MOD_WntLipid

The protein Wingless (Wg) is the archetype of the Wnt protein family (Cadigan,1997, Polakis,2000). Drosophila has several Wnt paralogues and vertebrates have large Wnt multigene families. Wnt proteins are secreted developmental regulators that bind to cell surface receptor complexes of the Frizzled family, LRP5/6 and Kremen1/2 (Pandur,2001, Mao,2002). Intracellular signalling by Frizzled receptors mainly occurs through the beta-catenin pathway. Wnt is glycosylated at conserved sites (Blasband,1992), which is absolutely required for binding to its receptor. Processing of Wnt in the Endoplasmic Reticulum is unusually slow so that secretion is "inefficient". The Drosophila protein Porcupine influences Wingless processing in the ER, so that porc mutant cells retain Wingless (Kadowaki,1996). A clue to the role of Porcupine was provided by the finding that it belongs to a family of transmembrane acyltransferase enzymes (Hofmann,2000). It has now been shown by mass spectrometry that secreted mouse Wnt3a and Drosophila Wnt8, when overexpressed, are partially palmitoylated on a conserved Cys residue: C77 and C51, respectively (Willert,2003). The region of interaction with Porcupine has been localised to a 23-residue peptide segment spanning this residue and Porcupine strongly facilitates Wnt glycosylation (Tanaka,2002). It is reasonable to infer that Porcupine is the acyltransferase.
Why is Wnt palmitoylated? One of the conserved N-glycosylation sites (MOD_N-GLC_1 and MOD_N-GLC_2) has the sequence NCS, while the second conserved site is CNxS, where the conserved Cys residues will form disulphide bonds, quite possibly with each other. Glycosylation occurs on exposed peptide segments, hence a disulphide-linked moiety will be unavailable for glycosylation. For Wnts, there must be a mechanism to prevent disulphide formation until glycosylation is complete. The palmitoylated Cys precedes the NCS glycosylated Asn by 11 residues. Palmitoylation will have two effects: (i) preventing Wnt from completing folding into the native structure, and (ii) targeting Wnt to the ER membrane where glycosyltransferase enzymes reside. After Wnt glycosylation, further processing should include deacylation to allow disulphide formation and folding to complete. Secretion of palmitoylated Wnt may be an artefact of overexpression, overloading the processing machinery. Mass spectrometry should then reveal that fully processed natural Wnt is not palmitoylated. Candidates for the deacylase may be the Palmitoyl protein thioesterases (PPT1/2), depalmitoylating enzymes found predomininantly in lysosomes but also in other compartments such as neuronal synaptosomes (Ahtiainen,2003). It cannot yet be excluded that secreted palmitoylated Wnt is indeed functional. Weak, indirect evidence is suggested by domains (SM00192; PF00057) found in LRP5/6 that are also found in LDL receptors, while the Dickkopf family of Wingless inhibitors have a domain that is related to colipases (Aravind,1998, Hofmann,2000), however current evidence indicates that Dickkopf proteins do not physically interact with Wnt itself. This emerging and tentative scheme needs further testing. The whole system seems unnecessarily complicated, unless it is part of a regulated mechanism used to modulate levels of Wnt secretion during development. Most palmitoylated proteins are intracellular but a few other extracellular proteins are found to be palmitoylated, including glypicans (Edgren,1997), surfactant protein C (ten Brinke,2001) and Apolipoprotein B (Zhao,2000). Exported signalling proteins of the hedgehog family are also palmitoylated (Pepinsky,1998, Chamoun,2001).