Mannosylphosphodolichol synthase overexpression supports angiogenesis.
Mannosylphospho dolichol synthase (DPMS) plays a critical role in Glc(3)Man(9)GlcNAc(2)-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 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 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-beta-(1,4)-GlcNAc)1-4-beta-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.
Zhang
Zhenbo
Z
Department of Biochemistry, School of Medicine, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico, USA.
Banerjee
Aditi
A
Baksi
Krishna
K
Banerjee
Dipak K
DK