Beahm Brendan J, Dehnert Karen W, Derr Nicolas L, Kuhn Joachim, Eberhart Johann K, Spillmann Dorothe, Amacher Sharon L, Bertozzi Carolyn R
Department of Chemistry and Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720 (USA).
Angew Chem Int Ed Engl. 2014 Mar 24;53(13):3347-52. doi: 10.1002/anie.201310569. Epub 2014 Feb 19.
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.
硫酸乙酰肝素（HS）和硫酸软骨素（CS）糖胺聚糖（GAG）是与蛋白聚糖相关的多糖，在动物体内具有重要功能。人们通过对生物合成酶进行基因操作对它们进行了广泛研究，但用于探究GAG功能的化学工具却很有限。HS和CS具有一个保守的木糖残基，该残基将多糖链连接到蛋白质主链上。在此我们报告，在斑马鱼胚胎中，通过给予UDP-4-叠氮基-4-脱氧木糖（UDP-4-XylAz）可将肽近端的木糖残基代谢替换为链终止性的4-叠氮基-4-脱氧木糖（4-XylAz）残基。UDP-4-XylAz破坏了HS和CS的生物合成，并导致发育异常，这让人联想到GAG生物合成和层粘连蛋白突变体。与蛋白质结合的4-XylAz的叠氮取代基通过与荧光环辛炔探针发生生物正交反应，能够在体内快速可视化链终止的机体部位。因此，UDP-4-XylAz补充了用于研究斑马鱼胚胎发育过程中GAG功能的基因工具。
