Active 1918 pandemic flu viral neuraminidase has distinct N-glycan profile and is resistant to trypsin digestion Zhengliang L. Wu * , Cheryl Ethen, Gregg E. Hickey, Weiping Jiang R&D Systems, Inc., 614 Mckinley place NE, Minneapolis, MN 55413, USA article info Article history: Received 14 December 2008 Available online 6 January 2009 Keywords: Glycosylation N-Glycosylation Neuraminidase 1918 pandemic flu Spanish flu Trypsin Tamiflu Relenza Stalk region Hemagglutinin Influenza N-Glycan abstract The 1918 pandemic flu virus caused one of the most deadly pandemics in human history. To search for unique structural features of the neuraminidase from this virus that might have contributed to its unu- sual virulence, we expressed this enzyme. The purified enzyme appeared as a monomer, a dimer and a tetramer, with only the tetramer being active and therefore biologically relevant. The monomer and the dimer could not be oligomerized into the tetramer in solution, suggesting that some unique structural features were required for oligomerization and activation. These features could be related to N-glycosyl- ation, because the tetramer displayed different N-glycans than the monomer and the dimer. Furthermore, the tetramer was found to be resistant to trypsin digestion, which may give the virus the capability to invade tissues that are normally not infected by influenza viruses and make the virus more robust for infection. Ó 2009 Elsevier Inc. All rights reserved. The 1918 pandemic flu virus had an extraordinary virulence and caused 50–100 million deaths world-wide [1]. In the laboratory, the resurrected virus has shown extreme lethality to mice and chicken embryos and displayed a high-growth phenotype in hu- man bronchial epithelial cells [2], which could be partially due to that its hemagglutinin (HA) can be activated in the absence of tryp- sin. Recently, small changes on the binding specificity of the HA and enzyme specificity of its neuraminidase (NA) are found to determine the host and tissue type that the virus infects [3–6]. Both HA and NA are envelope glycoproteins and are essential to the infectious cycle of an influenza virus. HA mediates initial attachment and fusion of viruses to host cells through binding to sialic acids on the cell membrane. NA removes sialic acids both on host cells and viral particles, thus facilitating the release of descendent viruses from infected cells and preventing them from self-aggregation. Accordingly, both NA and HA are found to be crit- ical determinants of the virulence for the 1918 virus [2,7,8]. Relative to HA, investigations into NA have led to results with more clinical application. The catalytic sites of NA have been found to be more rigid than HA, with only minor conformational changes seen upon inhibitor binding. This stability makes NA a logical target for drug intervention [9]. The recently marketed anti-influenza drugs Tamiflu and Relenza both are NA inhibitors [10]. NA is also reported to be more genetically stable than HA, as measured by point mutations over time [11]. Thus, NA-based vaccines are likely to be more effective for longer periods of time [12]. In molecular detail, NA is a type II membrane protein with three distinct domains: a globular head domain containing the enzyme active site, a highly variable stalk region [13,14], and an N-terminal signal anchor domain by which the enzyme is embedded in the viral envelope [15]. The head domain usually can be released from viral particles in tetrameric form through protease digestion in the stalk region [16–18]. Despite the pro- gress in drug development and structural understanding, the molecular mechanism for how NA contributed to the virulence of the 1918 virus is still not clear. In this report, we described the characterization of the neur- aminidase from the 1918 flu virus [19]. 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.12.139 Abbreviations: HA, hemagglutinin; NA, neuraminidase; Endo H, endo-b-N-acetyl- glucosaminidase H; Endo F1, endo-b-N-acetylglucosaminidase F1; Endo F3, endo-b- N-acetylglucosaminidase F3; PNGase F, peptide-N 4 -(N-acetyl-b-N-D-glucosaminyl) asparagine amidase F; RFU, relative fluorescent unit. * Corresponding author. Fax: +1 612 379 6580. E-mail address: Leon.wu@rndsystems.com (Z.L. Wu). Biochemical and Biophysical Research Communications 379 (2009) 749–753 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc