RRC ID 77093
Author Kawahara R, Usami T, Arakawa S, Kamo H, Suzuki T, Komatsu R, Hara H, Niwa Y, Shimizu E, Dohmae N, Shimizu S, Simizu S.
Title Biogenesis of fibrils requires C-mannosylation of PMEL.
Journal FEBS J
Abstract Premelanosome protein (PMEL), a melanocyte-specific glycoprotein, has an essential role in melanosome maturation, assembling amyloid fibrils for melanin deposition. PMEL undergoes several post-translational modifications, including N- and O-glycosylations, which are associated with proper melanosome development. C-mannosylation is a rare type of protein glycosylation at a tryptophan residue that might regulate the secretion and localization of proteins. PMEL has one putative C-mannosylation site in its core amyloid fragment (CAF); however, there is no report focusing on C-mannosylation of PMEL. To investigate this, we expressed recombinant PMEL in SK-MEL-28 human melanoma cells and purified the protein. Mass spectrometry analyses demonstrated that human PMEL is C-mannosylated at multiple tryptophan residues in its CAF and N-terminal fragment (NTF). In addition to the W153 or W156 residue (CAF), which lies in the consensus sequence for C-mannosylation, the W104 residue (NTF) was C-mannosylated without the consensus sequence. To determine the effects of the modifications, we deleted the PMEL gene by using CRISPR/Cas9 technology and re-expressed wild-type or C-mannosylation-defective mutants of PMEL, in which the C-mannosylated tryptophan was replaced with a phenylalanine residue (WF mutation), in SK-MEL-28 cells. Importantly, fibril-containing melanosomes were significantly decreased in W104F mutant PMEL-re-expressing cells compared with wild-type 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 C-mannosylation of PMEL is required for proper melanosome development by regulating PMEL-derived fibril formation.
Published 2023-8-8
DOI 10.1111/febs.16927
PMID 37552474
MeSH Amyloid* / chemistry Amyloidogenic Proteins / metabolism Glycoproteins / genetics Glycoproteins / metabolism Glycosylation Humans Melanosomes / genetics Melanosomes / metabolism Tryptophan* / genetics Tryptophan* / metabolism gp100 Melanoma Antigen / chemistry gp100 Melanoma Antigen / genetics gp100 Melanoma Antigen / metabolism
IF 4.392
DNA material CSII-CMV-MCS-IRES2-Bsd(RDB04385)
Human and Animal Cells MMAc(RCB0808) SK-MEL-28(RCB1930) 293T(RCB2202)