Constitutive activation and oncogenicity are mediated by loss of helical structure at the cytosolic boundary of thrombopoietin receptor mutant dimers.

Dimerization of the thrombopoietin receptor (TpoR) is necessary for receptor activation and downstream signaling through activated Janus kinase 2. We have shown previously that different orientations of the transmembrane (TM) helices within a receptor dimer can lead to different signaling outputs. Here we addressed the structural basis of activation for receptor mutations S505N and W515K that induce myeloproliferative neoplasms. We show using in vivo bone marrow reconstitution experiments that ligand-independent activation of TpoR by TM asparagine (Asn) substitutions is proportional to the proximity of the Asn mutation to the intracellular membrane surface. Solid-state NMR experiments on TM peptides indicate a progressive loss of helical structure in the juxtamembrane (JM) R/KWQFP motif with proximity of Asn substitutions to the cytosolic boundary. Mutational studies in the TpoR cytosolic JM region show that loss of the helical structure in the JM motif by itself can induce activation, but only when localized to a maximum of six amino acids downstream of W515, the helicity of the remaining region until Box 1 being required for receptor function. The constitutive activation of TpoR mutants S505N and W515K can be inhibited by rotation of TM helices within the TpoR dimer, which also restores helicity around W515. Together, these data allow us to develop a general model for activation of TpoR and explain the critical role of the JM W515 residue in the regulation of the activity of the receptor.

Tryptophan at the transmembrane-cytosolic junction modulates thrombopoietin receptor dimerization and activation.

Dimerization of single-pass membrane receptors is essential for activation. In the human thrombopoietin receptor (TpoR), a unique amphipathic RWQFP motif separates the transmembrane (TM) and intracellular domains. Using a combination of mutagenesis, spectroscopy, and biochemical assays, we show that W515 of this motif impairs dimerization of the upstream TpoR TM helix. TpoR is unusual in that a specific residue is required for this inhibitory function, which prevents receptor self-activation. Mutations as diverse as W515K and W515L cause oncogenic activation of TpoR and lead to human myeloproliferative neoplasms. Two lines of evidence support a general mechanism in which W515 at the intracellular juxtamembrane boundary inhibits dimerization of the TpoR TM helix by increasing the helix tilt angle relative to the membrane bilayer normal, which prevents the formation of stabilizing TM dimer contacts. First, measurements using polarized infrared spectroscopy show that the isolated TM domain of the active W515K mutant has a helix tilt angle closer to the bilayer normal than that of the wild-type receptor. Second, we identify second-site R514W and Q516W mutations that reverse dimerization and tilt angle changes induced by the W515K and W515L mutations. The second-site mutations prevent constitutive activation of TpoR W515K/L, while preserving ligand-induced signaling. The ability of tryptophan to influence the angle and dimerization of the TM helix in wild-type TpoR and in the second-site revertants is likely associated with its strong preference to be buried in the headgroup region of membrane bilayers.

Synthesis, purification, and characterization of single helix membrane peptides and proteins for NMR spectroscopy.

Membrane proteins function as receptors, channels, transporters, and enzymes. These proteins are generally difficult to express and purify in a functional form due to the hydrophobic nature of their membrane spanning sequences. Studies on membrane proteins with a single membrane spanning helix have been particularly challenging. Single-pass membrane proteins will often form dimers or higher order oligomers in cell membranes as a result of sequence motifs that mediate specific transmembrane helix interactions. Understanding the structural basis for helix association provides insights into how these proteins function. Nevertheless, nonspecific association or aggregation of hydrophobic membrane spanning sequences can occur when isolated transmembrane domains are reconstituted into membrane bilayers or solubilized into detergent micelles for structural studies by solid-state or solution NMR spectroscopy. Here, we outline the methods used to synthesize, purify, and characterize single transmembrane segments for structural studies. Two synthetic strategies are discussed. The first strategy is to express hydrophobic peptides as protein chimera attached to the maltose binding protein. The second strategy is by direct chemical synthesis. Purification is carried out by several complementary chromatography methods. The peptides are solubilized in detergent for solution NMR studies or reconstituted into model membranes for solid-state NMR studies. We describe the methods used to characterize the reconstitution of these systems prior to NMR structural studies to establish if there is nonspecific aggregation.

Recombinant influenza B virus HA and NA antigens administered in equivalent amounts are immunogenically equivalent and induce equivalent homotypic and broader heterovariant protection in mice than conventional and live influenza vaccines.

Influenza B virus is an important cause of acute upper respiratory disease in humans. Vaccination is the primary method of control of influenza related disease, yet vaccine methodology and production technology have not changed in over 40 years. In this study, we compare the efficacy of recombinant baculovirus produced protein based neuraminidase containing influenza B vaccines with conventional inactivated influenza vaccine (CIV) and live-attenuated influenza vaccine (LAIV) in a murine model. All HA containing vaccines stimulated antibody and protected against an infectious challenge with homotypic virus (B/Harbin/7/94), only recombinant protein based (rHA + rNA and rNA) vaccines containing immunogenic amounts of influenza neuraminidase (NA) protected against challenge with a significantly antigenically different heterovariant virus (B/Beijing/243/1997), as measured by a reduction in mean pulmonary virus titers. This report demonstrates with influenza B virus, in a side-by-side comparison with CIV and LAIV in a murine model system the superiority of vaccines containing immunogenic NA over currently approved CIV and LAIV vaccines.

Changing perspective on immunization against influenza.

Current vaccination strategies against influenza rely on decades old technology of strain selection and prolonged labor-intensive, embryonated chicken-egg based production methods. Although, containing both major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), the immunity engendered by these vaccines is dominated by the anti-HA response. Consequently, current vaccines are susceptible to failure resulting from significant antigenic drift or shift in the time elapsing from the selection of the vaccine candidate strain and wild-type virus exposure. Therefore, immunity may be of short duration. There must be a change in vaccine strategy to include immunization with both HA and NA to broaden the immune response against influenza. Inclusion of the more slowly evolving NA in a vaccine against influenza will reduce the vulnerability to antigenic changes in a potential emerging influenza virus. Alternative production technologies such as recombinant baculovirus and yeast should be explored to decrease vaccine production times.

Variation in the divalent cation requirements of influenza A virus N1 neuraminidases.

Enzymatic kinetic parameters of influenza A virus N1 neuraminidases (NA) chromatographically purified from several vaccine candidate strains were tested. With ionic strength held constant, Ca2+ or Mg2+ increased the initial rate of enzymatic activity. The 1934 and 1943 strains had statistically significant highest initial velocities, V(max)/K(m) and V(max). There were no significant differences among the influenza virus strains from 1947 to 1991. Measured K(m) for the 1943 strain (6.2 x 10(-5) M) was significantly higher than other strains (3.1-4.7 x 10(-5) M). V(max)/K(m) varied from 0.78 M(-1) s(-1) to 0.91 M(-1) s(-1) and V(max) varied from 3.0 s(-1) to 5.5 s(-1) before the addition of a divalent cation and increased approximately 2-fold for each of these kinetic parameters for each strain after the addition of exogenous Ca2+ or Mg2+. Dialysis reduced the initial velocity and immunogenicity of each strain with significant differences found among strains. Enzymatic activity and immunogenicity were partially restored by the addition of exogenous Ca2+. Nucleic acid sequence analysis could not predict these differences. Selection of vaccine strains must include analysis of antigenic changes, but also enzymatic studies and determination of the requirement of divalent cations to maintain immunogenicity and activity during production.

Immunization against influenza A virus: comparison of conventional inactivated, live-attenuated and recombinant baculovirus produced purified hemagglutinin and neuraminidase vaccines in a murine model system.

To simulate the 2003-2004 influenza season and compare available vaccination methods, immunologically naive mice were immunized with: influenza A virus hemagglutinin (rHA) and neuraminidase (rNA) from A/Panama/2007/99 H3N2 or A/Fujian/411/2002 H3N2 expressed by recombinant baculovirus, chromatographically purified, either as single antigens (rHA or rNA) or in combination (rHArNA); conventional inactivated monovalent (CIV) vaccines from each heterotypic strain; or a live-attenuated influenza (LAV) vaccine derived from the A/Panama/2007/99 strain. HA containing vaccines were highly immunogenic for the HA antigen, with no statistically significant differences among groups in the amount of homotypic anti-HA antibody induced. Little cross-reactive anti-HA antibody was induced by any vaccine, including LAV. Statistically, the greatest amount of anti-NA antibody was induced by the purified NA alone or in combination with purified HA; the least amount of anti-NA antibody was found in mice immunized with LAV or CIV. Immunization with vaccines immunogenic for both HA and NA resulted in an immune response to both surface glycoproteins that suppressed homotypic, closely related heterotypic infection and had a greater reduction in mPVT following an infectious challenge by a distantly related heterotypic strain. These studies suggest that vaccines immunogenic for both HA and NA offer an increased level of protection from influenza.

Variation in the divalent cation requirements of influenza a virus N2 neuraminidases.

Influenza virus N2 neuraminidases were chromatographically purified from several vaccine candidate strains from 1957 to 1994. Enzymatic kinetic parameters and immunogenicity were tested for each strain. For each NA tested, with ionic strength held constant, Ca(2+) or Mg(2+) increased the initial rate of enzymatic activity. Earlier N2-NA strains had the highest initial velocity, V(max)/K(m) and V(max). There were significant differences among the influenza virus strains in enzymatic activity before and after addition of Ca(2+) or Mg(2+): V(max)/K(m) varied from 0.54 M(-1) s(-1) to 0.88 M(-1) s(-1) and V(max) varied from 2.45 s(-1) to 4.3 s(-1) before the addition of a divalent cation; and increased approximately 2-fold each of these kinetic parameters for each strain after the addition of exogenous Ca(2+) or Mg(2+). Exhaustive dialysis with EDTA reduced the initial velocity of each strain with significant differences found among strains, with a range of 0.1% to 8% of original activity. Activity was partially restored by the addition of exogenous Ca(2+) or Mg(2+), varying from 8% to 60% of pre-dialysis levels, but original rates were not achieved. This reduction in enzymatic activity for the tested strains (i.e., A/Japan/57 and A/Johannesburg/94) was accompanied by a parallel decrease in NA-immunogenicity, with antibody response decreasing by as much as 76% as measured by NI titer, and ELISA titer decreasing by as much as 68%. The addition of Ca(2+) or Mg(2+) to the post-dialysis sample restored immunogenicity to as much as 80% of pre-dialysis NI titers and as much as 78% of pre-dialysis ELISA titers. Dialysis had the least effect on early strains as measured by enzymatic kinetic parameters and immunogenicity studies. Zn(2+) had a slight inhibitory effect on the activity of all tested strains. Review of the nucleic acid sequence of each of these strains could not predict their enzymatic activity, immunogenicity or response to dialysis. If immunity against neuraminidase is desirable in vaccination against influenza, selection of vaccine candidate strains must include not only analysis of antigenic changes and sequence analysis but also enzymatic studies and determination of the requirement of divalent cations to maintain immunogenicity and activity during production.