Development of an uricolytic treatment for HPRT-deficiency in animal models

Abstract

The evolutionary inactivation of the urate degradation pathway predisposes humans to hyperuricemia, a condition associated with a range of common disorders, including hypertension, metabolic disorders, kidney stones and or obesity. In addition, hereditary deficiency of the hypoxanthine phosphoribosyltransferase enzyme (HPRT), a key enzyme of the purine salvage pathway, causes urate overproduction, juvenile gout, and neurological disabilities as observed in Lesch-Nyhan disease (LND). The lack of purine bases recycling determines the increase of purine degradation rate, leading to hyperuricemia and hyperuricosuria. The severity of neurological phenotype, including neuromotor dysfunction, cognitive impairment and behavioral alterations with self-injury, is related to the degree of HPRT deficiency. The correlation of HPRT activity with the neurological phenotype is still unclear and an effective treatment is missing. Current therapeutic approach, based on inhibitors of urate production (uricostatic therapy), is not effective in refractory gout and does not improve neurological deficits in LND patients. Inhibition of the xanthine oxidase enzyme, efficiently prevents urate accumulation, but leads to an excess of the upstream metabolite hypoxanthine, which could determine the renal pathological phenotype. In most mammals, except humans and apes, urate is converted into more soluble allantoin in a three-step enzymatic pathway through the consecutive action of urate oxidase (Uox), HIU hydrolase (Urah), and OHCU decarboxylase (Urad). Restoration of uricolysis through enzyme therapy represents a promising treatment for severe hyperuricemia because it would act by facilitating rather than blocking purine degradation. Since the neurological manifestations are hypothesized to be related to the purine metabolite imbalance in LND phenotype, the uricolytic treatment, acting a normalization of purines, is predicted to have a positive effect also on neurological symptoms. The main goal of my PhD dissertation is to develop an enzymatic treatment to be tested in a mouse model of hyperuricemia. In the framework of this project, we expressed and purified separated Uox, Urah, and Urad proteins from Danio rerio. The functional and structural characterization of the enzymes used for uricolytic therapy production is a prerogative of this project. We obtained a pharmaceutical preparation of a triad of PEGylated proteins retaining native-like enzymatic activities and able to convert urate into allantoin without accumulation of potentially harmful intermediate metabolites. Although a preparation of three uricolytic enzymes has functional advantages with respect to the currently available drugs based on Uox alone (rasburicase/pegloticase), it has the disadvantages of being much more costly and time-consuming. The efficiency of production and chemical modification of these active ingredients could be improved by combining the three uricolytic enzymes in a single chimeric protein. Natural fusion enzymes of uricolytic pathway were analysed and in particular recombinant clones expressing the bifunctional Urad-Urah from diatoms (Phaeodactylum tricornutum) and bacterial Uox-Urad (Bacillus subtilis) were obtained. Both proteins were purified to homogeneity by affinity chromatography and functionally characterized by UV-vis absorbance and CD spectroscopy. The Urad-Urah enzyme, named allantoin synthase (PtAlls), demonstrated to be suitable for practical use and structure determination. The three-dimensional structure was determined at a 1.9 Å-resolution in collaboration with Sangkee Rhee, Seoul National University, Korea. Furthermore, PEG derivatization of allantoin synthase demonstrated that this enzyme can be used to replace two separated proteins in uricolytic therapy. PtAlls bi-domain protein was used as a basis for the fusion of DrUox domain to obtain the multifunctional chimeric enzyme. The tandem fusion of DrUox and PtAlls coding sequences was achieved through a naturally occurring protein linker, yielding a functional enzyme retaining the three activities. The kinetic properties of the chimeric enzyme were comparable to those measured for single proteins, respectively DrUox an PtAlls. The rational analysis of the fused domains suggested a domain arrangement not previously observed for natural uricolytic bi-domain proteins. For this construct a closed assembly could be achieved if the linker is sufficiently long and flexible to allow the association of both uricase and hydrolase tetramers with two decarboxylase dimers in between. To combine the three domains in a conformation similar to that of the natural PtAlls protein with a central tetrameric domain flanked by two dimeric domains, the uricase tetramer was transformed in a pseudo-dimeric assembly by tandemly fusing two monomers. Pseudo-dimeric uricase was efficiently produced in E. coli system, purified by affinity and the kinetic parameters were in accordance with those calculated for the native DrUox. The recombinant construct of the non-stop version of pseudo-dimeric uricase cloned in pET28 vector, was then used for PtAlls coding sequence sub-cloning to obtain the complete trifunctional uricolytic enzyme tagged with an His-tag at N terminus. The complete fusion protein was optimized by introducing various protein linkers with several degrees of flexibility between the two coding sequences. In all recombinant proteins containing a protein linker, a soluble and functional enzyme was obtained. However the N-terminal His-tag of the chimeric proteins was not well exposed, significantly affecting the efficiency of purification step. Best results in terms of protein expression, purification yield and enzymatic activity were achieved with the protein containing a rigid α-helical protein linker flanked by two flexible GGGGS units, herein named DrUox-Uox-rig-PtAlls. The solubility and the production protocol of DrUox-Uox-rig-PtAlls was optimized by substituting the N-terminal His-tag with the removable maltose-binding protein (MBP) tag. The fusion to the MBP protein improved both the solubility and purification yield without apparently affecting the enzymatic activities. The kinetic analysis of the enzyme retaining the MBP tag evidenced KM values compatible with those of single fusion units, while the kcat/KM were decrease by one order of magnitude for all the three activities. A three-step purification protocol was established to produce a sufficiently homogeneous sample to explore the structural conformation of DrUox-Uox-rig-PtAlls with negative staining EM technique. The predicted tetrameric organisation was confirmed by EM analysis. The overall domain arrangement was similar to that observed for PtAlls, with a central core (probably the bi-functional PtAlls protein) flanked by two dimeric Uox domains. In conclusion, two versions of chimeric Uox-Urad-Urah fusion proteins, able to metabolize urate to allantoin without intermediate accumulation, were produced by pursuing two different fusion strategies and obtaining two different domain arrangements. In particular, DrUox-nat-PtAlls construct revealed to be efficiently produced in E. coli cells as functional protein and purified to apparent homogeneity by a single purification step. Its therapeutic use for severe hyperuricemia is predicted to be advantageous compared to the existing uricolytic therapy based on the Uox alone since it would prevent the harmful accumulation of urate degradation intermediates. Moreover, as a single enzyme containing the three active domains, it could overcome limits of previous proposed uricolytic therapeutics in terms of protein production and purification.