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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

Effects of PSA on ldlD/N140 cell behavior.(A) mRNA levels of STX and PST genes in transfected cell lines were assessed by semi-quantitative (left panels) and quantitative (right panels) RT-PCR, with γ-tubulin as control. **, p = 0.01–0.05. (B) Western blot analysis of STX and PST expression using anti-His-tag antibody for selection of transfected cells and β-tubulin as loading control. (C) Immunofluorescence staining of PSA-NCAM. ldlD/N140, ldlD/N/S, and ldlD/N/P cells were cultured in serum-free medium with ITS+Gal. Cell nuclei were visualized by Hoechst staining. Size bars: 50 μm. (D) Proliferation (MTT) assays. ldlD/N/S, ldlD/N/P, and ldlD/N140 cells were cultured in serum-free medium containing ITS or ITS+Gal, and MTT assays were performed as described in Materials and Methods. Values shown are mean ± SD from three independent experiments. (E) Motility assays. ldlD/N/S and ldlD/N/P cells were cultured in serum-free medium containing ITS+Gal, and motility assays were performed as described in Fig 3B. Size bars: 10 μm. NS = not significant. (F) Migration assays. Cells were cultured and migratory cells were quantified as described in Fig 3E. Two independent experiments gave similar results. Magnification: 200 ×. *, p<0.05; NS = not significant.
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pone.0124237.g004: Effects of PSA on ldlD/N140 cell behavior.(A) mRNA levels of STX and PST genes in transfected cell lines were assessed by semi-quantitative (left panels) and quantitative (right panels) RT-PCR, with γ-tubulin as control. **, p = 0.01–0.05. (B) Western blot analysis of STX and PST expression using anti-His-tag antibody for selection of transfected cells and β-tubulin as loading control. (C) Immunofluorescence staining of PSA-NCAM. ldlD/N140, ldlD/N/S, and ldlD/N/P cells were cultured in serum-free medium with ITS+Gal. Cell nuclei were visualized by Hoechst staining. Size bars: 50 μm. (D) Proliferation (MTT) assays. ldlD/N/S, ldlD/N/P, and ldlD/N140 cells were cultured in serum-free medium containing ITS or ITS+Gal, and MTT assays were performed as described in Materials and Methods. Values shown are mean ± SD from three independent experiments. (E) Motility assays. ldlD/N/S and ldlD/N/P cells were cultured in serum-free medium containing ITS+Gal, and motility assays were performed as described in Fig 3B. Size bars: 10 μm. NS = not significant. (F) Migration assays. Cells were cultured and migratory cells were quantified as described in Fig 3E. Two independent experiments gave similar results. Magnification: 200 ×. *, p<0.05; NS = not significant.

Mentions: The polysialyltransferases STX and PST both catalyze transfer of multiple α2,8-linked sialic acid residues to glycans containing NeuNAc α2→3/6Galβ1→4GlcNAc→R[35]. To directly evaluate the role of PSA in modulating NCAM function, we generated ldlD-14-NCAM140-STX-His (ldlD/N/S) and ldlD-14-NCAM140-PST-His (ldlD/N/P) cells, which overexpressed STX and PST in ldlD/N140 cells, respectively (Fig 4A and 4B). Both of them showed stronger PSA-NCAM signals than did ldlD/N140 cells (Fig 4C).


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)

Effects of PSA on ldlD/N140 cell behavior.(A) mRNA levels of STX and PST genes in transfected cell lines were assessed by semi-quantitative (left panels) and quantitative (right panels) RT-PCR, with γ-tubulin as control. **, p = 0.01–0.05. (B) Western blot analysis of STX and PST expression using anti-His-tag antibody for selection of transfected cells and β-tubulin as loading control. (C) Immunofluorescence staining of PSA-NCAM. ldlD/N140, ldlD/N/S, and ldlD/N/P cells were cultured in serum-free medium with ITS+Gal. Cell nuclei were visualized by Hoechst staining. Size bars: 50 μm. (D) Proliferation (MTT) assays. ldlD/N/S, ldlD/N/P, and ldlD/N140 cells were cultured in serum-free medium containing ITS or ITS+Gal, and MTT assays were performed as described in Materials and Methods. Values shown are mean ± SD from three independent experiments. (E) Motility assays. ldlD/N/S and ldlD/N/P cells were cultured in serum-free medium containing ITS+Gal, and motility assays were performed as described in Fig 3B. Size bars: 10 μm. NS = not significant. (F) Migration assays. Cells were cultured and migratory cells were quantified as described in Fig 3E. Two independent experiments gave similar results. Magnification: 200 ×. *, p<0.05; NS = not significant.
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pone.0124237.g004: Effects of PSA on ldlD/N140 cell behavior.(A) mRNA levels of STX and PST genes in transfected cell lines were assessed by semi-quantitative (left panels) and quantitative (right panels) RT-PCR, with γ-tubulin as control. **, p = 0.01–0.05. (B) Western blot analysis of STX and PST expression using anti-His-tag antibody for selection of transfected cells and β-tubulin as loading control. (C) Immunofluorescence staining of PSA-NCAM. ldlD/N140, ldlD/N/S, and ldlD/N/P cells were cultured in serum-free medium with ITS+Gal. Cell nuclei were visualized by Hoechst staining. Size bars: 50 μm. (D) Proliferation (MTT) assays. ldlD/N/S, ldlD/N/P, and ldlD/N140 cells were cultured in serum-free medium containing ITS or ITS+Gal, and MTT assays were performed as described in Materials and Methods. Values shown are mean ± SD from three independent experiments. (E) Motility assays. ldlD/N/S and ldlD/N/P cells were cultured in serum-free medium containing ITS+Gal, and motility assays were performed as described in Fig 3B. Size bars: 10 μm. NS = not significant. (F) Migration assays. Cells were cultured and migratory cells were quantified as described in Fig 3E. Two independent experiments gave similar results. Magnification: 200 ×. *, p<0.05; NS = not significant.
Mentions: The polysialyltransferases STX and PST both catalyze transfer of multiple α2,8-linked sialic acid residues to glycans containing NeuNAc α2→3/6Galβ1→4GlcNAc→R[35]. To directly evaluate the role of PSA in modulating NCAM function, we generated ldlD-14-NCAM140-STX-His (ldlD/N/S) and ldlD-14-NCAM140-PST-His (ldlD/N/P) cells, which overexpressed STX and PST in ldlD/N140 cells, respectively (Fig 4A and 4B). Both of them showed stronger PSA-NCAM signals than did ldlD/N140 cells (Fig 4C).

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