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Expanding the diversity of chemical protein modification allows post-translational mimicry

Abstract

One of the most important current scientific paradoxes is the economy with which nature uses genes. In all higher animals studied, we have found many fewer genes than we would have previously expected. The functional outputs of the eventual products of genes seem to be far more complex than the more restricted blueprint. In higher organisms, the functions of many proteins are modulated by post-translational modifications (PTMs)1. These alterations of amino-acid side chains lead to higher structural and functional protein diversity and are, therefore, a leading contender for an explanation for this seeming incongruity. Natural protein production methods typically produce PTM mixtures within which function is difficult to dissect or control. Until now it has not been possible to access pure mimics of complex PTMs. Here we report a chemical tagging approach that enables the attachment of multiple modifications to bacterially expressed (bare) protein scaffolds: this approach allows reconstitution of functionally effective mimics of higher organism PTMs. By attaching appropriate modifications at suitable distances in the widely-used LacZ reporter enzyme scaffold, we created protein probes that included sensitive systems for detection of mammalian brain inflammation and disease. Through target synthesis of the desired modification, chemistry provides a structural precision and an ability to retool with a chosen PTM in a manner not available to other approaches. In this way, combining chemical control of PTM with readily available protein scaffolds provides a systematic platform for creating probes of protein–PTM interactions. We therefore anticipate that this ability to build model systems2 will allow some of this gene product complexity to be dissected, with the aim of eventually being able to completely duplicate the patterns of a particular protein’s PTMs from an in vivo assay into an in vitro system.

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Figure 1: Differential multi-site chemical protein modification.
Figure 2: Creating functional mimics of PSGL-1.
Figure 3: Binding of mimics to human P-selectin.
Figure 4: Use of mimics PSGL-LacZ, PSGL*-LacZ and GlcNAc-LacZ as probes.

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Acknowledgements

We thank Glycoform (Studentship to S.I.v.K., H.B.K.) and the Danish National Research Science Foundation (Fellowship to H.H.J.) for financial support; K. Drickamer for supply of DC-SIGN-R2 plasmid; and G. Bernardes, I. Davies, P. Wilainam, O. Pearce, K. Doores, A. French, T. P. Hughes, J. Errey, M. Squire, D. Gamblin and E. Scanlan for technical assistance.

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The work described has been patented by the University of Oxford with a view to commercial exploitation. If licensed, this affords royalties in line with standard University practice..

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This file contains Supplementary Figures S1-S7 and Legends, Supplementary Methods, Supplementary Tables S1-S3 and Legends, Supplementary Results, Supplementary Discussion, Supplementary Notes and additional references. (PDF 6287 kb)

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van Kasteren, S., Kramer, H., Jensen, H. et al. Expanding the diversity of chemical protein modification allows post-translational mimicry. Nature 446, 1105–1109 (2007). https://doi.org/10.1038/nature05757

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