The Hexosamine Biosynthetic Pathway: A role in nutrient regulation of growth signalling in the human placenta

Objective: Glucose or glucosamine-stimulated flux through the hexosamine biosynthetic pathway (HBP) leads to the post-translational addition of N-acetyl-glucosamine (GlcNAc) at Ser/Thr residues of intracellular proteins (O-GlcNAcylation), influencing their function by altering location, interactions and phosphorylation. We have previously shown that increased HBP flux inhibits trophoblast proliferation; here we investigate the pathways responsible.Methods: An O-GlcNAcylation site prediction algorithm, developed using information on amino acid sequences flanking published O-GlcNAcylated residues, was used to screen proteins known to influence trophoblast proliferation. BeWo cells were cultured in serum-free DMEM/F12 containing glucose (5-25mM) or glucosamine (0.5-10mM) for 24h, then switched to control medium for a further 48h. Cell number and viability were assessed by counting and trypan-blue exclusion. Global protein GlcNAcylation was confirmed by Western blotting using an anti-O-GlcNAc antibody. The localisation and GlcNAcylation status of tyrosine-protein phosphatase non-receptor type 11 (SHP-2) were assessed by immunocytochemistry and Western blotting using SHP-2 immunoprecipitates with an anti-O-GlcNAc antibody or wheat-germ agglutinin (WGA) respectively.Results: Protein GlcNAcylation increased with increasing glucosamine/glucose. 24h exposure to 10mM glucosamine led to BeWo cell death; lower concentrations (2.5-5mM) inhibited growth and cells returned to control medium remained in a slower proliferative cycle for at least 48h. 25mM glucose also inhibited proliferation. The prediction algorithm revealed potential O-GlcNAcylation sites in numerous transcription factors and signalling molecules, including Ser/Thr sites in SHP-2 that are close to Tyr residues needed for its activation. Blotting of SHP-2 immunoprecipitates with an anti-O-GlcNAc antibody or WGA demonstrated O-GlcNAcylation of SHP-2. Punctate SHP-2 membrane and cytoplasmic staining was present under all conditions tested suggesting glucose/glucosamine treatment does not affect its expression/localisation.Conclusion: SHP-2, a known regulator of trophoblast proliferation, is post-translationally modified by O-GlcNAc; regulation of transcription factors / signalling molecules by GlcNAcylation suggests a mechanism by which placental growth can be altered by nutrient availability.