Comprehensive Membrane N-Glycoproteomics Using Human Breast Cancer Cell Line Pairs

Abstract

Aberrant glycosylation of membrane proteins is a hallmark of cancer and a useful molecular marker for the diagnosis of breast cancer (BC). However, the molecular mechanisms by which altered glycosylation affects the malignant transformations associated with BC are poorly understood. Accordingly, we performed comparative membrane N-glycoproteomics using the human BC cell line pair, Hs578T, and its syngeneic normal cell line, Hs578Bst. A total of 359 N-glycoforms derived from 113 proteins were identified in both cell lines, of which 27 were found only in Hs578T cells. Significant changes in N-glycosylation were found in the lysosome-associated membrane protein 1 (LAMP1), the integrin family, and laminin. Confocal immunofluorescence microscopy images revealed the accumulation of lysosomes in the perinuclear space in cancer cells, which could be associated with marked changes in LAMP1 glycosylation, such as a decreased level of polylactosamine chains. Overall, the alterations in glycosylation may be involved in changes in the adhesion and degradation of BC cells.

Keywords: LC/MS/MS; N-glycoproteomics; glycosylation; membrane glycoproteins.

Fig. 1. Schematic workflow of N-glycoproteomics.

Fig. 2. Qualitative comparison of the N-glycopeptides identified by LC/MS/MS.

Fig. 3. Potential ability of the N-glycoproteins to distinguish between BC and control cell samples.

Fig. 4. Illustration of the PPI network of the 27 glycoproteins with site specific glycosylation changes.

Fig. 5. Changes in site-specific glycosylation in BC cells.

Fig. 6. Subcellular localization analysis of LAMP1 based on immunofluorescence staining in BC and control cells.