Identification of C-mannosylation in a receptor tyrosine kinase AXL

Area of Science:

• Biochemistry
• Molecular Biology
• Cancer Research

Background:

• C-mannosylation is a rare post-translational modification where mannose is attached to tryptophan residues in proteins.
• The biological roles of C-mannosylation remain largely unelucidated.
• AXL receptor tyrosine kinase is frequently overexpressed in various cancers, promoting tumor progression, invasion, drug resistance, and vasculogenic mimicry.

Purpose of the Study:

• To confirm C-mannosylation at Tryptophan 320 (Trp320) of AXL.
• To investigate the functional impact of AXL C-mannosylation on cancer cell behavior, specifically vasculogenic mimicry.
• To explore whether C-mannosylation affects AXL's downstream signaling pathways.

Main Methods:

• Mass spectrometry was employed to detect and confirm C-mannosylation of recombinant AXL.
• CRISPR/Cas9 gene editing was used to generate AXL-deficient breast cancer cells (MDA-MB-231).
• Re-expression of wild-type and C-mannosylation-deficient AXL mutants was performed in these engineered cells.

Main Results:

• C-mannosylation at Trp320 of AXL was experimentally confirmed using mass spectrometry analysis of purified recombinant AXL.
• Re-expression of C-mannosylation-deficient AXL led to a significant reduction in vasculogenic mimicry formation in MDA-MB-231 cells.
• Phosphorylation levels of AKT, a key downstream signaling molecule, were similar between cells expressing wild-type AXL and C-mannosylation-deficient AXL.

Conclusions:

• This study provides the first evidence of C-mannosylation occurring on a receptor tyrosine kinase, specifically AXL.
• AXL C-mannosylation appears to play a role in regulating AXL-mediated vasculogenic mimicry formation, independent of canonical AKT signaling.
• These findings suggest a novel mechanism by which C-mannosylation influences receptor tyrosine kinase function in cancer progression.