, 9Lindholm P. U. Göransson Johansson S. Claeson P. Gulbo J. Larsson R. Bohlin L. Backlund A. Mol. Cancer Ther. 2002; 1: 365-369Crossref PubMed Scopus (43) Google Scholar, 10Tam J.P. Lu Y.A. Yang J.L. Chiu K.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8913-8918Crossref PubMed Scopus (411) Google Scholar, 11Göransson U. Sjogren M. Svangard E. Claeson P. Bohlin L. J. Nat. Prod. 2004; 67: 1287-1290Crossref PubMed Scopus (125) Google Scholar, 12Witherup K.M. Bogusky M.J. Anderson P.S. Ramjit H. Ransom R.W. Wood T. Sardana M. J. Nat. Prod. 1994; 57: 1619-1625Crossref PubMed Scopus (229) Google Scholar) the ability of the cyclotides kalata B1 and kalata B2 to inhibit the development of the insect pests Helicoverpa punctigera and H. armigera suggests that their endogenous function is in plant defense (13Jennings C. West J. Waine C. Craik D. Anderson M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10614-10619Crossref PubMed Scopus (412) Google Scholar, 14Jennings C.V. Rosengren K.J. Daly N.L. Plan M. Stevens J. Scanlon M.J. Waine C. Norman D.G. Anderson M.A. Craik D.J. Biochemistry. 2005; 44: 851-860Crossref PubMed Scopus (198) Google Scholar). cyclic cystine knot N-terminal repeat 4-morpholineethanesulfonic acid asparaginyl endopeptidase vacuolar processing enzyme days postinfiltration endoplasmic reticulum matrix-assisted laser desorption/ionization-time of flight. Cyclotides are encoded as part of precursor proteins that have a highly conserved organization. Precursor cDNA clones derived from Oldenlandia affinis (Rubiaceae) (13Jennings C. West J. Waine C. Craik D. Anderson M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10614-10619Crossref PubMed Scopus (412) Google Scholar) and Viola odorata (Violaceae) (15Dutton J.L. Renda R.F. Waine C. Clark R.J. Daly N.L. Jennings C.V. Anderson M.A. Craik D.J. J. Biol. Chem. 2004; 279: 46858-46867Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) exhibit an N-terminal endoplasmic reticulum (ER) signal sequence followed by a pro-region, one or more cyclotide domains and a short hydrophobic C-terminal tail sequence. In precursors that contain multiple cyclotide domains each one is preceded by a repeated portion of the proregion (∼20 residues) that has been designated the N-terminal repeat (NTR) (13Jennings C. West J. Waine C. Craik D. Anderson M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10614-10619Crossref PubMed Scopus (412) Google Scholar). The sequence identity of the NTR between species is low but the motif appears to be a structurally conserved helix (15Dutton J.L. Renda R.F. Waine C. Clark R.J. Daly N.L. Jennings C.V. Anderson M.A. Craik D.J. J. Biol. Chem. 2004; 279: 46858-46867Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Fig. 1 shows the general organization of cyclotide precursor proteins and an expanded portion of the specific precursor that encodes kalata B1, designated Oak1 (for O. affinis kalata B1). Although the residues preceding the cyclotide domain are not highly conserved, an Asn (occasionally Asp) residue is located at the C terminus of the cyclotide domain across all precursor clones. The position of the Asn residue suggests that it is critical for processing, and possibly cyclization, of the cyclotide domain. Intein-based mechanisms have been used to cyclize cyclotides (16Kimura R.H. Tran A.-T. Camarero J.A. Angew. Chem. Int. Ed. Engl. 2006; 45: 973-976Crossref PubMed Scopus (95) Google Scholar) but the residues at the termini of the cyclotide domain are not reminiscent of those required for in vivo intein splicing, suggesting that an enzymatic mechanism is probably involved. Asparaginyl endopeptidases specifically cleave peptide bonds C-terminal to Asn and, less efficiently, after Asp and are widespread in plants where they are commonly called vacuolar processing enzymes (VPEs) and are involved in the activation and degradation of storage proteins (17Muntz K. Blattner F.R. Shutov A.D. J. Plant Physiol. 2002; 159: 1281-1293Crossref Scopus (45) Google Scholar). In the current study we present evidence for the role of a plant AEP in the backbone cyclization of cyclotides. We demonstrate that AEP activity is responsible for asparaginyl bond hydrolysis in cyclotide-containing plants and that this activity is linked to the cyclization of cyclotides in N. benthamiana. Although N. benthamiana does not endogenously produce circular proteins, it is capable of producing correctly folded and backbone-cyclized kalata B1 when transiently expressing the Oak1 precursor, in addition to C-terminally extended linear forms of the cyclotide. Analysis of the different cyclotide forms over time showed that linear kalata B1 was not cyclized in vivo despite the presence of a C-terminal Asn, suggesting that hydrolysis of the asparaginyl bond is necessary for cyclization. Knocking down or inhibiting AEP activity significantly reduced the amount of the cyclic protein produced and led to an accumulation of the longer linear cyclotide species. Overall the results appear to implicate an AEP in catalyzing both protein backbone hydrolysis and cyclization. Assay for Asparaginyl Bond Hydrolysis—O. affinis and V. tricolour leaf extracts in 50 mm sodium acetate buffer (pH 5.5) containing 50 mm NaCl and 1 mm EDTA were preincubated with protease inhibitors (0.2-5 mm in dimethyl sulfoxide) in 20 mm sodium acetate (pH 5.5) containing 0.1 mm EDTA and 0.1 m dithiothreitol at 30 °C for 1 h. Activity was measured against 200 at for 1 at 30 °C using a inhibitors were and of the Oak1 for the signal sequence (13Jennings C. West J. Waine C. Craik D. Anderson M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10614-10619Crossref PubMed Scopus (412) Google was by using and and the Plant Mol. Biol. PubMed Scopus Google Scholar) between the and the the Oak1 expression the Oak1 sequence and the and was from as a single and the of the Plant Mol. Biol. PubMed Scopus Google Scholar). of Oak1 in N. with the were in containing mm 20 mm and 20 at When the the were and in an of buffer mm mm with 200 The was at for N. benthamiana were with the as D. S. Plant J. 2002; PubMed Scopus Google Scholar). and of using extracts or 0.1 as were in and with the in containing a of proteins of known and This was with in acid with and a was the in was at the was at of the and a time was The low was at and were between and 50 at 20 were were the the protein from the of was used as an to which the amount of cyclotide proteins was The of the cyclotide proteins was expressed as the of the of the three of the to the for in protein expression between plants the were expressed as a of the of all the cyclotide proteins of in N. extracts from N. benthamiana days postinfiltration with in were in and reduced in 0.1 m with mm at °C for 30 were with acid and using for by from N. benthamiana in were by and by of kalata B1 from O. affinis and the protein from N. benthamiana were at buffer in were reduced in mm for at °C and 1 at °C and with in 0.1 m for h. The were using and by of the precursor was the in The of the were used to the sequence of the In or a was N. benthamiana 1 to of the leaf with the Oak1 expression as after the were with the protease inhibitor, and at for the leaf was days after of and was of the plants each treatment the leaf from the three different plants were for Oak1 in gene of in N. benthamiana was with the in N. M. K. T. S. M. M. Science. 2004; PubMed Scopus Google and the S. T. D. Plant J. Google Scholar) as a The were A. and with and at °C for of N. benthamiana plants was by the with a in the The plants were short at °C for days and to at plants were with each after each plant were with the Oak1 expression as and in for of was using the plant was using 1 of and with an for cDNA expression was compared using 1 of cDNA as a for with and specific The and have been N. M. K. T. S. M. M. Science. 2004; PubMed Scopus Google Scholar). The and were used to Analysis of Oak1 was as E. D. Scholar). leaf extracts were in and the protein was using the protein of the were by and to The was with against Oak1 of the Oak1 was used in the of plant against were used as were with an In AEP activity capable of asparaginyl bonds was in cyclotide-containing and plants using the short peptide of the asparaginyl bond was in both O. affinis (Rubiaceae) and V. tricolour (Violaceae) leaf extracts as an in at over addition of the which of was responsible for this activity the cleavage was after the plant extracts been preincubated with a of each a different of protease in Fig. and the ability of either the O. affinis or V. tricolour leaf extracts to cleave the but the and the specific AEP hydrolysis in a The observed is of which to the of M.A. H. M. K. Biochem. J. PubMed Scopus Google Scholar, F.R. 2006; 34: PubMed Scopus Google Scholar) and that AEP activity was responsible for hydrolysis in cyclotide-containing plants. AEP activity was against cyclotide kalata B1 linear kalata B1 and a protein to linear kalata B1 the C-terminal tail were with a of AEP from AEP) in the presence and absence of AEP was used because it has been to catalyze peptide ligation the processing of the precursor D.J. D.J. J.B. E. J. J. Biol. PubMed Scopus Google Scholar, A. PubMed Scopus Google Scholar, Nat. Mol. Biol. 1994; 1: Scopus Google Scholar). Although AEP was in it asparaginyl bonds after of the Fig. and B1 by N. plants are not for the study of cyclization. The plants are not to they endogenously of cyclotides to plant L. Google that in and the processing appear to to of the precursor protein and protein we expressed Oak1 in N. a plant that does not endogenously produce cyclotides and is not known to produce circular proteins, but which N. M. K. T. S. M. M. Science. 2004; PubMed Scopus Google Scholar). We A. the encoding Oak1 the of a and N. benthamiana cyclotide was at using of Oak1 in N. benthamiana produced a of proteins with to cyclic kalata B1 and to be linear forms of the cyclotide not commonly observed in O. of these were observed in in Fig. the to linear kalata B1, linear kalata B1 the N-terminal and linear kalata B1 the addition of the and residues that the C-terminal tail of Oak1 of N. benthamiana is and after treatment with the in Fig. the of each species to by that each residues and was an protein of kalata and were used to that the species identified in N. benthamiana to backbone-cyclized kalata in Fig. the protein from N. benthamiana with kalata B1 from O. affinis the linear after with and the as the protein was used to the which in to both linear kalata B1 and an of containing the were from an N. benthamiana by of these with linear kalata B1 the both and species in we at the of the linear to species in N. benthamiana is The is to as linear Analysis of the of cyclotide in N. benthamiana from Fig. shows the of the cyclotide protein species at the species present is The linear and species are in but cyclic kalata B1 and the N-terminal species are present in low the days the tail species as the amount of and linear Although the amount of kalata B1 this it does not in to the observed in the linear form. This suggests that in vivo linear kalata B1 a of the is the cyclic kalata B1 and that the Asn residue is not for cyclization. B1 of AEP activity Oak1 processing in vivo was using the of N. benthamiana were with or a 1 to of the with the the protease was to the and at for the days the leaf were each treatment leaf from three plants was for extracts of the were in acid and to with Oak1 to Oak1 processing and to cyclotide of the Oak1 precursor and were in the and as in Fig. suggesting that processing were not by the the of cyclotide proteins between the and expanded in kalata B1 was not in with and the of the linear cyclotide proteins was to the a from the short linear and proteins accumulation of the longer species. of in N. benthamiana of gene was used to N. benthamiana plants in which expression of the forms of in N. was was a of cDNA from N. benthamiana which is conserved in the N. benthamiana N. M. K. T. S. M. M. Science. 2004; PubMed Scopus Google Scholar). were with the of cDNA encoded in an in the plants were with the and to the plants after of both the and plants were with the Oak1 expression was from and to using a a to both N. benthamiana gene expression was in the plants but at the expression in the plants the in expression was precursor Oak1 leaf were to with against Fig. shows Oak1 protein in the plants compared with the plants. or in the amount of precursor was not of either expression cyclotide we compared the amount of kalata B1 in leaf extracts from plants days after of using each plant the of the cyclic kalata B1 was to an protein the of which we have to be to kalata B1 not cyclotide protein expression not significantly between the and plants but a in cyclic kalata B1 was observed in the plants compared with the controls in the a in cyclic kalata B1 was accompanied by a in the of the longer linear cyclotide protein species In this study we that AEP activity is required for the cyclization of cyclotides. suggest that AEP protein backbone cyclization by asparaginyl bond hydrolysis at the C terminus of the cyclotide domain with peptide bond These results an the but of protein cyclization in circular proteins are known to be stable A. E. M. 2002; Scopus Google Scholar, J. K. Tran D. Science. 1999; PubMed Scopus Google Scholar) and is evidence that cyclization can the and function of proteins their as R.J. H. L. Daly N.L. Rosengren K.J. D.J. Craik D.J. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar, J.P. Yang J.L. J. Biochem. PubMed Scopus Google Scholar). of in vivo biosynthesis of cyclotides has the to these features to drug as part of bioengineering activity capable of asparaginyl bonds was in cyclotide-containing plants and to have the as AEP We that specific hydrolysis of asparaginyl bonds in cyclotide-containing plants was by specificity asparaginyl and, to a bonds not all Asn residues in a M. L. Biochem. J. 1999; PubMed Scopus Google Scholar, R. R. K. Plant PubMed Scopus Google Scholar). A. cleave the and but not or M. K. N. M. M. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). these it was not that was to cleave cyclotide in their we in vivo to cyclization. N. benthamiana does not endogenously produce cyclotides and is not known to produce circular proteins. circular kalata B1 was in N. benthamiana the expression of the Oak1 precursor in the that the protein produced in N. benthamiana a cyclized backbone with the disulfide and as the of kalata B1 (6Daly N.L. Love S. Alewood P.F. Craik D.J. Biochemistry. 1999; 38: 10606-10614Crossref PubMed Scopus (196) Google Scholar, N.L. Clark R.J. Craik D.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) have that the structure and disulfide of cyclotide their The of kalata B1 with kalata B1 produced in N. benthamiana that the cyclotide produced in N. benthamiana has the as the The ability of N. benthamiana to produce correctly folded circular proteins that protein backbone cyclization is not a to cyclotide-containing plants but that it has to a in the the of kalata B1 in O. where the circular protein is expression of Oak1 in N. benthamiana circular kalata B1 as as a of proteins the linear cyclotide or the linear cyclotide domain an N-terminal C-terminal residues from the tail of the species identified were in the that formation of the cystine knot to C-terminal a the proteins was with C-terminal hydrolysis or of the longer linear proteins. Although it is not that C-terminal is involved in the endogenous of cyclotides, the of the processing in N. benthamiana with a to cyclization in the of each cyclotide species over days it that the linear and species were produced from the longer linear forms at a the of circular kalata This to that the linear kalata B1, were not as for cyclization. at linear kalata B1 was in the at a that the amount of circular kalata B1 produced and at a it was to it to the form. This that the asparaginyl bond is to the cyclization and that cyclization is linked to the hydrolysis of this it is that linear kalata B1 is the for cyclization but that in N. benthamiana it is by an cyclization can the of the N-terminal appears to be an processing producing a protein that be the time does not that the N-terminal is with cyclization for the linear this were the the linear be to at in the and we the to be produced the circular form. This is because the N-terminal processing have to the cyclization reaction to for the accumulation of over the circular appears the in the time and does not in to the in the suggesting that the cyclization to N-terminal the of the and of a in the of cyclic kalata B1 produced expression of Oak1 in N. benthamiana. The of AEP activity by the protease cyclic kalata B1 and a in the of the linear cyclotide proteins the longer species. the C-terminal observed in N. benthamiana it appear that the for cyclization is at as as the species. the cDNA of in a in of of expression in the plants. a in the of cyclic kalata B1 produced in the plants was observed together with a less the longer linear cyclotide species. processing of the Oak1 precursor in N. benthamiana was not by either that AEP activity was to the of cyclotide be that a in AEP activity a in cyclic kalata B1 by an the of an AEP in the cyclization process. The activity in the presence of a range of enzyme inhibitors that as in N. benthamiana N. M. K. T. S. M. M. Science. 2004; PubMed Scopus Google asparaginyl bond hydrolysis in cyclotide-containing plants is by an enzyme with of an cleavage of the C-terminal Asn bond of cyclotides it is by an This for the of this is that the conserved Asn to processing from the C terminus by a and that cyclization in a process. the that linear kalata B1 does not appear to be cyclized in vivo hydrolysis of the asparaginyl bond by an endopeptidase in the cyclization process. the accumulation of longer linear cyclotide species in plants that have AEP together with the of the C-terminal the asparaginyl bond in cyclotides as the of enzyme protein ligation of the precursor by the reaction to asparaginyl bond hydrolysis D.J. D.J. J.B. E. J. J. Biol. PubMed Scopus Google Scholar, Nat. Mol. Biol. 1994; 1: Scopus Google Scholar). In this the from peptide bond hydrolysis is for peptide bond in the of post-translational peptide bond formation in the of by the K. Yang 2004; PubMed Scopus Google Scholar, N. V. J. A. S. P. T. Science. 2004; PubMed Scopus Google Scholar, M.A. V. A. Science. 2006; PubMed Scopus Google Scholar). is that cyclization of cyclotides the mechanism the of a single are We that in the and plants, this was to This more of the extended the C-terminal a accumulation of the longer The mechanism of processing that at the terminus of the cyclotide domain to produce the extended linear cyclization but to processing at the C-terminal Asn and cyclization. In cyclotides are a and family of plant proteins L. M.L. R. U. Craik D.J. Plant 2005; PubMed Scopus Google Scholar). We have that are in catalyzing cyclization in a post-translational that appears to the cleavage of an asparaginyl bond in a linear with peptide bond are to the AEP involved in cyclization from a cyclotide-containing The ability of an enzyme to catalyze the of reaction when with a a mechanism for the of circular proteins, by their compared with linear The of linear to cyclotides in Plant Physiol. 2005; PubMed Scopus Google Scholar, J.P. R. Anderson M.A. Craik D.J. Plant 2006; PubMed Scopus Google Scholar) suggests that that located residues are to the cyclization as to to the biochemical of the of a in T. T. C. J.A. Yang Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google and is to circular in J. K. Tran D. Science. 1999; PubMed Scopus Google Scholar) suggests that circular proteins are probably more protein with termini in is The current as the for applications of protein cyclization, with to the bioengineering of proteins of and We for with and for with