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 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.

Risk factors for cholecystitis after stent placement in patients with distal malignant biliary obstruction.

BACKGROUND /PURPOSE: Limited data are available for acute cholecystitis after Self-Expandable Metallic Stent (SEMS) placement in patients with malignant distal biliary obstruction. We aimed to identify risk factors for cholecystitis. METHODS: This was a retrospective, single-center study of 280 patients (336 stents) who received endoscopic SEMS placement between May 2005 and April 2016. Clinical records were used to perform risk factor analyses. RESULTS: Of 336 SEMS placement procedures, 25 (7.4%) led to development of cholecystitis. Logistic regression analysis revealed three independent risk factors: covered SEMS (P = .014), tumor involvement to the cystic duct (P = .017), and presence of gallstones (P = .022). Median time to cholecystitis onset was shorter with covered SEMS than with uncovered SEMS (P = .034), and in patients with pancreatic cancer compared to those with other cancers (P = .001). Severe cholecystitis developed within 30 days after covered SEMS placement in three patients with pancreatic cancer without tumor involvement to the cystic duct. CONCLUSIONS: Use of covered SEMS might be a risk factor for cholecystitis onset within 30 days after placement. Clinicians should be aware of the risk for severe cholecystitis after covered SEMS placement, even if the tumor does not invade the cystic duct.