Role of nucleotide sugar pools in the inhibition of NCAM polysialylation by ammonia

Ammonia in animal cell cultures has been shown to specifically inhibit terminal sialylation of N- and O-linked oligosaccharides of glycoproteins. For example, we have previously shown that as little as 2.5 mM NH4Cl can decrease neural cell adhesion molecule (NCAM) polysialylation in both small cell lung cancer (SCLC) and Chinese hamster ovary (CHO) cells. Besides its potential involvement in SCLC metastasis, polysialic acid (PolySia) is a sensitive marker for measuring changes in sialylation. The role of UDP-N-acetylglucosamine (UDP-GlcNAc) in ammonia's inhibition of NCAM polysialylation was examined by adding glucosamine (GlcN) and uridine (Urd) to the cultures. This bypassed feedback inhibition of GlcN-6-P synthase and increased UDP-GlcNAc content by 25-fold in SCLC cells. After 3 days, PolySia levels were reduced to 10% of control with little effect on NCAM protein content. The extensive decrease in PolySia was confirmed in CHO cells. The effects of GlcN or Urd alone were less extensive, lending support to a specific role for UDP-GlcNAc in inhibition by ammonia. By comparison, 20 mM NH4Cl decreased PolySia content by 45% and increased UDP-GlcNAc in SCLC cells by 2-fold. The discrepancy between the {GlcN+Urd} and NH4Cl effects on UDP-GlcNAc and PolySia suggests that accumulation of UDP-GlcNAc is only partially responsible for decreased polysialylation in response to NH4Cl. In an attempt to increase NCAM polysialylation, N-acetylmannosamine and cytidine were added to cultures in order to circumvent the feedback inhibition of CMP-sialic acid synthesis. However, this only slightly increased PolySia levels and failed to counter ammonia's inhibition of NCAM polysialylation.