A Study Of Catalytic Antibodies Against β-amyloid Aggregates

Alzheimer’s disease(AD)is a neurodegenerative disease and it is still incurable.The accumulation of β-amyloid aggregates(especially Aβ42O)in the brain has been identified as one of the initiating and major pathological factors causing AD.In addition to their immunological activity,catalytic antibodies are antibodies with a catalytic function that catalyze certain chemical reactions of the targeted antigen.Therefore,antibodies against Aβ42 aggregates with protease activity are more effective in removing Aβ42O than normal antibodies.Based on the above theoretical foundation and our previous findings,this thesis screened a catalytic single-chain antibody against Aβ42O from a library of human single-chain antibodies and systematically investigated the structure and properties of this catalytic antibody and its efficacy against Aβ42 in vivo and in vitro,and the main findings obtained are as follows:1.The screened catalytic single-chain antibody HS72(126 amino acids in the heavy chain and 109 amino acids in the light chain)differs from the reported single-chain antibody HT7 by only 2 amino acid residues(Asp45 in HS72,His241 in HS72 correspond to Gly45 and Gly241 in HT7).Application of western blotting and enzyme-linked immunosorbent assay(ELISA)determined that HS72 is specific to Aβ42O and Aβ42P,and that the molecular weight range of its bounding to Aβ42O(14-68 kDa)is significantly wider than that of HT7(23-55 kDa).2.The ability of HS72 to catalyze the degradation of Aβ42 aggregates was determined by ELISA,western blot,and high-performance liquid chromatography(HPLC).After 10 days of incubation with HS72,the total Aβ42 level decreased by approximately 4.0%(caused by the aggregation of Aβ42M into Aβ42O),11%and 5.8%in the Aβ42M,Aβ42O and Aβ42F system,respectively,while both produced N-terminal and C-terminal truncated Aβ fragments,and at the same time,both had a corresponding amount of Aβ42M also dissociated from the Aβ42 aggregates.In contrast,HT7 had no catalytic effect on Aβ42 aggregates.These results demonstrate that HS72 not only catalyzes the degradation of Aβ42 aggregates,but also generates safe products.Based on the similarity between the primary structures of HS72 and HT7,the changes in the degree of Aβ42 aggregation of HS72/HT7 after incubation with four species of Aβ42 for 48 h were determined by thionin T-staining(ThT-F),and the results showed that,after 12 h,the effect of HS72 in decreasing the aggregation of the above four species of Aβ42 was significantly higher than that of HT7.This is obviously related to the dual efficacy of HS72,i.e.,in addition to the efficacy of HS72 in inhibiting Aβ42M aggregation and inducing the depolymerization of Aβ42 aggregates,similar to that of HT7,HS72 has the efficacy of catalyzing the degradation of Aβ42O,which promotes the depolymerization of Aβ42 aggregates to a great extent.3.HS72 has a similar binding affinity with HT7,both at the nM level,but the affinity of HS72 for Aβ42O is slightly lower than that of HT7.3D structural simulations and molecular docking reveal that the Asp45,His241 and Ser150 residues in the HS72 molecule formed a catalytic triad,which could bind to Aβ42O by an induced-fit mechanism,thus realizing the catalytic degradation of the target Aβ42 chain in Aβ42O by HS72,leading to the hydrolysis and breakage of the Aβ42 chain’s His13-His14 peptide bond of Aβ42 chain was hydrolyzed and broken.Thereby the catalytic mechanism of HS72 on Aβ42O as revealed also illustrates the necessity of HS72 having a slightly lower affinity for Aβ42O than HT7.The above results are consistent with the theory of enzyme catalysis and also establish that HS72 has the catalytic mechanism of a serine protease.4.In vitro,HS72 showed significant protective efficacy on the morphology and viability of both SH-SY5Y and HT22 neuronal cells,and was significantly more effective than that of HT7.This should be attributed to the multiple efficacies of HS72 on the degradation and inhibition of aggregation/induction of depolymerization of toxic Aβ42 aggregates,especially Aβ42O,which significantly reduced the neurotoxicity of Aβ42 aggregates.The safety of the degradation products of Aβ42O as well as that of the dissociated Aβ42M was also well verified here.In vivo,after the AD model rats were intravenously injected with HS72 or HT7(once a day)for 7 consecutive days,it was demonstrated by hematoxylin-eosin staining(HE)and immunohistochemistry(IHC)analyses that the morphology of the brain neuronal cells in the two groups improved to different degrees,while the β-amyloid plaque load of the hippocampal region in the HS72 and HT7 groups decreased by about 27.4%and 5.6%,suggesting that HS72/HT7 leads to the breakdown and clearance of Aβ42 aggregates through multiple interactions with Aβ42 aggregates as indicated in vivo.The results of animal experiments demonstrated that both HS72 and HT7 exerted neuroprotective efficacy against Aβ42 aggregates toxicity in vivo,with the results in the HS72administered group being significantly better than those in the HT7-administered group.5.Based on the existence of a specific interaction between matrines(Mat)and Aβ42O,the effect of Mat on HS72-catalyzed degradation of Aβ42 aggregates was further investigated.An enzyme activator is a compound or metal ion that promotes the catalytic reaction of an enzyme by binding to the enzyme molecule and changing the conformation of the enzyme molecule,thereby increasing the catalytic activity of the enzyme.However,Mat here does not act directly on HS72,but binds both to Aβ42O and to the HS72-Aβ42O complex,and these two binding effects are synergistic rather than antagonistic.The interactions of Mat with Aβ42O and with HS72-Aβ42O together promote the catalytic degradation of Aβ42O by HS72,and thus Mat acts as a facilitator or activator of a synergistic nature.For simplicity,the synergistic promotion or activation of the catalytic activity of HS72 by Mat will be referred to as synergistic in the following.The results of this part showed that although low concentrations of Mat(0.01-2.5 μM)had no significant inhibitory effect on the aggregation and toxicity of Aβ42,it had a synergistic effect on the catalytic effect of HS72:Mat increased the HS72 turnover number by enhancing the catalytic ability of HS72 catalytic groups and facilitating the release of the degradation products(Aβ fragments)by enhancing the catalytic ability of the HS72 catalytic group and promoting the release of the degradation product(Aβ fragment),thereby increasing the catalytic activity of HS72,and improving the neuroprotective efficacy of HS72 in vitro(SH-SY5Y and HT22 cells)and in vivo(AD mice),without affecting the specificity,binding and affinity of HS72 for Aβ42O.6.In view of the fact that single-domain antibodies exist naturally in living organisms and their smaller molecular weight makes it easier to cross the blood-brain barrier,this thesis further analyzes the nature and function of the heavy-chain variable region(VH)and light-chain variable region(VL)of the single-chain antibody HS72.The results showed that VH&VH or VH&VL were similar to HS72,with both catalytic and antibody effects,while VL&VL had only antibody effects,although their free combinations had integrated conformational diversity.Conformational analysis demonstrated that the catalytic motifs Asp45 and His241 in HS72 are still involved in the construction of the catalytic site in VH&VH or VH&VL,respectively.More importantly,although the protective effects of VH&VH or VH&VL on neuronal cells in vitro were slightly lower than that of HS72,their efficacy(reducing β-amyloid plaque load in hippocampal region by about 28.9%)in AD rats was equal to or slightly better than that of HS72,which may be related to the fact that the single-domain antibody crosses the blood-brain barrier more efficiently than the single-chain antibody.Taken together,This thesis leads to the following conclusions:(1)catalytic anti-Aβ42O single-chain antibodies are superior to the corresponding simple single-chain antibodies in removing Aβ42 aggregates and ameliorating the histopathological changes in the AD brain;(2)small-molecule natural products,such as Mat,enhance the efficacy of catalytic single-chain antibodies at low concentrations through synergistic interactions;(3)in vivo,the protective efficacy of single-domain antibodies against brain cells is slightly higher than that of the corresponding single-chain antibodies.Although catalytic antibodies such as HS72 are not yet therapeutic candidates for AD,they will provide some experimental basis and theoretical guidance for the pharmacological study of AD.