Identification of key residues and regions important for porcupine-mediated Wnt acylation
| Autor: | Jessica Rios-Esteves, Marilyn D. Resh, Brittany Haugen |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
Acylation
Amino Acid Motifs Molecular Sequence Data MBOAT Biology Biochemistry Fatty Acids Monounsaturated Mice Catalytic Domain Wnt3A Protein Membrane Biology Chlorocebus aethiops Enzyme Stability Animals Humans Point Mutation Protein palmitoylation Amino Acid Sequence Molecular Biology Tissue homeostasis Wnt signaling pathway Membrane Proteins Cell Biology PORCN HEK293 Cells Membrane protein COS Cells Fatty acylation Protein Processing Post-Translational Acyltransferases |
| Zdroj: | The Journal of biological chemistry. 289(24) |
| ISSN: | 1083-351X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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