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A Visualizable Chain-Terminating Inhibitor of Glycosaminoglycan Biosynthesis in Developing Zebrafish

Authors

  • Brendan J. Beahm,

    1. Department of Chemistry and Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720 (USA)
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  • Dr. Karen W. Dehnert,

    1. Department of Chemistry and Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720 (USA)
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  • Nicolas L. Derr,

    1. Departments of Molecular Genetics and Molecular and Cellular Biochemistry; Center for RNA Biology, Ohio State University, Columbus, OH 43210 (USA)
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  • Dr. Joachim Kuhn,

    1. Institut für Laboratoriums- und Transfusionmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Georgstraße 11, 32545 Bad Oeynhausen (Germany)
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  • Prof. Johann K. Eberhart,

    1. Department of Molecular Biosciences, University of Texas, Austin, TX 78713 (USA)
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  • Dr. Dorothe Spillmann,

    1. Department of Medical Biochemistry and Microbiology, The Biomedical Center, Uppsala University, Husargatan 3, PO Box 582, 75123 Uppsala (Sweden)
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  • Prof. Sharon L. Amacher,

    1. Departments of Molecular Genetics and Molecular and Cellular Biochemistry; Center for RNA Biology, Ohio State University, Columbus, OH 43210 (USA)
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  • Prof. Carolyn R. Bertozzi

    Corresponding author
    1. Department of Chemistry and Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720 (USA)
    • Department of Chemistry and Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720 (USA)===

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  • We thank J. Jewett, E. Sletten, B. Belardi, J. Hudak, T. Gallagher, S. Laughlin and B. Swarts for helpful discussions and for critical reading of the manuscript. This work was supported by NIH grants to C.R.B. (GM58867) and S.L.A. (GM61952). The lamb1ab1166 allele was isolated in a screen supported by NIH grant HD22486 (to Charles B. Kimmel).

Abstract

Heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycans (GAG) are proteoglycan-associated polysaccharides with essential functions in animals. They have been studied extensively by genetic manipulation of biosynthetic enzymes, but chemical tools for probing GAG function are limited. HS and CS possess a conserved xylose residue that links the polysaccharide chain to a protein backbone. Here we report that, in zebrafish embryos, the peptide-proximal xylose residue can be metabolically replaced with a chain-terminating 4-azido-4-deoxyxylose (4-XylAz) residue by administration of UDP-4-azido-4-deoxyxylose (UDP-4-XylAz). UDP-4-XylAz disrupted both HS and CS biosynthesis and caused developmental abnormalities reminiscent of GAG biosynthesis and laminin mutants. The azide substituent of protein-bound 4-XylAz allowed for rapid visualization of the organismal sites of chain termination in vivo through bioorthogonal reaction with fluorescent cyclooctyne probes. UDP-4-XylAz therefore complements genetic tools for studies of GAG function in zebrafish embryogenesis.

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