Exploration Of The Lectin's Binding Specificity For Glycan Based On Lectin Modification And Sugar Profile Analysis

Glycosylation modification is a ubiquitous post-translational modification which involved in various life processes of cells,including signal transduction,apoptosis,transcriptional regulation,and immune response.In addition,changes in glycosylation have been found in various symptoms including inflammatory response,tumor cancer,neurodegenerative diseases and so on.Lectins and anti-glycan antibodies,as the main research tools,both of them have shortcomings in specificity and affinity,which hinder the research process of the entire field.Therefore,it is of great significance to search for better glycosylation modification recognition tools and methodological studies to s improve tools.In the previous research of our research group,AANL(a lectin from fungi Agrocybe aegerita,high specific for Glc NAc)was subjected to carbohydrate recognition domain(CRD)homogenization,and successfully obtained the mutant lectin AANL6,which improved affinity and specificity of Glc NAc binding activity and used as a powerful tool for Glc NAc modification research.In this article,TL2(Tachylectin-2,a lectin isolated from Japanese horseshoe crabs capable of specifically binding Glc NAc and Gal NAc)was modified in a similar way based on the crystal structure of to obtain a more favorable sugar recognition tool.At second part of the research,glycan-binding spectrum of the galectin family was analyzedthrough a new methodnamed “end glycan collection&analysis” which developed by our lab.The analysis results give reasonableexplain the function overlap and specific of galectins family members and which is important for further function research.TL2 is structurally composed of five tandem similiaramino acid sequences and has five active domains.The CRD homogenization stratege,which was used for AANL modification successfully,was performed on TL2 modification in this paper.According to the sequence characteristics of TL2,two mutation schemes were designed,one to retain the amino acids in the ?-sheet and the other to homogenize the amino acids in the ?-sheet.We replaced all the key amino acids of CRD for the amino acid sequence of one of the CRDs,the mutants were named TL21-TL25 and TL2a-TL2 b according to different schemes.Finally,three TL2 mutants(TL22,TL2 b and TL2c)were successfully expressed and purified by Glc NAc-coupled affinity chromatography columns.The result verified that these three mutant lectins retained their affinity for Glc NAc.Through glcanchip detection,it was found that TL2 b and TL2 c have greatly improved the affinity of Glc NAc compared to TL2.Among them,TL2 b also increased the binding capacity of the mannose-terminated glycans.In the subsequent isothermal titration calorimetry(ITC)test,it was found that both TL22 and TL2 c lost the binding capacity to Gal NAc compared to TL2,which achieved our desired result: the mutant of TL2 increased the binding capacity of Glc NAc,at the same time,the affinity for Gal NAc is lost.To explore other options for glycan recognition tools,we analyzed members of the galectin family.Galectin is a class of lectins that specifically recognize ?-galactoglycan through highly conservative CRD.Galectin family lectins are involved in a variety of key functions in vivo,including cell signal transduction,apoptosis,cell differentiation,and immune response.Galectin has been reported to have ?-galactose recognition activity but the functional diversity of different galectins must be related to the different glycosylation modifications they recognize.However,there wasfewreports.We discarded the traditional histogram analysis method,and used the laboratory's innovative "terminal glycan collection analysis method" to reorganize,classify,and statistically plot the glycan array data of seven galectins.Several interestingroles were discovered by the new analysis method.It was found that galectin-1 and-9 did not show significant specificity for the recognition of different glycosylation ends,and the overall affinity was weaker than other members of the family,which tended to bind to the glycosylation modification of Gal?1-4Glc NAc terminal.And we clearly find that galectin-2,-4,-7,and-8 have good specificity.We also found the glycosylation modified terminals bound by these lectins have distinctive characteristics: galectin-2 mainly binds to the structure of Fuca1-2Gal?1-4Glc NAc,galectin-4 mainly binds to Gal NAc?1-3(Fuc?1-2)The structure of Gal?1-4Glc,Galectin-7 only binds to the structure of Fuca1-2Gal?1-4Glc NAc?1-3Gal?1-4Glc NAc,and galectin-8 mainly binds to Gal?1-4Glc NAc as a cyclic tandem repeating multi-branch structure.Galectin-3 is the most special case in that its affinity glycosylated terminal intersects with the other six galectin members analyzed.After the deep analysis of these glycan array data,we found several common glycans and specific glycans in galectins binding-glycans,which indicated the redundancy functions and specific functions of galectin family members.Among them,galectin-7 has the best specificity.After successfully expressing and purifying it in vitro,the influence between galectin-7 and fucosyltransferase was explored according to the characteristics of its preferred glycosylation modification.The results show that the stimulation of cells by galectin-7 is more likely to cause high expression of fucosyltransferase(Fut1-Fut7).Our "terminal glycan set analysis method" reanalyzes the galectin sugar profile and obtains important structural information for different galectin family members to identify sugar chains,which provides valuable data for further study of galectin's function.