Biosynthesis of circular proteins in plants

Backbone cyclisation has the potential to improve the thermodynamic and biochemical stability of proteins. The discovery of backbone cyclised (circular) proteins in nature has therefore generated a great deal of interest, both in understanding the basic biology of the cyclisation process and in its prospective application to bio-engineering. Naturally occurring circular proteins have now been identified in bacteria, plants and animals but their biosynthesis remains poorly understood, especially in eukaryotes. The broad aim of this study was to gain insight into natural mechanisms of cyclisation by examining the biosynthesis of the cyclotides, a large family of plant-derived circular proteins. The cyclotides are topologically complex circular proteins produced by plants from the Rubiaceae and Violaceae families. To date over 100 cyclotide sequences have been identified, but their total number is predicted to be in the thousands. Structurally, cyclotides are characterised by the combination of a cyclic backbone and a cystine knot arrangement of their three disulfide bonds. This framework is exceptionally stable and allows cyclotides to tolerate thermal, chemical and biological conditions that would degrade most linear proteins. The compact structure of cyclotides, while very stable, complicates detection of the proteins and has impeded efforts to characterise cyclotide expression levels in plants. As a result, in this project an alternative method for the detection of cyclotides was developed using MALDI-TOF mass spectrometry. The method facilitates both the detection and quantification of cyclotides in heterogenous mixtures, and challenges current assumptions that MALDI-TOF mass spectrometry is exclusively a qualitative technique. The analysis of cyclotides was also augmented by the development of a transient assay for the expression of cyclotide genes in Nicotiana benthamiana, a plant which does not endogenously produce cyclotides. Cyclotides are expressed as part of linear precursor proteins from which they must be excised before cyclisation can take place. Based on sequence conservation, processing at the N-terminus of the cyclotide domain does not appear to be specific but at the C-terminus occurs almost exclusively following an Asn or Asp residue. A major watershed in understanding this process was the finding presented that plants which do not naturally contain cyclotides are capable of producing the circular proteins when supplied with cyclotide precursor genes. This result indicates that the biochemical machinery necessary for protein cyclisation is not unique to cyclotide containing plants, but is common to plants in general. The production of cyclotides was less efficient in the transgenic system, leading to the production of linear cyclotide forms not characteristically observed in cyclotide containing plants. An analysis of the evolution of the linear species over time revealed an aberrant C-terminal processing mechanism that competed with the cyclisation process. This showed that the substrate for cyclisation was an extended linear cyclotide including part of the C-terminal tail and indicated that cleavage of the asparaginyl bond at the C-terminus of the cyclotide domain occurred together with cyclisation. Subsequent work was therefore directed towards identification of an enzyme capable of cleaving asparaginyl bonds. Only one class of enzymes, the legumains, are known to specifically hydrolyse asparaginyl, and occasionally aspartyl, bonds. These enzymes are common in plants where they are alternatively called asparaginyl endopeptidases and vacuolar processing enzymes (VPE). In vitro studies demonstrated that VPE activity was responsible for asparaginyl bond hydrolysis in cyclotide containing plants and was also capable of cleaving synthetic cyclotide substrates at the C-terminal asparaginyl bond of the cyclotide domain. The C-terminal Asn residue was not targeted when part of a full length precursor protein, suggesting that VPE activity was limited to the late stages of processing. Two approaches were used to assess the effects of decreased VPE activity on cyclotide biosynthesis in vivo. Both the application of a VPE inhibitor directly into plant leaves and virus induced gene silencing of VPEs caused a decrease in the levels of circular protein produced upon expression of the cyclotide precursor in N. benthamiana. The decease in VPE activity also correlated with an increase in the linear cyclotide forms, consistent with previous findings that cyclisation occurs together with cleavage of an extended linear substrate. The identification of a VPE from a cyclotide containing plant was the first step towards characterising this mechanism. Further inhibitor studies pointed towards the involvement of a cysteine protease in processing upstream of the cyclotide domain and subsequent non specific trimming to the N-terminal cleavage site. In summary this study has provided important insights into the processing and cyclisation of cyclotides in plants. A mechanism for backbone cyclisation is proposed in which cleavage at the C-terminal asparaginyl bond of the cyclotide domain is coupled to cyclisation in a transpeptidation reaction catalysed by a VPE.