Study On The Structure-Activity Relationship Of Angelica Polysaccharide With Galectin-3 As A Target In Inhibiting Leukemia Cell Apoptosis

ObjectivesAngelica sinensis（Olive）Diels is a famous traditional Chinese medicine of treating hematologic and gynecological diseases for centuries.Polysaccharide is a major ingredient in Angelica sinensis with significant bioactivities,including antioxidant,antitumor,antiaging,antihepatotoxic,immunomodulatory,and neuroprotective effects.It has been reported that angelica polysaccharides can significantly inhibit the proliferation of leukemia cells and induce their apoptosis,which has played a unique role in the treatment of leukemia.Our previous study also showed that angelica polysaccharide（especially AAPS-2Ⅰ）had a direct inhibitory effect on the proliferation of leukemia cells,and it was stronger than the neutral polysaccharide from Angelica sinensis.Our previous experiments also suggested that Galectin 3（Gal-3）may be the target of angelica polysaccharides to induce apoptosis of leukemic cells.In this research,we attempted to 1)analysize the structural characteristics of AAPS-2Ⅰ;2)establish method of preparing of AAPS-2Ⅰpolysaccharide fragments;3)screen AAPS-2Ⅰpolysaccharide fragments that can bind to Gal-3 protein;4)determin the proliferation inhibitory and apoptosis-inducing effects of AAPS-2Ⅰand its fragments on leukemia K562 cells;5)comprehensive analysize the structural characteristics of AAPS-2Ⅰactive fragments and elucidate the structure-activity relationship,and further to demonstrate the critically antileukemia-dependent structure of angelica polysaccharides.From above,the study will reveal the pharmacodynamic mechanism of Angelica sinensis polysaccharides,providing more modern biological experimental basis for the clinical application of traditional Chinese medicine and the development of new drugs.Methods（1）HPSEC、HPLC and NMR technologies were applied to analysis the structure characteristics of AAPS-2Ⅰ.HPSEC was used to determine the purity and molecular weight of AAPS-2Ⅰ.Total sugar content,uronic acid content and protein content of AAPS-2Ⅰwere respectively determined by phenol-sulfuric acid method,sulfuric acid-carbazole method and Coomassie blue method.HPLC was used to analysize the monosaccharide composition.Sugar residue type,linking site were studied using NMR spectroscopy.（2）AAPS-2Ⅰdegradation fragments were respectively prepared by enzymatic hydrolysis and partial acid hydrolysis.（3）Solid phase binding assay and MST analysis were used to determine the affinity of AAPS-2Ⅰand its degradation fragments with Gal-3.（4）CCK-8 method was used to determine the inhibitory ratios of AAPS-2Ⅰand its degradation fragments on leukemia K562 cells.AnnexinV-FITC/PI double staining method was used to detect the apoptosis ratio of leukemia cells after polysaccharide treatment.（5）The monosaccharide composition of AAPS-2Ⅰactive fragment was determined using HPLC,and the structure of AAPS-2Ⅰactive fragment was analyzed by NMR.Results（1）HPSEC results showed that AAPS-2Ⅰwas a homogeneous polysaccharide with a molecular weight of 6.5×10⁴ Da.The total sugar content,uronic acid content and protein content of AAPS-2Ⅰwere 96.37%,36.87%and 1.08%,respectively.Its monosaccharide composition and molar ratio were Man:Rha:GalA:Glc:Gal:Ara（0.2:0.4:0.2:1.0:3.9:2.4）.In addition,trace amounts of GlcA and Fuc were also found in AAPS-2Ⅰ.NMR analysis revealed that the AAPS-2Ⅰstructure mainly containedα-L-1,5-Araf,α-L-1,3,5-Araf,T-α-Araf,β-D-1,4-Galp,β-D-1,3-Galp,T-α-Glcp andα-L-1,2-Rhap,seven kinds of sugar residues.（2）The preparation process of AAPS-2Ⅰenzymolysis and acid hydrolysis fragment was established,and the AAPS-2Ⅰwas hydrolyzed with galactosidase,mannanase,pectinase and rhamnoses respectively.10 digested fragments of Galase-1,Galase-2,Galase-3,Galase-4,Manase-1,Manase-2,Pecase-1,Pecase-2,Rhaase-1 and Rhaase-1 were obtained.Furthermore,AAPS-2Ⅰwere hydrolyzed with 0.5,1.0 and 2.0M TFA and three acidic hydrolysis fragments AAPS-2Ⅰ-0.5-R,AAPS-2Ⅰ-1-R and AAPS-2Ⅰ-2-R were obtained.After purification,13 homogeneous degradation fragments were obtained with molecular weights from 831.7 Da to 2.5×10⁴ Da.（3）In eight polysaccharides from Angelica sinensis,AAPS-2Ⅰ、AAPS-2Ⅱ和AAPS-1Ⅰcould bind to Gal-3 using MST assay.The dissociation constants（Kd）were 93.5±33μM,510.3±106μM and 166±4.46μM,respectively.Galase-4,Pecase-1 and Rhaase-2 of AAPS-2Ⅰenzymatic hydrolysis fragments could combine with Gal-3 with Kd of 24.5±12μM,166.7±120μM and 596±146μM,respectively.The acid-hydrolyzed fragments all had no binding effect to Gal-3.The solid-phase binding experiments further confirmed that Galase-4,Pecase-1,and Rhaase-2 binding to Gal-3 with IC₅₀0 of 179±18 mg/L,182±17mg/L and 189±29 mg/L,respectively.（4）CCK-8 assay was used to determine K562 cells inhibitory effects of eight polysaccharides from Angelica sinensis and AAPS-2 I degradation fragment.The results showed that AAPS-2Ⅰhad the strongest inhibitory activity among eight homogenous polysaccharides.Its digested fragment Galase-4 could further increase the inhibitory effect,with IC₅₀0 0.39 mg/L.Flow cytometry was used to determine the effects of polysaccharides on the apoptosis of K562 cells.The results showed that AAPS-2Ⅰhad significant apoptosis induction on K562 cells.Its hydrolysis fragment Galase-4 could further promote the apoptosis rate of leukemia K562 cells in a concentration-dependent manner.（5）The possible sugar chain structure of AAPS-2Ⅰcontains→2)-Rhap-（1→3）-Galp-（1→）-T-Glcp,→5)-Arap-（1→4）-Galp-（1→and→5）-Araf-（1→3,5）-Araf-（1→3）-T-Araf.AAPS-2Ⅰenzymatic hydrolysis fragment Galase-4contaning→3)-Galp-（1→4）-Manp-（1→as main chain,→4）-Glcp-（1→5）)-Araf-（1→as in the side chain,demonstrated the strongest affinity interaction with Gal-3.The main sugar chain linkage of AAPS-2Ⅰ-0.5-R was→4）-Manp-（1→4）-Glcp-（1→）-GlcpA.By comparing the monosaccharide composition and the sugar chain connection of AAPS-2Ⅰand its active fragments and the acid hydrolyzed fragments,the structure-activity relationship of angelica polysaccharides against leukemia was preliminarily determined.The activity of acidic polysaccharides might be determined by the structure of the sugar chain,but no significant relationship with its molecular weight;GlcA and Rha are not essential sugar residues;→3)-Galp-（1→4）-Manp-（1→and→5）-Araf-（1→are important active units;Acidification could damage the structure of active polysaccharide.ConclusionsIn this study,the structural features of AAPS-2Ⅰwere elucidated,which mainly containsα-L-1,5-Araf,β-D-1,4-Galp,β-D-1,3-Galp andα-L-1,2-Rhap sugar residues.The preparation processes of AAPS-2Ⅰenzymolysis and acid hydrolysis fragments were established and thirteen kinds of AAPS-2Ⅰdegraded fragments were obtained.AAPS-2Ⅰhad the most significant inhibitory effect on leukemia K562 cells,and it could induce apoptosis of leukemia cells by binding to Gal-3.Compared with AAPS-2Ⅰ,the binding affinity to Gal-3,the apoptotic rate to K562 cells and inhibitory effect on proliferation were further enhanced by Galase-4,indicating that Galase-4 was the main active unit.By further analyzing the structure of the active fragment,it was found that the effect of angelica polysaccharides on the apoptosis of leukemic cells was independent of the molecular weight and was mainly determined by the structure of the sugar chain.Main chain of→3）-Galp-（1→4）-Manp-（1→and branch of→5）-Araf-(1→is responsible for the apoptosis-inducing effects of angelica polysaccharides.