Abstract
Selective exo-enzymatic labeling (or SEEL) uses recombinant glycosyltransferases and nucleotide-sugar analogues to allow efficient labeling of cell surface glycans. SEEL can circumvent many of the possible issues associated with metabolic labeling, including low incorporation of sugar precursors, and allows for sugars to be added selectively to different types of glycans by virtue of the inherent specificity of the glycosyltransferases. Here we compare the labeling of sialoglycoproteins in undifferentiated and differentiated human erythroleukemia cells (HEL) using SEEL using the sialyltransferases ST6Gal1 and ST3Gal1, which label N- and O-glycans, respectively. Our results show that the profile of glycoproteins detected varies between undifferentiated HEL cells and those differentiated to megakaryocytes, with a shift to more N-linked sialoglycoproteins in the differentiated cells. The efficiency of SEEL for both sialyltransferases in HEL cells was greatly increased with prior neuraminidase treatment highlighting the necessity for the presence of available acceptors with this labeling method. Following metabolic labeling or SEEL, tagged glycoproteins were enriched by immunoprecipitation and identified using mass spectrometry. The proteomic findings demonstrated that the detection of many glycoproteins is markedly improved by SEEL labeling, and that unique glycoproteins can be identified using either ST6Gal1 or ST3Gal1. Furthermore, this analysis enabled the identification of increased surface expression of several sialylated cell adhesion molecules, including the known megakaryocytic markers integrinβ3 and CD44, upon differentiation of HEL cells to adherent megakaryocytes.
Highlights
The ability to label glycans using chemical glycobiology methods has ushered in new opportunities to investigate the functions of these molecules in living systems [1,2,3]
We demonstrate that labeling with different sialyltransferases produces distinct profiles of sialoglycoproteins in human erythroleukemia (HEL) cells, and that SEEL facilitates the detection of glycoproteins that are not readily visualized by metabolic labeling
Human erythroleukemia (HEL) cells grown in suspension can be differentiated to adherent megakaryocytes by phorbol 12-myristate 13-acetate (PMA) treatment, providing an opportunity to analyze how the profile of sialoglycoproteins changes upon differentiation with various labeling methods [13,14,15]
Summary
The ability to label glycans using chemical glycobiology methods has ushered in new opportunities to investigate the functions of these molecules in living systems [1,2,3]. Because of the more selective nature of SEEL-based labeling, we propose that this method would allow glycoproteins bearing specific types of glycans to be enriched and identified by proteomic analysis. Such an approach would provide an advantageous and complementary way to identify glycoproteins whose glycosylation is sensitive in the context of human diseases or upon differentiation to various cell lineages. We demonstrate that labeling with different sialyltransferases produces distinct profiles of sialoglycoproteins in HEL cells, and that SEEL facilitates the detection of glycoproteins that are not readily visualized by metabolic labeling. The potential of SEEL and neuraminidase-coupled SEEL to analyze the sialylation status and cell surface expression of sialoglycoproteins, respectively, is discussed
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