Báo cáo khoa học: Odorant binding protein has the biochemical properties of a scavenger for 4-hydroxy-2-nonenal in mammalian nasal mucosa

Odorant Binding Protein Has The Biochemical Properties Of A Scavenger For 4-Hydroxy-2-Nonenal In Mammalian Nasal Mucosa

Tác giả: Stefano Grolli, Elisa Merli, Virna Conti, Erika Scaltriti and Roberto Ramoni

Lĩnh vực: Dipartimento di Produzioni Animali, Biotecnologie Veterinarie, Qualità e Sicurezza degli Alimenti, Università degli Studi di Parma, Italy

Nội dung tài liệu:
Nghiên cứu này khám phá khả năng liên kết và khả năng kháng hóa chất của protein gắn mùi (OBP) đối với 4-hydroxy-2-nonenal (HNE), một sản phẩm phụ phản ứng hóa học của quá trình peroxy hóa lipid. Kết quả cho thấy OBP có thể liên kết HNE với ái lực tương đương các phối tử OBP điển hình và vẫn giữ được một phần đáng kể khả năng liên kết của nó sau khi bị biến đổi hóa học bởi aldehyde. Các thí nghiệm sơ bộ trong một mô hình đơn giản hóa biểu mô mũi cho thấy OBP ngoại bào có thể bảo vệ tính sống của một dòng tế bào biểu mô có nguồn gốc từ vách mũi bò khi tiếp xúc với lượng aldehyde độc hại. Những phát hiện này gợi ý rằng OBP, có mặt ở nồng độ millimolar, có thể làm giảm độc tính của HNE bằng cách loại bỏ một phần đáng kể aldehyde khỏi chất nhầy mũi, do đó góp phần chống lại tổn thương tế bào do stress oxy hóa.

Mục lục chi tiết:

• Keywords
• Correspondence
• Abbreviations
• Odorant binding proteins (OBP) are soluble lipocalins produced in large amounts in the nasal mucosa of several mammalian species. Although OBPs can bind a large variety of odorous compounds, direct and exclusive involvement of these proteins in olfactory perception has not been clearly demonstrated. This study investigated the binding properties and chemical resistance of OBP to the chemically reactive lipid peroxidation end-product 4-hydroxy-2-nonenal (HNE), in an attempt to establish a functional relationship between this protein and the molecular mechanisms combating free radical cellular damage. Experiments were carried out on recombinant porcine and bovine OBPs and results showed that both forms were able to bind HNE with affinities comparable with those of typical OBP ligands (Ka = 4.9 and 9.0 µm for porcine and bovine OBP, respectively). Furthermore, OBP functionality, as determined by measuring the binding of the fluorescent ligand 1-aminoanthracene, was partially lost only when incubating HNE levels and exposure time to HNE exceeded physiological values in nasal mucosa. Finally, preliminary experiments in a simplified model resembling nasal epithelium showed that extracellular OBP can preserve the viability of an epithelial cell line derived from bovine turbinates exposed to toxic amounts of the aldehyde. These results suggest that OBP, which is expressed at millimolar levels, might reduce HNE toxicity by removing from the nasal mucus a significant fraction of the aldehyde that is produced as a consequence of direct exposure to the oxygen present in inhaled air.
• Reactive oxygen species (ROS) are short-lived radical intermediates generated as a consequence of oxidative metabolism. They can react with virtually all classes of biological molecules and are responsible for most of the cellular damage caused by oxidative stress. In the case of reactions with membrane polyunsaturated fatty acids (PUFA), ROS initiate a lipid peroxidation process that gives rise to a large number of toxic low molecular mass aldehydes as end products, including the 4-hydroxyalkenals [1,2]. These molecules may be responsible for significant loss of biological activity in proteins and nucleic acids by reacting in a Michael-type addition with the nucleophilic groups (-SH, -NH2 and imidazole) of amino acids and nucleotides [1,2]. Inactivation of 4-hydroxyalkenals in vivo is achieved by several enzymatic systems [3], with a predominant role played by glutathione S-transferases (GSTs) which catalyse a Michael addition of glutathione to the aldehyde double bond [4]. Furthermore, 4-hydroxyalkenal cytotoxicity is possible if it is exported via the
• OBP as a scavenger for HNE
• Protein purification and functional characterization
• Direct binding test to detect HNE-OBP reversible binding complexes
• Determination of the dissociation constant of HNE-OBP complexes
• Western blotting and ligand-binding assay of HNE-modified OBP
• Titration of HNE covalent adducts in OBP
• Biochemical characterization of OBP exposed to HNE under conditions simulating oxidative stress in nasal mucosa
• Evaluation of the protective role of OBP against HNE cytotoxicity in a simplified model simulating the nasal epithelium
• Discussion
• Experimental procedures
• Materials
• OBP purification and functionality test with AMA
• Spectrophotometric assay for the detection of HNE-OBP binding complexes
• Competitive binding test to determine the dissociation constant for HNE-OBP complexes
• Immunoblotting of HNE-modified OBP
• Titration of HNE-reacting residues in OBP
• Biochemical characterization of OBP exposed to HNE in a test tube assay simulating conditions of oxidative stress in nasal mucosa
• Evaluation of the protective role of OBP against HNE cytotoxicity in a simplified model simulating nasal mucosa
• Preparation of immobilized bovine OBP (i-bOBP)
• Cell culture and treatment
• Acknowledgments
• References