SUMMARY Dengue is the most common vector-borne viral disease, causing nearly 400 million infections yearly. Currently there are no approved therapies. Antibody epitopes that elicit weak humoral responses may not be accessible by conventional B cell panning methods. To demonstrate an alternative strategy to generating a therapeutic antibody, we employed a non-immunodominant, but functionally relevant, epitope in domain III of the E protein, and engineered by structure-guided methods an antibody directed to it. The resulting antibody, Ab513, exhibits high-affinity binding to, and broadly neutralizes, multiple genotypes within all four serotypes. To assess therapeutic relevance of Ab513, activity against important human clinical features of dengue was investigated. Ab513 mitigates thrombocytopenia in a humanized mouse model, resolves vascular leakage, reduces viremia to nearly undetectable levels, and protects mice in a maternal transfer model of lethal antibody-mediated enhancement. The results demonstrate that Ab513 may reduce the public health burden from dengue.

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Heparinase II depolymerizes heparin and heparan sulfate glycosaminoglycans, yielding unsaturated oligosaccharide products through an elimination degradation mechanism. This enzyme cleaves the oligosaccharide chain on the nonreducing end of either glucuronic or iduronic acid, sharing this characteristic with a chondroitin ABC lyase. We have determined the first structure of a heparin-degrading lyase, that of heparinase II from Pedobacter heparinus (formerly Flavobacterium heparinum), in a ligand-free state at 2.15 Å resolution and in complex with a disaccharide product of heparin degradation at 2.30 Å resolution. The protein is composed of three domains: an N-terminal ␣-helical domain, a central twolayered ␤-sheet domain, and a C-terminal domain forming a twolayered ␤-sheet. Heparinase II shows overall structural similarities to the polysaccharide lyase family 8 (PL8) enzymes chondroitin AC lyase and hyaluronate lyase. In contrast to PL8 enzymes, however, heparinase II forms stable dimers, with the two active sites formed independently within each monomer. The structure of the N-terminal domain of heparinase II is also similar to that of alginate lyases from the PL5 family. A Zn 2؉ ion is bound within the central domain and plays an essential structural role in the stabilization of a loop forming one wall of the substrate-binding site. The disaccharide binds in a long, deep canyon formed at the top of the N-terminal domain and by loops extending from the central domain. Based on structural comparison with the lyases from the PL5 and PL8 families having bound substrates or products, the disaccharide found in heparinase II occupies the "؉1" and "؉2" subsites. The structure of the enzyme-product complex, combined with data from previously characterized mutations, allows us to propose a putative chemical mechanism of heparin and heparan-sulfate degradation.

D1R 1-545, an active subdomain of the large subunit of vaccinia virus mRNA capping enzyme possessing ATPase, RNA 5-triphosphatase, and guanylyltransferase activities, was expressed in Escherichia coli and shown to be functionally equivalent to the heterodimeric enzyme (Myette, J. R., and Niles, E. G. (1996) J. Biol. Chem. 271, 11936 -11944). A detailed characterization of the phosphohydrolytic activities of D1R demonstrates that, in addition to ATPase and RNA 5-triphosphatase activities, the capping enzyme also possesses a general nucleoside triphosphate phosphohydrolase activity that lacks a preference for the nucleoside base or sugar. Nucleoside triphosphate and mRNA saturation kinetics are markedly different, with RNA exhibiting a K m and turnover number 100-and 10-fold less, respectively, than those values measured for any NTP. The linear competitive inhibition of RNA 5-triphosphatase activity by ATP, and the relative manner by which both ATPase and RNA 5-triphosphatase activities are inhibited by specific oligonucleotides, kinetically demonstrate that each activity is carried out at a common active site. Direct UV photo-cross-linking of either 32 P-radiolabeled ATP or 23-mer triphosphorylated RNA, followed by cyanogen bromide cleavage of the photo-linked enzyme, localizes the major binding site for both ATP and RNA to a region between amino acids 1 and 221. The inability of ATP to competitively inhibit either EϳGMP formation or the transfer of GMP to RNA kinetically differentiates the phosphohydrolase active site from the guanylyltransferase active site.The vaccinia virus mRNA capping enzyme catalyzes three of the four reactions required for cap I formation, including RNA 5Ј-triphosphatase (1, 2), guanylyltransferase, and (guanine-7-)methyltransferase activities (3-5). The RNA 5Ј-triphosphatase, nucleoside triphosphate phosphohydrolase, and guanylyltransferase active sites have been mapped to a 60-kDa NH 2 -terminal domain of the D1R subunit (7,8,10), while the methyltransferase resides in a independent domain comprised of the carboxyl terminus of D1R together with D12L (9 -11). In an accompanying report (12), kinetic analyses demonstrated that the D1R 1-545 subdomain expressed in Escherichia coli is functionally equivalent to the full-size capping enzyme with respect to RNA 5Ј-triphosphatase, ATPase, and guanylyltransferase activities, indicating that the methyltransferase domain exerts little influence on catalysis carried out at the active sites within D1R . In addition to triphosphorylated RNA, the mRNA capping enzyme employs ATP and GTP as substrates for phosphohydrolysis (1,6,23,24), raising the question whether both the nucleoside triphosphate phosphohydrolase activity and the RNA 5Ј-triphosphatase activity are carried out at the same active site.In this report, we describe the kinetic properties of the RNA 5Ј-triphosphatase and nucleoside triphosphate phosphohydrolase activities of the vaccinia virus mRNA capping enzyme. Through competitive inhibition analyses, we demonstrate that both activities ...

The soil bacterium Flavobacterium heparinum produces several enzymes that degrade heparan sulfate glycosaminoglycans (HSGAGs) in a sequence-specific manner. Among others, these enzymes include the heparinases and an unusual glycuronidase that hydrolyzes the unsaturated Delta4,5 uronic acid at the nonreducing end of oligosaccharides resulting from prior heparinase eliminative cleavage. We report here the molecular cloning of the Delta4,5 glycuronidase gene from the flavobacterial genome and its recombinant expression in Escherichia coli as a highly active enzyme. We also report the biochemical and kinetic characterization of this enzyme, including an analysis of its substrate specificity. We find that the Delta4,5 glycuronidase discriminates on the basis of both the glycosidic linkage and the sulfation pattern within its saccharide substrate. In particular, we find that the glycuronidase displays a strong preference for 1-->4 linkages, making this enzyme specific to heparin/heparan sulfate rather than 1-->3 linked glycosaminoglycans such as chondroitin/dermatan sulfate or hyaluronan. Finally, we demonstrate the utility of this enzyme in the sequencing of heparinase-derived HSGAG oligosaccharides.