Study On The Mechanism Of Galectin-3 Oligomerization/Liquid-liquid Phase Separation

Galectin-3(Gal-3)is the only chimeric galectin,consisting of a C-terminal carbohydrate-recognition domain(CRD)and N-terminal tail(NT)peptide chain.Gal-3 is secreted extracellular via nonclassical pathways.Gal-3 mediates a spectrum of cell processes,including cell surface receptor clustering and lattice formation,the formation of lipid rafts/microdomains,the adhesion of cells to the ECM,and cell agglutination by binding to glycoconjugates(also known as Gal-3 receptors)on the cell surface and ECM.These cell activities all depend on the oligomerization of Gal-3.However,a Gal-3 molecule has only one sugar binding site,and how it achieves multivalency has been confusing.In recent years,a special molecular interaction mode-liquid-liquid phase separation(LLPS)has been proposed.LLPS is a unique molecular interaction mode.Molecules relies on weak binding force to gather to form droplets,and the molecules inside and outside the droplets exchange quickly.Given that Gal-3 contains disordered sequences related to the occurrence of LLPS and oligomerization,we suspect that Gal-3 may undergo dynamic assembly like LLPS,instead of forming fixed oligomers.This thesis takes this as a starting point to study the mechanism of Gal-3 oligomerization and how proline in NT regulates the oligomerization and function of Gal-3,and the detail contents and results are as follows:Firstly,LLPS of Gal-3 under high salt.The amino acid sequence of Gal-3 showed that Gal-3 has the possibility of LLPS.Changing the salt concentration of solution is the simplest and most commonly used method to explore whether proteins can develop LLPS.Therefore,we chose to change the salt concentration in Gal-3 solution to study whether Gal-3 could occur LLPS.Using microscope imaging,microplate reader detection,and dynamic light scattering(DLS)detection,the results all showed that Gal-3 occurred LLPS in high salt conditions.By using the truncated and mutated proteins of Gal-3 to study the role of NT and CRD,it was found that NT is necessary and NT-NT interaction drives Gal-3 LLPS under high salt conditions.Second,glycan-mediated Gal-3 LLPS.Although Gal-3 occur LLPS in high salt,such a high salt concentration cannot be achieved under physiological conditions.Since Gal-3 exerts its biological function through the interaction with glycan,we are investigating whether glycan can mediate Gal-3 LLPS.Fluorescence recovery after photobleaching(FRAP),DLS,flow cytometry and other techniques were used to analyze glycan-mediated Gal-3 oligomerization.The results showed that glycan can induce Gal-3 to undergo LLPS in solution.Gal-3 oligomerization mediated by glycan is also LLPS-like(herein referred to LLPS-like)on the surface of the cell membrane,between cells,and on the damaged inner membrane.Subsequently,the characteristics of the condensation/droplets were studied using microscope imaging,DLS,western blot and other technologies.The results showed that the size and composition of the droplets depend on the concentration and type of glycoprotein and the concentration of Gal-3.Thirdly,the mechanism of glycan-mediated Gal-3 LLPS.The glycan-mediated Gal-3 LLPS was studied from the two processes of triggering and aggregation.1)The triggering process was studied from the perspective of glycoprotein and Gal-3 protein.The results showed that:i)the removal of the glycosylated D5 domain in CD146 or the digestion of N-glycan chains on the surface of CD146 by PNGase F could not trigger the LLPS of Gal-3;ii)the removal of the CRD or the mutating sugar binding site R186of Gal-3,LLPS cannot occur either.The above results indicate that the glycan in glycoprotein triggers Gal-3 LLPS.2)Using flow cytometry,FRAP,MST and other techniques to study the aggregation process between Gal-3 molecules.The results show that:i)NT and CRD are jointly involved in glycan-mediated Gal-3 LLPS;ii)After the CRD_S face in Gal-3 molecule is combined with the glycan chain on the glycoprotein,NT is released and the CRD_F face is exposed.Gal-3mainly depends on intermolecular NT-CRD_F interaction for aggregation;iii)NT interacts with the L203 site in CRD_F,and one NT can bind to multiple CRD,resulting in a cascade effect similar to polymerization,leading to Gal-3 LLPS.Fourthly,the regulation of proline on Gal-3 LLPS and function.Gal-3 NT is rich in proline,the content is as high as about 25%,but its function and mechanism of action are still unclear.At present,the role of proline in Gal-3 and its mechanism have not been systematically studied.Therefore,the first 14 prolines in NT were mutated respectively,and the effects of proline mutation on the triggering and aggregation process were analyzed by using MST,ITC,sugar chip and other techniques.The results showed that a single proline mutation(especially P37A,P50A,P55A,P64A and P67A)significantly altered the binding footprint and affinity of CRD to glycan,and differentially regulate NT-CRD interaction.Further study the effect of proline mutation on Gal-3oligomerization/LLPS.The results show that the same proline mutation has different effects on the Gal-3 oligomerization/LLPS mediated by different glycoproteins,while different proline mutations have different effects on the Gal-3 oligomerization/LLPS mediated by the same glycoprotein.The oligomerization of Gal-3 mediates a series of cell activities,s including the formation of cell surface receptor clusters and network lattices,the formation of lipid rafts,the adhesion of cells to ECM,and cell aggregation.Therefore,the effects of proline mutation on cell migration,activation,endocytosis and erythrocyte agglutination induced by Gal-3 were analyzed.The results showed that Gal-3 ultimately regulates various cellular functions of Gal-3 by regulating its oligomerization/LLPS,and each cellular function requires different proline.In summary,this thesis is the first to find that glycan can induce Gal-3oligomerization/LLPS,and the mechanism of Gal-3 oligomerization/LLPS is explored from the perspective of LLPS.This thesis is the first to systematically study and report the regulation of Gal-3 oligomerization/LLPS and function by a series of proline residues in NT.The results of the study provide a new theoretical model for the Gal-3 oligomerization and new ideas for the mechanism of proline in a variety of diseases.