Target Effects Of BoNT-LH (N)-Elafin Fusion Expression On Mucin Secretion In Airway Epithelial Cells

Objetive To recombine the light chain of botulinum neurotoxin which can cleave SNARE complex associated secretion, N-terminal domain of heavy chain of botulinum neurotoxin which is involved in intracellular membrane translocation, and elastase-specific inhibitor (Elafin). To obtain the fusion protein BoNT-LH(N)-Elafin, and to observe the inhibitory effects of the fusion protein BoNT-LH(N)-Elafin on the processes of mucin synthesis and secretion. To analysis the possible functionary targets of signal pathways and transcriptional factors.Methods (1) pMD18-T-BoNT-LH(N) and pEGFP-N1-Elafin were constructed. BoNT-LH(N) and Elafin fragments were amplificated from pMD18-T-BoNT-LH(N) and pEGFP-N1-Elafin through PCR reaction. The fusion gene fragment of BoNT-LH(N)-Elafin was obtained by platnium pfx polymerase enzyme and PCR reaction. The BoNT-LH(N)-Elafin fusion gene was cloned into Pichia yeast expression vector pPIC9k. The recombinant vector was then transfected into GS115 Pichia yeast strain, and the fusion protein was purified by cation-exchange chromatography. The fusion protein was identified by western blotting. The anti-elastase activity was detected by a special method. The cleavage effect of the fusion protein on SNARE complex was assessed by western blotting. (3) 16HBE airway epithelial cells were cultured and exposed to cigarette smoke extract (CSE) and lipopolysaccharide (LPS). The levels of MUC5AC intracellular protein and mRNA were detected by immunofluorescence and RT-PCR. MUC5AC secretion level was assessed by ELISA. The protein levels of phosphorylation Jun N-terminal kinase (p-JNK) , phosphorylation extracellular signal-regulated kinase (p-ERK) and p-P38 were detected by western blot. The cleavage effect of the fusion protein on SNARE complex was also assessed by western blotting. The transcription activities of activiator protein-1(AP-1) and nuclear factorκB (NF-κB )were detected by luciferase reporter gene detection system.Results (1) pPIC9K-BoNT-LH(N)-Elafin was identificated by restriction enzyme reaction and DNA sequencing and successfully constructed. (2) BoNT-LH(N)-Elafin was abundantly secreted by GS115 and was highly purified by cation-exchange chromatography. The fusion protein significantly inhibited the elastase activity, and SNARE complex (SNAP23,syntaxin 2 and VAMP-2) were cleaved by it. (3) In CSE or LPS group, there was an obvious increase of MUC5AC protein production and mRNA expression, with elevation of p-JNK production, p-ERK production, SNARE complex (SNAP23,syntaxin 2 and VAMP-2), AP-1 activity and NF-κB activity, all significantly higher than that in control group. BoNT-LH(N)-Elafin reduced MUC5AC protein and mRNA level, decreased p-JNK , p-ERK production and SNARE complex (SNAP23,syntaxin 2 and VAMP-2), and inhibited AP-1 activity and NF-κB activity, compared with single CSE-stimulated group or single LPS-stimulated group. p-P38 had no significant change after CSE or LPS stimulation or BoNT-LH(N)-Elafin treatment.Conclusion (1) CSE and LPS increase MUC5AC mucin synthesis and secretion, and BoNT-LH(N)-Elafin can inhibit MUC5AC mucin synthesis and secretion induced by CSE and LPS; (2) BoNT-LH(N)-Elafin may down-regulate AP-1 and NF-κB activiation via effects on mitogen-activated protein kinases, and cleave SNARE complex (SNAP23,syntaxin 2 and VAMP-2). (3) Fusion protein BoNT-LH(N)-Elafin can decease mucin synthesis by inhibiting elastase, down-regulate MUC5AC gene transcription by inhibiting the signal pathways and transcriptional factors, and inhibit mucin section by blocking exocytosis process. The fusion protein may provide extensive potency in clinical appilication.