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Quantitative investigation of human cell surface N-glycoprotein dynamics.

ABSTRACT: Surface glycoproteins regulate nearly every extracellular event and they are dynamic for cells to adapt to the ever-changing extracellular environment. These glycoproteins contain a wealth of information on cellular development and disease states, and have significant biomedical implications. Systematic investigation of surface glycoproteins will result in a better understanding of surface protein functions, cellular activities and the molecular mechanisms of disease. However, it is extraordinarily challenging to specifically and globally analyze surface glycoproteins. Here we designed the first method to systematically analyze surface glycoprotein dynamics and measure their half-lives by integrating pulse-chase labeling, selective enrichment of surface glycoproteins, and multiplexed proteomics. The current results clearly demonstrated that surface glycoproteins with catalytic activities were more stable than those with binding and receptor activities. Glycosylation sites located outside of any domain had a notably longer median half-life than those within domains, which strongly suggests that glycans within domains regulate protein interactions with other molecules while those outside of domains mainly play a role in protecting the protein from degradation. This method can be extensively applied to biological and biomedical research.

SUBMITTER: Xiao H

PROVIDER: S-EPMC5458730 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Quantitative investigation of human cell surface <i>N</i>-glycoprotein dynamics.

Xiao Haopeng H Wu Ronghu R

Chemical science 20160815 1

Surface glycoproteins regulate nearly every extracellular event and they are dynamic for cells to adapt to the ever-changing extracellular environment. These glycoproteins contain a wealth of information on cellular development and disease states, and have significant biomedical implications. Systematic investigation of surface glycoproteins will result in a better understanding of surface protein functions, cellular activities and the molecular mechanisms of disease. However, it is extraordinar ...[more]

PMID: 28616130

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Project description:Cell surface proteins have been shown to be effective therapeutic targets. In addition, shed forms of these proteins and secreted proteins can serve as biomarkers for diseases, including cancer. Thus, identification of cell surface and secreted proteins has been a prime area of interest in the proteomics field. Most cell surface and secreted proteins are known to be glycosylated, and therefore, a proteomics strategy targeting these proteins was applied to obtain proteomic profiles from various thyroid cancer cell lines that represent the range of thyroid cancers of follicular cell origin. In this study, we oxidized the carbohydrates of secreted proteins and those on the cell surface with periodate and isolated them via covalent coupling to hydrazide resin. The glycoproteins obtained were identified from tryptic peptides and N-linked glycopeptides released from the hydrazide resin using two-dimensional liquid chromatography-tandem mass spectrometry in combination with the gas phase fractionation. Thyroid cancer cell lines derived from papillary thyroid cancer (TPC-1), follicular thyroid cancer (FTC-133), Hurthle cell carcinoma (XTC-1), and anaplastic thyroid cancer (ARO and DRO-1) were evaluated. An average of 150 glycoproteins were identified per cell line, of which more than 57% are known cell surface or secreted glycoproteins. The usefulness of the approach for identifying thyroid cancer associated biomarkers was validated by the identification of glycoproteins (e.g., CD44, galectin 3 and metalloproteinase inhibitor 1) that have been found to be useful markers for thyroid cancer. In addition to glycoproteins that are commonly expressed by all of the cell lines, we identified others that are only expressed in the more well-differentiated thyroid cancer cell lines (follicular, Hurthle cell and papillary), or by cell lines derived from undifferentiated tumors that are uniformly fatal forms of thyroid cancer (i.e., anaplastic). On the basis of the results obtained, a set of glycoprotein biomarker candidates for thyroid cancer is proposed.

Project description:The plasma membrane proteome plays a crucial role in inter- and intracellular signaling, cell survival, and cell identity. As such, it is a prominent target for pharmacological intervention. The relatively low abundance of this subproteome in conjunction with challenging extractability and solubility still hampers its comprehensive analysis. Here, we combined a chemical glycoprotein-tagging strategy with mass spectrometry to enable comprehensive analysis of the cell-surface glycoproteome. To benchmark this workflow and to provide guidance for cell line selection for functional experiments, we generated an inventory of the N-linked cell-surface glycoproteomes of 15 standard laboratory human cell lines and three primary lymphocytic cell types. On average, about 900 plasma membrane and secreted proteins were identified per experiment, including more than 300 transporters and ion channels. Primary cells displayed distinct expression of surface markers and transporters underpinning the importance of carefully validating model cell lines selected for the study of cell surface-mediated processes. To monitor dynamic changes of the cell-surface proteome in a highly multiplexed experiment, we employed an isobaric mass tag-based chemical labeling strategy. This enabled the time-resolved analysis of plasma membrane protein presentation during differentiation of the monocytic suspension cell line THP-1 into macrophage-like adherent cells. Time-dependent changes observed in membrane protein presentation reflect functional remodeling during the phenotypic transition in three distinct phases: rapid surface presentation and secretion of proteins from intracellular pools concurrent with rapid internalization of no longer needed proteins and finally delayed presentation of newly synthesized macrophage markers. Perturbation of this process using marketed receptor tyrosine kinase inhibitors revealed dasatinib to severely compromise macrophage differentiation due to an off-target activity. This finding suggests that dynamic processes can be highly vulnerable to drug treatment and should be monitored more rigorously to identify adverse drug effects.

Dataset Information

Quantitative investigation of human cell surface N-glycoprotein dynamics.

Publications

Quantitative investigation of human cell surface <i>N</i>-glycoprotein dynamics.

Similar Datasets

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