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

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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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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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Acknowledgement

The editorial assistance of Ms. Laura M. Bretaña is greatly appreciated. The work was partly supported by the funds from the University of Puerto Rico Medical sciences Campus and the grants from the Department of Defense DAMD17-03-1-0754, NIH U54-CA096297and the Susan G. Komen for the cure BCTR58206 (DKB) and the NIH/NCRR/RCMI grant G12-RR03035 (KB).

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Authors and Affiliations

  1. Department of Anatomy and Cell Biology, School of Medicine, Universidad Central del Caribe, Bayamón, PR, 00930-3100, USA

    Krishna Baksi

  2. Department of Biochemistry, School of Medicine, University of Puerto Rico, Medical Sciences Campus, San Juan, PR, 00936-5067, USA

    Zhenbo Zhang, Aditi Banerjee & Dipak K. Banerjee

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  1. Krishna Baksi
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  2. Zhenbo Zhang
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  4. Dipak K. Banerjee
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Correspondence to Dipak K. Banerjee.

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Baksi, K., Zhang, Z., Banerjee, A. et al. Cloning and expression of mannosylphospho dolichol synthase from bovine adrenal medullary capillary endothelial cells. Glycoconj J 26, 635–645 (2009). https://doi.org/10.1007/s10719-008-9214-9

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