Functional Ubiquitin Conjugates with Lysine-ε-Amino-Specific Linkage by Thioether Ligation of Cysteinyl-Ubiquitin Peptide Building Blocks

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• 作者：Ji Eun Jung ; Hans-Peter Wollscheid ; Andreas Marquardt ; Marilena Manea ; Martin Scheffner ; Michael Przybylski
• 刊名：Bioconjugate Chemistry
• 出版年：2009
• 出版时间：June 17, 2009
• 年：2009
• 卷：20
• 期：6
• 页码：1152-1162
• 全文大小：386K
• 年卷期：v.20,no.6(June 17, 2009)
• ISSN：1520-4812

文摘

The modification of ubiquitin to defined oligo-ubiquitinated conjugates has received considerable interest due to the finding that isomeric oligo-ubiquitin conjugates exhibit distinct differences in their biochemical functions, depending on the specific lysine-ε-amino linkage used for conjugate formation. Here, we report the design and development of a thioether linkage-based approach for the synthesis of oligo-ubiquitin conjugates with lysine-specific branching by thioether ligation of a linear ubiquitin peptide containing a C-terminal cysteine residue as the “donor” component, with a corresponding lysine-ε-amino-branched haloacyl-activated ubiquitin “acceptor” peptide. This approach was successfully used for the synthesis of a lysine-63-linked diubiquitin conjugate by ligation of the modified ubiquitin(1−52)-Cys- donor peptide to the N-terminal Arg-54 residue of the branched Lys-63-linked acceptor peptide, ubiquitin(54−76)₂. Advantages of the present approach are as follows: (i) the conjugation reaction is performed in solution using suitable preformed donor ubiquitin peptides with a C-terminal Cys residue, and (ii) different corresponding N-chloroacetylated ubiquitin acceptor peptides containing the branched Lys residue are employed, providing broad applicability to the preparation of isomeric oligo-ubiquitin conjugates. The Lys-63-diubiquitin conjugate 7 described here was purified by semipreparative HPLC, and its structure and homogeneity ascertained by HPLC and high-resolution MALDI and electrospray-mass spectrometry. CD spectra and molecular modeling indicate a conformationally stable structure of the conjugate with spatial separation of the ubiquitin parts of the Lys-63 linkage. Moreover, the activity of the thioether-linked diubiquitin conjugate was ascertained by in vitro autoubiquitination assay. These results indicate the feasibility of this approach for the preparation of functional oligo-ubiquitin conjugates.