Improvement Of Polymerization State And Deimmunization Of Candida Uricase Using Bioinformatics

Uricase is a peroxisomal enzyme involved in purine metabolism which converts uric acid?UA?to allantoin that is easily excreted by the kidney.But as a result of multiple independent evolutionary events,the uricase gene in higher primates,including human,is silenced or pseudogenized.As a consequence,UA levels are 3 to 10 times higher in human than in other mammals possessing a functional uricase,which may lead to hyperuricemia.And hyperuricemia can result in urate precipitation with the formation of crystals that are involved in gout,chronic kidney disease?CKD?and various cardiovascular diseases.Currently,available anti-hyperuricemia drugs include uricosuric agents that promote UA excretion and inhibitors of xanthine oxidase?XO?that block urate formation.But they could only be used for particular hyperuricemia-associated diseases because of drug toxicity and tolerance.Uricase is a unique anti-hyperuricemia drug acting on urate and is effective in diverse forms of hyperuricemia in virtue of removing pre-existing urate crystals in joints with negligible drug-drug interactions.Up to date there are two kinds of uricase that have been approved for management of plasma UA level: rasburicase and pegloticase.Rasburicase is indicated for the prevention and treatment of TLS which can be a potentially life-threatening emergency.Though it is significantly more effective than conventional drugs,the enthusiasm for its use has been dampened by serious allergic reactions.Pegloticase is a medication for the treatment of severe,treatment-refractory,chronic gout.As a polyethylene glycol-conjugated form of uricase,pegloticase has elevated thermal stability and longer serum half-life,but has a high discontinuation rate?30-50%?due to development of anti-drug antibodies with the resultant loss of efficacy and risk of infusion reactions.All of these adverse effects are associated with immunogenicity,which is considered as a major challenge for therapeutic proteins.Immunogenicity is caused by diverse interacting factors including intrinsic causes such as specific amino acid sequence,posttranslational modifications,and extrinsic factors including route of administration,dosage and,in particular,the patients' genetic profile.At present,there is a common consensus that one of the highest risk factors for drug immunogenicity is the property of proteins to aggregate.Even the presence of sub-visible nano-and microparticles may be immunogenic.However,aggregation mechanisms are highly complex and the investigation requires experimental efforts,manpower and material cost.Some in-silico methods using conformational flexibility,electrostatic and hydrophobic protein surface properties are supposed to be best suited for prediction of protein aggregation.In our previous study,we find that Candida uricase is a promising candidate for therapeutic use as its superior soluability and activity.But it aggregated in a very short time after being purified,which created difficulties for the following analyzation and deimmunnizing modifications.Considering that aggregation of therapeutic proteins could cause immunogenicity,we decided to optimize its property using bioinformatics,in order to improve the polymerization state and decrease its immunogenicity.This study is carried out in two steps.Firstly,in order to improve its aggregation propensity,its three-dimensional structure was homology modeled to reveal the causes of polymerization and guide the design of sequence mutations to eliminate them.Mutants with specific mutations were expressed and purified,and their influences on polymerization state were evaluated.Secondly,as the mutant with improved polymerization state was obtained,its immunogenicity was predicted.The T cells recognize epitopes in the mutant were substituted with human sequence for deimmunization,and their effects were evaluated by immunizing mice with mutants containing these substitutions.In this paper,the above content is divided into three parts.Part one,design and expression of mutant with improved polymerization state.The three-dimensional structure of Candida uricase was simulated by the SWISS-MODEL server using uricase from Aspergillus flavus as a template,which had a high homology identity?65.93%?with it.In addition,quality of the model was evaluated by RAMPAGE Verify₃D,ERRAT and ProSA,and results of them showed the good quality of the model.According to the 3D model and related references,2 mutantions were designed to inhibite the formation of inter chain disulfide bonds and eliminate hydrophobic interactions between homotetramers: C249 S and ?L302.Then wild type uricase and mutants containing either or both of the 2 mutations were expressed by E.coli.All the purified protein had purity over 95% and normal enzymatic activity.Part two,characterization and evaluation of wild type uricase and mutants.Amino acid sequences of the 4 proteins were validated by HPLC-MS.Polymerization analysis showed that the polymers could be eliminated by reduction and no polymer were formed in C249 S and ?L302&C249S,which indicate that formation of inter-chain disulfide bonds is the main inducing factor of aggregation,and C249 S substitution significantly improve the aggregation state of uricase.Results also show that deletion of Leu promoted the formation of plolymers and has no effect on polymerization after Cys249 was substituted with Ser.In addition,deletion of Leu increases uricase tetramers' s specific activity,and neither of the 2 mutation have any effect on the secondary structure and thermal stability of uricase.Taken together,mutant ?L302&C249S has improved polymerization state and enzymatic activity and could be used for the following deimmunization research.Beyond that,we also find that the polymers are formed with partial disulfide bonds rather than all of them and without altering the structure of tetramers,according to results of SEC,SDS-PAGE,disulfide bond analysis,CD,thermal stability and enzymatic activity test.Part three,design and evaluation of deimmunization.Though mutant ?L302&C249S has acquired improved properties for therapeutic use,immunogenicity remains a major obstacle.With the development of bioinformatics,a great deal of T cell epitope mapping tools was established.In this study,immunogenicity of mutant ?L302&C249S was predicted by TEPITOPEpan,and results reveale that 2 T cell epitopes existed in it.Then the 2 epitopes were subsitituted with corresponding human sequence after homologous comparison with BLAST.The new sequence was retested by TEPITOPEpan and no T cell epitope were found.Using the same method described in part one,a mutant “Deimmunized” with one T cell epitope substituted with human sequence was expressed and purified.Its sequence was also validated by HPLC-MS.The mutant had similar polymerization state and thermal stability.Enzymatic characterization showed that the mutant had a decreased enzymatic efficiency,and its enzymatic activity decreased to 75% when tested under 37? and pH7.0.Antibody titer detection showed that serum immunized by mutant “Deimmunized” had lower titer,which suggested that the mutant had a lower immunogenicity.Results also showed that incubation with serum containing specific antibody had no effect on its acticity.In conclusion,the deimmunizing design achieves the desired results despite has some negative effect on activity.In our future research,we will optimize the mutation to recover its activity,and deimmunize the other sites of T cell epitopes in order to obtain a mutant with more low immunogenicity.In summary,based on bioinformatics tools,we successfully improve the polymerization state of Candida uricase,and furthermore carry out the deimmunization research which achieves the desired results.This study promotes the development of therapeutic application of Candida uricase,and sheds light on other researches involving therapeutic use of foreign proteins.