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Promotion of cell migration by neural cell adhesion molecule (NCAM) is enhanced by PSA in a polysialyltransferase-specific manner.

Guan F, Wang X, He F - PLoS ONE (2015)

Bottom Line: We cloned the genes for NCAM-140 and for polysialyltransferases STX and PST (responsible for PSA synthesis) from normal murine mammary gland epithelial (NMuMG) cells and transfected them into ldlD-14 and human breast cancer cells MCF-7.The effect of PSA on NCAM-mediated cell proliferation, motility, migration and adhesion was studied.We found that NCAM-140 significantly promoted cell proliferation, motility and migration, while polysialylation of NCAM-140 catalyzed by STX, but not by PST, enhanced NCAM-mediated cell migration, but not cell proliferation or motility.

View Article: PubMed Central - PubMed

Affiliation: The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.

ABSTRACT
Neural cell adhesion molecule 140 (NCAM-140) is a glycoprotein and always highly polysialylated in cancer. Functions of polysialic acid (PSA) that binds to N-glycan termini on NCAM remain unclear. ldlD-14 cells, a CHO cell mutant deficient in UDP-Gal 4-epimerase, are useful for structural and functional studies of Gal-containing glycoproteins because their abnormal glycosylation can be converted to normal status by exogenous addition of galactose (Gal). We cloned the genes for NCAM-140 and for polysialyltransferases STX and PST (responsible for PSA synthesis) from normal murine mammary gland epithelial (NMuMG) cells and transfected them into ldlD-14 and human breast cancer cells MCF-7. The effect of PSA on NCAM-mediated cell proliferation, motility, migration and adhesion was studied. We found that NCAM-140 significantly promoted cell proliferation, motility and migration, while polysialylation of NCAM-140 catalyzed by STX, but not by PST, enhanced NCAM-mediated cell migration, but not cell proliferation or motility. In addition, PSA catalyzed by different polysialyltransferases affected the adhesion of NCAM to different extracellular matrix (ECM) components.

No MeSH data available.


Related in: MedlinePlus

Modification of glycosylation in ldlD/N140 cells.(A) and (B) MALDI-TOF/TOF-MS spectra of N-glycans in ITS- or ITS+Gal-cultured ldlD/N140 cells. ldlD/N140 cells were cultured in serum-free medium supplemented with ITS (A) or ITS+Gal (B). Derivatized N-Glycans were separated, desalted, and characterized by MALDI-TOF-MS as described in Materials and Methods. Representative spectra from triplicate experiments are shown. Detailed glycan structures were analyzed using the GlycoWorkbench program. Proposed structures and their m/z values are shown for each peak. (C) Western blot analysis of ldlD/N140 cells cultured in serum-free medium with ITS (lane 1), ITS+5 μM Gal (lane 2), and ITS+20 μM Gal (lane 3). NCAM was analyzed by western blotting with β-tubulin as loading control. HRP-conjugated goat anti-mouse IgG was used as secondary antibody. (D) Flow cytometric analysis of glycan patterns using GSL-II (a), anti-NCAM antibody (b), and anti-PSA-NCAM antibody (c).
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pone.0124237.g002: Modification of glycosylation in ldlD/N140 cells.(A) and (B) MALDI-TOF/TOF-MS spectra of N-glycans in ITS- or ITS+Gal-cultured ldlD/N140 cells. ldlD/N140 cells were cultured in serum-free medium supplemented with ITS (A) or ITS+Gal (B). Derivatized N-Glycans were separated, desalted, and characterized by MALDI-TOF-MS as described in Materials and Methods. Representative spectra from triplicate experiments are shown. Detailed glycan structures were analyzed using the GlycoWorkbench program. Proposed structures and their m/z values are shown for each peak. (C) Western blot analysis of ldlD/N140 cells cultured in serum-free medium with ITS (lane 1), ITS+5 μM Gal (lane 2), and ITS+20 μM Gal (lane 3). NCAM was analyzed by western blotting with β-tubulin as loading control. HRP-conjugated goat anti-mouse IgG was used as secondary antibody. (D) Flow cytometric analysis of glycan patterns using GSL-II (a), anti-NCAM antibody (b), and anti-PSA-NCAM antibody (c).

Mentions: Modification of glycosylation in ldlD-14 cells by addition of Gal was confirmed by MALDI-TOF/TOF-MS/MS. Since sialic acids are easily lost during mass spectrometric ionization, the glycans released from ldlD/N140 cells cultured with ITS or ITS+Gal were purified, derivatized and analyzed. Proposed N-glycan structures and their molecular weights are shown in Fig 2A and 2B. Cells cultured with ITS had no Gal or sialic acid residues on any of the annotated N-glycans (Fig 2A). In contrast, cells cultured with ITS+Gal presented two unique structures (m/z values 2465.120, 3147.336), annotated as N-glycans with terminal Gal and sialic acid (Fig 2B). This result indicated that ldlD/N140 cells can express NCAM under ITS condition, and express PSA-NCAM under ITS +Gal condition.


Promotion of cell migration by neural cell adhesion molecule (NCAM) is enhanced by PSA in a polysialyltransferase-specific manner.

Guan F, Wang X, He F - PLoS ONE (2015)

Modification of glycosylation in ldlD/N140 cells.(A) and (B) MALDI-TOF/TOF-MS spectra of N-glycans in ITS- or ITS+Gal-cultured ldlD/N140 cells. ldlD/N140 cells were cultured in serum-free medium supplemented with ITS (A) or ITS+Gal (B). Derivatized N-Glycans were separated, desalted, and characterized by MALDI-TOF-MS as described in Materials and Methods. Representative spectra from triplicate experiments are shown. Detailed glycan structures were analyzed using the GlycoWorkbench program. Proposed structures and their m/z values are shown for each peak. (C) Western blot analysis of ldlD/N140 cells cultured in serum-free medium with ITS (lane 1), ITS+5 μM Gal (lane 2), and ITS+20 μM Gal (lane 3). NCAM was analyzed by western blotting with β-tubulin as loading control. HRP-conjugated goat anti-mouse IgG was used as secondary antibody. (D) Flow cytometric analysis of glycan patterns using GSL-II (a), anti-NCAM antibody (b), and anti-PSA-NCAM antibody (c).
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Related In: Results  -  Collection

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pone.0124237.g002: Modification of glycosylation in ldlD/N140 cells.(A) and (B) MALDI-TOF/TOF-MS spectra of N-glycans in ITS- or ITS+Gal-cultured ldlD/N140 cells. ldlD/N140 cells were cultured in serum-free medium supplemented with ITS (A) or ITS+Gal (B). Derivatized N-Glycans were separated, desalted, and characterized by MALDI-TOF-MS as described in Materials and Methods. Representative spectra from triplicate experiments are shown. Detailed glycan structures were analyzed using the GlycoWorkbench program. Proposed structures and their m/z values are shown for each peak. (C) Western blot analysis of ldlD/N140 cells cultured in serum-free medium with ITS (lane 1), ITS+5 μM Gal (lane 2), and ITS+20 μM Gal (lane 3). NCAM was analyzed by western blotting with β-tubulin as loading control. HRP-conjugated goat anti-mouse IgG was used as secondary antibody. (D) Flow cytometric analysis of glycan patterns using GSL-II (a), anti-NCAM antibody (b), and anti-PSA-NCAM antibody (c).
Mentions: Modification of glycosylation in ldlD-14 cells by addition of Gal was confirmed by MALDI-TOF/TOF-MS/MS. Since sialic acids are easily lost during mass spectrometric ionization, the glycans released from ldlD/N140 cells cultured with ITS or ITS+Gal were purified, derivatized and analyzed. Proposed N-glycan structures and their molecular weights are shown in Fig 2A and 2B. Cells cultured with ITS had no Gal or sialic acid residues on any of the annotated N-glycans (Fig 2A). In contrast, cells cultured with ITS+Gal presented two unique structures (m/z values 2465.120, 3147.336), annotated as N-glycans with terminal Gal and sialic acid (Fig 2B). This result indicated that ldlD/N140 cells can express NCAM under ITS condition, and express PSA-NCAM under ITS +Gal condition.

Bottom Line: We cloned the genes for NCAM-140 and for polysialyltransferases STX and PST (responsible for PSA synthesis) from normal murine mammary gland epithelial (NMuMG) cells and transfected them into ldlD-14 and human breast cancer cells MCF-7.The effect of PSA on NCAM-mediated cell proliferation, motility, migration and adhesion was studied.We found that NCAM-140 significantly promoted cell proliferation, motility and migration, while polysialylation of NCAM-140 catalyzed by STX, but not by PST, enhanced NCAM-mediated cell migration, but not cell proliferation or motility.

View Article: PubMed Central - PubMed

Affiliation: The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.

ABSTRACT
Neural cell adhesion molecule 140 (NCAM-140) is a glycoprotein and always highly polysialylated in cancer. Functions of polysialic acid (PSA) that binds to N-glycan termini on NCAM remain unclear. ldlD-14 cells, a CHO cell mutant deficient in UDP-Gal 4-epimerase, are useful for structural and functional studies of Gal-containing glycoproteins because their abnormal glycosylation can be converted to normal status by exogenous addition of galactose (Gal). We cloned the genes for NCAM-140 and for polysialyltransferases STX and PST (responsible for PSA synthesis) from normal murine mammary gland epithelial (NMuMG) cells and transfected them into ldlD-14 and human breast cancer cells MCF-7. The effect of PSA on NCAM-mediated cell proliferation, motility, migration and adhesion was studied. We found that NCAM-140 significantly promoted cell proliferation, motility and migration, while polysialylation of NCAM-140 catalyzed by STX, but not by PST, enhanced NCAM-mediated cell migration, but not cell proliferation or motility. In addition, PSA catalyzed by different polysialyltransferases affected the adhesion of NCAM to different extracellular matrix (ECM) components.

No MeSH data available.


Related in: MedlinePlus