Importance of a Factor VIIIc-Like Glycoprotein Expressed in Capillary Endothelial Cells (eFactor VIIIc) in Angiogenesis

Banerjee, Dipak K.; Oliveira, Caio C.; Tavárez, José J.; Katiyar, Viswa N.; Saha, Subiman; Martínez, Juan A.; Banerjee, Aditi; Sánchez, Aurymar; Baksi, Krishna

doi:10.1007/978-1-4419-7877-6_24

Cited by 6 publications

(3 citation statements)

References 22 publications

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“…cAMP turned out to be an activator of angiogenesis and a positive regulator of N-linked glycosylation, especially that of the DPMS (18) β-Adrenoreceptor is a trimeric G-protein coupled receptor and when activated the rate of LLO biosynthesis and its turnover are increased. As a result, the N-glycosylation of capillary endothelial cells Factor VIIIc (eFVIIIc) is enhanced (19). cDNA cloning of capillary endothelial cell DPMS and its deduced amino acid sequence indicated the presence of a consensus sequence for phosphorylation by cAMP-dependent protein kinase (20; GenBank #GQ367549).…”

Section: Introductionmentioning

confidence: 99%

Mannosylphosphodolichol synthase overexpression supports angiogenesis

Zhang

Banerjee

Baksi

et al. 2009

Biocatalysis and Biotransformation

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Mannosylphospho dolichol synthase (DPMS) plays a critical role in Glc3Man9GlcNAc2-PP-Dol (lipid-linked oligosaccharide, LLO) biosynthesis, an essential intermediate in asparagine-linked (N-linked) protein glycosylation. We have observed earlier that phosphorylation of DPMS increases the catalytic activity of the enzyme by increasing the Vmax as well as the enzyme turnover (kcat) without significantly changing the Km for GDP-mannose. As a result, LLO biosynthesis, turnover and protein N-glycosylation are increased. This is manifested in increased proliferation of capillary endothelial cells, i.e., angiogenesis. We have then asked if the phosphorylation event or the up-regulation of the DPMS due to over production of the enzyme is a key factor in up-regulating angiogenesis? This question has been answered by isolating a stable capillary endothelial cell clone overexpressing the DPMS gene. Our results indicate that the DPMS overexpressing clone has a high level DPMS mRNA judged by QRT-PCR. The clone also expresses nearly four-times higher DPMS protein over the clone transfected with pEGFP-N1 vector only (i.e., control) as analyzed by western blotting. Most importantly, the overexpressing DPMS clone has ~108% higher DPMS activity than that of the vector control. Immunofluorescence microscopy with Texas-Red conjugated WGA indicates a high level expression of GlcNAc-β-(1,4)-GlcNAc)1-4-β-GlcNAc-NeuAc glycans on the external surface of the capillary endothelial cells overexpressing DPMS. Increased cellular proliferation and accelerated healing of the wound induced by a mechanical stress of the DPMS overexpressing clone unequivocally supports DPMS for angiogenesis.

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Section: Introductionmentioning

confidence: 99%

Mannosylphosphodolichol synthase overexpression supports angiogenesis

Zhang

Banerjee

Baksi

et al. 2009

Biocatalysis and Biotransformation

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“…β-Adrenoreceptor is a trimeric G-protein coupled receptor and when activated the rate of LLO biosynthesis and turnover are increased. As a result the N -glycosylation of Factor VIIIc and other cellular proteins is enhanced [24, 25]. Factor VIIIc in capillary endothelial cells ( i.e.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2009

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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.

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“…We have observed that proliferation of capillary endothelial cells as well as the capillary lumen formation is upregulated when the cells are treated with 8Br-cAMP [35, 30, 40]. Upregulated mannosylphospho dolichol synthase (DPMS) activity increases the synthesis and turnover of Glc 3 Man 9 GlcNAc 2 -PP-Dol (lipid-linked oligosaccharide, LLO) and consequently the N -glycosylation of Factor VIIIC (eFVIIIc); enhancing angiogenesis [41]. Tunicamycin, a glucosamine-containing pyrimidine nucleoside and a competitive inhibitor of GPT inhibits protein N -glycosylation by blocking the LLO synthesis in the ER (42).…”

Section: Discussionmentioning

confidence: 99%

Balancing life with glycoconjugates: Monitoring unfolded protein response-mediated anti-angiogenic action of tunicamycin by Raman spectroscopy

Longas

Kotapati

Prasad

et al. 2012

Pure and Applied Chemistry

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Asparagine-linked protein glycosylation is a hallmark for glycoprotein structure and function. Its impairment by tunicamycin [a competitive inhibitor of N-acetylglucosaminyl 1-phosphate transferase (GPT)] has been known to inhibit neo-vascularization (i.e., angiogenesis) in humanized breast tumor due to an induction of ER stress-mediated unfolded protein response (UPR). The studies presented here demonstrate that (i) tunicamycin (i) inhibits capillary endothelial cell proliferation in a dose dependent manner; (ii) treated cells are incapable of forming colonies upon its withdrawal; and (iii) tunicamycin treatment causes nuclear fragmentation. Tunicamycin-induced ER stress-mediated UPR event in these cells was studied with the aid of Raman spectroscopy, in particular, the interpretation of bands at 1672, 1684 and 1694 cm−1, which are characteristics of proteins and originate from C=O stretching vibrations of mono-substituted amides. In tunicamycin-treated cells these bands decreased in area as follows: at 1672 cm−1 by 41.85% at 3 h and 55.39% at 12 h; at 1684 cm−1 by 20.63% at 3 h and 40.08% at 12 h; and also at 1994 cm−1 by 33.33% at 3 h and 32.92% at 12 h, respectively. Thus, in the presence of tunicamycin, newly synthesized protein chains fail to arrange properly into their final secondary and/or tertiary structures, and the random coils they form had undergone further degradation.

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Importance of a Factor VIIIc-Like Glycoprotein Expressed in Capillary Endothelial Cells (eFactor VIIIc) in Angiogenesis

Cited by 6 publications

References 22 publications

Mannosylphosphodolichol synthase overexpression supports angiogenesis

Mannosylphosphodolichol synthase overexpression supports angiogenesis

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

Balancing life with glycoconjugates: Monitoring unfolded protein response-mediated anti-angiogenic action of tunicamycin by Raman spectroscopy

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