{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,22]],"date-time":"2026-02-22T06:39:27Z","timestamp":1771742367498,"version":"3.50.1"},"reference-count":50,"publisher":"Wiley","issue":"22","license":[{"start":{"date-parts":[[2023,8,18]],"date-time":"2023-08-18T00:00:00Z","timestamp":1692316800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["JP18K06137"],"award-info":[{"award-number":["JP18K06137"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100009578","name":"Mizutani Foundation for Glycoscience","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100009578","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["febs.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["The FEBS Journal"],"published-print":{"date-parts":[[2023,11]]},"abstract":"<jats:p>Premelanosome protein (PMEL), a melanocyte\u2010specific glycoprotein, has an essential role in melanosome maturation, assembling amyloid fibrils for melanin deposition. PMEL undergoes several post\u2010translational modifications, including <jats:italic>N<\/jats:italic>\u2010 and <jats:italic>O<\/jats:italic>\u2010glycosylations, which are associated with proper melanosome development. <jats:italic>C<\/jats:italic>\u2010mannosylation is a rare type of protein glycosylation at a tryptophan residue that might regulate the secretion and localization of proteins. PMEL has one putative <jats:italic>C<\/jats:italic>\u2010mannosylation site in its core amyloid fragment (CAF); however, there is no report focusing on <jats:italic>C<\/jats:italic>\u2010mannosylation of PMEL. To investigate this, we expressed recombinant PMEL in SK\u2010MEL\u201028 human melanoma cells and purified the protein.\u00a0Mass spectrometry analyses demonstrated that human PMEL is <jats:italic>C<\/jats:italic>\u2010mannosylated at multiple tryptophan residues in its CAF and N\u2010terminal fragment (NTF). In addition to the W<jats:sup>153<\/jats:sup> or W<jats:sup>156<\/jats:sup> residue (CAF), which lies in the consensus sequence for <jats:italic>C<\/jats:italic>\u2010mannosylation, the W<jats:sup>104<\/jats:sup> residue (NTF) was <jats:italic>C<\/jats:italic>\u2010mannosylated without the consensus sequence. To determine the effects of the modifications, we deleted the <jats:italic>PMEL<\/jats:italic> gene by using CRISPR\/Cas9 technology and re\u2010expressed wild\u2010type or <jats:italic>C<\/jats:italic>\u2010mannosylation\u2010defective mutants of PMEL, in which the <jats:italic>C<\/jats:italic>\u2010mannosylated tryptophan was replaced with a phenylalanine residue (WF mutation), in SK\u2010MEL\u201028 cells. Importantly, fibril\u2010containing melanosomes were significantly decreased in W104F mutant PMEL\u2010re\u2010expressing cells compared with wild\u2010type PMEL, observed using transmission electron microscopy. Furthermore, western blot and immunofluorescence analysis suggested that the W104F mutation may cause mild endoplasmic reticulumretention, possibly associated with early misfolding, and lysosomal misaggregation, thus reducing functional fibril formation. Our results demonstrate that <jats:italic>C<\/jats:italic>\u2010mannosylation of PMEL is required for proper melanosome development by regulating PMEL\u2010derived fibril formation.<\/jats:p>","DOI":"10.1111\/febs.16927","type":"journal-article","created":{"date-parts":[[2023,8,8]],"date-time":"2023-08-08T15:32:18Z","timestamp":1691508738000},"page":"5373-5394","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Biogenesis of fibrils requires <i>C<\/i>\u2010mannosylation of <scp>PMEL<\/scp>"],"prefix":"10.1111","volume":"290","author":[{"given":"Ryota","family":"Kawahara","sequence":"first","affiliation":[{"name":"Department of Applied Chemistry, Faculty of Science and Technology Keio University  Yokohama Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tomoko","family":"Usami","sequence":"additional","affiliation":[{"name":"Department of Applied Chemistry, Faculty of Science and Technology Keio 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