Preliminary Study Of The Interaction Of Heparin Oligosaccharides With The Antimicrobial Motif Of Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) is a linear cationic neuropeptide consisting of28amino acids. It is widely distributed throughout the body with pleiotropic functions. VIP exerts attractive neuroprotective actions with neurotransmitter, neuromodulatory and neurotrophic properties. VIP’s potent anti-inflammatory, antioxidant and anti-apoptosis functions are involved in the neuroprotective progress. It is reported that VIP has a intimate relationship with many neurodevelopmental and neuroinflammatory disorders including Down syndrome, foetal alcohol syndrome, Parkinson’s Disease (PD) and Alzheimer’s Disease (AD). It was found that interactions of heparin and VIP play a great role in the neuroprotective progress of VIP. VIP was thought to preserve neurons by inducing native brain mast cells to secrete numerous neuroprotective substances, such as nerve growth factor (NGF) and heparin, heparin is capable of functional uncoupling of VIP to its receptors, thus VIP increases intracellular c-AMP levels, and c-AMP is the signal transduction molecular that mediates the intracellular actions of VIP. In this study, we preliminarily explored the interaction of heparin oligosaccharides with VIP represented by it’s antimicrobial activity in vitro. We constructed the E.coli strain to recombinantly express VIP, and we finally obtained VIP and15N labeled VIP with purity over95%, which laid the foundation of the structural research on the interaction of VIP with heparin oligosaccharides.1Intein mediated expression and purification of VIP and15N-VIPThe strategy of Intein-Mediated Purification with an Affinity Chitin-binding Tag (IMPACT) was used for the preparation of VIP and15N-VIP. This strategy is fusing the target protein with intein and chitin binding domain (CBD). The fusion protein affinity binding to chitin beads mediated by CBD and the target protein separating from the affinity tag mediated by intein, and intein is an engineered protein splicing element capable of the inducible self-cleavage activity. The aim gene was inserted to the expression vector pTWINl between Sap Ⅰ and Pst Ⅰ sites to construct the recombinant plasmid pTWINl-VIP, and the fusion protein CBD-Ssp Dnab Intein-VIP was expressed by the plasmid. The self-cleavage activity of intein was induced by the change of temperature and pH of buffer. Specifically, the cDNA sequence which encoding the28amino acids of VIP was designed based on the codon preference of E.coli. The Sap Ⅰ restriction site and the termination codon were added to the3’end of the cDNA sequence, while the Pst Ⅰ restriction site was added to the5’end. The designed sequence was inserted into PUC57vector and the recombinant vector PUC57-VIP was manually synthesized. The target gene was PCR amplified using the PUC57-VIP vector as template, and then the PCR product was digested by Sap Ⅰ and Pst Ⅰ. The expression vector pTWINl was digested by the same restriction enzyme. Finally, the insert gene and the linear vector were ligated by T4DNA Ligase. DNA sequencing indicated that the recombinant vector was successfully constructed.The engineering bacteria expressed about30kD fusion protein compared with uninduced cells.IPTG effect:The impact of IPTG concentration on the expression of fusion protein was investigated, Only when at the concentration of0.3mM, the expression level increased slightly.Temperature effect:Induction temperature is critical for the expression and structure formation of proteins. The expression quantity and cleavage efficiency of the fusion protein were investigated under different induction temperatures of15℃,25℃and30℃. SDS-PAGE indicated that the quantity of soluble protein was increased when lowering the induction temperature and the fusion protein was not cleaved in vivo under the induction temperature of15℃or25℃, while approximately77%fusion protein was cleaved in vivo under the induction temperature of30℃. The fusion protein could not be cleaved under the induction temperature of15℃or25℃, while the on column cleavage efficiency was68%when the temperature was30℃. Therefore, the cells should be induced with0.3mM IPTG under30℃for3h.pH effect:The impact of the buffer pH on the cleavage efficiency of the fusion protein was investigated, and results indicated pH6.0is the best one. The recombinant product was desalted by mini-dialysis kit and ultrafiltered by ultrafiltration tubes with the MWCO of10kD and2kD, respectively to increase the sample’s purity.270.8μg of VIP was obtained with the purity over95%from1L fermentation broth. LC-IT-TOF MS indicated the molecular weight of the sample was extremely close to the theoretical value of VIP.I5N labeled fusion protein was expressed using the constructed strain in15N labeled M9medium (15N-M9). In vivo cleavage always existed under the induction temperature of15℃,25℃and30℃, while the quantity of fusion protein increased and the on column cleavage efficiency was about56%when the temperature was30℃. After desalt and ultrafiltration,100.μg of15N-VIP with purity over95%was obtained from1L fermentation broth. LC-IT-TOF MS indicated the molecular weight of the sample was3366.76Da and it was consistent with the theoretical value of15N-VIP.2Preparation of heparin oligosaccharidesHeparin sodium was digested with heparinase I under optimized conditions.40μL of heparinase I was added to solution containing100mg of heparin sodium and incubated at30℃for6h. Heparin oligosaccharides were separated by Bio gel P10column with0.2M NH4HCO3at the flow rate of0.16mL/min. Samples were desalted by PD MidiTrap G-10column. ESI-MS indicated that the molecular weight of the separated oligosaccharides were consistent with their theoretical values.3Inhibition of heparin oligosaccharides on the antimicrobial activity of VIPRadical diffusion assay (RDA) is a sensitive and effective method for detecting the antimicrobial activity of molecules. C. albicans was used as the tested bacteria for detecting of the impact of heparin oligosaccharides on the antimicrobial activity of VIP. Experiments were divided into the following groups as:(1) effect of equal molar of heparin oligosaccharides on C. albicans,(2) effect of equal molar of heparin oligosaccharides with VIP (500μg/mL) on the antimicrobial activity,(3) effect of different molar of heparin octasaccharides on the antimicrobial activity of VIP (500μ/mL). Results showed that RDA could sensitively detect the antimicrobial activity of VIP and the impact level on the activity. Statistical analysis of the diameter of the inhibition zone indicated that (1) heparin oligosaccharides had no antimicrobial activity against C. albicans (2) heparin oligosaccharides could visibly inhibit the antimicrobial activity of VIP except dp2and dp4, and dp8was the outstanding one (3) the inhibition action of dp8on the antimicrobial activity of VIP reduced when the mole of dp8decreased, and the inhibition action vanished when the ratio of dp8over VIP decreased to about1:6ratio.In general, this study demonstrated that heparin oligosaccharides could interact with VIP, and the binding motif is the antimicrobial activity motif of VIP, dp8is the smallest fragment to effectively interact with VIP. The E.coli strain expressing VIP was successfully constructed. Interactions of heparin with VIP play a great role in the neuroprotective action of VIP. The results can lay foundation for the future study of interaction of heparin oligosaccharides with VIP on molecular level, and for the exploration of the mechanism of the neuroprotective action of VIP.