Biogenesis of fibrils requires C-mannosylation of PMEL

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 W-153 or W-156 residue (CAF), which lies in the consensus sequence for C-mannosylation, the W-104 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.

Automated summary

PMEL is a melanocyte-specific glycoprotein involved in melanosome maturation. It undergoes C-mannosylation at multiple tryptophan residues in its core amyloid fragment and N-terminal fragment. The C-mannosylation of PMEL is required for proper melanosome development and fibril formation.