Báo cáo khoa học: Molecular identification of adrenal inner zone antigen as a heme-binding protein

Molecular Identification Of Adrenal Inner Zone Antigen As A Heme-Binding Protein

Tác giả: Li Min, Natallia V. Strushkevich, Ivan N. Harnastai, Hiroko Iwamoto, Andrei A. Gilep, Hiroshi Takemori, Sergey A. Usanov, Yasuki Nonaka, Hiroshi Hori, Gavin P. Vinson, Mitsuhiro Okamoto

Lĩnh vực: Hóa sinh, Sinh lý học

Nội dung tài liệu: Nghiên cứu này mô tả việc xác định danh tính của kháng nguyên vùng trong tuyến thượng thận (IZA) là một protein gắn kết heme. Các nhà khoa học đã tinh chế protein IZA và nghiên cứu các đặc tính phân tử của nó, bao gồm cả phổ hấp thụ UV-vis và phổ EPR, cho thấy IZA có khả năng gắn kết với heme loại b. Các nghiên cứu đột biến định hướng đã xác định các gốc axit amin quan trọng trong việc neo giữ heme vào túi kỵ nước của protein. Hơn nữa, nghiên cứu cũng khám phá vai trò của IZA trong phản ứng hydroxyl hóa steroid 21, cho thấy sự kết hợp của IZA với CYP21 có thể tăng cường hoạt tính của enzyme này.

Mục lục chi tiết:

• Keywords
• Correspondence
• *Note
• The adrenal inner zone antigen (IZA), which reacts specifically with a monoclonal antibody raised against the fasciculata and reticularis zones of the rat adrenal, was previously found to be identical with a protein variously named 25-Dx and membrane-associated progesterone receptor. IZA was purified as a glutathione S-transferase-fused or His6-fused protein, and its molecular properties were studied. The UV-visible absorption and EPR spectra of the purified protein showed that IZA bound a heme chromophore in high-spin type. Analysis of the heme indicated that it is of the b type. Site-directed mutagenesis studies were performed to identify the amino-acid residues that bind the heme to the protein. The results suggest that two Tyr residues, Tyr107 and Tyr113, and a peptide stretch, D99-K102, were important for anchoring the heme into a hydrophobic pocket. The effect of IZA on the steroid 21-hydroxylation reaction was investigated in COS-7 cell expression systems. The results suggest that the coexistence of IZA with CYP21 enhances 21-hydroxylase activity.
• Distinguished histologically, the three zones in the mammalian adrenal cortex have distinct functions. In man, the outermost zona glomerulosa secretes aldosterone, the intermediate zona fasciculata, cortisol, and the innermost zona reticularis is the main site for dehydroepiandrosterone formation, whereas in the rat, corticosterone is the main product of the fasciculata and reticularis, with little if any dehydroepiandrosterone. The molecular mechanisms underlying the functional differentiation of the three zones have been a focus of numerous investigations [1-3]. To facilitate the study of zonal function, Laird et al. [4] produced a monoclonal antibody that recognizes an antigen, named inner zone antigen (IZA), which is present in the zonae fasciculata/reticularis in the rat, but not in the zona glomerulosa. Here we call this antigen, which was originally identified in rat tissue, ‘rIZA1’. The monoclonal antibody was capable of inhibiting dose-dependently adrenal 21-hydroxylation of progesterone and 18-hydroxylation
• Abbreviations
• Results and Discussion
• Molecular properties of adrenal inner zone antigen
• Fig. 1. Primary structure of IZA reveals a heme/steroid-binding domain.
• Fig. 2. IZA1 contains a protoheme.
• Fig. 3. IZA1 contains a type b heme.
• Fig. 4. The heme-binding capacities of the wild-type hIZA1 and mutants.
• Fig. 5. Intracellular localization of rIZA1, rat CYP11B1 and human cytochrome b5.
• Fig. 6. Effects of IZA1 on the CYP21 reactions.
• Experimental procedures
• Materials
• Construction of plasmids
• Expression and purification of IZA1
• Preparation of mutated hIZA1s used for measuring heme-binding capacity
• Spectrophotometric analysis
• Preparation of apo-rIZA1
• EPR measurements
• Cells culture, immunofluorescence microscopy, and steroid secretion
• Reporter assays
• In vitro reconstitution assay
• Acknowledgements
• References
• Supplementary material