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

Baksi, Krishna; Zhang, Zhenbo; Banerjee, Aditi; Banerjee, Dipak K.

doi:10.1007/s10719-008-9214-9

Cited by 10 publications

(13 citation statements)

References 34 publications

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“…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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“…However, among diversities, the DPMS seem to have a unity. Dpm has now been cloned and sequenced from 34 species including the capillary endothelial cells from bovine adrenal medulla (GenBank identifier GQ367549) [54]. The DNA sequence of all of them has a motif [RR(K)xxS] to be phosphorylated by cAMP-dependent protein kinase (PKA).…”

Section: Scope Of Reviewmentioning

confidence: 99%

N-glycans in cell survival and death: Cross-talk between glycosyltransferases

Banerjee

2012

Biochimica et Biophysica Acta (BBA) - General Subjects

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Asparagine-linked (N-linked) protein glycosylation is one of the most important protein modifications. N-glycans with “high mannose”, “hybrid”, or “complex” type sugar chains participate in a multitude of cellular processes. These include cell–cell/cell–matrix/receptor–ligand interaction, cell signaling/growth and differentiation, to name a few. Many diseases such as disorders of blood clotting, congenital disorder of glycosylation, diseases of blood vessels, cancer, neo-vascularization, i.e., angiogenesis essential for breast and other solid tumor progression and metastasis are associated with N-glycan expression. Biosynthesis of N-glycans requires multiple steps and multiple cellular compartments. Following transcription and translation the proteins migrate to the endoplasmic reticulum (ER) lumen to acquire glycan chain(s) with a defined glycoform, i.e., a tetradecasaccharide. These are further modified, i.e., edited in ER lumen and in Golgi prior to moving to their respective destinations. The tetradecasaccharide is pre-assembled on a poly-isoprenoid lipid called dolichol, and becomes an essential component of the supply chain. Therefore, dolichol cycle synthesizing the lipid-linked oligosaccharide (LLO) is a hallmark for all N-linked glycoproteins. It is expected that there is a great deal of crosstalk between the participating glycosyltransferases and any missed step would express defective N-glycans that could have fatal consequences. The positive impact of the structurally altered N-glycans could lead to discovery of an N-glycan signature for a disease and/or help developing glycotherapeutic treating cancer or other human diseases. The purpose of this review is to identify the gaps of N-glycan biology and help developing appropriate technology for biomedical applications. This article is part of a Special Issue entitled Glycoproteomics.

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“…unpublished observation). The recombinant protein has also been purified from E. coli transformed with S. cerevisiae DPMS gene as well as the gene from bovine capillary endothelial cells [24,25]. The enzyme from higher eukaryotes is most active in the presence of Mn 2+ but that from yeast and archaea prefer Mg 2+ .…”

Section: Dpms In Glycosylationmentioning

confidence: 99%

“…The consequence is activation of “angiogenic switch” and increased cellular proliferation. It is important to note that bDPMS has a PKA motif (RKIIS 165 R) [25] (Figure 4d). The underline mechanism includes: (i) no changes in the expression of Bcl-2 (a pro-angiogenic protein) as well as caspase-3, and -9 (pro-apoptotic proteins) activities in 8Br-cAMP treated cells; and (ii) 4.5–4.8 fold and 1.5–1.6 fold reduced expression of ER chaperones GRP-78/Bip and GRP-94 whereas 1.4–1.6 fold increased expression of cytoplasmic chaperones HSP-70 and HSP-90 in 8Br-cAMP treated cells.…”

Section: Impact Of Dpms In Biological Functions and In Human Healthmentioning

confidence: 99%

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Dolichol phosphate mannose synthase: a Glycosyltransferase with Unity in molecular diversities

et al. 2017

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N-glycans provide structural and functional stability to asparagine-linked (N-linked) glycoprotein, and add flexibility. Glycan biosynthesis is elaborative, multi-compartmental and involves many glycosyltransferases. Failure to assemble N-glycans leads to phenotypic changes developing infection, cancer, congenital disorders of glycosylation (CDGs) among others. Biosynthesis of N-glycans begins at the endoplasmic reticulum (ER) with the assembly of dolichol-linked tetra-decasaccharide (Glc3Man9GlcNAc2-PP-Dol) where dolichol phosphate mannose synthase (DPMS) plays a central role. DPMS is also essential for GPI anchor biosynthesis as well as for O- and C-mannosylation of proteins in yeast and in mammalian cells. DPMS has been purified from several sources and its gene has been cloned from 39 species (e.g., from protozoan parasite to human). It is an inverting GT-A folded enzyme and classified as GT2 by CAZy (carbohydrate active enZyme; http://www.cazy.org). The sequence alignment detects the presence of a metal binding DAD signature in DPMS from all 39 species but finds cAMP-dependent protein phosphorylation motif (PKA motif) in only 38 species. DPMS also has hydrophobic region(s). Hydropathy analysis of amino acid sequences from bovine, human, S. crevisiae and A. thaliana DPMS show PKA motif is present between the hydrophobic domains. The location of PKA motif as well as the hydrophobic domain(s) in the DPMS sequence vary from species to species. For example, the domain(s) could be located at the center or more towards the C-terminus. Irrespective of their catalytic similarity, the DNA sequence, the amino acid identity, and the lack of a stretch of hydrophobic amino acid residues at the C-terminus, DPMS is still classified as Type I and Type II enzyme. Because of an apparent bio-sensing ability, extracellular signaling and microenvironment regulate DPMS catalytic activity. In this review, we highlight some important features and the molecular diversities of DPMS.

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

Cited by 10 publications

References 34 publications

Mannosylphosphodolichol synthase overexpression supports angiogenesis

Mannosylphosphodolichol synthase overexpression supports angiogenesis

N-glycans in cell survival and death: Cross-talk between glycosyltransferases

Dolichol phosphate mannose synthase: a Glycosyltransferase with Unity in molecular diversities

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