Identification of Key Residues and Regions Important for Porcupine-mediated Wnt Acylation*

By Jessica Rios-Esteves, Brittany K. Haugen, M. Resh. 2014

75 citations recorded by the source corpus.

Background: The acyltransferase Porcupine transfers a monounsaturated fatty acid, palmitoleate, to Wnt proteins. Results: Structure-function analysis of Wnt3a and Porcupine identified key regions necessary for stability, activity, and signaling. Conclusion: Residues in predicted transmembrane domain 9 within Porcupine and surrounding Ser-209 in Wnt3a are essential for palmitoleoylation. Significance: This analysis provides insight into mechanisms operative within the membrane-bound O-acyltransferase family. 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.

Subfields: Molecular Biology

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In Molecular Biology

1. Hans Clevers
2. R. Nusse
3. M. Resh
4. R. Hendrickson
5. Matthew M. Miele
6. J. Asciolla
7. Chul-Jin Lee
8. A. Banerjee

Citations in the corpus

• Notum is required for neural and head induction via Wnt deacylation, oxidation and inactivation (2015, unknown): “This form of O-acylation, which has been best demonstrated for themouse Wnt3a, conjugates a mono-unsaturated palmitoleic acid onto the hydroxyl group of a conserved serine residue (serine 209 of Wnt3a), likely through the action of a Wnt-specific O-acyltransferase called Porcupine in the ER (Rios-Es”
• In vitro reconstitution of Wnt acylation reveals structural determinants of substrate recognition by the acyltransferase human Porcupine (2018, background): “In contrast, N301A and H336A mutants of hPORCN, which are thought to be catalytically deficient mutants (4, 22, 24), showed significantly decreased enzymatic activities (Fig.”
• Structural model of PORCN illuminates disease-associated variants and drug binding sites (2021, methodology): “However, mutation of Asn306 has no effect on the catalytic activity of PORCN in cell-based assays (Proffitt and Virshup, 2012; Rios-Esteves et al., 2014) but markedly decreased activity in a purified system where only PAM-CoA was available as a”
• An in vitro fatty acylation assay reveals a mechanism for Wnt recognition by the acyltransferase Porcupine (2017, methodology): “Several of the reported FDH-associated missense mutations in human PORCN have been analyzed using a cell-based Wnt acylation assay, and have been shown to exhibit decreased acylation activity as well as decreased protein stability (15).”
• Wnt/ b -Catenin Signaling, Disease, and Emerging Therapeutic Modalities (2017, background): “During synthesis, Wnt proteins, 40 kDa in size and rich in cysteines, are modified by attachment of a lipid, an acyl group termed palmitoleic acid (Willert et al., 2003; Rios-Esteves et al., 2014; Takada et al., 2006; Rios-Esteves andResh, 2013).”