Generation of monoclonal antibodies against a soluble form of lectin-like oxidized low-density lipoprotein receptor-1 and development of a sensitive chemiluminescent enzyme immunoassay.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), expressed prominently in atherosclerotic lesions, is cleaved and released as a soluble LOX-1 (sLOX-1), which is a specific biomarker to diagnose acute coronary syndrome (ACS) at an early stage. Although sLOX-1 levels in patient's blood were successfully measured with our previously established enzyme-linked immunosorbent assay (ELISA), the assay was not sensitive enough to detect normal serum levels of sLOX-1 in healthy human subjects. We therefore developed sensitive and specific monoclonal antibodies (mAbs) against sLOX-1 in order to establish a more sensitive immunoassay. Mice were immunized with recombinant human LOX-1 extracellular domain. mAbs were subsequently generated by standard myeloma cell fusion techniques with a novel screening method using time-resolved fluorescence immunoassay. Using two anti-human sLOX-1 mAbs and alkaline phosphatase as a label, a sandwich chemiluminescent enzyme immunoassay (CLEIA) was developed. In total, nine mAbs were obtained. The dissociation constant (K(d)) values of these mAbs for sLOX-1 were 0.12-1.32 nM. Characteristics of these mAbs were estimated and the best combination for CLEIA was selected. The newly established CLEIA could determine sLOX-1 levels as low as 8 pg/mL, and thus, was sensitive enough to measure serum sLOX-1 levels in normal human subjects and to evaluate subtle differences. Values for sLOX-1 measured by monoclonal CLEIA and polyclonal ELISA were highly correlated (r(2)=0.7594, p<0.0001). Area under the curve values of the receiver-operating characteristic curves in detecting ACS were 0.948 and 0.978 for monoclonal CLEIA and polyclonal ELISA, respectively. Thus, a more sensitive sLOX-1 CLEIA was established using newly developed mAbs against sLOX-1. In addition to its advantage in early diagnosis of ACS, this assay may also be useful in predicting cardiovascular disease risk in disease-free subjects.

The role of cysteine 116 in the active site of the antitumor enzyme L-methionine gamma-lyase from Pseudomonas putida.

The cysteinyl residue at the active site of L-methionine gamma-lyase from Pseudomonas putida (MGL_Pp) is highly conserved among the heterologous MGLs. To determine the role of Cys116, we constructed 19 variants of C116X MGL_Pp by saturation mutagenesis. The Cys116 mutants possessed little catalytic activity, while their affinity for each substrate was almost the same as that of the wild type. Especially, the C116S, C116A, and C116H variants composed active site catalytic function as measured by the kinetic parameter k(cat) toward L-methionine. Furthermore, the mutagenesis of Cys116 also affected the substrate specificity of MGL_Pp at the active center. Substitution of Cys116 for His led to a marked increase in activity toward L-cysteine and a decrease in that toward L-methionine. Propargylglycine inactivated the WT MGL, C116S, and C116A mutants. Based on these results, we postulate that Cys116 plays an important role in the gamma-elimination reaction of L-methionine and in substrate recognition in the MGLs.

Structure of the antitumour enzyme L-methionine gamma-lyase from Pseudomonas putida at 1.8 A resolution.

l-Methionine gamma-lyase (EC 4.4.1.11, MGL_Pp) from Pseudomonas putida is a multifunctional enzyme, which belongs to the gamma-family of pyridoxal-5'-phosphate (PLP) dependent enzymes. In this report, we demonstrate that the three-dimensional structure of MGL_Pp has been completely solved by the molecular replacement method to an R-factor of 20.4% at 1.8 A resolution. Detailed information of the overall structure of MGL_Pp supplies a clear picture of the substrate- and PLP-binding pockets. Tyr59 and Arg61 of neighbouring subunits, which are strongly conserved in other gamma-family enzymes, contact the phosphate group of PLP. These residues are important as the main anchor within the active site. Lys240, Asp241 and Arg61 of one partner monomer and Tyr114 and Cys116 of the other partner monomer form a hydrogen-bond network in the MGL active site which is specific for MGLs. It is also suggested that electrostatic interactions at the subunit interface are involved in the stabilization of the structural conformation. The detailed structure will facilitate the development of MGL_Pp as an anticancer drug.