Active 1918 pandemic flu viral neuraminidase has distinct N-glycan profile and is resistant to trypsin digestion

Interface dynamics explain assembly dependency of influenza neuraminidase catalytic activity

“…Tetramers are the functional and catalytic active forms of iNAs; their monomer and dimer counterparts are inactive (Wu et al, 2009). This is in contrast to non-iNAs, which are active as monomers.…”

“…The importance of the iNA-specific loop insertion in facilitating the iNA assembly or in stabilizing the integrity of its tetramers must be put into perspective, because free head domains of iNA tetramers tend to dissociate (Schmidt et al, 2011). Additional mechanisms of oligomerization mediated by the membrane anchoring region and the glycosylation motives in the stalk region play a role for group 1 iNA tetramer integrity and complement the mechanistic effect of the enabling loop for the tetramerization (Wu et al, 2009). In group 2 iNAs the N-glycosylation site at N200 has been postulated as an additional factor promoting tetramer stability (Air, 2012; Xu et al, 2008).…”

“…For subtype N1 iNA a systematic investigation of stalk length variations identified both transmembrane region and the catalytic head as factors contributing to the tetramer assembly (da Silva, Nordholm, Madjo, Pfeiffer, & Daniels, 2013). An analysis of the 1918 pandemic N1 confirmed that iNA indeed requires tetramer assembly to exhibit enzymatic activity (Wu, Ethen, Hickey, & Jiang, 2009). The importance of tetramerization is further emphasized by the efforts to develop a plasmid expression platform for recombinant iNA with a suitable tetramerization domain in order to stabilize the quaternary structure (Schmidt, Attwood, Mohr, Barrett, & McKimm-Breschkin, 2011).…”

Influenza neuraminidase

“…Unfortunately, when protein was crystallized for structure determination the enzyme activity of the protein was either not determined or not reported, leaving many unanswered questions, such as whether the Ca 2+ binding sites are important to activity. Hopefully more laboratories will follow the example of the McKimm-Breschkin and Wu groups in reporting the activity of their expressed protein 42,48 . A compilation of available data is included as Supplementary Table 1.…”

Glycoscience aids in biomarker discovery

“…Large glycopeptides are not only harder to detect by mass spectrometry (due to decreased ionization efficiency as well as instrumental limitations) but can also incorporate multiple sites of glycosylation, severely complicating or obfuscating site-specific analysis. This is exacerbated by the well-known phenomenon of glycoprotein trypsin resistance, which occurs when glycosylation in close proximity to a proteolytic cleavage site sterically hinders the proteolytic activity, resulting in a missed cleavage (64,65). One strategy to avoid such issues is to subject a glycoprotein of interest to a customized digestion scheme consisting of either sequential or simultaneous digestion by multiple specific proteases.…”