Structure-Function Relationship Between Ginseng Pectin And Galectin-8

Pectin,an acidic hetero-polysaccharide present in the cell wall of plants,has various biological activities,such as tumor growth inhibition,anti-biotic and immune regulation.However,its mechanism of action is not well understood.In recent years,it has been reported that pectin can antagonize galectin-mediated functions,opening a new avenue of study.Galectins are a class of lectins with a carbohydrate recognition domain(CRD)that binds ?-galactosides.Galectin-8(GAL8),a tandem repeat type galectin,is involved in numerous physiological and pathological processes in which cell adhesion is required.Overexpression of GAL8 causes diseases,such as osteoporosis.GAL8 can also promote tumor cell proliferation and angiogenesis in some cancers.However,there are few studies that have reported on inhibitors/modulators of GAL8,especially pectic-based agents.In this thesis,ginseng pectin was used as a model to study interactions between pectin and GAL8,as well as its structure-activity relationship.The specific research results are summarized as:?.Preparation and structural analysis of ginseng pectin fractions and fragments.Initially,ginseng pectin(WGPA)was separated into four RG-I and four HG fractions using ion exchange chromatography,gel filtration and pectinase digestion.RG-I fraction 2A-Dp was partially hydrolyzed by TFA into three sub-fractions.2D-NMR spectroscopy shows that Ara is mainly connected via ?-1,5 linkages in 2A-Dp;some are branched at C3,whereas all Gal residues have ?-1,4 linkages.Fragments 2A-Dp-4 and 2A-Dp-12 only have ?-1,4-Gal in their side chains.?.Preparation and structural analysis of pectic oligosaccharides.In order to obtain the side chains,a series of galacto-oligosaccharides with DP(degrees of polymerization)from 2 to 15 were prepared by partial acid hydrolysis of potato galactan that is rich in ?-1,4-Gal.By analyzing 2D-NMR spectra,changes in resonance distributions were correlated with the degree of polymerization.To obtain HG fragments,a series of oligo-galacturonans with DP from 2 to 6 were prepared by enzymatic hydrolysis using sunflower pectin that is rich in ?-1,4-GalA.GAL8 and five of its mutants were constructed by molecular cloning and recombinant proteins were expressed.?.Comparison of interactions between ginseng pectic fractions and GAL8.The interaction of various pectin fractions with GAL8 was assessed by using the G8 H inhibition assay,and binding affinity was quantified by using the BLI method.For the first time,we found that pectins could bind to GAL8,with RG-I pectin fractions showing the highest affinity.?.Structure-activity relationshisp between RG-I pectin and GAL8.To delineate how these species interact,we investigated binding at the level of pectin and protein.In terms of pectin structure,the inhibition of WGPA fractions was first estimated from GAL8-induced hemagglutination.These results showed that RG-I-rich pectin fractions had greater inhibition activity than HG-rich pectin.Using RG-I pectin fraction 2A-Dp and its three fragments,we found that the Gal side chains are vital for RG-I binding to GAL8,with Ara residues playing no role in this interaction.Interactions between RG-I side chains and GAL8 were then examined by using a series of galacto-oligosaccharides(GOs).Results showed that the binding affinity of GOs for GAL8 was comparable to that of LacNAc.Moreover,the backbone of RG-I did not interact with GAL8.By analyzing these data,we concluded that the RG-I backbone acts as a skeletal support,with the actual binding affinity of RG-I pectin to GAL8 arising from side chain galactose residues.On the protein side,we investigated the interaction between GAL8 mutants and RG-I fraction 2A-Dp.Results from lactose inhibition on the binding of RG-I to GAL8 indicate that RG-I pectin binds to the classical lactose binding site in the CRD.The binding affinity of 2A-Dp to two truncated CRDs of GAL8 and two mutants(R69H and R233H)showed that RG-I pectin can bind to both N-CRD and C-CRD,with the affinity for N-CRD being slightly higher.?.Crystallographic study.In this thesis,the co-crystal structure of GAL8 and natural pectic oligosaccharides was obtained by screening and optimizing co-crystallization conditions.This allowed for us to study the interaction between pectic oligosaccharides and a galectin for the first time by using X-ray crystallography.After anamorphic structure analysis by X-ray diffraction,we found that the non-reducing end and the second galactose of the galacto-oligosaccharides form a stable hydrogen bond with the Arg 45,Arg 69 and Glu 89 of GAL8.This paper also reported for the first time that GAL8N(1-186aa)forms a new ?-sheet structure when it binds to the galacto-tetramer(GO4).In summary,this thesis is focused on structure-activity relationships between GAL8 and ginseng pectin fractions.We found here that pectin can bind and inhibit GAL8,RG-I(not HG)domains are the active component,and galactose side chains in RG-I are the major active structural units.In addition,we report here the co-crystal structure of natural oligosaccharides and a galectin for the first time.This research will lay the foundation for development of pectin-based GAL8 antagonists for potential use as therapeutics against cancer.