Glycoconjugate Journal

, Volume 26, Issue 6, pp 635–645

Cloning and expression of mannosylphospho dolichol synthase from bovine adrenal medullary capillary endothelial cells

  • Krishna Baksi
  • Zhenbo Zhang
  • Aditi Banerjee
  • Dipak K. Banerjee
Article

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 Mr 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.

Keywords

Angiogenesis Mannosylphospho dolichol synthase EC 2.41.83 cAMP Lipid-linked oligosaccharide N-linked glycoprotein Breast cancer 

Abbreviations

EMEM

minimal essential medium with Earle’s salt

PBS

phosphate-buffer-saline

NP-40

Nonident P-40

cAMP

adenosine 3′,5′-cyclic monophosphate

RNA

ribonucleic acid

mRNA

messenger ribonucleic acid

DNA

deoxyriboynucleic acid

PKA

cAMP-dependent protein kianse

ER

endoplasmic reticulum

DPMS

mannosylphospho dolichol synthase

PCR

polymerase chain reaction

RT-PCR

reverse transcription-polymerase chain reaction

EDTA

ethylenediamine tetraacetic acid

Dol-P-Man

dolichol-P-mannose

GDP

guanosine diphosphate

DTT

dithiothretol

Dol-P

dolichyl monophosphate

DMSO

dimethyl sulfoxide

SDS

sodium dodecylsulfate

PAGE

polyacrylamide gel electrophoresis

LLO

lipid-linked oligosaccharide

Glc3Man9GlcNAc2-PP-Dol

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Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Krishna Baksi
    • 1
  • Zhenbo Zhang
    • 2
  • Aditi Banerjee
    • 2
  • Dipak K. Banerjee
    • 2
  1. 1.Department of Anatomy and Cell Biology, School of MedicineUniversidad Central del CaribeBayamónUSA
  2. 2.Department of Biochemistry, School of MedicineUniversity of Puerto RicoSan JuanUSA

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