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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 effect of the glycosyltransferase activity of LH3 on HT-1080 cell proliferation. The cells were grown under the conditions described in experimental procedures. Student's t-test (one-tailed) was used for the statistical analysis, P< 0.05 was taken as a significant change. The changes of cell numbers deviating significantly from control cells are indicated by stars. (A) Extracellular effect in the presence of the wild-type LH3 N-terminal fragment or its glycosyltransferase-deficient form (DXD fragment) in cell medium. Untreated cells were used as controls. Significant inhibition of cell proliferation was observed with the DXD fragment-treated cells. (B) Intracellular effect by stably overexpressing full-length LH-deficient LH3 (clone H14-3). Remarkable acceleration of cell growth was seen, compared to the pcDNA3 vector (clone 9) trans-fected cells, when the glycosyltransferase activities of LH3 (not LH activity of LH3) increased in HT-1080 cells (clone H14-3).
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fig05: The effect of the glycosyltransferase activity of LH3 on HT-1080 cell proliferation. The cells were grown under the conditions described in experimental procedures. Student's t-test (one-tailed) was used for the statistical analysis, P< 0.05 was taken as a significant change. The changes of cell numbers deviating significantly from control cells are indicated by stars. (A) Extracellular effect in the presence of the wild-type LH3 N-terminal fragment or its glycosyltransferase-deficient form (DXD fragment) in cell medium. Untreated cells were used as controls. Significant inhibition of cell proliferation was observed with the DXD fragment-treated cells. (B) Intracellular effect by stably overexpressing full-length LH-deficient LH3 (clone H14-3). Remarkable acceleration of cell growth was seen, compared to the pcDNA3 vector (clone 9) trans-fected cells, when the glycosyltransferase activities of LH3 (not LH activity of LH3) increased in HT-1080 cells (clone H14-3).

Mentions: As demonstrated recently [25, 26], LH3 is secreted from cells and is located on the cell surface and in the extracellular space in some tissues. This extracellular localization differentiates LH3 from the other LH isoforms, although the functions of LH3 in the extracellular space are not known. In order to study the extracellular functions we exposed cell surfaces to medium containing an excess amount of the glycosyltransferase-deficient LH3 N-terminal fragment, and then determined the consequences of the treatment to the cell. The same amount of a LH3 N-terminal fragment with GGT activity was used as a control. We treated HT-1080 cells with a purified 30 kD LH3 N-terminal fragment deficient in the glycosyltransferase activities (the DXD fragment) by adding the fragment (0–4 μg/ml) into the medium. Our preliminary experiments revealed that the effect was concentration dependent, a 3–4 μg/ml concentration of the DXD fragment arresting cell growth already after 24 hrs incubation (see later in Fig. 5A), 3 μg/ml reducing the cell number by 12% and 4 μg/ml by 34%, when compared to the LH3 N-terminal fragment. A fragment concentration of 3 μg/ml was used in further studies.


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 effect of the glycosyltransferase activity of LH3 on HT-1080 cell proliferation. The cells were grown under the conditions described in experimental procedures. Student's t-test (one-tailed) was used for the statistical analysis, P< 0.05 was taken as a significant change. The changes of cell numbers deviating significantly from control cells are indicated by stars. (A) Extracellular effect in the presence of the wild-type LH3 N-terminal fragment or its glycosyltransferase-deficient form (DXD fragment) in cell medium. Untreated cells were used as controls. Significant inhibition of cell proliferation was observed with the DXD fragment-treated cells. (B) Intracellular effect by stably overexpressing full-length LH-deficient LH3 (clone H14-3). Remarkable acceleration of cell growth was seen, compared to the pcDNA3 vector (clone 9) trans-fected cells, when the glycosyltransferase activities of LH3 (not LH activity of LH3) increased in HT-1080 cells (clone H14-3).
© Copyright Policy
Related In: Results  -  Collection

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

fig05: The effect of the glycosyltransferase activity of LH3 on HT-1080 cell proliferation. The cells were grown under the conditions described in experimental procedures. Student's t-test (one-tailed) was used for the statistical analysis, P< 0.05 was taken as a significant change. The changes of cell numbers deviating significantly from control cells are indicated by stars. (A) Extracellular effect in the presence of the wild-type LH3 N-terminal fragment or its glycosyltransferase-deficient form (DXD fragment) in cell medium. Untreated cells were used as controls. Significant inhibition of cell proliferation was observed with the DXD fragment-treated cells. (B) Intracellular effect by stably overexpressing full-length LH-deficient LH3 (clone H14-3). Remarkable acceleration of cell growth was seen, compared to the pcDNA3 vector (clone 9) trans-fected cells, when the glycosyltransferase activities of LH3 (not LH activity of LH3) increased in HT-1080 cells (clone H14-3).
Mentions: As demonstrated recently [25, 26], LH3 is secreted from cells and is located on the cell surface and in the extracellular space in some tissues. This extracellular localization differentiates LH3 from the other LH isoforms, although the functions of LH3 in the extracellular space are not known. In order to study the extracellular functions we exposed cell surfaces to medium containing an excess amount of the glycosyltransferase-deficient LH3 N-terminal fragment, and then determined the consequences of the treatment to the cell. The same amount of a LH3 N-terminal fragment with GGT activity was used as a control. We treated HT-1080 cells with a purified 30 kD LH3 N-terminal fragment deficient in the glycosyltransferase activities (the DXD fragment) by adding the fragment (0–4 μg/ml) into the medium. Our preliminary experiments revealed that the effect was concentration dependent, a 3–4 μg/ml concentration of the DXD fragment arresting cell growth already after 24 hrs incubation (see later in Fig. 5A), 3 μg/ml reducing the cell number by 12% and 4 μg/ml by 34%, when compared to the LH3 N-terminal fragment. A fragment concentration of 3 μg/ml was used in further studies.

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