Lectin-based glycoproteomics reveals a novel role of O-glycan truncation in the induction of melanogenesis

Glycosylation is implicated in melanogenesis; however, the precise molecular mechanisms involved remain largely unexplored. In this study, we aimed to elucidate the molecular mechanisms through which glycosylation regulates melanin production. Comparative lectin-based glycan profiling of high- and low-pigmented cells revealed that truncated O-glycans, particularly core 1 O-GalNAc structures (Galβ1-3GalNAcα-O-Ser/Thr) recognized by peanut agglutinin (PNA), were prominently expressed in highly pigmented cells. Gene expression analysis demonstrated a strong inverse correlation between melanin levels and the activity of the core-2 β1,6-N-acetylglucosaminyltransferase-1 (C2GnT1), a key enzyme involved in O-glycan elongation. Suppression of C2GnT1 by specific siRNA or inhibition with benzyl-α-GalNAc (BAG) significantly enhanced melanin production through the induction of O-glycan truncation. Global proteomic analysis identified approximately 8,200 proteins in BAG-treated melanoma cells, among which 759 proteins were significantly upregulated relative to untreated cells, including many membrane-associated endoplasmic reticulum (ER)-associated proteins involved in pigment metabolic processes. PNA lectin affinity-enriched glycoproteomics further identified 422 HexNAc(1)Hex(1)-modified glycopeptides increased following BAG treatment, including 13 membrane glycoproteins potentially associated with melanogenic regulation. Among these, the chloride channel protein CLCC1 emerged as a candidate regulator. CLCC1 knockdown enhanced melanin production and induced endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) production. Similarly, BAG treatment could induce O-glycan truncation of CLCC1 and also promote ER stress and ROS production in melanoma cells, suggesting that CLCC1 glycosylation contributes to melanogenesis regulation. Taken together, our findings demonstrate a novel role for O-glycan truncation in melanogenesis, partly through ER homeostasis-associated pathways involving CLCC1 and potentially other O-glycosylated proteins.