Structure Elucidation Of Pumpkin Acidic Polysaccharides And The Interaction With Functional Proteins

Two acidic polysaccharides of different structure were isolated from pumpkin.They were water-extracted acidic polysaccharides named WAP and alkali-extracted acidic polysaccharides named AAP.The detailed structure of WAP and AAP was resolved in this study.Sulfated derivative of WAP(SWAP)was prepared and its interaction with tau protein involved in Alzheimer's disease was characterized in comparision with heparin.Besides,interaction between SWAP and two anticoagulation-related protein(antithrombin III and heparin cofactor ?)was also studied to understand the mechanism of anticoagulant activity of SWAP.What's more,interaction between AAP and galectin-3 was analysed and the inhibition effect of AAP on galectin-3 was testified at the cellular level.(1)Crude polysaccharide WP was obtained from pumpkin lyophilized podwer by hot water extraction and 80%alcohol precipitation.The primary acidic component WAP with a molecular weight of 8.32×104Da was purified from WP by ion exchange and secondary alcohol precipitaiotn.WAP was mainly composed of galacturonic acid with a high degree of esterification(67.91%).The main chain was polygalacturonan linked by ?-(1-4)-linkage confiremed by nuclear magnetic resonance(NMR).Atomic force microscope was appied to characterize the conformation of WAP in water.WAP was distributed as chain conformation,rarely branched,with an average chain length of 160.8 nm and chain height of 0.28?1.4 nm.(2)Crude polysaccharide AP was obtained from pumpkin residue podwer using alkali extraction and 80%alcohol precipitation.The primary acidic component AAP with a molecular weight of 2.26×104 Da was purified from AP by ion exchange and secondary alcohol precipitaiotn.AAP was mainly composed of rhamnose,galacturonic acid,galactose and arabinose,with a molecular ratio of 7.4:25:28:2.6.AAP had a low degree of esterification of 5.4%.The main chain was polygalacturonan connected by ?-(1-4)-linkage and rhamnogalacturonan in the form of[?4)-a-D-galacturonic acid-(1?2)-a-L-rhamnose-(1?]confiremed by NMR.The side chain was galactan connected by ?-(1-4)-linkage with a little arabinose attached.(3)Sulfated derivative SWAP was prepared from WAP with a sulfation group content of 16.7%.The degree of substation was 1.81 and the molecular weight was 2.47×104 Da.The substitution postion of sulfation group on SWAP was C-2 and C-3 identified by NMR.The binding affinity of SWAP-tau K18 interaction was 1.15×10-8 M,higher than heparin.The binding site of SWAP on tau K18 was identified by NMR,which was similar to heparin.The main binding site was R2 domain of tau K18.The result indicated that SWAP resembled heparin in the interaction with tau,suggesting its potential inhibitory effect on the metastasis of tau.(4)Interactions between SWAP and two anticoagulation-related protein(antithrombin III and cofactor II)were analyzed by surface plasmon resonance(SPR).The binding affinity between SWAP and antithrombin III was 1.71 × 10-4 M,which was a weak binding lower than heparin.The binding affinity between SWAP and heparin cofactor II was 1.39×10-7 M,which was a tight binding similar to heparin.It indicated that the inhibiton activity of SWAP on thrombin was mostly mediated by heparin cofactor ?.SWAP significantly prolonged the activated partial thromboplastin time(APTT)and prothrombin time(PT),while had little effect on thrombin time(TT),revealing that the anticoagulant activity of SWAP was accomplished through endogenous pathway.Alkali extracted polysaccharide AAP interacted with galectin-3 with a binding affinity of 1.26×10-6 M.The inhibitory effect of AAP on galectin-3 was testified at cellular level,which was higher than galactose and lactose.