Comparative efficiencies of C-terminal signals of native glycophosphatidylinositol (GPI)-anchored proproteins in conferring GPI-anchoring

Every protein fated to receive the glycophosphatidylinositol (GPI) anchor p ost-translational modification has a C-terminal CPI-anchor attachment signa l sequence. This signal peptide varies with respect to length, content, and hydrophobicity. With the exception of predictions based on an upstream ami no acid triplet termed omega --> omega + 2 which designates the site of GPI uptake, there is no information on how the efficiencies of different nativ e signal sequences compare in the transamidation reaction that catalyzes th e substitution of the GPI anchor for the C-terminal peptide. In this study we utilized the placental alkaline phosphatase (PLAP) minigene, miniPLAP, a nd replaced its native 3' end-sequence encoding omega -2 to the C-terminus with the corresponding C-terminal sequences of nine other human GPI-anchore d proteins. The resulting chimeras then were fed into an in vitro processin g microsomal system where the cleavages leading to mature product from the nascent preproprotein could be followed by resolution on an SDS-PAGE system after immunoprecipitation. The results showed that the native signal of ea ch protein differed markedly with respect to transamidation efficiency, wit h the signals of three proteins out-performing the others in GPI-anchor add ition and those of two proteins being poorer substrates for the CPI transam idase. The data additionally indicated that the hierarchical order of effic iency of transamidation did not depend solely on the combination of permiss ible residues at omega --> omega + 2. (C) 2001 Wiley-Liss, Inc.