Wnt3 Is Lipidated at Conserved Cysteine and Serine Residues in Zebrafish Neural Tissue

Abstract

Wnt proteins are a family of hydrophobic cysteine-rich secreted glycoproteins that regulate a gamut of physiological processes involved in embryonic development and tissue homeostasis. Wnt ligands are post-translationally lipidated in the endoplasmic reticulum (ER), a step essential for its membrane targeting, association with lipid domains, secretion and interaction with receptors. However, at which residue(s) Wnts are lipidated remains an open question. Initially it was proposed that Wnts are lipid-modified at their conserved cysteine and serine residues (C77 and S209 in mWnt3a), and mutations in either residue impedes its secretion and activity. Conversely, some studies suggested that serine is the only lipidated residue in Wnts, and substitution of serine with alanine leads to retention of Wnts in the ER. In this work, we investigate whether in zebrafish neural tissues Wnt3 is lipidated at one or both conserved residues. To this end, we substitute the homologous cysteine and serine residues of zebrafish Wnt3 with alanine (C80A and S212A) and investigate their influence on Wnt3 membrane organization, secretion, interaction and signaling activity. Collectively, our results indicate that Wnt3 is lipid modified at its C80 and S212 residues. Further, we find that lipid addition at either C80 or S212 is sufficient for its secretion and membrane organization, while the lipid modification at S212 is indispensable for receptor interaction and signaling.

Microscope Alignment Using Real-Time Imaging FCS

“…The light sheet microscope used in this work is a home-built, cylindrical lens-based selective plane illumination microscope (SPIM) system with continuous illumination and detection as reported earlier [18, 19, 7, 20, 21]. We employed an identical setup according to our latest report [21]. Illumination was achieved by a 488 nm laser (06-MLD, Cobolt) coupled into a single-mode optical fibre (kineFLEX, Qioptiq, USA) which expands the beam 1.3 times, sufficient to fill the back aperture of the illumination objective.…”

“…The light sheet microscope used in this work is a home-built, cylindrical lens-based selective plane illumination microscope (SPIM) system with continuous illumination and detection as reported earlier [18, 19, 7, 20, 21]. We employed an identical setup according to our latest report [21].…”

Corrigendum: WNT5B in Physiology and Disease

“…All the cysteines in Wnt are engaged in disulfide bonds (DOI 10.1126/science.1222879, 10.1074/jbc.m114.575027). However, a new reference describes WNT palmitoylation in zebrafish WNT3A (Dhasmana et al, 2021).…”

“…Mammalian WNT proteins are palmitoylated at conserved serine residues by a special palmitoyl transferase, Porcupine (PORCN), in the endoplasmic reticulum (Takada et al, 2006;Galli et al, 2007;Rios-Esteves et al, 2014). Zebrafish WNT3 is lipidated at both cysteine and serine residues (Dhasmana et al, 2021). The activity of PORCN is essential for the secretion of WNT ligands.…”

“…Then, the seven-transmembrane protein Wntless/Evi (Wls) in the endoplasmic reticulum escorts mature hydrophobic WNT proteins to be secreted at the plasma membrane or released in exosomes, leading to both autocrine and paracrine effects (Banziger et al, 2006;Routledge and Scholpp, 2019). Accordingly, the following reference has been added to the original article: Dhasmana, D., Veerapathiran, S., Azbazdar, Y., Nelanuthala, A. V. S., Teh, C., Ozhan, G., et al (2021). Wnt3 is lipidated at conserved cysteine and serine residues in zebrafish neural tissue.…”