Structural basis of the divergent oxygenation reactions catalyzed by the rieske nonheme iron oxygenase carbazole 1,9a-dioxygenase.

Carbazole 1,9a-dioxygenase (CARDO), a Rieske nonheme iron oxygenase (RO), is a three-component system composed of a terminal oxygenase (Oxy), ferredoxin, and a ferredoxin reductase. Oxy has angular dioxygenation activity against carbazole. Previously, site-directed mutagenesis of the Oxy-encoding gene from Janthinobacterium sp. strain J3 generated the I262V, F275W, Q282N, and Q282Y Oxy derivatives, which showed oxygenation capabilities different from those of the wild-type enzyme. To understand the structural features resulting in the different oxidation reactions, we determined the crystal structures of the derivatives, both free and complexed with substrates. The I262V, F275W, and Q282Y derivatives catalyze the lateral dioxygenation of carbazole with higher yields than the wild type. A previous study determined the crystal structure of Oxy complexed with carbazole and revealed that the carbonyl oxygen of Gly178 hydrogen bonds with the imino nitrogen of carbazole. In these derivatives, the carbazole was rotated approximately 15, 25, and 25°, respectively, compared to the wild type, creating space for a water molecule, which hydrogen bonds with the carbonyl oxygen of Gly178 and the imino nitrogen of carbazole. In the crystal structure of the F275W derivative complexed with fluorene, C-9 of fluorene, which corresponds to the imino nitrogen of carbazole, was oriented close to the mutated residue Trp275, which is on the opposite side of the binding pocket from the carbonyl oxygen of Gly178. Our structural analyses demonstrate that the fine-tuning of hydrophobic residues on the surface of the substrate-binding pocket in ROs causes a slight shift in the substrate-binding position that, in turn, favors specific oxygenation reactions toward various substrates.

Improvement of mass spectrometry analysis of glycoproteins by MALDI-MS using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid.

Protein glycosylation analysis is important for elucidating protein function and molecular mechanisms in various biological processes. We previously developed a glycan analysis method using a 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid liquid matrix (3-AQ/CHCA LM) and applied it to the quantitative glycan profiling of glycoproteins. However, information concerning glycosylation sites is lost; glycopeptide analysis is therefore required to identify the glycosylation sites in glycoproteins. Human epidermal growth factor receptor 2 (HER2) is a glycoprotein that plays a role in the regulation of cell proliferation, differentiation, and migration. Several reports have described the structure of HER2, but the structures of N-glycans attached to this protein remain to be fully elucidated. In this study, 3-AQ/CHCA LM was applied to tryptic digests of HER2 to reveal its N-glycosylation state and to evaluate the utility of this LM in characterizing glycopeptides. Peptide sequence coverage was considerably improved compared to analysis of HER2 using α-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid. Most of the peaks observed using only this LM were localized at the inner or outer regions of sample spots. Furthermore, five of the peptide peaks that were enriched within the inner region were confirmed to be glycosylated by MS/MS analysis. Three glycosylation sites were identified and their glycan structures were elucidated. The reduction in sample complexity by on-target separation allowed for higher sequence coverage, resulting in effective detection and characterization of glycopeptides. In conclusion, these results demonstrate that MS-based glycoprotein analysis using 3-AQ/CHCA is an effective method to identify glycosylation sites in proteins and to elucidate the glycan structures of glycoproteins in complex samples.

Rapid quantitative profiling of N-glycan by the glycan-labeling method using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid.

Protein glycosylation is a crucial phenomenon for understanding protein functions, since its patterns and degree are associated with many biological processes, such as intercellular signaling and immune response. We previously reported a novel glycan-labeling method using a 3-ainoquinoline/α-cyano-4-hydroxycinnamic acid (3-AQ/CHCA) liquid matrix for highly sensitive detection by matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS). In the present study, we examined the practicality of this method for qualitative and quantitative glycan profile analysis. We first investigated the reproducibility of the data for 16 N-glycans prepared from human epidermal growth factor receptor type 2 (HER2). All of the data obtained in intra-assays and interassays were highly correlated with statistical significance (R(2) > 0.9, p < 0.05). In addition, the HER2 glycosylation pattern differed significantly between different breast cancer cell lines SK-BR-3 and BT474 in a comparative analysis of profile data. Finally, the quantitative capability of this method was examined by using PA-labeled monosialylated N-glycan as an internal standard (IS). Using IS for AQ-labeled neutral and sialylated standard glycans, the ion peak intensity was highly linear (R(2) > 0.9) from 0.5 to 5000 fmol. Furthermore, using IS for HER2 N-glycans, all of the N-glycans were highly linear with their dilution factors (R(2) > 0.9). These results suggest that our developed AQ labeling method enabled rapid qualitative and quantitative analyses of glycans. This glycan analysis method should contribute to the field of biomarker discovery and biomedicine in applications such as quality control of biotechnology-based drugs.

Intact-cell-based surface plasmon resonance measurements for ligand affinity evaluation of a membrane receptor.

Toward future applications to the discovery of drugs against membrane receptors on pathological cells, an intact-cell-based surface plasmon resonance (SPR) methodology has been developed. The injection of a suspension of epidermal carcinoma A431 cells (5×10(7)cells/ml), as an analyte, generated clear SPR responses to epidermal growth factor (EGF) immobilized on the sensor chip. Because the responses were competitively reduced by the free ligand EGF, added to the analyte cell suspension, they certainly reflect the specific interaction of the immobilized EGF with the extracellular region of its receptor, which is highly expressed on the surface of the A431 cells.

Inhibitory effect of a dimerization-arm-mimetic peptide on EGF receptor activation.

A cyclic decapeptide was chemically synthesized that mimics the loop structure of a beta-hairpin arm of the EGF receptor, which is highly involved in receptor dimerization upon activation by ligand binding. This peptide was revealed to reduce dimer formation of the receptor in a detergent-solubilized extract of epidermoid carcinoma A431 cells and to inhibit receptor autophosphorylation at less than 10 microM in the intact cells.

Fmoc-based solid phase chemical synthesis of 71-meric neuregulin 1-beta1, an epidermal growth factor-like domain.

The human neuregulin 1-beta1 (NRG1-beta1, amino acid residues 176-246) was chemically synthesized by Fmoc-based solid phase peptide synthesis (SPPS) followed by folding in a redox buffer. The biological activity of the synthesized NRG1-beta1 was confirmed by ligand-induced tyrosine phosphorylation on Chinese hamster ovary (CHO) cells expressing ErbB-4.

Crystallization and preliminary X-ray diffraction studies of the ferredoxin reductase component in the Rieske nonhaem iron oxygenase system carbazole 1,9a-dioxygenase.

Carbazole 1,9a-dioxygenase (CARDO), which consists of an oxygenase component (CARDO-O) and the electron-transport components ferredoxin (CARDO-F) and ferredoxin reductase (CARDO-R), catalyzes dihydroxylation at the C1 and C9a positions of carbazole. CARDO-R was crystallized at 277 K using the hanging-drop vapour-diffusion method with the precipitant PEG 8000. Two crystal types (types I and II) were obtained. The type I crystal diffracted to a maximum resolution of 2.80 A and belonged to space group P4(2)2(1)2, with unit-cell parameters a = b = 158.7, c = 81.4 A. The type II crystal was obtained in drops from which type I crystals had been removed; it diffracted to 2.60 A resolution and belonged to the same space group, with unit-cell parameters a = b = 161.8, c = 79.5 A.