Streptavidin-Conjugated C3 Protein Mediates the Delivery of Mono-Biotinylated RNAse A into Macrophages

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• 作者：Maren Lillich ; Xi Chen ; Tanja Weil ; Holger Barth ; J枚rg Fahrer
• 刊名：Bioconjugate Chemistry
• 出版年：2012
• 出版时间：July 18, 2012
• 年：2012
• 卷：23
• 期：7
• 页码：1426-1436
• 全文大小：431K
• 年卷期：v.23,no.7(July 18, 2012)
• ISSN：1520-4812

文摘

The C3 toxin produced by Clostridium botulinum (C3bot) catalyzes the mono-ADP-ribosylation of the small GTPases Rho A, B and C, resulting in their inactivation. Recently, a specific endocytotic uptake mechanism of C3bot was identified in macrophages and myeloid leukemia cells. Here, we present a novel delivery system based upon a mutant C3bot devoid of ADP-ribosylation activity (C3Mut) and wild-type streptavidin (Stv). The C3Mut moiety mediates endocytosis into macrophages, whereas Stv functions as an adaptor protein for attaching biotinylated molecules to facilitate their subsequent internalization. First, a bioconjugate consisting of recombinant C3Mut and Stv was generated via a thioether linkage that tightly interacted with biotinylated bovine serum albumin as demonstrated by dot blot analysis. We then showed the internalization of C3Mut-Stv into J774A.1 macrophages by confocal microscopy and observed translocation into the cytosol using cell fractionation. The C3Mut-Stv bioconjugate did not affect cell viability. Next, we prepared mono-biotinylated RNase A, which was attached to the C3Mut-Stv transporter, and demonstrated its C3Mut-Stv-mediated delivery into the cytosol of J774A.1 cells. Finally, C3Mut-Stv also promoted the efficient uptake of mono-biotinylated lysozyme into J774A.1 cells, highlighting its versatility. This study intriguingly demonstrates the use of the novel C3Mut-Stv delivery system for protein transduction and may provide a basis for future applications, in particular, of cytotoxic RNase A mutants.