Ha Hyung Kim

Melanoma is a highly aggressive skin cancer with strong metastatic potential and frequent chemoresistance. Aberrant N-glycosylation plays a critical role in tumor progression and drug resistance; however, cisplatin-induced alterations in melanoma surface N-glycans remain poorly understood. Thus, this study aimed to perform comprehensive structural and quantitative profiling of surface N-glycans in G-361 (low metastatic potential), A375P (moderate), and SK-MEL-28 (high) melanoma cells following cisplatin treatment using LC–MS/MS. A total of 25, 39, and 28 surface N-glycans were identified in untreated G-361, A375P, and SK-MEL-28 cells, respectively, whereas 40, 37, and 40 N-glycans were detected after cisplatin treatment. Absolute quantification revealed a marked increase in total N-glycan abundance in G-361 (3.2 to 8.4 pmol) and A375P (5.7 to 10.9 pmol), whereas SK-MEL-28 maintained homeostasis (3.9 to 4.0 pmol). Surface N-glycans were predominantly high-mannose and complex-type glycans with extensive sialylation and fucosylation. Cisplatin induced distinct cell line-specific remodeling: G-361 accumulated high-mannose glycans with reduced Neu5Gc-containing sialylation; A375P showed increased glycan branching and Neu5Ac-containing sialylation; and SK-MEL-28 maintained a stable glycan distribution but exhibited increased Neu5Gc-containing sialylation. These results demonstrate adaptive N-glycosylation responses to cisplatin in melanoma cells with distinct metastatic potentials and provide a glycomic framework for understanding cisplatin-associated glycan alterations.

Cytotoxic T-lymphocyte-associated antigen-4 (CTLA4)-Ig is a fusion protein used to treat autoimmune diseases. In glycoproteins, sialic acids are linked to galactose via either α2,6 or α2,3 linkages, with the latter being more rigid and crucial for extending the serum half-life of biotherapeutics. However, site-specific identification of α2,6 and/or α2,3 linkages in glycoproteins remains a significant analytical challenge. In this study, we performed site-specific N-glycan analysis with sialyl-linkage isomers in a CTLA4-Ig mutant (CTLA4-Ig^MT) produced in Chinese hamster ovary (CHO) cells co-overexpressing N-acetylglucosaminyltransferase-IV and α2,6-sialyltransferase. Since CHO cells produce α2,3-linked glycoproteins, wild-type CTLA4-Ig (CTLA4-Ig^WT) was used as a control. Following Pronase digestion, N-glycopeptides were enriched and analyzed using nano-liquid chromatography-quadrupole-Orbitrap-tandem mass spectrometry. Retention time analysis revealed that α2,6-linked N-glycopeptides exhibit lower hydrophobicity than their α2,3-linked counterparts. Fragmentation analysis showed that fragment ion intensity ratios ([sialic acid]⁺/[N-acetylglucosamine]⁺) are lower for α2,6 linkages. To improve the accuracy, multiple linear regression was applied to adjust fragment ion intensities, deriving compensation coefficients for N-acetylglucosamine, galactose, and sialic acid. Using this approach, structural alterations in CTLA4-Ig^MT were identified, including 6 additionally generated, 11 partially converted, and 6 completely converted N-glycans, yielding an overall α2,6 linkage proportion of 56.7%. Notably, α2,6 linkages were identified at N76 and N108 in the CTLA4 region but were absent at N207 in the Fc region. This study is the first to determine α2,6 and/or α2,3 linkages at three N-glycosylation sites in CTLA4-Ig, providing a strategy for distinguishing sialyl-linkage isomers.

Yongwei Piao, So Yoon Yun, Zhicheng Fu, Ji Min Jang, Moon Jung Back, Ha Hyung Kim, and Dae Kyong Kim. Mol. Cells 2023;46:558-72. https://doi.org/10.14348/molcells.2023.0097