Cloning and expression of mannosylphospho dolichol synthase from bovine adrenal medullary capillary endothelial cells.
Mannosylphospho dolichol synthase (DPMS) is a critical enzyme in the biosynthesis of lipid-linked oligosaccharide (LLO; Glc(3)Man(9)GlcNAc(2)-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.
Baksi
Krishna
K
Department of Anatomy and Cell Biology, School of Medicine, Universidad Central del Caribe, Bayamón, PR 00930-3100, USA.
Zhang
Zhenbo
Z
Banerjee
Aditi
A
Banerjee
Dipak K
DK
D000311:Adrenal Glands / Q000098:blood supply / Q000166:cytology*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D049109:Cell Proliferation; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D008364:Mannosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D018919:Neovascularization, Physiologic / Q000502:physiology; D010766:Phosphorylation; D016415:Sequence Alignment
D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase