Cloning and expression of mannosylphospho dolichol synthase from bovine adrenal medullary capillary endothelial cells |
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Authors: | Krishna Baksi Zhenbo Zhang Aditi Banerjee Dipak K Banerjee |
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Institution: | (1) Department of Anatomy and Cell Biology, School of Medicine, Universidad Central del Caribe, Bayamón, PR 00930-3100, USA;(2) Department of Biochemistry, School of Medicine, University of Puerto Rico, Medical Sciences Campus, San Juan, PR 00936-5067, USA |
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Abstract: | Mannosylphospho dolichol synthase (DPMS) is a critical enzyme in the biosynthesis of lipid-linked oligosaccharide (LLO; Glc3Man9GlcNAc2-PP-Dol), a pre-requisite for asparagine-linked (N-linked) protein glycosylation. We have shown earlier that DPMS is important
for angiogenesis, i.e., endothelial cell proliferation. This is true when cAMP is used for intracellular signaling. During cAMP signaling, DPMS
is activated and ER stress is reduced. To understand the activation of DPMS at the molecular level we have isolated a cDNA
clone for the DPMS gene (bDPMS) from the capillary endothelial cells of bovine adrenal medulla. DNA sequencing and the deduced
amino acid sequence have established that bDPMS has a motif to be phosphorylated by cAMP-dependent protein kinase (PKA). Based
on the sequence information Serine 165 has been found to be the phosphorylation target in bDPMS. Hydropathy Index when plotted
against amino acid number indicates the presence of a hydrophobic region around the amino acid residues 120–160, supporting
that bDPMS has one membrane spanning region. The recombinant bDPMS has now been purified as His-tag protein with an apparent
molecular weight of M
r 33 kDa. Additionally, we show here that overexpression of DPMS is indeed angiogenic. The capillary endothelial cells proliferate
at a higher rate carrying the DPMS overexpression plasmid over the parental cells or the vector. |
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Keywords: | Angiogenesis Mannosylphospho dolichol synthase EC 2 41 83 cAMP Lipid-linked oligosaccharide N-linked glycoprotein Breast cancer |
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