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Role of Nucleotide Sugar Pools in the Inhibition of NCAM Polysialylation by Ammonia
Author(s) -
Zanghi James A.,
Mendoza Thomas P.,
Schmelzer Albert E.,
Knop Richard H.,
Miller William M.
Publication year - 1998
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp9800945
Subject(s) - polysialic acid , sialic acid , neural cell adhesion molecule , chemistry , biochemistry , glucosamine , chinese hamster ovary cell , cytidine , cell , cell adhesion , enzyme , receptor
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 NH 4 Cl 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 NH 4 Cl decreased PolySia content by 45% and increased UDP‐GlcNAc in SCLC cells by 2‐fold. The discrepancy between the {GlcN+Urd} and NH 4 Cl effects on UDP‐GlcNAc and PolySia suggests that accumulation of UDP‐GlcNAc is only partially responsible for decreased polysialylation in response to NH 4 Cl. 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.