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Journal of Biochemistry Advance Access originally published online on March 15, 2008
Journal of Biochemistry 2008 144(1):39-49; doi:10.1093/jb/mvn039
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© 2008 The Japanese Biochemical Society

SUMO Assay with Peptide Arrays on Solid Support: Insights into SUMO Target Sites

Klaus Schwamborn1,*, Puck Knipscheer2,{dagger}, Evert van Dijk1, Willem J. van Dijk2, Titia K. Sixma2, Rob H. Meloen1,3 and Johannes P.M. Langedijk1

1Pepscan Therapeutics BV, Zuidersluisweg 2, 8243 RC Lelystad; 2Department of Molecular Carcinogenesis, the Netherlands Cancer Institute and Center for Biomedical Genetics, Plesmanlaan, Amsterdam; and 3Academic Biomedical Centre, University of Utrecht, Yalelaan 1, Utrecht, the Netherlands

*To whom correspondence should be addressed. Tel: +31 320 225300, Fax: +31 320 225301, E-mail: k.schwamborn{at}pepscan.com

Received February 11, 2008; Accepted March 10, 2008


  Abstract

The modification of proteins by SUMO (small ubiquitin-like modifier) regulates various cellular processes. Sumoylation often occurs on a specific lysine residue within the consensus motif {psi}KxE/D. However, little is known about the specificity and selectivity of SUMO target sites. We describe here a SUMO assay with peptide array on solid support for the simultaneous characterization of hundreds of different SUMO target sites. This approach was used to characterize known SUMO substrates. The position of the motif within the peptide and the amino acids flanking the acceptor site affected the efficiency of SUMO modification. Interestingly, a sequence of only four amino acids, corresponding to the SUMO consensus motif without flanking amino acids, was a bona fide target site. Analysis of a peptide library for all variants of the {psi}KxE/D consensus motif revealed that the first and third positions in the tetrapeptide preferably contain aromatic amino acid residues. Furthermore, by adding the SUMO E3 ligase PIAS1 to the reaction mixture, we show specific enhancement of the modification of a PIAS1-dependent SUMO substrate in this system. Overall, our results demonstrate that the sumoylation assay with peptide array on solid support can be used for the high-throughput characterization of SUMO target sites, and provide new insights into the composition, selectivity and specificity of SUMO target sites.

Key Words: assay, E3 ligase, peptide library, pepscan, SUMO (small ubiquitin-like modifier)

{dagger}Present address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.


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