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Targeted identification of metastasis-associated cell-surface sialoglycoproteins in prostate cancer.

Yang L, Nyalwidhe JO, Guo S, Drake RR, Semmes OJ - Mol. Cell Proteomics (2011)

Bottom Line: A selective enrichment of sialoglycoproteins was confirmed.When compared with global proteomic analysis of the same cells, the proportion of identified glycoprotein and cell-surface proteins were on average threefold higher using the selective capture approach.Our approach effectively targeted surface sialoglycoproteins and efficiently identified proteins that underlie the metastatic potential of the ML2 cells.

View Article: PubMed Central - PubMed

Affiliation: Leroy T. Canoles Cancer Research Center, Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, Virginia 23507, USA.

ABSTRACT
Covalent attachment of carbohydrates to proteins is one of the most common post-translational modifications. At the cell surface, sugar moieties of glycoproteins contribute to molecular recognition events involved in cancer metastasis. We have combined glycan metabolic labeling with mass spectrometry analysis to identify and characterize metastasis-associated cell surface sialoglycoproteins. Our model system used syngeneic prostate cancer cell lines derived from PC3 (N2, nonmetastatic, and ML2, highly metastatic). The metabolic incorporation of AC(4)ManNAz and subsequent specific labeling of cell surface sialylation was confirmed by flow cytometry and confocal microscopy. Affinity isolation of the modified sialic-acid containing cell surface proteins via click chemistry was followed by SDS-PAGE separation and liquid chromatography-tandem MS analysis. We identified 324 proteins from N2 and 372 proteins of ML2. Using conservative annotation, 64 proteins (26%) from N2 and 72 proteins (29%) from ML2 were classified as extracellular or membrane-associated glycoproteins. A selective enrichment of sialoglycoproteins was confirmed. When compared with global proteomic analysis of the same cells, the proportion of identified glycoprotein and cell-surface proteins were on average threefold higher using the selective capture approach. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that the vast majority of glycoproteins overexpressed in the metastatic ML2 subline were involved in cell motility, migration, and invasion. Our approach effectively targeted surface sialoglycoproteins and efficiently identified proteins that underlie the metastatic potential of the ML2 cells.

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Related in: MedlinePlus

Functional pathway analysis of glycoproteins overexpressed in PC3-N2. Ingenuity Pathway Analysis (IPA) was employed to functionally map the cell-surface glycoproteins overexpressed in PC3-N2 cells. The network shows the biological functions (Fx) that have been associated with these glycoproteins in the context of disease. “Apoptosis of tumor cell lines” is the top listed function, as determined by the number of overexpressed glycoproteins (highlighted in blue) associated with this activity. Individual proteins with known biochemical activities are highlighted with class-specific shapes (indicated in the legend).
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Figure 6: Functional pathway analysis of glycoproteins overexpressed in PC3-N2. Ingenuity Pathway Analysis (IPA) was employed to functionally map the cell-surface glycoproteins overexpressed in PC3-N2 cells. The network shows the biological functions (Fx) that have been associated with these glycoproteins in the context of disease. “Apoptosis of tumor cell lines” is the top listed function, as determined by the number of overexpressed glycoproteins (highlighted in blue) associated with this activity. Individual proteins with known biochemical activities are highlighted with class-specific shapes (indicated in the legend).

Mentions: We next subjected the selected group of differentially expressed cell-surface glycoproteins to analysis via IPA. Among the 36 cell-surface glycoproteins unique to the nonmetastatic N2 cells (Table III), many proteins, such as, complement decay-accelerating factor (CD55), Catenin β-1 (CTNNB1), focal adhesion kinase 1 (PTK2), Myosin-VI (MYO6), A kinase anchor protein 1 (AKAP1), and symplekin (SYMPK), can positively or negatively regulate apoptosis pathway (Fig. 6) and cell adhesion (catenin Δ-1; CTNND1/disks large homolog 5;DLG5/symplekin; SYMPK). Other proteins overexpressed reflect basic cell functions involving vesicular transport, lipid metabolism and mRNA processing and splicing.


Targeted identification of metastasis-associated cell-surface sialoglycoproteins in prostate cancer.

Yang L, Nyalwidhe JO, Guo S, Drake RR, Semmes OJ - Mol. Cell Proteomics (2011)

Functional pathway analysis of glycoproteins overexpressed in PC3-N2. Ingenuity Pathway Analysis (IPA) was employed to functionally map the cell-surface glycoproteins overexpressed in PC3-N2 cells. The network shows the biological functions (Fx) that have been associated with these glycoproteins in the context of disease. “Apoptosis of tumor cell lines” is the top listed function, as determined by the number of overexpressed glycoproteins (highlighted in blue) associated with this activity. Individual proteins with known biochemical activities are highlighted with class-specific shapes (indicated in the legend).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3108840&req=5

Figure 6: Functional pathway analysis of glycoproteins overexpressed in PC3-N2. Ingenuity Pathway Analysis (IPA) was employed to functionally map the cell-surface glycoproteins overexpressed in PC3-N2 cells. The network shows the biological functions (Fx) that have been associated with these glycoproteins in the context of disease. “Apoptosis of tumor cell lines” is the top listed function, as determined by the number of overexpressed glycoproteins (highlighted in blue) associated with this activity. Individual proteins with known biochemical activities are highlighted with class-specific shapes (indicated in the legend).
Mentions: We next subjected the selected group of differentially expressed cell-surface glycoproteins to analysis via IPA. Among the 36 cell-surface glycoproteins unique to the nonmetastatic N2 cells (Table III), many proteins, such as, complement decay-accelerating factor (CD55), Catenin β-1 (CTNNB1), focal adhesion kinase 1 (PTK2), Myosin-VI (MYO6), A kinase anchor protein 1 (AKAP1), and symplekin (SYMPK), can positively or negatively regulate apoptosis pathway (Fig. 6) and cell adhesion (catenin Δ-1; CTNND1/disks large homolog 5;DLG5/symplekin; SYMPK). Other proteins overexpressed reflect basic cell functions involving vesicular transport, lipid metabolism and mRNA processing and splicing.

Bottom Line: A selective enrichment of sialoglycoproteins was confirmed.When compared with global proteomic analysis of the same cells, the proportion of identified glycoprotein and cell-surface proteins were on average threefold higher using the selective capture approach.Our approach effectively targeted surface sialoglycoproteins and efficiently identified proteins that underlie the metastatic potential of the ML2 cells.

View Article: PubMed Central - PubMed

Affiliation: Leroy T. Canoles Cancer Research Center, Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, Virginia 23507, USA.

ABSTRACT
Covalent attachment of carbohydrates to proteins is one of the most common post-translational modifications. At the cell surface, sugar moieties of glycoproteins contribute to molecular recognition events involved in cancer metastasis. We have combined glycan metabolic labeling with mass spectrometry analysis to identify and characterize metastasis-associated cell surface sialoglycoproteins. Our model system used syngeneic prostate cancer cell lines derived from PC3 (N2, nonmetastatic, and ML2, highly metastatic). The metabolic incorporation of AC(4)ManNAz and subsequent specific labeling of cell surface sialylation was confirmed by flow cytometry and confocal microscopy. Affinity isolation of the modified sialic-acid containing cell surface proteins via click chemistry was followed by SDS-PAGE separation and liquid chromatography-tandem MS analysis. We identified 324 proteins from N2 and 372 proteins of ML2. Using conservative annotation, 64 proteins (26%) from N2 and 72 proteins (29%) from ML2 were classified as extracellular or membrane-associated glycoproteins. A selective enrichment of sialoglycoproteins was confirmed. When compared with global proteomic analysis of the same cells, the proportion of identified glycoprotein and cell-surface proteins were on average threefold higher using the selective capture approach. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that the vast majority of glycoproteins overexpressed in the metastatic ML2 subline were involved in cell motility, migration, and invasion. Our approach effectively targeted surface sialoglycoproteins and efficiently identified proteins that underlie the metastatic potential of the ML2 cells.

Show MeSH
Related in: MedlinePlus