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The glycosyltransferase activities of lysyl hydroxylase 3 (LH3) in the extracellular space are important for cell growth and viability.

Wang C, Kovanen V, Raudasoja P, Eskelinen S, Pospiech H, Myllylä R - J. Cell. Mol. Med. (2009)

Bottom Line: We have demonstrated that LH3 is found not only intracellularly, but also on the cell surface and in the extracellular space, suggesting additional functions for LH3.Here we show that the targeted disruption of LH3 by siRNA causes a marked reduction of both glycosyltransferase activities, and the overexpression of LH3 in HT-1080 cells increases hydroxylation of lysyl residues and the subsequent galactosylation and glucosylation of hydroxylysyl residues.The overexpression of a glycosyltransferase-deficient mutant or targeted disruption of LH3 by siRNA in cells results in abnormal cell morphology followed by cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Biocenter Oulu, University of Oulu, Oulu, Finland.

ABSTRACT
Lysyl hydroxylase (LH) isoform 3 is a post-translational enzyme possessing LH, collagen galactosyltransferase (GT) and glucosyltransferase (GGT) activities. We have demonstrated that LH3 is found not only intracellularly, but also on the cell surface and in the extracellular space, suggesting additional functions for LH3. Here we show that the targeted disruption of LH3 by siRNA causes a marked reduction of both glycosyltransferase activities, and the overexpression of LH3 in HT-1080 cells increases hydroxylation of lysyl residues and the subsequent galactosylation and glucosylation of hydroxylysyl residues. These data confirm the multi-functionality of LH3 in cells. Furthermore, treatment of cells in culture medium with a LH3 N-terminal fragment affects the cell behaviour, rapidly leading to arrest of growth and further to lethality if the fragment is glycosyltransferase-deficient, and leading to stimulation of proliferation if the fragment contains LH3 glycosyltransferase activities. The effect is reversible, the cells recovering after removal of the glycosyltransferase-deficient fragment. The findings were confirmed by overexpressing the full-length LH3 in native or mutated forms in the cells. The data indicate that the increase in proliferation depends on the glycosyltransferase activity of LH3. The overexpression of a glycosyltransferase-deficient mutant or targeted disruption of LH3 by siRNA in cells results in abnormal cell morphology followed by cell death. Our data clearly indicate that the deficiency of LH3 glycosyltransferase activities, especially in the extracellular space, causes growth arrest revealing the importance of the glycosyltransferase activities of LH3 for cell growth and viability, and identifying LH3 as a potential target for medical applications, such as cancer therapy.

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The binding of the LH3 N-terminal fragment and its glycosyltransferase-deficient counterpart on HT-1080 cell surfaces. (A) Western blot analysis after incubation with the hLH3N1 antibody: lanes 1–3 are cell pellets from untreated cells, DXD fragment treated cells and wild-type LH3 N-terminal fragment treated cells, respectively, demonstrating that both fragments (30 kD) bound to cell membranes; lanes 4–6 are medium samples purified on a Nickel column from the untreated cells, DXD fragment-treated cells and LH3 N-terminal fragment-treated cells, respectively, showing the same amount of the fragments (30 kD) used in all experiments. Lanes 7–9 are cell lysates purified on a Nickel column from untreated cells, DXD fragment treated cells and LH3 N-terminal fragment-treated cells, indicating trace amount of the fragments (30 kD) taken into the cells. (B) Immunofluorescence staining of non-permeabilized cells (A–C: untreated; D–F: DXD treatment; G–I: LH3N treatment) by the affinity-purified PLOD3 antibody (green colour, A, D and G), Wheat germ agglutinin (cell surface marker, red colour, B, E and H), and Hoechst 33258 (nuclei marker, blue colour). Parts C, F and I show the overlapping of the LH3 staining with the WGA staining on the cell surface (yellow colour, indicated by arrowheads). Untreated cells (C) are used as a background control, showing a small amount of endogenous LH3 on the cell surface. The cells treated with the DXD fragment (F) or the LH3N fragment (I) show much more overlap of the LH3 with the WGA, indicating that both fragments are bound to the cell surface.
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fig03: The binding of the LH3 N-terminal fragment and its glycosyltransferase-deficient counterpart on HT-1080 cell surfaces. (A) Western blot analysis after incubation with the hLH3N1 antibody: lanes 1–3 are cell pellets from untreated cells, DXD fragment treated cells and wild-type LH3 N-terminal fragment treated cells, respectively, demonstrating that both fragments (30 kD) bound to cell membranes; lanes 4–6 are medium samples purified on a Nickel column from the untreated cells, DXD fragment-treated cells and LH3 N-terminal fragment-treated cells, respectively, showing the same amount of the fragments (30 kD) used in all experiments. Lanes 7–9 are cell lysates purified on a Nickel column from untreated cells, DXD fragment treated cells and LH3 N-terminal fragment-treated cells, indicating trace amount of the fragments (30 kD) taken into the cells. (B) Immunofluorescence staining of non-permeabilized cells (A–C: untreated; D–F: DXD treatment; G–I: LH3N treatment) by the affinity-purified PLOD3 antibody (green colour, A, D and G), Wheat germ agglutinin (cell surface marker, red colour, B, E and H), and Hoechst 33258 (nuclei marker, blue colour). Parts C, F and I show the overlapping of the LH3 staining with the WGA staining on the cell surface (yellow colour, indicated by arrowheads). Untreated cells (C) are used as a background control, showing a small amount of endogenous LH3 on the cell surface. The cells treated with the DXD fragment (F) or the LH3N fragment (I) show much more overlap of the LH3 with the WGA, indicating that both fragments are bound to the cell surface.

Mentions: We first determined whether the LH3 N-terminal fragment and/or its DXD mutated form bound to the cell surface when added to the cell medium. HT-1080 cells were incubated with the fragments (3 μg/ml) for 3 hrs at 37°C. The fragments remaining in the medium served as a molecular weight control. The cell lysate and the cell pellet were collected after sonication and centrifugation. The his-tagged proteins in lysate and medium were concentrated by Ni-NTA column. All fractions were re-suspended in a SDS sample loading buffer, and analysed by Western blotting with the antibody hLH3N1 against the purified human LH3 N-terminal fragment (Fig. 3A). The antibody detected a major 30 kD band in the cell pellet fraction, indicating that the fragments were bound to the surface of the HT-1080 cells (Fig. 3A, lane 1–3). The same-sized bands in medium confirmed that similar amounts of both fragments were used in the experiment (Fig. 3A, lane 4–6). A faint band was also observed in the cell lysate after Nickel purification, indicating that a trace amount of the fragment was internalized in the cell (Fig. 3A, lane 7–9) and suggesting that endocytosis is associated with the phenomenon. Both fragments, mutated and wild-type, behaved similarly. Furthermore, immuno-fluorescence stainings of non-permeabilized HT-1080 cells treated with the DXD fragment and with the LH3 N-terminal fragment revealed an overlay of the LH3 staining with the cell surface marker, so confirming that the fragments were bound to the cell surface (Fig. 3B, F, I).


The glycosyltransferase activities of lysyl hydroxylase 3 (LH3) in the extracellular space are important for cell growth and viability.

Wang C, Kovanen V, Raudasoja P, Eskelinen S, Pospiech H, Myllylä R - J. Cell. Mol. Med. (2009)

The binding of the LH3 N-terminal fragment and its glycosyltransferase-deficient counterpart on HT-1080 cell surfaces. (A) Western blot analysis after incubation with the hLH3N1 antibody: lanes 1–3 are cell pellets from untreated cells, DXD fragment treated cells and wild-type LH3 N-terminal fragment treated cells, respectively, demonstrating that both fragments (30 kD) bound to cell membranes; lanes 4–6 are medium samples purified on a Nickel column from the untreated cells, DXD fragment-treated cells and LH3 N-terminal fragment-treated cells, respectively, showing the same amount of the fragments (30 kD) used in all experiments. Lanes 7–9 are cell lysates purified on a Nickel column from untreated cells, DXD fragment treated cells and LH3 N-terminal fragment-treated cells, indicating trace amount of the fragments (30 kD) taken into the cells. (B) Immunofluorescence staining of non-permeabilized cells (A–C: untreated; D–F: DXD treatment; G–I: LH3N treatment) by the affinity-purified PLOD3 antibody (green colour, A, D and G), Wheat germ agglutinin (cell surface marker, red colour, B, E and H), and Hoechst 33258 (nuclei marker, blue colour). Parts C, F and I show the overlapping of the LH3 staining with the WGA staining on the cell surface (yellow colour, indicated by arrowheads). Untreated cells (C) are used as a background control, showing a small amount of endogenous LH3 on the cell surface. The cells treated with the DXD fragment (F) or the LH3N fragment (I) show much more overlap of the LH3 with the WGA, indicating that both fragments are bound to the cell surface.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3822511&req=5

fig03: The binding of the LH3 N-terminal fragment and its glycosyltransferase-deficient counterpart on HT-1080 cell surfaces. (A) Western blot analysis after incubation with the hLH3N1 antibody: lanes 1–3 are cell pellets from untreated cells, DXD fragment treated cells and wild-type LH3 N-terminal fragment treated cells, respectively, demonstrating that both fragments (30 kD) bound to cell membranes; lanes 4–6 are medium samples purified on a Nickel column from the untreated cells, DXD fragment-treated cells and LH3 N-terminal fragment-treated cells, respectively, showing the same amount of the fragments (30 kD) used in all experiments. Lanes 7–9 are cell lysates purified on a Nickel column from untreated cells, DXD fragment treated cells and LH3 N-terminal fragment-treated cells, indicating trace amount of the fragments (30 kD) taken into the cells. (B) Immunofluorescence staining of non-permeabilized cells (A–C: untreated; D–F: DXD treatment; G–I: LH3N treatment) by the affinity-purified PLOD3 antibody (green colour, A, D and G), Wheat germ agglutinin (cell surface marker, red colour, B, E and H), and Hoechst 33258 (nuclei marker, blue colour). Parts C, F and I show the overlapping of the LH3 staining with the WGA staining on the cell surface (yellow colour, indicated by arrowheads). Untreated cells (C) are used as a background control, showing a small amount of endogenous LH3 on the cell surface. The cells treated with the DXD fragment (F) or the LH3N fragment (I) show much more overlap of the LH3 with the WGA, indicating that both fragments are bound to the cell surface.
Mentions: We first determined whether the LH3 N-terminal fragment and/or its DXD mutated form bound to the cell surface when added to the cell medium. HT-1080 cells were incubated with the fragments (3 μg/ml) for 3 hrs at 37°C. The fragments remaining in the medium served as a molecular weight control. The cell lysate and the cell pellet were collected after sonication and centrifugation. The his-tagged proteins in lysate and medium were concentrated by Ni-NTA column. All fractions were re-suspended in a SDS sample loading buffer, and analysed by Western blotting with the antibody hLH3N1 against the purified human LH3 N-terminal fragment (Fig. 3A). The antibody detected a major 30 kD band in the cell pellet fraction, indicating that the fragments were bound to the surface of the HT-1080 cells (Fig. 3A, lane 1–3). The same-sized bands in medium confirmed that similar amounts of both fragments were used in the experiment (Fig. 3A, lane 4–6). A faint band was also observed in the cell lysate after Nickel purification, indicating that a trace amount of the fragment was internalized in the cell (Fig. 3A, lane 7–9) and suggesting that endocytosis is associated with the phenomenon. Both fragments, mutated and wild-type, behaved similarly. Furthermore, immuno-fluorescence stainings of non-permeabilized HT-1080 cells treated with the DXD fragment and with the LH3 N-terminal fragment revealed an overlay of the LH3 staining with the cell surface marker, so confirming that the fragments were bound to the cell surface (Fig. 3B, F, I).

Bottom Line: We have demonstrated that LH3 is found not only intracellularly, but also on the cell surface and in the extracellular space, suggesting additional functions for LH3.Here we show that the targeted disruption of LH3 by siRNA causes a marked reduction of both glycosyltransferase activities, and the overexpression of LH3 in HT-1080 cells increases hydroxylation of lysyl residues and the subsequent galactosylation and glucosylation of hydroxylysyl residues.The overexpression of a glycosyltransferase-deficient mutant or targeted disruption of LH3 by siRNA in cells results in abnormal cell morphology followed by cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Biocenter Oulu, University of Oulu, Oulu, Finland.

ABSTRACT
Lysyl hydroxylase (LH) isoform 3 is a post-translational enzyme possessing LH, collagen galactosyltransferase (GT) and glucosyltransferase (GGT) activities. We have demonstrated that LH3 is found not only intracellularly, but also on the cell surface and in the extracellular space, suggesting additional functions for LH3. Here we show that the targeted disruption of LH3 by siRNA causes a marked reduction of both glycosyltransferase activities, and the overexpression of LH3 in HT-1080 cells increases hydroxylation of lysyl residues and the subsequent galactosylation and glucosylation of hydroxylysyl residues. These data confirm the multi-functionality of LH3 in cells. Furthermore, treatment of cells in culture medium with a LH3 N-terminal fragment affects the cell behaviour, rapidly leading to arrest of growth and further to lethality if the fragment is glycosyltransferase-deficient, and leading to stimulation of proliferation if the fragment contains LH3 glycosyltransferase activities. The effect is reversible, the cells recovering after removal of the glycosyltransferase-deficient fragment. The findings were confirmed by overexpressing the full-length LH3 in native or mutated forms in the cells. The data indicate that the increase in proliferation depends on the glycosyltransferase activity of LH3. The overexpression of a glycosyltransferase-deficient mutant or targeted disruption of LH3 by siRNA in cells results in abnormal cell morphology followed by cell death. Our data clearly indicate that the deficiency of LH3 glycosyltransferase activities, especially in the extracellular space, causes growth arrest revealing the importance of the glycosyltransferase activities of LH3 for cell growth and viability, and identifying LH3 as a potential target for medical applications, such as cancer therapy.

Show MeSH
Related in: MedlinePlus