Effective annual influenza vaccination requires frequent changes in vaccine composition due to both antigenic shift for different subtype hemagglutinins (HAs) and antigenic drift in a particular HA. Here we present a broadly neutralizing human monoclonal antibody with an unusual binding modality. The antibody, designated CT149, was isolated from convalescent patients infected with pandemic H1N1 in 2009. CT149 is found to neutralize all tested group 2 and some group 1 influenza A viruses by inhibiting low pH-induced, HA-mediated membrane fusion. It promotes killing of infected cells by Fc-mediated antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. X-ray crystallographic data reveal that CT149 binds primarily to the fusion domain in HA2, and the light chain is also largely involved in binding. The epitope recognized by this antibody comprises amino-acid residues from two adjacent protomers of HA. This binding characteristic of CT149 will provide more information to support the design of more potent influenza vaccines.
A multifunctional enzyme, G h , is a GTP-binding protein that couples to the ␣ 1B -adrenoreceptor and stimulates phospholipase C-␦1 but also displays transglutaminase 2 (TG2) activity. G h /TG2 has been implicated to play a role in cell motility. In this study we have examined which function of G h /TG2 is involved in this cellular response and the molecular basis. Treatment of human aortic smooth muscle cell with epinephrine inhibits migration to fibronectin and vitronectin, and the inhibition is blocked by the
Phospholipase C-g1(PLC-g1) is known to play an essential role in various cellular responses, such as proliferation and tumorigenesis, and PLC-g1-speci®c inhibitors are commonly employed to investigate the mechanism of the PLC-g1-mediated signaling pathway. In this study, we developed a single chain antibody fragment (scFv) as a blocker for PLC-g1 mediated signaling. scFv, designated F7-scFv, speci®cally bound to PLC-g1 with high a nity (K d =1.9610 78 M) in vitro. F7-scFv also bound to PLC-g1 in vivo and altered the distribution pattern of PLC-g1 from the cytoplasm to the intracellular aggregates, where F7-scFv was localized. Moreover, F7-scFv interrupted the EGF-induced translocation of PLC-g1 from the cytosol to the membrane ru e and attenuated EGF-induced inositol phosphates generation and intracellular calcium mobilization. These results indicate that F7-scFv blocks EGF-induced PLCg1 activation by causing sequestering of PLC-g1 into intracellular aggregates, and may therefore be useful in studies of the PLC-g1-mediated signaling pathway. Oncogene (2001) 20, 7954 ± 7964.
There are several broadly neutralizing monoclonal antibodies that neutralize influenza viruses with different mechanisms from traditional polyclonal antibodies induced by vaccination. CT149, which is one of the broadly neutralizing antibodies, was also previously reported to neutralize group 2 and some of group 1 influenza viruses (13 out of 13 tested group 2 viruses and 5 out of 11 group 1 viruses). In this study, we developed another antibody with the aim of compensating partial coverage of CT149 against group 1 influenza viruses. CT120 was screened among different antibody candidates and mixed with CT149. Importantly, although the binding sites of CT120 and CT149 are close to each other, the two antibodies do not interfere. The mixture of CT120 and CT149, which we named as CT-P27, showed broad efficacy by neutralizing 37 viruses from 11 different subtypes, of both group 1 and 2 influenza A viruses. Moreover, CT-P27 showed
in vivo
therapeutic efficacy, long prophylactic potency, and synergistic effect with oseltamivir in influenza virus-challenged mouse models. Our findings provide a novel therapeutic opportunity for more efficient treatment of influenza.
Phosphorylation of phospholipase C-delta(1) (PLC-delta(1)) in vitro and in vivo was investigated. Of the serine/threonine kinases tested, protein kinase C (PKC) phosphorylated the serine residue(s) of bacterially expressed PLC-delta(1) most potently. It was also demonstrated that PLC-delta(1) directly bound PKC-alpha via its pleckstrin homology (PH) domain. Using deletion mutants of PLC-delta(1) and synthetic peptides, Ser35 in the PH domain was defined as the PKC mediated in vitro phosphorylation site of PLC-delta(1). In vitro phosphorylation of PLC-delta(1) by PKC stimulated [(3)H]PtdIns(4,5)P(2) hydrolyzing activity and [(3)H]Ins(1,4,5)P(3)-binding of the PLC-delta(1). On the other hand, endogenous PLC-delta(1) was constitutively phosphorylated and phosphoamino acid analysis revealed that major phosphorylation sites were threonine residues in quiescent cells. The phosphorylation level and the species of phosphoamino acid were not changed by various stimuli such as PMA, EGF, NGF, and forskolin. Using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, we determined that Thr209 of PLC-delta(1) is one of the constitutively phosphorylated sites in quiescent cells. The PLC activity was potentiated when constitutively phosphorylated PLC-delta(1) was dephosphorylated by endogenous phosphatase(s) in vitro. Additionally, coexpression with PKC-alpha reduced serine phosphorylation of PLC-delta(1) detected by an anti-phosphoserine antibody and PLC-delta(1)-dependent basal production of inositol phosphates in NIH-3T3 cells, suggesting PKC-alpha activates phosphatase or inactivates another kinase involved in PLC-delta(1) serine phosphorylation to modulate the PLC-delta(1) activity in vivo. Taken together, these results suggest that PLC-delta(1) has multiple phosphorylation sites and phosphorylation status of PLC-delta(1) regulates its activity positively or negatively depends on the phosphorylation sites.
The down-regulation of the epidermal growth factor (EGF) receptor is critical for the termination of EGF-dependent signaling, and the dysregulation of this process can lead to oncogenesis. In the present study, we suggest a novel mechanism for the regulation of EGF receptor down-regulation by phospholipase C-⑀. The overexpression of PLC-⑀ led to an increase in receptor recycling and decreased the down-regulation of the EGF receptor in COS-7 cells. Adaptor protein complex 2 (AP2) was identified as a novel binding protein that associates with the PLC-⑀ RA2 domain independently of Ras. The interaction of PLC-⑀ with AP2 was responsible for the suppression of EGF receptor down-regulation, since a perturbation in this interaction abolished this effect. Enhanced EGF receptor stability by PLC-⑀ led to the potentiation of EGF-dependent growth in COS-7 cells. Finally, the knockdown of PLC-⑀ in mouse embryo fibroblast cells elicited a severe defect in EGF-dependent growth. Our results indicated that PLC-⑀ could promote EGF-dependent cell growth by suppressing receptor down-regulation.
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