Experimental and Computational Studies on Uricase and Its Bio- Conjugation With Bovine Serum Albumin For Hyperuricemia

Abstract

Hyperuricemia is a significant risk factor for many health conditions like gout, obesity, diabetes, hyperlipidemia, hypertension, and renal disease. Hyperuricemia is generally caused by increased blood uric acid due to a high intake of purine-rich food, decreased renal uric acid removal, or combining the two. Hyperuricemia is described as high blood uric acid level, which further results in the deposition of urate crystals in the joints and kidneys. When the blood uric acid concentration in adult men is above 7.0 mg/dL and in adult women of 6.0 mg/dL, they are said to have hyperuricemia (Maiuolo et al. 2016). Hyperuricemia conditions, including refractory gout, are treated by uricases which effectively eliminate pre-existing uric acid crystals in the joints. Uricases have few drug-drug interactions. Though only uricases effectively treat refractory gout, the current uricase formulations are not appropriate for long-term use (Yang et al. 2012). Uricase is a naturally occurring enzyme (urate oxidase, E.C.1.7.3.3) that catalyzes the conversion of uric acid to allantoin and is a promising therapy for hyperuricemia. Rasburicase and pegloticase are the two major uricase formulations that have been approved for the treatment of hyperuricemia. However, unfortunately, prolonged intake of native form of uricase causes severe immunoreactions due to its foreignness (Garay et al. 2012). In the present research work, we made efforts to use bioinformatics tools to characterize uricase protein sequences from different sources computationally. These protein sequences were subjected to multiple sequence alignment, homology search, domain architecture, motif search, and physiochemical properties. Multiple sequence analysis and homology search results revealed that the amino acid sequences of all the selected sequences have a high degree of similarity. The phylogenetic analysis of all the selected sequences from diverse sources of organisms revealed distinct clusters and demonstrated sequence similarity based on the source of the organism. Each sequence contains six motifs, and each of the twenty-five motifs is unique to its group of uricase sources. The computational physicochemical features of all the selected uricase proteins gave a complete understanding of their properties, namely pI, EC, Ai, ii Ii, GRAVY, and are in the nature of basic properties of these enzymes with 33 kDa- 39 kDa molecular weight. The amino acid valine has the highest average frequency of 8.79 percent in all the selected sources, indicating that it plays a critical role in the formation of uricase. Literature survey shows that several Bacillus species can produce uricase with 25-30 U/ml of activity. The Bacillus fastidious uricase was commercialized by Sigma- Aldrich (product 94310, 9 U/mg) and used for various applications (Pustake et al. 2019a). To expand the usefulness of uricase, it is essential to screen more economical producers of unique properties of novel Bacillus uricase, considering the significance of the enzyme in treating hyperuricemia. The detection and identification of new strains capable of producing uricase have a high demand in the medical field. In this work, an attempt has been made to provide a comprehensive description of computational-based structural, functional, and phylogenetic analyses of uricase enzymes from various Bacillus species. Uricase protein sequences were analyzed for multiple sequence alignment, phylogenetic analysis, motif assessment, domain architecture review, basic physicochemical property understanding, and in-silico identification of uricase amino acid composition. Further, the structural and functional properties of uricase were analyzed. From the analysis, it has been observed that the selected Bacillus uricase proteins are active in an acidic to a neutral environment. CFSSP and PSIPRED were used to predict the secondary structure of uricase, which revealed that it is abundant in alpha helices and sheets. The tertiary structure model of the Bacillus simplex (WP_063232385.1) uricase protein was predicted and validated. Also, all Bacillus species of uricase enzyme and their corresponding genes showed a strong correlation from the phylogenetic comparison of the selected taxa. Due to the antigenicity issue, the clinical application of uricase as an anti- hyperuricemia agent is limited. To develop less immunogenic uricase, in-silico mutagenesis of B-cell and T-cell epitopes have been proposed. The linear B-cell epitopes of Arthrobacter globiformis (Ag)-uricase and Bacillus fastidious (Bf)-uricase were predicted using the Emini surface accessibility, Parker hydrophilicity, and Karplus & Schulz flexibility methods. T159W, D169C, N264W, and Y203D mutations in Ag-uricase resulted in a decreased antigenic probability, whereas S139V, iii K215W, G216F, and I172P mutations in Bf-uricase resulted in a decreased antigenic probability. Uric acid had a binding affinity of -48.71 kcal/mol for the catalytic pocket of Ag-uricase and Bf-uricase models, respectively. This energy is stabilized further in the mutant model by -6.36 kcal/mol for Ag-uricase and -1.45 kcal/mol for Bf-uricase. According to the 100ns MD simulation, both muteins are stable and retained their native-like structural characteristics. The outcome of the above analysis can be a guide for the experimental development of uricase to treat gout and related diseases. Modifications of proteins are the critical biological tools for the production of a wide variety of proteins. Uricase from Bacillus fastidious was successfully conjugated to bovine serum albumin to improve its therapeutic properties. Various molar ratios of bovine serum albumin and glutaraldehyde were conjugated with uricase, and the maximum enzymatic activity of 91.85 percent was obtained at a ratio of 1:6 (mg/ml) uricase: BSA with 0.5 % glutaraldehyde concentration. As determined by the TNBSA assay, the degree of modification indicates that a 1:6 molar ratio of uricase and BSA could result in 76.69 percent of the enzymatic activity. The stability of the conjugated and native uricases was compared at different temperatures (20°C to 60 °C). Likewise, pH stability was investigated at pH values of 7.2 and 9.0. Both native and modified uricase at optimum pH 9.0 shows better retention in enzyme activity after 48 hrs of incubation, which indicates a steady decrease in enzyme activity. The findings of this study indicate that conjugated uricase is effective under physiological conditions, suggesting that it may be a helpful drug for treating hyperuricemia. Considering the potency of the drug for hyperuricemia, this work aims to study the structure, function, and physiochemical properties of uricase by in-silico analysis, and to obtain uricase mutein, an enzyme with reduced immunogenicity, by in-silico mutagenesis. This study also aims to understand the various chemical modifications of the enzyme to enhance its efficacy in treating the disease.