Active site competition is the mechanism for the inhibition of lipoprotein-associated phospholipase A2 by detergent micelles or lipoproteins and for the efficacy reduction of darapladib.

Lipoprotein associated phospholipase A2 (Lp-PLA2) has been characterized for its interfacial activation as well as inhibition by detergent micelles and lipoprotein particles. The enzyme has been shown to bind on the surfaces of hydrophobic aggregates, such as detergent micelles, lipoprotein particles and even polystyrene latex nanobeads. Binding to hydrophobic aggregates stimulates the activity of Lp-PLA2 but may not be the necessary step for catalysis. However, at higher concentrations, detergent micelles, latex nanobeads or lipoprotein particles inhibit Lp-PLA2 possibly by blocking the access of substrates to the active site. The competition mechanism also blocks inhibitors such as darapladib binding to Lp-PLA2 and reduces the efficacy of the drug. Darapladib has very low solubility and mainly exists in solutions as complexes with detergents or lipoprotein particles. The inhibition of Lp-PLA2 by darapladib is dependent on many factors such as concentrations of detergents or lipoproteins, incubation time, as well as the order of mixing reaction components. The in vitro Lp-PLA2 activity assays used in clinical studies may not accurately reflect the residual Lp-PLA2 activity in vivo. Darapladib has been found mainly bound on HDL and albumin when it is incubated with human serum. However, Lp-PLA2 is more sensitive to darapladib when bound on LDL and relatively resistant to darapladib when bound on HDL. Therefore, high cholesterol levels may decrease the efficacy of darapladip and cause the drug to be less effective in high risk patients. Our study will help to design better inhibitors for Lp-PLA2. The discoveries also contribute to understanding the mechanism of interfacial activation and inhibition for Lp-PLA2 and provide a new concept for researchers in building better kinetic model for interfacial enzymes.

Association with lipids or detergents is essential for preservation of the active structure of lipoprotein-associated phospholipase A2.

Recombinant lipoprotein-associated phospholipase A2 (rLp-PLA2) expressed in HEK293 cells has a propensity to form oligomers in the absence of detergents. Dilution of rLp-PLA2 in the absence of detergents results in irreversible inactivation of the enzyme. The monomeric rLp-PLA2 may expose its hydrophobic interfacial binding region or substrate binding compartment to water and that may cause structural collapsing of the enzyme. Formation of self-aggregate, complex with binding partners or association with detergent micelles is to block the access of aqueous solvent to the hydrophobic substrate binding site and therefore prevents the structural collapsing. Dilution inactivation of the enzyme can be prevented in the presence of LDL or HDL suggesting that Lp-PLA2 association with lipoprotein particles (LDL and HDL) is necessary for Lp-PLA2 to maintain its enzymatic activity in human plasma. Formation of higher affinity complex gave better protection of rLp-PLA2 structure and activity. The method can be harnessed to detect the interaction between rLp-PLA2 and components of lipoprotein particles. Apo(a), ApoB 100 and ApoA1 were found to protect the enzyme from inactivation at roughly the similar level (˜80 ±â€¯5%) comparing to human serum albumin control (˜40%). One mg/ml pig brain phospholipid showed 100% protection under the same conditions.

Characterization of PLAC® tests in the quantization of lipoprotein associated phospholipase A2 for assessment of cardiovascular diseases.

BACKGROUND: PLAC® mass test (diaDexus, Inc.) does not detect all Lp-PLA2 proteins in the circulation. The total circulating Lp-PLA2 mass can be quantized by using the CHAPS modified PLAC® mass test. To compare the difference of the PLAC® mass, CHAPS modified PLAC® mass and PLAC® activity tests in risk assessment of CVD, the 3 Lp-PLA2 quantization methods were characterized using a collection of serum and plasma from CVD patents and matched non-symptomatic controls. Improvement on risk assessment for ischemic stroke by Lp-PLA2 and lipids were also investigated. METHODS: Ninety one human sera and plasma from elderly patients with first CVD incidents and 78 matched controls were collected at clinics. Lp-PLA2 was assessed by PLAC® mass, CHAPS modified PLAC® mass and PLAC® activity tests and data were subjected to statistical analyses. Correlation with lipid cholesterols or Apo proteins was compared for all formats of PLAC® tests. Ratios of Lp-PLA2 by different PLAC® tests to different lipids were assessed for synergistic enhancement in the indication of ischemic stroke. RESULTS: The PLAC® mass test was superior to other formats of PLAC® tests in the assessment of CVD and is independent of lipids. The Lp-PLA2 by the CHAPS modified PLAC® mass test has no separation between the CVD and control groups. CONCLUSIONS: Both PLAC® mass and PLAC® activity tests are effective but the CHAPS modified PLAC® mass test has no or less utility in the risk assessment of CVD. The ratio of Lp-PLA2 by either PLAC® mass or PLAC® activity over ApoA1 or (Apo A1 + Apo B) synergistically enhance the risk assessment power for ischemic stroke.

Biochemical differences in the mass and activity tests of lipoprotein-associated phospholipase A2 explain the discordance in results between the two assay methods.

OBJECTIVES: There are two platforms for the detection of Lp-PLA2 in sera or plasmas: by its enzymatic activity (PLAC® activity test) and by its mass concentration (PLAC® mass test). It has been long recognized that these two platforms are not correlated well. The underlying cause for this is therefore investigated by the biochemical characterization of the two PLAC tests. DESIGN & METHODS: Human sera with and without the treatment by detergent were fractionated by using a Superose-6 column in phosphate buffered saline and the phospholipid associated Lp-PLA2 was assessed by both PLAC mass and activity tests. The Lp-PLA2 values of the two PLAC tests were compared under such conditions. RESULTS: Fractionation of sera and plasmas indicates that the association of Lp-PLA2 with phospholipids, especially LDL and other large size phospholipid vesicles, may block the detection of the enzyme by antibodies in the immunoassay format under the conditions of the PLAC mass test. Inclusion of high concentration (>CMC, critical micelle concentration) of detergents in the assay buffer of PLAC mass test dissociates Lp-PLA2 from phospholipid vesicles and results in the full detection of all Lp-PLA2 in sera or plasmas for concentration. Such assay modification significantly improves the correlation between the PLAC mass and PLAC activity tests. CONCLUSIONS: PLAC mass test only detects a small portion of the total Lp-PLA2, mainly the Lp-PLA2 associated with HDL. This is the main cause of the discordance and poor correlation between the PLAC mass and activity tests. Our results demonstrate the PLAC activity test is more accurate in assessing the total level of circulating Lp-PLA2.

Enhancing the secretion of recombinant proteins by engineering N-glycosylation sites.

N-glycosylation is important for the folding and quality control of membrane and secretory proteins. We used mutagenesis to introduce N-glycosylation sequons in recombinant proteins to improve their secretion in HEK293 cells. Seven recombinant proteins, with or without endogenous N-glycosylation sequons, were tested by this method. Our results indicate that N-glycosylation sequons located at the N- or C-terminal are glycosylated at high rates and thus the N- and C-terminal may be convenient sites for effectively attaching oligosaccharide chains. More importantly, introduction of oligosaccharide chains at such positions has been found to improve the secretion levels for the majority of the recombinant proteins in our studies, regardless of endogenous N-glycosylation, presumably by improving their folding in the endoplasmic reticulum.

B7-h4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer.

Using cDNA database mining strategies and real-time quantitative reverse transcription-PCR, we identified B7-H4 as a novel gene that is overexpressed in ovarian and breast cancer tissues when compared with normal tissues. The gene encodes a protein of 282 amino acids with a signal sequence, an immunoglobulin domain, and a COOH-terminal hydrophobic transmembrane domain. Immunohistochemistry experiments show plasma membrane staining in serous ovarian and breast cancer, confirming the tissue specificity and cell surface localization. We have developed a sensitive dual monoclonal antibody sandwich ELISA to analyze the level of B7-H4 protein in >2,500 serum samples, ascites fluids, and tissue lysates. High levels of B7-H4 protein were detected in ovarian cancer tissue lysates when compared with normal tissues. B7-H4 was present at low levels in all sera but showed an elevated level in serum samples from ovarian cancer patients when compared with healthy controls or women with benign gynecologic diseases. The median B7-H4 concentration in endometrioid and serous histotypes was higher than in mucinous histotypes, consistent with results of immunohistochemical staining. The multivariate logistic regression analysis of B7-H4 and CA125 measured in the same sample set resulted in an area under the curve (AUC) of 0.86 for all stages and 0.86 for stage I/II patients, which was significantly higher than the AUC for either marker alone. In early-stage patients, the sensitivity at 97% specificity increased from 52% for CA125 alone to 65% when used in combination with B7-H4. We conclude that B7-H4 is a promising new biomarker for ovarian carcinoma.

B7-h4 is highly expressed in ductal and lobular breast cancer.

PURPOSE: This study was designed to investigate the expression of B7-H4 protein, a member of the B7 family that is involved in the regulation of antigen-specific immune responses, in normal breast and in primary and metastatic breast carcinomas. EXPERIMENTAL DESIGN: Archival formalin-fixed tissue blocks from breast cancers and normal somatic tissues were evaluated for B7-H4 expression by immunohistochemistry with manual and automated image analysis. The proportion of B7-H4-positive cells and the intensity of B7-H4 staining were compared with histologic type, grade, stage, hormone receptor status, and HER-2/neu status. RESULTS: B7-H4 was detected in 165 of 173 (95.4%) primary breast cancers and in 240 of 246 (97.6%) metastatic breast cancers. B7-H4 staining intensity was greater in invasive ductal carcinomas [24.61 relative units (RU)] and in invasive lobular carcinomas (15.23 RU) than in normal breast epithelium (4.30 RU, P = 0.0003). Increased staining intensity was associated with negative progesterone receptor status (P = 0.014) and history of neoadjuvant chemotherapy (P = 0.004), and the proportion of B7-H4-positive cells was associated with negative progesterone receptor (P = 0.001) and negative HER-2/neu (P = 0.024) status. However, there was no statistically significant relationship between the proportion of B7-H4-positive cells or staining intensity and grade, stage, or other clinicopathologic variables. Low levels of B7-H4 expression were also detected in epithelial cells of the female genital tract, lung, pancreas, and kidney, but B7-H4 was generally absent in most other normal somatic tissues. CONCLUSIONS: The nearly ubiquitous expression of B7-H4 in breast cancer, independent of tumor grade or stage, suggests a critical role for this protein in breast cancer biology.

Species-specific urokinase receptor ligands reduce glioma growth and increase survival primarily by an antiangiogenesis mechanism.

Species-specific urokinase receptor (uPAR) ligands with improved pharmacokinetics were generated by site-specific mutagenesis and amino-terminal pegylation. These molecules were used to probe the role of uPAR in brain tumor progression and angiogenesis. The ligands blocked endothelial cell tube formation in Matrigel in a species-specific manner and reduced both baseline and uPA amino-terminal fragment-stimulated cell migration on vitronectin gradients. Treatment of U87MG gliomas implanted orthotopically in mice with single species-specific or combination uPAR ligands resulted in significant decreases in tumor size, which translated to increases in survival time, and which were most significant when the murine-specific ligand was included. Further analysis of tumors showed that the reduced sizes were correlated with a decrease in tumor cell proliferation and mean vessel density and an increase in tumor cell apoptosis. In addition, a large increase in collagen deposition was observed in the treated groups. Statistical analysis showed that the combination therapy demonstrated a clear synergy as compared to the individual agent treatments. These results suggest that the major role of the uPAR system in brain tumor progression is in the stromal compartment and particularly in neovascularization, a hallmark of invasive brain tumors.