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. 2013 Jan 4;288(1):644-53.
doi: 10.1074/jbc.M112.424150. Epub 2012 Nov 13.

Assembly of subtype 1 influenza neuraminidase is driven by both the transmembrane and head domains

Diogo V da Silva  1 , Johan Nordholm, Ursula Madjo, Annika Pfeiffer, Robert Daniels
Affiliations

Assembly of subtype 1 influenza neuraminidase is driven by both the transmembrane and head domains

Diogo V da Silva et al. J Biol Chem. .

Abstract

Neuraminidase (NA) is one of the two major influenza surface antigens and the main influenza drug target. Although NA has been well characterized and thought to function as a tetramer, the role of the transmembrane domain (TMD) in promoting proper NA assembly has not been systematically studied. Here, we demonstrate that in the absence of the TMD, NA is synthesized and transported in a predominantly inactive state. Substantial activity was rescued by progressive truncations of the stalk domain, suggesting the TMD contributes to NA maturation by tethering the stalk to the membrane. To analyze how the TMD supports NA assembly, the TMD was examined by itself. The NA TMD formed a homotetramer and efficiently trafficked to the plasma membrane, indicating the TMD and enzymatic head domain drive assembly together through matching oligomeric states. In support of this, an unrelated strong oligomeric TMD rescued almost full NA activity, whereas the weak oligomeric mutant of this TMD restored only half of wild type activity. These data illustrate that a large soluble domain can force assembly with a poorly compatible TMD; however, optimal assembly requires coordinated oligomerization between the TMD and the soluble domain.

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Figures

FIGURE 1.
FIGURE 1.
NA requires a minimal stalk length for assembly and function. A, schematic representation of influenza neuraminidase (Protein Data Bank code 3BEQ; WSN33 numbering) with schematic depictions of the stalk region and the TMD within the signal anchor sequence. The position of the Myc epitope (myc), disulfide bonds (red), and N-linked glycans (black forks) are included. B, distribution of unique N1 stalks with the indicated length from the different human and avian type A influenza strains. The available N1 sequences were grouped by host and their hemagglutinin (H) subtype, and the number of unique stalks at each length was calculated. C, similarity of NA activity profiles from influenza A/WSN/33 (H1N1) particles and lysates from 293T cells transfected with NA-WT-Myc exposed to EDTA, the reducing agent DTT, elevated temperature, and the detergent DDM. NA-Y386F-Myc is an inactive mutant used as control. D, depiction of the stalk deletions in NA WSN33 that fuse residue 34 with 39, 47, 57, or 67. E, immunoblots of lysates from cells transfected with the indicated NA stalk deletions resolved by non-reducing (NR) and reducing (RD) SDS-PAGE. The percentage of NA-WT-Myc activity for each construct was normalized to the NA-WT levels on reducing blots (n = 3). Note the oxidative aggregates (Oxagg) for NA-TM67–453-Myc. F, oligomeric state of the stalk deletions in NA were determined by BN-PAGE immunoblots.
FIGURE 2.
FIGURE 2.
Removal of the stalk domain restores secreted NA activity. A, soluble NA constructs with stalk truncations were created by attaching a cleavable ss to residue 32, 39, 47, 57, or 67. B, reducing (RD) immunoblots of the soluble (S) and membrane protein (P) fractions of total vesicles (Tot) prepared from 293T cells transfected with NA-WT-Myc and ssNA32–453-Myc that were subjected to Na2CO3 extraction as well as whole cell lysates (WCL) and media. C, cell retained (lysates) and secreted (media) ssNA32–453-Myc analyzed in comparison with NA-WT-Myc by nonreducing (NR) and reducing (RD) SDS-PAGE. Note the NA-WT-Myc signal loss upon reduction. The percentage of NA-WT-Myc activity is shown (n = 3) and the oxidized dimers (Oxdi), monomers (Oxmon). and aggregates (OxAgg) are depicted. D, lysates and media from cells transfected with the indicated stalk truncations were analyzed by nonreducing and reducing SDS-PAGE. The activity rate for each construct was normalized for the secreted protein levels in the reducing samples and calculated with respect to ssNA57–453-Myc (n = 3). Oxidized (Oxdi) and SDS-resistant (SDSRdi) dimers are indicated.
FIGURE 3.
FIGURE 3.
The NA TMD forms a tetramer and traffics through the Golgi. A, NA-WT and NA1–74 transfected cell lysates were deglycosylated with PNGase F and Endo-H, separated by reducing (RD) SDS-PAGE and immunoblotted. The number of N-linked glycans and Endo-H resistant (EHR) forms are indicated. B, immunoblots of NA-WT-Myc, NA-I48C-Myc and NA-N50C-Myc, expressed in 293T cells and separated by nonreducing (NR) and reducing SDS-PAGE. Oxidized dimers (Oxdi), trimers (Oxtri), and tetramers dimers (Oxtet) are designated by arrowheads. C, NA1–74-Myc and NA1–74-I48C-Myc transfected cell lysates were deglycosylated with PNGase F and resolved by nonreducing (−DTT) and reducing (+DTT) SDS-PAGE.
FIGURE 4.
FIGURE 4.
Optimal stabilization of the stalk domain is achieved by tetramerization. A, depiction of the 32-residue GCN4 soluble dimer, trimer, and tetramer coiled-coil domains that were inserted after the signal peptidase cleavage (SPase) site following the signal sequence of ssNA32–453. B, immunoblots of lysates and media from cells transfected with ssNA32–453-Myc containing the dimer (Di), trimer (Tri), and tetramer (Tet) domains. The activity rate for each construct was normalized to the secreted protein levels in the reducing (RD) samples and calculated with respect to ssTetNA32–453-Myc (n = 3). The oxidized dimers (Oxdi) and monomers (Oxmon) are depicted. C, the oligomeric state of the secreted ssNA32–453-Myc, ssDiNA32–453-Myc, ssTriNA32–453-Myc, and ssTetNA32–453-Myc were analyzed by BN-PAGE immunoblots. The filled circle is thought to represent the native tetrameric species compared with the open circle. The secreted ssNA32–453-Myc was only observed when five times more sample was loaded likely due to its aggregation (denoted by an asterisk).
FIGURE 5.
FIGURE 5.
Quantification of the stalk and TMD oligomerization. A, schematic representation of the GALLEX system for measuring TMD interactions with schematics of the TMD segments. The N- and C-terminal orientations are depicted, and the asterisk indicates the G83I mutation in GpA. B, anti-MBP immunoblots illustrating the expression levels of the TMD constructs that were quantified using the GALLEX system (in E). Expressed constructs are indicated with filled circles. C, NA2–74 forms an intermolecular disulfide bonded dimer (Oxdi) in the bacterial periplasm through Cys-49, resulting in SDS-resistant trimers (SDSRtri) and tetramers (SDSRtet) that are sensitive to reduction with DTT. The monomers (mon) are also indicated. D, representative immunoblots displaying the expression levels and SDSRtet formation of NA2–74, GpA-NA24–74 and G83I-NA24–74 from the SU101 E. coli used to quantify their interactions (in E). E, bar graph displaying the relative β-galactosidase activity, which inversely correlates to the strength of the TMD segment interaction (n = 3).
FIGURE 6.
FIGURE 6.
Membrane tethering and TMD oligomerization coordinate optimal NA assembly. A, the indicated NA-TMD chimeras were transfected into 293T cells and analyzed by non-reducing (NR) and reducing (RD) SDS-PAGE immunoblots, and the percentage of NA-WT-Myc activity is displayed (n = 3). The oxidized dimers (Oxdi) and reduced monomers (RDmon) are indicated by arrowheads. B, oligomeric state of the NA TMD chimeras was assessed by BN-PAGE immunoblots. The tetrameric species (Tet) and the higher order oligomers (asterisk) are indicated. C, frequency of N-linked glycosylation sites within the unique N1 stalk domains from human, avian, and swine influenza A with respect to stalk length. The total number of unique stalks (N) and the number of stalks without N-linked glycosylation sites are indicated. a.a., amino acids.
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