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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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Functional pathway analysis of glycoproteins overexpressed in PC3-ML2. Ingenuity Pathway Analysis (IPA) was employed to functionally map the cell-surface glycoproteins overexpressed in PC3-ML2 cells. The network shows the biological functions (Fx) that have been associated with these glycoproteins in the context of disease. “Cell movement” and “invasion” are the top listed functions, as determined by the number of glycoproteins (highlighted in blue) associated with these activities. Individual proteins with known biochemical activities are highlighted with class-specific shapes (indicated in the legend).
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Figure 7: Functional pathway analysis of glycoproteins overexpressed in PC3-ML2. Ingenuity Pathway Analysis (IPA) was employed to functionally map the cell-surface glycoproteins overexpressed in PC3-ML2 cells. The network shows the biological functions (Fx) that have been associated with these glycoproteins in the context of disease. “Cell movement” and “invasion” are the top listed functions, as determined by the number of glycoproteins (highlighted in blue) associated with these activities. Individual proteins with known biochemical activities are highlighted with class-specific shapes (indicated in the legend).

Mentions: Conversely, the 44 cell-surface glycoproteins unique to the metastatic ML2 cells (Table IV), a large number of proteins are involved in cell movement, migration, and cell invasion. There are 11 proteins involved in cell movement (Fig. 7). Notably, six proteins, Basigin (BSG), Periostin (POSTN), Glucose-6-phosphate isomerase (GPI), Calreticulin (CALR), Leucine-rich repeat-containing protein 15 (LRRC15), and Myristoylated alanine-rich protein kinase C substrate (MARCKS) are all involved in the invasion of tumor cell lines. An additional five proteins, Junction plakoglobin (JUP), Tyrosine-protein phosphatase nonreceptor type 1 (PTPN1), Niemann-Pick C1 protein (NPC1), Sodium-coupled neutral amino acid transporter 2 (SLC38A2), and Adenylyl cyclase-associated protein 1 (CAP1), are involved in cellular migration and/or movement. Six of the aforementioned proteins (SLC38A2, JUP, PTPN1, POSTN, CALR, and BSG) and Pinin (PNN), CUB domain-containing protein 1 (CDCP1), DNA-directed RNA polymerase I subunit RPA49 (POLR1E), and Collagen α-1(VI) chain (COL6A1), are involved in growth/colony formation. Cumulatively, the types of glycoproteins that were differentially expressed in the N2 and ML2 cells functionally reflect their respective nonmetastatic and metastatic phenotypes.


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-ML2. Ingenuity Pathway Analysis (IPA) was employed to functionally map the cell-surface glycoproteins overexpressed in PC3-ML2 cells. The network shows the biological functions (Fx) that have been associated with these glycoproteins in the context of disease. “Cell movement” and “invasion” are the top listed functions, as determined by the number of glycoproteins (highlighted in blue) associated with these activities. 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 7: Functional pathway analysis of glycoproteins overexpressed in PC3-ML2. Ingenuity Pathway Analysis (IPA) was employed to functionally map the cell-surface glycoproteins overexpressed in PC3-ML2 cells. The network shows the biological functions (Fx) that have been associated with these glycoproteins in the context of disease. “Cell movement” and “invasion” are the top listed functions, as determined by the number of glycoproteins (highlighted in blue) associated with these activities. Individual proteins with known biochemical activities are highlighted with class-specific shapes (indicated in the legend).
Mentions: Conversely, the 44 cell-surface glycoproteins unique to the metastatic ML2 cells (Table IV), a large number of proteins are involved in cell movement, migration, and cell invasion. There are 11 proteins involved in cell movement (Fig. 7). Notably, six proteins, Basigin (BSG), Periostin (POSTN), Glucose-6-phosphate isomerase (GPI), Calreticulin (CALR), Leucine-rich repeat-containing protein 15 (LRRC15), and Myristoylated alanine-rich protein kinase C substrate (MARCKS) are all involved in the invasion of tumor cell lines. An additional five proteins, Junction plakoglobin (JUP), Tyrosine-protein phosphatase nonreceptor type 1 (PTPN1), Niemann-Pick C1 protein (NPC1), Sodium-coupled neutral amino acid transporter 2 (SLC38A2), and Adenylyl cyclase-associated protein 1 (CAP1), are involved in cellular migration and/or movement. Six of the aforementioned proteins (SLC38A2, JUP, PTPN1, POSTN, CALR, and BSG) and Pinin (PNN), CUB domain-containing protein 1 (CDCP1), DNA-directed RNA polymerase I subunit RPA49 (POLR1E), and Collagen α-1(VI) chain (COL6A1), are involved in growth/colony formation. Cumulatively, the types of glycoproteins that were differentially expressed in the N2 and ML2 cells functionally reflect their respective nonmetastatic and metastatic phenotypes.

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