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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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Overexpression of LH3 cDNAs in HT-1080 cells. Cells were transfected with different human LH3 cDNAs in a pcDNA3 vector with a 6×His-tag at the N-terminal after the signal peptide. His-tag antibodies were used to detect the overexpressed LH3 and its mutated variants by Western blot analysis in HT-1080 cell lysates after Nickel purification [24]. The molecular weight of the LH3 bands corresponds to about 85 kD. (A) The transfected cells were kept under constant selection pressure with G418 at 750 μg/ml. (B) The LH3 protein produced from the single stably transfected clones.
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fig01: Overexpression of LH3 cDNAs in HT-1080 cells. Cells were transfected with different human LH3 cDNAs in a pcDNA3 vector with a 6×His-tag at the N-terminal after the signal peptide. His-tag antibodies were used to detect the overexpressed LH3 and its mutated variants by Western blot analysis in HT-1080 cell lysates after Nickel purification [24]. The molecular weight of the LH3 bands corresponds to about 85 kD. (A) The transfected cells were kept under constant selection pressure with G418 at 750 μg/ml. (B) The LH3 protein produced from the single stably transfected clones.

Mentions: In order to verify that LH3 modifies lysyl residues in collagens in cellulo, we have generated wild-type, LH-deficient and glycosyltransferase-deficient human LH3 cDNA constructs in a mammalian pcDNA3 expression vector, and successfully transfected them separately into HT-1080 cells. These cells produce mainly type IV collagen [34, 35] that is highly hydroxylated and glycosylated [6, 10], and thus a good read-out molecule for the study. Western blot analysis showed clearly the overexpression of human LH3 and its mutated variants in the transfected HT-1080 cells under constant selection pressure (Fig. 1A) or with stably transfected clones (Fig. 1B). Stably transfected clones were easily established with all constructs except the glycosyltransferase-deficient mutant (Fig. 1B). The transfected cell lines were used to analyse cellular LH, GT and GGT activities as well as the amount of lysyl modifications in collagenous proteins.


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)

Overexpression of LH3 cDNAs in HT-1080 cells. Cells were transfected with different human LH3 cDNAs in a pcDNA3 vector with a 6×His-tag at the N-terminal after the signal peptide. His-tag antibodies were used to detect the overexpressed LH3 and its mutated variants by Western blot analysis in HT-1080 cell lysates after Nickel purification [24]. The molecular weight of the LH3 bands corresponds to about 85 kD. (A) The transfected cells were kept under constant selection pressure with G418 at 750 μg/ml. (B) The LH3 protein produced from the single stably transfected clones.
© Copyright Policy
Related In: Results  -  Collection

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

fig01: Overexpression of LH3 cDNAs in HT-1080 cells. Cells were transfected with different human LH3 cDNAs in a pcDNA3 vector with a 6×His-tag at the N-terminal after the signal peptide. His-tag antibodies were used to detect the overexpressed LH3 and its mutated variants by Western blot analysis in HT-1080 cell lysates after Nickel purification [24]. The molecular weight of the LH3 bands corresponds to about 85 kD. (A) The transfected cells were kept under constant selection pressure with G418 at 750 μg/ml. (B) The LH3 protein produced from the single stably transfected clones.
Mentions: In order to verify that LH3 modifies lysyl residues in collagens in cellulo, we have generated wild-type, LH-deficient and glycosyltransferase-deficient human LH3 cDNA constructs in a mammalian pcDNA3 expression vector, and successfully transfected them separately into HT-1080 cells. These cells produce mainly type IV collagen [34, 35] that is highly hydroxylated and glycosylated [6, 10], and thus a good read-out molecule for the study. Western blot analysis showed clearly the overexpression of human LH3 and its mutated variants in the transfected HT-1080 cells under constant selection pressure (Fig. 1A) or with stably transfected clones (Fig. 1B). Stably transfected clones were easily established with all constructs except the glycosyltransferase-deficient mutant (Fig. 1B). The transfected cell lines were used to analyse cellular LH, GT and GGT activities as well as the amount of lysyl modifications in collagenous proteins.

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