Expression of blood clotting Factor VIII:C gene in capillary endothelial cells

Banerjee, Dipak K.; Tavárez, José J.; Oliveira, Caio C.

doi:10.1016/0014-5793(92)80831-z

Cited by 12 publications

(6 citation statements)

References 25 publications

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“…The Protein A-Sepharose antigen-antibody complexes were washed twice with NET buffer, twice with washing buffer, and once with phosphate-buffered saline, pH 7.4. The immune complexes were released by boiling at 100°C for 5 min and analyzed by SDS-PAGE (12%) followed by autoradiography (48).…”

Section: Construction Of the Expression Vector Bl21(de3)pdpm1-9 Andmentioning

confidence: 99%

In Vitro Phosphorylation by cAMP-dependent Protein Kinase Up-regulates Recombinant Saccharomyces cerevisiae Mannosylphosphodolichol Synthase

Banerjee

Carrasquillo

Hughey

et al. 2005

Journal of Biological Chemistry

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DPM1 is the structural gene for mannosylphosphodolichol synthase (i.e. Dol-P-Man synthase, DPMS) in Saccharomyces cerevisiae. Earlier studies with cDNA cloning and sequence analysis have established that 31-kDa DPMS of S. cerevisiae contains a consensus sequence (YRRVIS 141 ) that can be phosphorylated by cAMPdependent protein kinase (PKA). We have been studying the up-regulation of DPMS activity by protein kinase A-mediated phosphorylation in higher eukaryotes, and used the recombinant DPMS from S. cerevisiae in this study to advance our knowledge further. DPMS catalytic activity was indeed enhanced severalfold when the recombinant protein was phosphorylated in vitro. The rate as well as the magnitude of catalysis was higher with the phosphorylated enzyme. A similar increase in the catalytic activity was also observed when the in vitro phosphorylated recombinant DPMS was assayed as a function of increasing concentrations of exogenous dolichylmonophosphate (Dol-P). Kinetic studies indicated that there was no change in the K m for GDPmannose between the in vitro phosphorylated and control recombinant DPMS, but the V max was increased by 6-fold with the phosphorylated enzyme. In vitro phosphorylated recombinant DPMS also exhibited higher enzyme turnover (k cat ) and enzyme efficiency (k cat /K m ). SDS-PAGE followed by autoradiography of the 32 P-labeled DPMS detected a 31-kDa phosphoprotein, and immunoblotting with anti-phosphoserine antibody established the presence of a phosphoserine residue in in vitro phosphorylated recombinant DPMS. To confirm the phosphorylation activation of recombinant DPMS, serine 141 in the consensus sequence was replaced with alanine by PCR site-directed mutagenesis. The S141A DPMS mutant exhibited more than half-a-fold reduction in catalytic activity compared with the wild type when both were analyzed after in vitro phosphorylation. Thus, confirming that S. cerevisiae DPMS activity is indeed regulated by the cAMP-dependent protein phosphorylation signal, and the phosphorylation target is serine 141.Mannosylphosphodolichol (Dol-P-Man), 1 a mannosyl donor in the assembly of the precursor oligosaccharide-lipid Glc 3 Man 9 -GlcNAc 2 -PP-Dol in N-glycosylation of proteins, in the synthesis of glycosylphosphatidylinositol (GPI) anchors, in O-glycosylation of proteins in yeast, and in C-mannosylation of Trp-7 in human ribonuclease 2 (RNase 2) is formed by the transfer of mannose from GDP-mannose to the polyisoprenoid-lipid, dolichylmonophosphate (Dol-P; 1-7). Dol-P-Man is synthesized at the cytoplasmic face of the endoplasmic reticulum (ER) membrane (8 -10), and catalyzed by mannosylphosphodolichol synthase (DPMS; Dol-P-Man synthase, EC 2.4.1.83). Dol-P-Man synthase deficiency has been observed in a Class E Thy-1 lymphoma patient and is unable to elongate Man 5 GlcNAc 2 -PP-Dol to Man 9 GlcNAc 2 -PP-Dol, a pre-requisite for Glc 3 Man 9 GlcNAc 2 -PP-Dol synthesis (11). We have also made a similar observation using in vitro studies with amphomycin, a lipopeptide antibiotic from Streptomyces ...

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Section: Construction Of the Expression Vector Bl21(de3)pdpm1-9 Andmentioning

confidence: 99%

In Vitro Phosphorylation by cAMP-dependent Protein Kinase Up-regulates Recombinant Saccharomyces cerevisiae Mannosylphosphodolichol Synthase

Banerjee

Carrasquillo

Hughey

et al. 2005

Journal of Biological Chemistry

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“…Factor VIIIc in capillary endothelial cells ( i.e. , eFVIIIc) has ~18% N-linked glycan and its expression precedes the cell proliferation [26, 27]. Dol-P-Man synthesis when evaluated either by metabolic labeling of cells with 2-[ 3 H]mannose or by measuring the synthase activity in isolated microsomes following isoproterenol treatment, the level of the enzyme activity was high.…”

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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“…Factor VIIIC is a 270 kDa N-linked glycoprotein constitutively expressed in these capillary endothelial cells [28]; it maintains a temporal relationship with the cell proliferation [25]. When examined by flow cytometry, a 10% increase in the expression of Factor VIIIC was observed in capillary endothelial cells treated with 8Br-cAMP for 32 h (Fig.…”

Section: Expression Of Surface Glycans and Factor Viiic In Capillary mentioning

confidence: 94%

“…The cell lysates were centrifuged at 100,000 × g for 40 min at 4 • C (using a Beckman 50 Ti rotor), and the supernatants were kept frozen until analyzed. Factor VIIIC in cell lysates and in the conditioned media were immunoprecipitated with anti-Factor VIIIC monoclonal antibody and analyzed by SDS-PAGE followed by autoradiography [28].…”

Section: Biosynthesis Of Factor Viiicmentioning

confidence: 99%

Potentiation of angiogenic switch in capillary endothelial cells by cAMP: A cross-talk between up-regulated LLO biosynthesis and the HSP-70 expression

et al. 2006

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During tumor growth and invasion, the endothelial cells from a relatively quiescent endothelium start proliferating. The exact mechanism of switching to a new angiogenic phenotype is currently unknown. We have examined the role of intracellular cAMP in this process. When a non-transformed capillary endothelial cell line was treated with 2 mM 8Br-cAMP, cell proliferation was enhanced by approximately 70%. Cellular morphology indicated enhanced mitosis after 32-40 h with almost one-half of the cell population in the S phase. Bcl-2 expression and caspase-3, -8, and -9 activity remained unaffected. A significant increase in the Glc(3)Man(9)GlcNAc(2)-PP-Dol biosynthesis and turnover, Factor VIIIC N-glycosylation, and cell surface expression of N-glycans was observed in cells treated with 8Br-cAMP. Dol-P-Man synthase activity in the endoplasmic reticulum membranes also increased. A 1.4-1.6-fold increase in HSP-70 and HSP-90 expression was also observed in 8Br-cAMP treated cells. On the other hand, the expression of GRP-78/Bip was 2.3-fold higher compared to that of GRP-94 in control cells, but after 8Br-cAMP treatment for 32 h, it was reduced by 3-fold. GRP-78/Bip expression in untreated cells was 1.2-1.5-fold higher when compared with HSP-70 and HSP-90, whereas that of the GRP-94 was 1.5-1.8-fold lower. After 8Br-cAMP treatment, GRP-78/Bip expression was reduced 4.5-4.8-fold, but the GRP-94 was reduced by 1.5-1.6-fold only. Upon comparison, a 2.9-fold down-regulation of GRP-78/Bip was observed compared to GRP-94. We, therefore, conclude that a high level of Glc(3)Man(9)GlcNAc(2)-PP-Dol, resulting from 8Br-cAMP stimulation up-regulated HSP-70 expression and down-regulated that of the GRP-78/Bip, maintained adequate protein folding, and reduced endoplasmic reticulum stress. As a result capillary endothelial cell proliferation was induced.

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Expression of blood clotting Factor VIII:C gene in capillary endothelial cells

Cited by 12 publications

References 25 publications

In Vitro Phosphorylation by cAMP-dependent Protein Kinase Up-regulates Recombinant Saccharomyces cerevisiae Mannosylphosphodolichol Synthase

In Vitro Phosphorylation by cAMP-dependent Protein Kinase Up-regulates Recombinant Saccharomyces cerevisiae Mannosylphosphodolichol Synthase

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

Potentiation of angiogenic switch in capillary endothelial cells by cAMP: A cross-talk between up-regulated LLO biosynthesis and the HSP-70 expression

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