Dataset Information

Heavy Heparin: A Stable Isotope-Enriched, Chemoenzymatically-Synthesized, Poly-Component Drug.

ABSTRACT: Heparin is a highly sulfated, complex polysaccharide and widely used anticoagulant pharmaceutical. In this work, we chemoenzymatically synthesized perdeuteroheparin from biosynthetically enriched heparosan precursor obtained from microbial culture in deuterated medium. Chemical de-N-acetylation, chemical N-sulfation, enzymatic epimerization, and enzymatic sulfation with recombinant heparin biosynthetic enzymes afforded perdeuteroheparin comparable to pharmaceutical heparin. A series of applications for heavy heparin and its heavy biosynthetic intermediates are demonstrated, including generation of stable isotope labeled disaccharide standards, development of a non-radioactive NMR assay for glucuronosyl-C5-epimerase, and background-free quantification of in?vivo half-life following administration to rabbits. We anticipate that this approach can be extended to produce other isotope-enriched glycosaminoglycans.

SUBMITTER: Cress BF

PROVIDER: S-EPMC6461503 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Heavy Heparin: A Stable Isotope-Enriched, Chemoenzymatically-Synthesized, Poly-Component Drug.

Cress Brady F BF Bhaskar Ujjwal U Vaidyanathan Deepika D Williams Asher A Cai Chao C Liu Xinyue X Fu Li L M-Chari Vandhana V Zhang Fuming F Mousa Shaker A SA Dordick Jonathan S JS Koffas Mattheos A G MAG Linhardt Robert J RJ

Angewandte Chemie (International ed. in English) 20190401 18

Heparin is a highly sulfated, complex polysaccharide and widely used anticoagulant pharmaceutical. In this work, we chemoenzymatically synthesized perdeuteroheparin from biosynthetically enriched heparosan precursor obtained from microbial culture in deuterated medium. Chemical de-N-acetylation, chemical N-sulfation, enzymatic epimerization, and enzymatic sulfation with recombinant heparin biosynthetic enzymes afforded perdeuteroheparin comparable to pharmaceutical heparin. A series of applicati ...[more]

PMID: 30870573

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Project description:The non-receptor tyrosine kinase SRC is frequently deregulated in human colorectal cancer (CRC), and SRC increased activity has been associated with poor clinical outcomes. In nude mice engrafted with human CRC cells, SRC over-expression favors tumor growth and is accompanied by a robust increase in tyrosine phosphorylation in tumor cells. How SRC contributes to this tumorigenic process is largely unknown. We analyzed SRC oncogenic signaling in these tumors by means of a novel quantitative proteomic analysis. This method is based on stable isotope labeling with amino acids of xenograft tumors by the addition of [(13)C(6)]-lysine into mouse food. An incorporation level greater than 88% was obtained in xenograft tumors after 30 days of the heavy lysine diet. Quantitative phosphoproteomic analysis of these tumors allowed the identification of 61 proteins that exhibited a significant increase in tyrosine phosphorylation and/or association with tyrosine phosphorylated proteins upon SRC expression. These mainly included molecules implicated in vesicular trafficking and signaling and RNA binding proteins. Most of these proteins were specific targets of SRC signaling in vivo, as they were not identified by analysis via stable isotope labeling by amino acids in cell culture (SILAC) of the same CRC cells in culture. This suggests that oncogenic signaling induced by SRC in tumors significantly differs from that induced by SRC in cell culture. We next confirmed this notion experimentally with the example of the vesicular trafficking protein and SRC substrate TOM1L1. We found that whereas TOM1L1 depletion only slightly affected SRC-induced proliferation of CRC cells in vitro, it drastically decreased tumor growth in xenografted nude mice. We thus concluded that this vesicular trafficking protein plays an important role in SRC-induced tumor growth. Overall, these data show that SILAC analysis in mouse xenografts is a valuable approach for deciphering tyrosine kinase oncogenic signaling in vivo.

Dataset Information

Heavy Heparin: A Stable Isotope-Enriched, Chemoenzymatically-Synthesized, Poly-Component Drug.

Publications

Heavy Heparin: A Stable Isotope-Enriched, Chemoenzymatically-Synthesized, Poly-Component Drug.

Similar Datasets

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