Recombinant human lactoperoxidase and eosinophil peroxidase

Human lactoperoxidase (hLPO) and eosinophil peroxidase (hEPO) are structurally and functionally related mammalian heme enzymes and play an important role in the unspecific immune defense system. Human LPO is synthesized and secreted by ductal epithelial cells of the mammary gland and other exocrine glands, whereas EPO is a predominant protein in eosinophils and is released at sites of parasite invasion. Both oxidoreductases predominantly catalyze the oxidation of halides and thiocyanate thereby producing antimicrobial oxidant products. On the other hand both hLPO and hEPO are discussed to contribute to pathogenesis. As example, hLPO seems to be involved in oxidative activation of certain drugs and thus in mammary carcinogenesis, whereas hEPO is involved in allergic eosinophilic inflammatory disease pathology (e.g. asthma). However, our knowledge on both proteins is very limited due to the lack of suitable sources for purification. Based on our successful protocolls on expression of homologous myeloperoxidase and bovine LPO in Chinese hamster ovary cell lines, we have recently also succeeded in cloning and expression of recombinant (r) hLPO. Additionally, stable transfectants for rhEPO are already obtained. This, for the first time, will enable a comprehensive functional, structural and mutational analysis of human LPO and EPO. In detail we intend to produce rhLPO and rhEPO in high yield thereby testing additional and alternative cloning strategies and mammalian expression systems. Comprehensive application of a variety of biochemical and biophysical methods (UV-Vis and resonance Raman spectroscopy, X-ray crystallography, steady- state and presteady-state kinetics and spectroelectrochemical methods) in combination with site-directed mutagenesis will provide substantial mechanistic details on accessibility, binding and oxidation sites of two- (e.g. halides, thiocyanate etc.) and one-electron (nitrite, aromatic substrates etc.) donors, the kinetics of interconversion of redox intermediates of the halogenation and peroxidase cycle, heme structure and reactivity (covalent modification, distortion etc.) and, generally, modulation of redox-activity by the protein matrix. The new recombinant model peroxidases will expedit our understanding of the differences within the human peroxidases. Moreover, they should provide the basis for future studies on (i) their (patho)physiological role, (ii) their contribution to drug metabolism, (iii) the effect(s) of observed polymorphism, and, (iv) for (computational) drug design. The planned work will be performed in close cooperation with internationally well-known scientists, namely Prof. Smulevich in Florence (resonance Raman spectroscopy), Prof. Battistuzzi in Modena (spectro- electrochemistry) and Prof. Fita in Barcelona (X-ray crystallography).

Human lactoperoxidase (hLPO) and eosinophil peroxidase (hEPO) are structurally and functionally related mammalian heme enzymes and play an important role in the unspecific immune defense system. Human LPO is synthesized and secreted by ductal epithelial cells of the mammary gland and other exocrine glands, whereas EPO is a predominant protein in eosinophils and is released at sites of parasite invasion. Both oxidoreductases predominantly catalyze the oxidation of halides and thiocyanate thereby producing antimicrobial oxidant products. On the other hand both hLPO and hEPO are discussed to contribute to pathogenesis. As example, hLPO seems to be involved in oxidative activation of certain drugs and thus in mammary carcinogenesis, whereas hEPO is involved in allergic eosinophilic inflammatory disease pathology (e.g. asthma). However, our knowledge on both proteins is very limited due to the lack of suitable sources for purification. Based on our successful protocolls on expression of homologous myeloperoxidase and bovine LPO in Chinese hamster ovary cell lines, we have recently also succeeded in cloning and expression of recombinant (r) hLPO. Additionally, stable transfectants for rhEPO are already obtained. This, for the first time, will enable a comprehensive functional, structural and mutational analysis of human LPO and EPO. In detail we intend to produce rhLPO and rhEPO in high yield thereby testing additional and alternative cloning strategies and mammalian expression systems. Comprehensive application of a variety of biochemical and biophysical methods (UV-Vis and resonance Raman spectroscopy, X-ray crystallography, steady- state and presteady-state kinetics and spectroelectrochemical methods) in combination with site-directed mutagenesis will provide substantial mechanistic details on accessibility, binding and oxidation sites of two- (e.g. halides, thiocyanate etc.) and one-electron (nitrite, aromatic substrates etc.) donors, the kinetics of interconversion of redox intermediates of the halogenation and peroxidase cycle, heme structure and reactivity (covalent modification, distortion etc.) and, generally, modulation of redox-activity by the protein matrix. The new recombinant model peroxidases will expedit our understanding of the differences within the human peroxidases. Moreover, they should provide the basis for future studies on (i) their (patho)physiological role, (ii) their contribution to drug metabolism, (iii) the effect(s) of observed polymorphism, and, (iv) for (computational) drug design. The planned work will be performed in close cooperation with internationally well-known scientists, namely Prof. Smulevich in Florence (resonance Raman spectroscopy), Prof. Battistuzzi in Modena (spectro- electrochemistry) and Prof. Fita in Barcelona (X-ray crystallography).