The Design And Biological Functions Of Lectin Mutants Based On Crystal Structure

Glycosylation is the most abundant and common posttranslational protein modification.The glycosylation of protein is divided into N-linked glycosylation and O-linked glycosylation.Currently,it has been reported to play important roles in many cellular processes,such as signal transduction,cell cycle progression,and transcriptional regulation.Among them,GlcNAc(N-acetylglucosamine)terminal glycosylation is researched extensively.However,facile detection of this glycosylation is greatly hampered due to the low specificity or sensitivity of anti-glycan antibodies and lectins.Our lab has purified a GlcNAc-binding lectin named AANL from Agrocybe aegirita.To improve the specificity and sensitivity,we restructured AANL based on its'crystal structure so that it can be a more powerful tool to identify terminal GlcNAc glycosylation.Apart 1:The design and glycan-binding profile of AANL mutants.By analyzing the amino acid sequence and crystal structure of AANL,we found that six CRDs(carbohydrate recognition domain)of AANL were homologous,but the amino acid sequence of each CRD wasn't the same.And all CRDs were located in the edge,which are loose.So we speculated that mutating CRD would have little effect on its'structure.To improve the specificity and sensitivity of AANL,we designed a CRD-homogenization strategy which changed the key amino acids of 6 CRDs into same sequence and restructured six mutant lectins,named as AANL1-6.Two of which were expressed successfully called AANL3 and AANL6.They could be purified by Sepherose-GlcNAc column,so AANL3 and AANL6 could still bind to GlcNAc.The Kd value of the two mutants and AANL binding to GlcNAc were at the same order of magnitude.Glycan microarray from CFG(Consortium for Functional Glycomics)suggested that AANL6 lost the binding of some truncated O-glycans compared with AANL.All results suggested that AANL6 kept GlcNAc-binding activity and improved the specificity.Cell binding assays which included 6 types of erythrocytes,3 types of stem cell lines,4 types of cancer cell lines,lymphocytes and human sperms further proved that AANL6 was more specific than AANL.Compared with AANL,AANL3lost the binding of rat erythrocyte,T,B,He La,Huh7 and TPC115 cell and AANL6couldn't bind to these cells but human sperms.When analyzing the glycan profiles from2 cancer cell lines and human sperm surfaces,we found that abundant high mannose type glycans and terminal sialic acid glycans were presented on cancer cell surfaces,while human sperm surface contains a lot of terminal GlcNAc glycans.All these results indicated that AANL6 were more specific than AANL.Apart 2:Using AANL6 as a tool to identify terminal GlcNAc glycans.AANL6 could be used to identify O-linked N-acetylglucosamine modification(O-GlcNAcylation).O-GlcNAcylation is a ubiquitous dynamic carbohydrate modification in almost all metazoans mainly located in nucleus and cytoplasm.O-GlcNAcylation plays an important role in many cellular processes,such as signal transduction,cell cycle progression,and transcriptional regulation.All lectins could bind to three tumor cells when these cells were treated with permeabilization.Immunofluorescence analysisresults were in line with flow cytometry analysis.AANL strongly interacted with glycans on cell membrane,nucleus membrane and in cytoplasm and AANL3bound to the glycans mainly in cytoplasm.AANL6 only bound to glycans in nucleus mainly.After PNGase F pretreatment,AANL6 still kept positive signals in nucleus.However,AANL and AANL3 staining was almost completely removed.It indicated that AANL6 might recognize a lot of O-GlcNAcylation in nucleus.In addition,SPR(surface plasmon resonance)assays demonstrated that AANL6 had a micromolar affinity for O-GlcNAcylated peptides.In complex tissue sample,AANL6 staining indicated that terminal GlcNAc glycans were highly expressed in testis,brain,heart and kidney tissue from mouse,lower signals were shown in liver and lung.This result was consistent with the other reasearchs.It was still a challenge to identify a lot of O-GlcNAcylation in liver tissue.Our lab has identified 23 O-GlcNAcylated sites in liver tissue by AANL enrichment.By POROS-_AANL6 affinity chromatography enrichment,there was evident tailed peak.By mass spectrometry analysis,79 high confident and 21putative O-GlcNAcylated sites were identified on 85 peptides mapped on 54 proteins.Most of which were new.Meanwhile,the enrichment of AANL6 was higher than wild-type AANL and O-GlcNAc antibody(CTD110.6).In conclusion,the lectin-mutation strategy was successful and a mutant AANL named AANL6 was identified as a powerful tool for O-GlcNAcylated peptide enrichment.AANL6 could also be used to identify terminal GlcNAcN-glycans(N-?GlcNAc glycans)of monoclonal antibody.More and more researchs have found that the glycosylation of antibody affects its'function or stability.Currently,glycosylation has been a standard for antibody quality control.To dectect the specificity of AANL6binding to glycosylated antibody,we used another glycan microarray in which all 100glycans are from antibody.The results indicated that AANL6 and AANL3 were more specific for N-?GlcNAc glycans than AANL.And AANL6 bound N-?GlcNAc glycans with the highest specificity.Then,we analyzed the N-glycan of two monoclonal antibodies(anti-PD1 and anti-VEGF)from phase II clinical trials by MALDI-MS and conformed that abundant N-?GlcNAc glycans covered on antibody.Detected by lectin elisa and lectin blot,we proved that AANL6 could bind to trace of antibody and had a higher sensitivity and specificity than AANL.And this binding depended on the N-glycans from antibody.Most importantly,AANL6 binding was linearly dependent on the concentration of GlcNAcylated antibody.So it may be used to identify the GlcNAcylated antibody quantitatively.To enrich N-?GlcNAc glycans in serum by using"filter aided sample preparation"(FASP)-based method,AANL6 could enrich a lot of N-?GlcNAc glycans and the total percentage of two N-?GlcNAc glycans(H3N4F1 and H4N4F1)were over 50%.The results of AANL enrichment was similar to no enrichment which kept the binding profile of a main sialic acid terminal N-glycan named H5N4S2.Meanwhile,we collected peptides after glycopeptides were deglycosylated by PNGase F treatment.The MS analysis showed that AANL6 identified more proteins than AANL.With the quantitative analysis of proteome between AANL6 and AANL enrichment,we identified 26 proteins enriched in AANL6 enrichment,they might be modified with one or multiple N-?GlcNAc glycans.In conclusion,AANL6 was more proper to identify or enrich GlcNAcylated proteins than AANL in serum because of its specificity and enrichment efficiency.The GlcNAcylated level of proteins in serum were related to many diseases,so it would be potential to be used in disease diagnosis.Using AANL6as an enriched tool,total 11 proteins were identified as biomarker candidates for early non-small cell lung cancer.Apart 3:The dimerization of AANL6 and the binding profie of AANL6-dimerTo increase the GlcNAc binding avidity of AANL6,a linker was used to dimerize AANL6 monomer.Two AANL6-dimers,named PA6 and EAA6,were purified successful.The result detected by ITC(isothermal titration calorimetry)showed that PA6 and EAA6 had a higher avidity than AANL6.PA6 was selected to perform other assays.PA6 kept some binding profiles of AANL6 including cells and antibodies.Glycan microassay showed that although the concentration of PA6 was 20 times less,compared with AANL6,the maximum fluorescence value was still close to AANL6.And PA6 bound to terminal GlcNAcN-glycans with a higher specificity than AANL6.The results indicated that PA6 bound to terminal GlcNAc glycans with a higher avidity and specificity than AANL6.So PA6 might be useful for identifying terminal GlcNAc glycans including O-GlcNAc modification and N-?GlcNAc glycans.