Hye-Yoon Jeon

Sensitive array-based assay for determination of serological protein kinase A autoantibody levels based on its antigen protein activation.

OBJECTIVES We investigated the effect of cPKAα conformational states during protein immobilization on an array platform for cPKA autoantibody assays for sensitive and high-throughput profiling of protein kinase A (PKA) autoantibody levels in human sera. DESIGN AND METHODS We prepared activated human cPKAα protein arrays by addition of cofactors including ATP, MgCl2, and Triton X-100 to incubation buffer. Anti-human cPKAα antibody or PKA autoantibody levels in human sera were analyzed using activated human cPKAα protein arrays. RESULTS Activation of cPKAα with ATP, Mg(2+), and Triton X-100 enhanced the sensitivity of the assay by increasing the signal/noise ratio and lowering the limit of detection. cPKAα activation also enhanced the sensitivity of cPKA autoantibody detection in human sera. We successfully applied this assay to determine cPKA autoantibody levels in human sera from normal individuals (n=30) and hepatic cancer patients (n=30). CONCLUSIONS Our results demonstrate that cPKAα activation enhanced the sensitivity of array-based PKA autoantibody assays, and that this assay is suitable for high-throughput analyses of cPKA autoantibodies in human sera.

Immunoprofiling of the Tryptophan-Rich Antigen Family in Plasmodium vivax

ABSTRACT Tryptophan-rich antigens (TRAgs) are an antigen family that has been identified in human and rodent malaria parasites. TRAgs have been proposed as candidate antigens for potential vaccines. The Plasmodium vivax TRAg (PvTRAg) family includes 36 members. Each PvTRAg contains a tryptophan-rich (TR) domain in the C-terminal region. In this study, we recombinantly expressed all 36 PvTRAgs using a cell-free expression system, and, for the first time, profiled the IgG antibody responses against all PvTRAgs in the sera from 96 vivax malaria patients and 40 healthy individuals using protein microarray technology. The mean seropositive rate for all PvTRAgs was 60.3%. Among them, nine PvTRAgs were newly identified in this study and showed a seropositive rate of >50%. Five of them, PvTRAg_13, PvTRAg_15, PvTRAg_16, PvTRAg_26, and PvTRAg_29, produced higher levels of IgG antibody, even in low-endemicity countries. In addition, the results of an immunofluorescence analysis suggest that PvTRAgs are, at least in part, associated with caveola-vesicle complexes, a unique structure of P. vivax-infected erythrocytes. The mechanism of formation and the function of these abundant membrane structures are not known. Further investigation aimed at determining the functions of these proteins would lead to a better understanding of the blood-stage biology of P. vivax.

C-reactive protein as a parameter for defining normal blood samples in identification and evaluation of serological biomarkers

Selecting normal control samples is critical for the identification and evaluation of serum biomarker proteins; however, currently few inclusion parameters have been identified. In this paper, we investigated whether serum C-reactive protein (CRP) levels are a suitable inclusion parameter to define normal controls. We analyzed serum α-fetoprotein (AFP), cytokeratin 19 fragment (CYFRA 21-1), intercellular adhesion molecule 1 (ICAM-1), and fibronectin expression levels from normal individuals (n=97) and patients with hepatocellular carcinoma (n=36), lung cancer (n=50), and colorectal cancer (n=30). Receiver operating characteristic (ROC) analysis of the biomarker proteins was performed using four different control groups (C1–C4) with increasing serum CRP levels. The biomarker expression levels were higher in all three sets of cancer patients when compared with control groups C1 through C3. The AUC, sensitivity, and specificity values of the biomarkers dramatically decreased with increasing CRP levels in the control groups. These results suggest that the serum CRP level affects downstream ROC analysis, and that CRP levels could be used as a control inclusion parameter to define normal samples for identifying and evaluating serum cancer biomarkers.

Identification of transglutaminase 2 kinase substrates using a novel on-chip activity assay

Transglutaminase 2 (TG2) is an enzyme that plays a critical role in a wide variety of cellular processes through its multifunctional activities. TG2 kinase has emerged as an important regulator of apoptosis, as well as of chromatin structure and function. However, systematic investigation of TG2 kinase substrates is limited due to a lack of a suitable TG2 kinase activity assays. Thus, we developed a novel on-chip TG2 kinase activity assay for quantitative determination of TG2 kinase activity and for screening TG2 kinase substrate proteins in a high-throughput manner. Quantitative TG2 kinase activity was determined by selective detection of substrate protein phosphorylation on the surface of well-type amine arrays. The limit of detection (LOD) of this assay was 4.34μg/ml. We successfully applied this new activity assay to the kinetic analysis of 27 TG2-related proteins for TG2 kinase activity in a high-throughput manner and determined Michaelis-Menten constants (Km) of these proteins. We used the Km values and cellular locations of the TG2-related proteins to construct a substrate affinity map for TG2 kinase. Therefore, this on-chip TG2 kinase activity assay has a strong potential for the systematic investigation of substrate proteins and will be helpful for studying new physiological functions.

Correction for Wang et al., “Immunoprofiling of the Tryptophan-Rich Antigen Family in Plasmodium vivax”

Volume 83, no. 8, p. 3083–3095, 2015, [https://doi.org/10.1128/IAI.03067-14][1]. Page 3090: Figure 4B should appear as shown below. The original figure contained a duplication in 26H gel and 26R gel, as the result of an error in figure preparation. ![Figure][2] [1]: /lookup/doi/10.1128/

On-chip dual enzyme activity assay to investigate regulation of the transamidase and kinase activities of transglutaminase 2

Transglutaminase 2 (TGase2), a multifunctional enzyme exhibiting both transamidase and kinase activity, is involved in a variety of cellular processes and diseases. However, details of the regulation of TGase2 have not been reported due to the lack of a suitable assay to examine both activities on the same platform under near-physiologic conditions. Thus, we developed an on-chip dual enzyme activity assay for TGase2 to simultaneously monitor the transamidase and kinase activities. Reaction mixtures specific for each enzyme activity were applied onto osteopontin arrays, and enzyme activity was monitored by sequential probing with Cy5-strepavidin and Pro-Q Diamond stain. This approach was used to determine the optimal concentrations of ATP, Mg2+, and Ca2+ for dual-activity assays. The optimized assay was then used to investigate regulation of TGase2 transamidase and kinase activities by various cofactors that could potentially affect its conformation. Monothiol- and disulfide-containing compounds differentially regulated TGase2 transamidase and kinase activities. Acetylation of TGase2 activated the kinase activity but had no effect on the transamidase activity. Phosphorylation and dephosphorylation of TGase2 reciprocally regulated the transamidase and kinase activities. The new approach described here is thus useful for screening potential regulators of TGase2 transamidase and kinase and investigating the pathogenesis of TGase2-associated diseases.

Dammarenediol-II Prevents VEGF-Mediated Microvascular Permeability in Diabetic Mice.

Diabetic retinopathy is a major diabetic complication predominantly caused by vascular endothelial growth factor (VEGF)‐induced vascular permeability in the retina; however, treatments targeting glycemic control have not been successful. Here, we investigated the protective effect of dammarenediol‐II, a precursor of triterpenoid saponin biosynthesis, on VEGF‐induced vascular leakage using human umbilical vein endothelial cells (HUVECs) and diabetic mice. We overproduced the compound in transgenic tobacco expressing Panax ginseng dammarenediol‐II synthase gene and purified using column chromatography. Analysis of the purified compound using a gas chromatography–mass spectrometry system revealed identical retention time and fragmentation pattern to those of authentic standard dammarenediol‐II. Dammarenediol‐II inhibited VEGF‐induced intracellular reactive oxygen species generation, but it had no effect on the levels of intracellular Ca2+ in HUVECs. We also found that dammarenediol‐II inhibited VEGF‐induced stress fiber formation and vascular endothelial‐cadherin disruption, both of which play critical roles in modulating endothelial permeability. Notably, microvascular leakage in the retina of diabetic mice was successfully inhibited by intravitreal dammarenediol‐II injection. Our results suggest that the natural drug dammarenediol‐II may have the ability to prevent diabetic microvascular complications, including diabetic retinopathy. Copyright