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Cellular metabolism of unnatural sialic acid precursors

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Abstract

Carbohydrates, in addition to their metabolic functions, serve important roles as receptors, ligands, and structural molecules for diverse biological processes. Insight into carbohydrate biology and mechanisms has been aided by metabolic oligosaccharide engineering (MOE). In MOE, unnatural carbohydrate analogs with novel functional groups are incorporated into cellular glycoconjugates and used to probe biological systems. While MOE has expanded knowledge of carbohydrate biology, limited metabolism of unnatural carbohydrate analogs restricts its use. Here we assess metabolism of SiaDAz, a diazirine-modified analog of sialic acid, and its cell-permeable precursor, Ac4ManNDAz. We show that the efficiency of Ac4ManNDAz and SiaDAz metabolism depends on cell type. Our results indicate that different cell lines can have different metabolic roadblocks in the synthesis of cell surface SiaDAz. These findings point to roles for promiscuous intracellular esterases, kinases, and phosphatases during unnatural sugar metabolism and provide guidance for ways to improve MOE.

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Acknowledgments

We thank Sunil Laxman and Benjamin Tu for guidance on LC-MS/MS analysis. We thank Maciej Kukula at the Shimadzu Center for Advanced Analytical Chemistry (SCAAC) at the University of Texas at Arlington for aiding us with mass spectrometry identification of NeuAc-9-P. We thank Yibing Wang, Randy Parker, Amberlyn Wands, Akiko Fujita, and Fan Yang for experimental assistance and thank Akiko Fujita and Amberlyn Wands for comments on the manuscript. We acknowledge support from the National Institutes of Health (NIH R01GM090271), the Cancer Prevention and Research Institute of Texas (CPRIT RP110080), and the Welch Foundation (I-1686). NDP was supported by a predoctoral fellowship from the NIH (F30AG040909) and ACR received support from a training grant from the NIH (T32GM007062).

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    1. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA

      Nam D. Pham, Charles S. Fermaintt, Andrea C. Rodriguez, Janet E. McCombs, Nicole Nischan & Jennifer J. Kohler

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    1. Nam D. Pham
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    2. Charles S. Fermaintt
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    Correspondence to Jennifer J. Kohler.

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    Charles S. Fermaintt and Andrea C. Rodriguez contributed equally to this work.

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    Pham, N.D., Fermaintt, C.S., Rodriguez, A.C. et al. Cellular metabolism of unnatural sialic acid precursors. Glycoconj J 32, 515–529 (2015). https://doi.org/10.1007/s10719-015-9593-7

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