In vitro acellular method to reveal O-fucosylation on EGF-like domains.

ABSTRACT

A hundred of human proteins have one or more EGF-like domains (EGF-LD) bearing the O-fucosylation consensus motif C2X4(S/T)C3 but to date, only a few of them have been shown to be O-fucosylated. The protein O-fucosyltransferase (POFUT1) specifically recognizes correctly folded EGF-LD of the human EGF (hEGF) type and transfers fucose on serine or threonine residue within the O-fucosylation motif. Here, we propose a strategy for a rapid screening for ability of any EGF-LD to be O-fucosylated, using copper-catalyzed azide-alkyne cycloaddition (CuAAC). By an oligonucleotide hybridization approach, double-stranded fragments encoding any EGF-LD can be first rapidly cloned into the prokaryotic vector pET-25b to promote its targeting to periplasm and formation of the three conserved disulfide bonds. After protein production and purification, an in vitro POFUT1-mediated O-fucosylation can be performed with azido GDP-fucose. Successful transfer of O-fucose is finally revealed by blotting technique after CuAAC. In this study, we specially focused on mouse NOTCH1 EGF12 and EGF26, which are both known to be O-fucosylated although having different binding affinities towards POFUT1. Indeed, we clearly showed here that addition of O-fucose by POFUT1 was much more efficient for EGF26 than for EGF12. This experimental approach is rapid and sufficiently sensitive to reveal propensity of any EGF-LD to be O-fucosylated; it is thus useful prior to perform structure-function studies on target proteins containing one or several EGF-LD.