Research And Application Of A New Method For Enzyme-regulated Anchorage On Cell Membrane

In cellular microenvironment,numerous biomolecules such as enzymes,proteases and cytokines,play a vital role in the regulation of intercellular connection and communication,biomolecular transporting,cellular immunity,cell adhesion,cell polarity,cell migration,and cell differentiation.Adverse alterations in the expression levels of these biomolecules would cause dysfunctions in microenvironment,pathological changes in cell and even abnormal body functions,including normal cell canceration,type I diabetes,sulfocysteinuria,and cardiovascular diseases.Therefore,there is still a need for the developing in situ detecting techniques that enable to monitor the activity of these biomolecules in the native cellular environment.Presently,cell surface sensors have been demonstrated as effective in the monitoring of biomolecules in the cellular environment.Typically,each sensing unit was decorated on plasma membrane by appending a membrane-anchoring moiety,such as hydrophobic fatty chain or peptide.Interestingly,it was found that the membrane-inserting peptide(MIP)is a new type of effective tool to anchor biomacromolecule on the cell surface compared to hydrophobic fatty chains.However,these elements that anchor cell membranes suffer from poor selectivity among different cell membranes,which might cause unpredictable disturbs to normal cells during cell membrane engineering and disease diagnosis and treatment.In this paper,based on the mechanism of MIP anchoring to cell membrane,we engineer the MIP as enzyme-activated membrane-inserting peptide probes(eaMIPs)that allow in situ tracking the activity of target enzymes in local environment.The membrane-inserting capacity of the MIP motif in each eaMIP is caged by appending a chemical moiety that enables to disturb the structure of MIP motif or neutralize the positive charges in MIP motif.Moreover,we constructed a platform of enzyme-mediated lipid anchorage onto cell membrane based on the transferase NMT catalyzed the substrate peptide myristoylation.The specific work is as follow:1.Investigation of MMP2-activated anchorage of eaMIP1 on plasma membranes.Taking MMP2 as a triggering enzyme model,the substrate sequence(LGLAG)of MMP2 and negatively charged sequence(EEEEE)were appended to the C-terminus of MIP to obtain an enzyme-activable probe eaMIP1.Next,we investigated whether eaMIPl could selectively anchor on the plasma membranes of cell lines with different MMP2 expression levels,and elaborated that this is because of the negatively charged tail decreased electrostatic interaction between peptide probes and plasma membranes.When the eaMIPl are incubated with target cells,the caging moiety in each eaMIPl will be removed by MMP2 in cellular environment,leading to the generation of active MIPs.It was determined that the ability of MIP to anchor cell membranes can be regulated by neutralizing its positive charge,and its activation is related to the expression of cell surface enzymes.2.Study of ALP-dependent anchorage of eaMIP2 on plasma membranes.Analogously,taking ALP as the triggering enzyme,eaMIP2 was designed by phosphorylating the Serll residue in MIP motif.Next,we explored whether eaMIP2 could selectively anchor on the plasma membranes of cell lines with different ALP expression levels,and explored its reason.For the eaMIP2,serine phosphorylation hampered the formation of a helices,thereby weakening the ability of peptides to drill cell membranes.And restoring its membrane-inserting ability is positively correlated with the expression level of ALP.Finally,the distinguishing of cells was achieved by lighting up different tumor cells with distinct fluorescence signal patterns,which provides an alternative tool for clinical diagnosis,biochemical research,and membrane engineering.3.Exploration of NMT-mediated lipid anchors on cell membranes.Based on the characteristic of myristoylation product that has a hydrophobic tail of C14 saturated fatty acids and has a strong hydrophobic interaction with the phospholipid bilayer of the cell membrane,we used the pET28-NMT recombinant plasmid constructed by our group to successfully express NMT protein with catalytic activity in vitro,and confirmed the possibility of NMTmediated lipid anchoring to cell membranes.The diversified design and synthesis of substrate peptides lay a foundation for the construction of a sensing platform for the detection and analysis of different targets in the microenvironment.