Dataset Information

N-glycosylation of AXL receptor tyrosine kinase regulates its stability, phosphorylation and oncogenic function

ABSTRACT: AXL, a receptor tyrosine kinase implicated in tumor progression, undergoes post-translational modifications that regulate its activity and stability. In this study, we have analyzed AXL immunoprecipitated from cell lines by liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based glycoproteomics and show that AXL is extensively N-glycosylated in breast and ovarian cancer cells, existing predominantly as two isoforms corresponding to high-mannose and complex-type glycans. The extracellular cleaved soluble form of AXL (sAXL) primarily carries complex N-glycans. LC-MS/MS–based glycoproteomics analysis identified five glycosylation sites (Asn43, Asn157, Asn198, Asn339, and Asn345) with extensive microheterogeneity, including sialylation, fucosylation, and bisecting GlcNAc modifications. Overall, our results demonstrate that N-glycosylation is essential for AXL stability, localization, and oncogenic signaling, offering new insights into how glycosylation regulates receptor tyrosine kinases function in cancer.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Xinyan Wu

LAB HEAD: Xinyan Wu

PROVIDER: PXD071027 | Pride | 2026-06-15

REPOSITORIES: Pride

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Publications

N-Glycosylation of AXL Receptor Tyrosine Kinase Regulates Its Stability, Phosphorylation, and Oncogenic Function.

Wang Li L Li Lingrui L Wang Jidong J Garapati Kishore K Li Feifei F Khan Md Kamrul Hasan MKH Kong Xiangyi X Bakshi Hamid H Jiang Dan D Zheng Shiya S Chen Lifeng L Yang Jasper J Hou Xiaonan X Kaufmann Scott H SH Weroha S John SJ Wang Jing J Pandey Akhilesh A Wu Xinyan X

Molecular & cellular proteomics : MCP 20260427 6

AXL, a receptor tyrosine kinase implicated in tumor progression, undergoes post-translational modifications that regulate its activity and stability. In this study, we demonstrated that AXL is extensively N-glycosylated in breast and ovarian cancer cells, existing predominantly as two isoforms corresponding to high-mannose and complex-type glycans. The extracellular cleaved soluble form of AXL (sAXL) primarily carries complex N-glycans and relies on glycosylation maturation. Functional analyses ...[more]

PMID: 42055283

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Project description:Recent advances in software-driven glycopeptide identification in LC-MS/MS-based N-glycoproteomics have facilitated biochemical studies reporting thousands of intact N-glycopeptides, i.e. N-glycan-conjugated peptides, but the automated identification process remains to be scrutinized. Herein, we explore the efficiency of site-specific glycoprofiling using the PTM-centric search-engine Byonic relative to manual expert annotation. To allow an appropriately deep comparison, the study utilised typical glycoproteomics acquisition and data analysis strategies, but of a single glycoprotein, the uncharacterised N-glycosylated (Asn160, Asn268 and Asn302) human basigin. Detailed site-specific reference glycoprofiles of purified basigin were manually established using ion trap CID-MS/MS and high-resolution Q-Exactive Orbitrap HCD-MS/MS acquisition of tryptic N-glycopeptides and released N-glycans. The basigin N-glycosylation sites, which showed extensive micro- and macro-heterogeneity, were then glycoprofiled using Byonic with or without a background of complex peptides using Q-Exactive Orbitrap HCD-MS/MS data. The glycoprofiling efficiencies were assessed against the site-specific reference glycoprofiles and target and decoy proteome databases. The search criteria and confidence thresholds (Byonic scores) recommended by the vendor provided very high glycoprofiling accuracy and coverage (both >80%) and low peptide FDRs (<1%). The data complexity, search parameters including search space (proteome/glycome size), mass tolerance and peptide modifications, and confidence thresholds affected the glycoprofiling efficiency and analysis time. Automated identification of peptide modifications (methionine oxidation/carbamidomethylation) that coincide with monosaccharide mass differences (Fuc/Hex/HexNAc) and accurately distinguishing isobaric (Hex1NeuAc1-R/Fuc1NeuGc1-R) or near-isobaric (NeuAc1-R/Fuc2-R) monosaccharide sub-compositions remain challenging, arguing particular attention to such “difficult-to-identify” N-glycopeptides. The presented analysis provides valuable insights into automated glycopeptide identification; knowledge that facilitates further developments in FDR-based glycoproteomics.

			Action	DRS
	1_Glu_Try.raw	Raw
	2_Chy_Tryp.raw	Raw
	AXL-GluC.raw	Raw
	AXL-LysC.raw	Raw
	AXL_AspN.pdResult	Other