Mannosylphosphodolichol synthase overexpression supports angiogenesis

Abstract

Mannosylphosphodolichol synthase (DPMS) plays a critical role in Glc₃Man₉GlcNAc₂-PP-Dol (lipid-linked oligosaccha-ride, LLO) biosynthesis, an essential intermediate in asparagine-linked (N-linked) protein glycosylation. We observed earlier that phosphorylation of DPMS increases the catalytic activity of the enzyme by increasing the V_max as well as the enzyme turnover (k_cat) without significantly changing the K_m 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 upregulation of DPMS due to overproduction of the enzyme is the key factor in upregulating angiogenesis? This question has been answered by isolating a stable capillary endothelial cell clone overexpressing the gene encoding DPMS. Our results indicate that the DPMS-overexpressing clone has a high level of DPMS mRNA as judged by QRT-PCR. The clone also expresses nearly four times more DPMS protein than 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 the vector control. Immunofluorescence microscopy with Texas Red-conjugated wheat germ agglutinin indicates a high level of 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 mechanical stress of the DPMS-overexpressing clone unequivocally supports a role of DPMS in angiogenesis.