The Fabrication And Application Of Lectin Microarray:the Construction Of The Human Lectin Microarray And The Optimization Of The Plant Lectin Microarray

Independent of the template,the process of sugar synthesis depends on Enzymes and produces different glycan chains,with a variety of polymerization degree,structure and monosaccharide types.Glycoproteins play critical roles in cytomembrane and extracellular matrix,including cell growth,cell division,cell-cell recognition,cells adhesion and signal transduction.Disorder of glycosylation induces many diseases,such as autoimmune disease,sterility infertility and cancer.The favorite strategis for structural and functional study of glycan chains are mass spectrum,glycan microarray and lectin microarray.Most of the current lectin microarrays are primarily constructed from plant lectins,which are not well suited for studies of human glycosylation because of the extreme complexity of human glycans.There are many human lectins and lectin-like proteins those can both recognize glycans and play significant important roles in many biological processes.One main point of this research is to construct a human lectin microarray and identify the activity of human lectin and human lectin-like protiens on microarray.Entry clones of 93 human lectin and lectin-like proteins were selected from human coding sequences of entry clone library.Yeast library for overexpressing human lectins was constructed by the Gateway system.Glutathione affinity capture was applied to purify 60 human lectins and lectin-like poteins.Contact printing arrayer was used to print human lectin microarray on the surface of polymer-Slide H.Mammal cells and yeast cells were tested and 43 combined human lectins and lectin-like proteins showed positive binding,indicated at least those protein are active.To test the applicability of the human lectin microarray,we took sperm as an example and set to analyze glycosylation of human sperm surface with human lectin microarray.Fixed human sperm were labeled with Propidium iodide and then incubated with human lectin microarray.Five human lectins,include galectin-1,galectin-7,galectin-8,ERGIC-53 and GalNAc-T6 showed binding with sperm and they recognize Gal?1-4Glc,Gal?1-3GlcNAc,Sia?1-3Gal?1-4Glc,Mannose and GalNAc respectively.Flow cytometry and fluorescence microscope confirmed the binding of those 5 human lectins with sperm,and different human lectin binds at different site of sperm surface.Sperm proteins were enrich by galectin-1 affinity and identified with mass spectrum.Several galectin-1 binding sperm glycoproteins were identified,such as Mucin-6,Angiotensin converting enzyme,HSP90,HSP70 and Tubulin ?-4B chain.Following up functional study showed that galectin-8 significantly could improve the incidence of acrosome reaction.Our laboratory has worked in the field of lectin microarray for more than ten years.We have constructed and applied lectin microarray a variety of applications.We are pretty aware of the drawbacks of the current version of plant lectin microarray that we have.Thus,we set of optimize the lectin microarray by getting rid of functionless and redundant lectins,56 plant lectins were left.Contact printing arrayer and polymer-Slide H were used to print plant lectin microarray.To explore the application of the new version of lectin microarray,we have tested single glycoprotein,complex glycoproteins and vesicles-such as IgG,ovalbumin,transferrin,serum and exosomes.We have also tested purified histone on the lectin microarray.Further more,we have now optimized and standardized a series of lectin microarray application.To sum up,we have constructed the first human lectin microarray,with 71.7% of the human lectins and lectin-like proteins are active.Using the human lectin microarray,we identified that several lectins binds to human sperm surface,these lectins are specific to sialic acid,galactoside and mannose.In addition,we have also improved the construction of plant lectin microarray and standardized the operation steps of glycoprotein analysis with lectin microarray.It is possible to take advantage of the human lectin microarray and the plant lectin microarray,combining with mass spectrum and glycan microarray,thus providing effective and powerful tools for glycosylation related studies.