Magdalena Surman1, Dorota Hoja-Łukowicz2, Sabina Szwed3, Anna Drożdż4, Ewa Stępień5, Małgorzata Przybyło6. 1. Department of Glycoconjugate Biochemistry, Institute of Zoology and Biomedical Research, Jagiellonian University in Kraków, ul. Gronostajowa 9, Kraków, Poland. Electronic address: magdalena.surman@doctoral.uj.edu.pl. 2. Department of Glycoconjugate Biochemistry, Institute of Zoology and Biomedical Research, Jagiellonian University in Kraków, ul. Gronostajowa 9, Kraków, Poland. Electronic address: dorota.hoja-lukowicz@uj.edu.pl. 3. Department of Glycoconjugate Biochemistry, Institute of Zoology and Biomedical Research, Jagiellonian University in Kraków, ul. Gronostajowa 9, Kraków, Poland. Electronic address: sabina.szwed@doctoral.uj.edu.pl. 4. Department of Medical Physics, M. Smoluchowski Institute of Physics, Jagiellonian University in Kraków, ul. Łojasiewicza 11, Kraków, Poland. Electronic address: anna.drozdz@uj.edu.pl. 5. Department of Medical Physics, M. Smoluchowski Institute of Physics, Jagiellonian University in Kraków, ul. Łojasiewicza 11, Kraków, Poland. Electronic address: e.stepien@uj.edu.pl. 6. Department of Glycoconjugate Biochemistry, Institute of Zoology and Biomedical Research, Jagiellonian University in Kraków, ul. Gronostajowa 9, Kraków, Poland. Electronic address: malgorzata.przybylo@uj.edu.pl.
Abstract
AIMS: Numerous studies confirmed the involvement of extracellular vesicles in cancer development and progression. The present study was designed to investigate the glycan composition of ectosomes derived by human cutaneous melanoma (CM) cell lines with the use of lectins. MAIN METHODS: Ectosomes released by primary (WM115, WM793) and metastatic (WM266-4, WM1205Lu) CM cells were isolated from conditioned media by sequential centrifugation. Proteins from ectosomes, the whole cell extracts and membrane fractions were probed with a panel of lectins using Western Blot and flow cytometry and compared in terms of disease stage and glycosignature. KEY FINDINGS: Ectosomal proteins revealed enrichment (mainly with fucose and complex type N-glycans with bisecting GlcNAc) or depletion of specific glycoepitopes in comparison to the parental cell membranes. Moreover, similar lectin binding patterns were observed between related cell lines. It is the first study to characterize the glycosylation of ectosome proteins released by CM cells. SIGNIFICANCE: Our data indirectly supports the findings that ectosomes derive from particular regions of the cell membrane contain a unique glycan composition, which could serve as a specific sorting signal. If proven correct, the hypothesis that glycan-based protein sorting is a major mechanism for protein incorporation into ectosomes may provide new means to control vesicular content and have possible clinical implications.
AIMS: Numerous studies confirmed the involvement of extracellular vesicles in cancer development and progression. The present study was designed to investigate the glycan composition of ectosomes derived by humancutaneous melanoma (CM) cell lines with the use of lectins. MAIN METHODS: Ectosomes released by primary (WM115, WM793) and metastatic (WM266-4, WM1205Lu) CM cells were isolated from conditioned media by sequential centrifugation. Proteins from ectosomes, the whole cell extracts and membrane fractions were probed with a panel of lectins using Western Blot and flow cytometry and compared in terms of disease stage and glycosignature. KEY FINDINGS: Ectosomal proteins revealed enrichment (mainly with fucose and complex type N-glycans with bisecting GlcNAc) or depletion of specific glycoepitopes in comparison to the parental cell membranes. Moreover, similar lectin binding patterns were observed between related cell lines. It is the first study to characterize the glycosylation of ectosome proteins released by CM cells. SIGNIFICANCE: Our data indirectly supports the findings that ectosomes derive from particular regions of the cell membrane contain a unique glycan composition, which could serve as a specific sorting signal. If proven correct, the hypothesis that glycan-based protein sorting is a major mechanism for protein incorporation into ectosomes may provide new means to control vesicular content and have possible clinical implications.