Network


Latest external collaboration on country level. Dive into details by clicking on the dots.

Hotspot


Dive into the research topics where Lee Ho Wang is active.

Publication


Featured researches published by Lee Ho Wang.


Journal of Biological Chemistry | 1996

Role of Val509 in Time-dependent Inhibition of Human Prostaglandin H Synthase-2 Cyclooxygenase Activity by Isoform-selective Agents

Qiupeng Guo; Lee Ho Wang; Ke-He Ruan; Richard J. Kulmacz

Prostaglandin H synthase (PGHS), a key enzyme in prostanoid biosynthesis, exists as two isoforms. PGHS-1 is considered a basal enzyme; PGHS-2 is associated with inflammation and cell proliferation. A number of highly selective inhibitors for PGHS-2 cyclooxygenase activity are known. Inhibition by these agents involves an initial reversible binding, followed by a time-dependent transition to a much higher affinity enzyme-inhibitor complex, making these agents potent and poorly reversible PGHS-2 inhibitors. To investigate the PGHS-2 structural features that influence the time-dependent action of the selective inhibitors, we have constructed a three-dimensional model of human PGHS-2 by homologous modeling. Examination of the PGHS-2 model identified Val509 as a cyclooxygenase active site residue, that was not conserved in PGHS-1. Recombinant human PGHS-2 with Val509 mutated to either Ile (the corresponding residue in PGHS-1), Ala, Glu, or Lys was expressed by transient transfection of COS-1 cells to evaluate the effects of the mutations on cyclooxygenase activity and on inhibition by four agents reported to be selective for PGHS-2 (NS398, nimesulide, DuP697, and SC58125). All the recombinant proteins were of the expected mass. The mutants exhibited 45-210% of wild-type cyclooxygenase activity, with Km values for arachidonate of 2.1-7.6 μM (wild-type PGHS-2, 3.8 μM), indicating that changes in position 509 had modest effects on cyclooxygenase catalysis. Each of the agents inhibited wild-type PGHS-2 in a time-dependent fashion, and all but nimesulide did the same for the V509A mutant. In contrast, the V509E and V509I PGHS-2 mutants, like recombinant human PGHS-1, did not show time-dependent inhibition with any of the agents, and the V509K mutant responded in a time-dependent manner only to DuP697. Reversible inhibition was still observed with Val509 mutants that did not show time-dependent inhibition. Thus, the side chain structure at position 509 markedly influenced the ability of PGHS-2 to undergo the time-dependent transition without removing inhibitor or substrate binding. These results indicate that Val509 in PGHS-2 has a major role in the structural transition that underlies time-dependent inhibition by the isoform-selective agents.


Journal of Biological Chemistry | 1999

Expression, Purification, and Spectroscopic Characterization of Human Thromboxane Synthase

Pei Yung Hsu; Ah Lim Tsai; Richard J. Kulmacz; Lee Ho Wang

Thromboxane A2(TXA2) is a potent inducer of vasoconstriction and platelet aggregation. Large scale expression of TXA2 synthase (TXAS) is very useful for studies of the reaction mechanism, structural/functional relationships, and drug interactions. We report here a heterologous system for overexpression of human TXAS. The TXAS cDNA was modified by replacing the sequence encoding the first 28 amino acid residues with a CYP17 amino-terminal sequence and by adding a polyhistidine tag sequence prior to the stop codon; the cDNA was inserted into the pCW vector and co-expressed with chaperonins groES and groEL in Escherichia coli. The resulting recombinant protein was purified to electrophoretic homogeneity by affinity, ion exchange, and hydrophobic chromatography. UV-visible absorbance (UV-Vis), magnetic circular dichroism (MCD), and electron paramagnetic resonance (EPR) spectra indicate that TXAS has a typical low spin cytochrome P450 heme with an oxygen-based distal ligand. The UV-Vis and EPR spectra of recombinant TXAS were essentially identical to those of TXAS isolated from human platelets, except that a more homogenous EPR spectrum was observed for the recombinant TXAS. The recombinant protein had a heme:protein molar ratio of 0.7:1 and a specific activity of 12 μmol of TXA2/min/mg of protein at 23u2009°C. Furthermore, it catalyzed formation of TXA2, 12-hydroxy-5,8,10-heptadecatrienoic acid, and malondialdehyde in a molar ratio of 0.94:1.0:0.93. Spectral binding titrations showed that bulky heme ligands such as clotrimazole bound strongly to TXAS (K d ∼0.5 μm), indicating ample space at the distal face of the heme iron. Analysis of MCD and EPR spectra showed that TXAS was a typical low spin hemoprotein with a proximal thiolate ligand and had a very hydrophobic distal ligand binding domain.


Journal of Biological Chemistry | 1996

Identification of Thromboxane A2 Synthase Active Site Residues by Molecular Modeling-guided Site-directed Mutagenesis

Lee Ho Wang; Nena Matijevic-Aleksic; Pei Yung Hsu; Ke-He Ruan; Kenneth K. Wu; Richard J. Kulmacz

Human thromboxane A2 synthase (TXAS) exhibits spectral characteristics of cytochrome P450 but lacks monooxygenase activity. Its distinctive amino acid sequence makes TXAS the sole member of family 5 in the P450 superfamily. To better understand the structure-function relationship of this unusual P450, we have recently constructed a three-dimensional model for TXAS using P450BM-3 as the template (Ruan, K.-H., Milfeld, K., Kulmacz, R. J., and Wu, K. K. (1994) Protein Eng. 7, 1345-1551) and have identified a potential active site region. The catalytic roles of several putative active site residues were evaluated using selectively mutated recombinant TXAS expressed in COS-1 cells. Mutation of Ala-408 to Glu or Arg-413 to Gly led to a complete loss of enzyme activity despite expression of mutant protein levels equivalent to that of the wild-type TXAS. Mutation of Ala-408 to Gly or Leu retained the enzyme activity at levels of 30 or 40%, respectively. This suggests that Ala-408 provides a hydrophobic environment for substrate binding. Mutation of Arg-413 to Lys or Gln completely abolished the enzyme activity, indicating that this residue is essential to catalytic activity and supports its identification as an active site residue. Mutation of Arg-410 to Gly or Glu-433 to Ala resulted in >50% reduction in the enzyme activity without appreciably altering mutant protein expression, consistent with a more subtle effect of these residues on TXAS catalytic efficiency. Mutation of residues predicted to be involved in binding the heme prosthetic group, including the heme thiolate ligand Cys-480, Arg-478, Phe-127, and Asn-110, each markedly reduced the expressed protein level and abolished enzyme activity. This suggests that proper heme binding is important to synthesis or stability of recombinant TXAS. Mutation of Ile-346, which corresponds to P450cam-Thr-252, an essential amino acid involved in dioxygen bond scission, to Thr increased the enzymatic activity by 40%, suggesting that oxygen bond cleavage is not a rate-limiting step in thromboxane A2 biosynthesis. The present results from site-directed mutagenesis support the overall structure of the TXAS active site predicted by homology modeling and have allowed refinement of the position of bound substrate.


Biochimica et Biophysica Acta | 1995

Differential expression of thromboxane A synthase and prostaglandin H synthase in megakaryocytic cell line

Nevenka Matijevic-Aleksic; Sudershan K. Sanduja; Lee Ho Wang; Kenneth K. Wu

We determined the expression of isoforms of prostaglandin H synthase (PGHS) and thromboxane A synthase (TXAS) in a human megakaryocyte cell line (MEG-01. The basal levels of full-length TXAS mRNA and the 60 kDa TXAS protein were high when compared to those of PGHS-1 and PGHS-2 in uninduced cells. Despite a high TXAS level, uninduced MEG-01 cells synthesized only a small amount of thromboxane A2 (TXA2) due to limited PGHS-1 or PGHS-2 expressions. Following PMA induction there was little change in TXAS. PGHS-2 mRNA was significantly increased at only 3 h of PMA treatment and the level declined rapidly, whereas PGHS-1 mRNA and protein levels were concordantly stimulated. Induction of PGHS-1 reached plateau on day 3 of PMA treatment. Analysis of arachidonate metabolism in cells induced by PMA for 3 and 5 days showed a high level of PGH2 synthesis which exceeded the TXAS capacity for TXA2 synthesis. Only traces of PGHS-2 mRNA and alternate-spliced TXAS mRNA were detected in human platelets. We conclude that TXAS and PGHS are differentially expressed in MEG-01 during PMA-induced differentiation.


Biochemistry | 2009

Characterization of the peroxidase mechanism upon reaction of prostacyclin synthase with peracetic acid. Identification of a tyrosyl radical intermediate.

Hui Chun Yeh; Gary J. Gerfen; Jinn Shyan Wang; Ah Lim Tsai; Lee Ho Wang

Prostacyclin synthase (PGIS) is a membrane-bound class III cytochrome P450 that catalyzes an isomerization of prostaglandin H(2), an endoperoxide, to prostacyclin. We report here the characterization of the PGIS intermediates in reactions with other peroxides, peracetic acid (PA), and iodosylbenzene. Rapid-scan stopped-flow experiments revealed an intermediate with an absorption spectrum similar to that of compound ES (Cpd ES), which is an oxo-ferryl (Fe(IV)O) plus a protein-derived radical. Cpd ES, formed upon reaction with PA, has an X-band (9 GHz) EPR signal of g = 2.0047 and a half-saturation power, P(1/2), of 0.73 mW. High-field (130 GHz) EPR reveals the presence of two species of tyrosyl radicals in Cpd ES with their g-tensor components (g(x), g(y), g(z)) of 2.00970, 2.00433, 2.00211 and 2.00700, 2.00433, 2.00211 at a 1:2 ratio, indicating that one is involved in hydrogen bonding and the other is not. The line width of the g = 2 signal becomes narrower, while its P(1/2) value becomes smaller as the reaction proceeds, indicating migration of the unpaired electron to an alternative site. The rate of electron migration ( approximately 0.2 s(-1)) is similar to that of heme bleaching, suggesting the migration is associated with the enzymatic inactivation. Moreover, a g = 6 signal that is presumably a high-spin ferric species emerges after the appearance of the amino acid radical and subsequently decays at a rate comparable to that of enzymatic inactivation. This loss of the g = 6 species thus likely indicates another pathway leading to enzymatic inactivation. The inactivation, however, was prevented by the exogenous reductant guaiacol. The studies of PGIS with PA described herein provide a mechanistic model of a peroxidase reaction catalyzed by the class III cytochromes P450.


Pharmacogenetics and Genomics | 2012

Functional analysis of human thromboxane synthase polymorphic variants.

Chung Ying K. Chen; Elizabeth M. Poole; Cornelia M. Ulrich; Richard J. Kulmacz; Lee Ho Wang

Background Thromboxane A synthase (TXAS) metabolizes the cyclooxygenase product prostaglandin (PG) H2 into thromboxane H2 (TXA2), a potent inducer of blood vessel constriction and platelet aggregation. Nonsynonymous polymorphisms in the TXAS gene have the potential to alter TXAS activity and affect TXA2 generation. Objectives The aim of this study was to assess the functional effects of genetic variants in the TXAS protein, including K258E, L357V, Q417E, E450K, and T451N. Methods Wild-type TXAS and the variant proteins were expressed in a bacterial system and purified by affinity and hydroxyapatite chromatography. The two characteristic catalytic activities of TXAS were assayed in each of the purified recombinant proteins: isomerization of PGH2 to TXA2 and fragmentation of PGH2 to 12-hydroxyheptadecatrienoic acid and malondialdehyde. Results All of the variants showed both isomerization and fragmentation activities. The Km values of the variants ranged from 27 to 52 µmol/l PGH2 (wild-type value: 32 &mgr;mol/l PGH2); the Vmax values of the variants ranged from 18 to 40 U/mg (wild-type value: 41 U/mg). The kinetic differences were largest for the L357V variant, whose Vmax/Km ratio was just 27% of the wild-type value. Conclusion The increased Km and decreased Vmax values observed with L357V suggest that this variant may generate less TXA2 at the low levels of PGH2 expected in vivo, raising the possibility of attenuated signaling through the thromboxane pathway.


FEBS Journal | 2008

Spectroscopic characterization of the oxyferrous complex of prostacyclin synthase in solution and in trapped sol–gel matrix

Hui Chun Yeh; Pei Yung Hsu; Ah Lim Tsai; Lee Ho Wang

Prostacyclin synthase (PGIS) is a member of the cytochrome P450 family in which the oxyferrous complexes are generally labile in the absence of substrate. At 4u2003°C, the on‐rate constants and off‐rate constants of oxygen binding to PGIS in solution are 5.9u2003×u2003105u2003m−1·s−1 and 29u2003s−1, respectively. The oxyferrous complex decays to a ferric form at a rate of 12u2003s−1. We report, for the first time, a stable oxyferrous complex of PGIS in a transparent sol–gel monolith. The encapsulated ferric PGIS retained the same spectroscopic features as in solution. The binding capabilities of the encapsulated PGIS were demonstrated by spectral changes upon the addition of O‐based, N‐based and C‐based ligands. The peroxidase activity of PGIS in sol–gel was three orders of magnitude slower than that in solution owing to the restricted diffusion of the substrate in sol–gel. The oxyferrous complex in sol–gel was observable for 24u2003h at room temperature and displayed a much red‐shifted Soret peak. Stabilization of the ferrous–carbon monoxide complex in sol–gel was observed as an enrichment of the 450‐nm species over the 420‐nm species. This result suggests that the sol–gel method may be applied to other P450s to generate a stable intermediate in the di‐oxygen activation.


Journal of Biological Chemistry | 1996

Sequence determination of an extremely acidic rat dentin phosphoprotein.

Helena H. Ritchie; Lee Ho Wang


Journal of Biological Chemistry | 1992

Primary structure of human thromboxane synthase determined from the cDNA sequence.

Kazuteru Ohashi; Ke-He Ruan; Richard J. Kulmacz; Kenneth K. Wu; Lee Ho Wang


European Journal of Oral Sciences | 1998

Six decades of dentinogenesis research. Historical and prospective views on phosphophoryn and dentin sialoprotein.

Helena H. Ritchie; David G. Ritchie; Lee Ho Wang

Collaboration


Dive into the Lee Ho Wang's collaboration.

Top Co-Authors

Avatar

Richard J. Kulmacz

University of Texas Health Science Center at Houston

View shared research outputs
Top Co-Authors

Avatar

Ah Lim Tsai

University of Texas Health Science Center at Houston

View shared research outputs
Top Co-Authors

Avatar

Ke-He Ruan

University of Texas Health Science Center at Houston

View shared research outputs
Top Co-Authors

Avatar

Kenneth K. Wu

University of Texas Health Science Center at Houston

View shared research outputs
Top Co-Authors

Avatar

Pei Yung Hsu

University of Texas Health Science Center at Houston

View shared research outputs
Top Co-Authors

Avatar
Top Co-Authors

Avatar

Hui Chun Yeh

University of Texas Health Science Center at Houston

View shared research outputs
Top Co-Authors

Avatar

Vladimir Berka

University of Texas Health Science Center at Houston

View shared research outputs
Top Co-Authors

Avatar

Wen Liu

University of Texas Health Science Center at Houston

View shared research outputs
Top Co-Authors

Avatar

Chung Ying K. Chen

University of Texas Health Science Center at Houston

View shared research outputs
Researchain Logo
Decentralizing Knowledge