title: Identification of key residues and regions important for porcupine-mediated Wnt acylation.
abstract: Wnts comprise a family of lipid-modified, secreted signaling proteins that control embryogenesis, as well as tissue homeostasis in adults. Post-translational attachment of palmitoleate (C16:1) to a conserved Ser in Wnt proteins is catalyzed by Porcupine (Porcn), a member of the membrane bound O-acyltransferase (MBOAT) family, and is required for Wnt secretion and signaling. Moreover, genetic alterations in the PORCN gene lead to focal dermal hypoplasia, an X-linked developmental disorder. Despite its physiological importance, the biochemical mechanism governing Wnt acylation by Porcn is poorly understood. Here, we use a cell-based fatty acylation assay that is a direct readout of Porcn acyltransferase activity to perform structure-function analysis of highly conserved residues in Porcn and Wnt3a. In total, 16-point mutations in Porcn and 13 mutations in Wnt3a were generated and analyzed. We identified key residues within Porcn required for enzymatic activity, stability, and Wnt3a binding and mapped these active site residues to predicted transmembrane domain 9. Analysis of focal dermal hypoplasia-associated mutations in Porcn revealed that loss of enzymatic activity arises from altered stability. A consensus sequence within Wnt3a was identified (CXCHGXSXXCXXKXC) that contains residues that mediate Porcn binding, fatty acid transfer, and Wnt signaling. We also showed that Ser or Thr, but not Cys, can serve as a fatty acylation site in Wnt, establishing Porcn as an O-acyltransferase. This analysis sheds light into the mechanism by which Porcn transfers fatty acids to Wnt proteins and provides insight into the mechanisms of fatty acid transfer by MBOAT family members.
authors.affiliation: From the Cell Biology Program and the Gerstner Sloan-Kettering Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center, New York, New York 10065.
authors.forename: Jessica
authors.initials: J
authors.lastname: Rios-Esteves
authors.affiliation: From the Cell Biology Program and the Gerstner Sloan-Kettering Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center, New York, New York 10065.
authors.forename: Brittany
authors.initials: B
authors.lastname: Haugen
authors.affiliation: From the Cell Biology Program and the Gerstner Sloan-Kettering Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center, New York, New York 10065 reshm@mskcc.org.
authors.forename: Marilyn D
authors.initials: MD
authors.lastname: Resh
mesh_terms: D000215:Acylation; D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D020134:Catalytic Domain; D002522:Chlorocebus aethiops; D004795:Enzyme Stability; D005229:Fatty Acids, Monounsaturated / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D017354:Point Mutation; D011499:Protein Processing, Post-Translational; D060509:Wnt3A Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D005229:Fatty Acids, Monounsaturated; D008565:Membrane Proteins; D060509:Wnt3A Protein; C008757:palmitoleic acid; D000217:Acyltransferases; C491187:Porcn protein, mouse
keywords: Enzyme Mutation; Fatty Acid; Membrane Protein; Protein Palmitoylation; Wnt Signaling