Measurement of Lipoprotein-Associated Phospholipase A2 by Use of 3 Different Methods: Exploration of Discordance between ELISA and Activity Assays

BACKGROUND Lipoprotein-associated phospholipase A2 (Lp-PLA2), an enzyme associated with inflammation, is used as a biomarker for cardiovascular disease risk. Both the concentration and activity of Lp-PLA2 have been shown to be clinically relevant. However, there is a discordance between the serum concentration of Lp-PLA2 measured by the standard ELISA-based immunoassays and the activity of this enzyme, leading to substantial discordance in risk categorization depending on assay format. METHODS We developed 2 LC-MS/MS–based assays to quantify serum Lp-PLA2 activity (multiple reaction monitoring detection of product) and concentration [stable isotope standards and capture by antipeptide antibody (SISCAPA) immunoaffinity], and we investigated their correlation to commercially offered colorimetric activity and immunometric concentrations assays. Associations between Lp-PLA2 and lipoproteins and the effect of selected detergents in liberating Lp-PLA2 were evaluated by use of immunoprecipitation and Western blot analyses. RESULTS Serum Lp-PLA2 concentrations measured by quantitative SISCAPA-mass spectrometry were substantially higher than concentrations typically measured by immunoassay and showed an improved agreement with Lp-PLA2 activity. With detergents, liberation of Lp-PLA2 from lipoprotein complexes dramatically increased the amount of protein detected by immunoassay and improved the agreement with activity measurements. CONCLUSIONS Quantitative analysis of Lp-PLA2 concentration and activity by LC-MS/MS assays provided key insight into resolving the well-documented discordance between Lp-PLA2 concentration (determined by immunoassay) and activity. Quantitative detection of Lp-PLA2 by immunoassay appears to be strongly inhibited by interaction of Lp-PLA2 with lipoprotein. Together, the results illustrate the advantages of quantitative LC-MS/MS for measurement of Lp-PLA2 concentration (by SISCAPA) and activity (by direct product detection).

On the Meaning of Affinity Limits in B-Cell Epitope Prediction for Antipeptide Antibody-Mediated Immunity

B-cell epitope prediction aims to aid the design of peptide-based immunogens (e.g., vaccines) for eliciting antipeptide antibodies that protect against disease, but such antibodies fail to confer protection and even promote disease if they bind with low affinity. Hence, the Immune Epitope Database (IEDB) was searched to obtain published thermodynamic and kinetic data on binding interactions of antipeptide antibodies. The data suggest that the affinity of the antibodies for their immunizing peptides appears to be limited in a manner consistent with previously proposed kinetic constraints on affinity maturation in vivo and that cross-reaction of the antibodies with proteins tends to occur with lower affinity than the corresponding reaction of the antibodies with their immunizing peptides. These observations better inform B-cell epitope prediction to avoid overestimating the affinity for both active and passive immunization; whereas active immunization is subject to limitations of affinity maturation in vivo and of the capacity to accumulate endogenous antibodies, passive immunization may transcend such limitations, possibly with the aid of artificial affinity-selection processes and of protein engineering. Additionally, protein disorder warrants further investigation as a possible supplementary criterion for B-cell epitope prediction, where such disorder obviates thermodynamically unfavorable protein structural adjustments in cross-reactions between antipeptide antibodies and proteins.

Online High-Flow Peptide Immunoaffinity Enrichment and Nanoflow LC-MS/MS: Assay Development for Total Salivary Pepsin/Pepsinogen

BACKGROUND Detection limit challenges associated with measuring low-abundance protein biomarkers can be addressed with hybrid immunoaffinity–mass spectrometric assays, such as antipeptide antibody capture followed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Popular assay formats use magnetic bead–based immunoaffinity enrichment and nanoflow LC-MS/MS or high-flow immunoaffinity chromatography coupled online to conventional LC-MS/MS. As a proof of principle, we describe a novel online immunoaffinity LC-MS/MS configuration that combines high-flow peptide immunoaffinity enrichment and nanoflow LC-MS/MS. METHODS We configured and validated an assay for the measurement of total pepsin/pepsinogen from human saliva that uses a pepsinogen standard. Saliva was heat-inactivated to quench residual enzymatic activity and then digested with endoproteinase AspN. Online immunoaffinity enrichment using an antipeptide antibody directed against the pepsin C-terminal sequence, DRANNQVGLAPVA, was linked to nanoflow liquid chromatography and selected reaction monitoring mass spectrometry. We used the assay to measure pepsin/pepsinogen concentrations in human saliva from presumed healthy volunteers. RESULTS Heat inactivation at 100 °C for 25 min stabilized the target peptide. The final assay had <15% interassay relative error and <15% interassay CV across a range of 4.08–2980 pmol/L human pepsinogen (0.165–120 μg/L). Low but quantifiable signals were observed in some samples from presumed normal healthy volunteers ranging from 4.3 to 16.6 pmol/L (0.17–0.67 μg/L) total salivary pepsin/pepsinogen. CONCLUSIONS This assay approach provides a high-sensitivity platform for protein bioanalysis in the low picomolar range. It bears the potential to deliver additional data on the salivary occurrence of pepsin/pepsinogen with greater confidence than previously.

Pneumocystis Encodes a Functional S-Adenosylmethionine Synthetase Gene

ABSTRACT S-Adenosylmethionine (AdoMet) synthetase (EC 2.5.1.6) is the enzyme that catalyzes the synthesis of AdoMet, a molecule important for all cellular organisms. We have cloned and characterized an AdoMet synthetase gene (sam1) from Pneumocystis spp. This gene was transcribed primarily as an ∼1.3-kb mRNA which encodes a protein containing 381 amino acids in P. carinii or P. murina and 382 amino acids in P. jirovecii. sam1 was also transcribed as part of an apparent polycistronic transcript of ∼5.6 kb, together with a putative chromatin remodeling protein homologous to Saccharomyces cerevisiae, CHD1. Recombinant Sam1, when expressed in Escherichia coli, showed functional enzyme activity. Immunoprecipitation and confocal immunofluorescence analysis using an antipeptide antibody showed that this enzyme is expressed in P. murina. Thus, Pneumocystis, like other organisms, can synthesize its own AdoMet and may not depend on its host for the supply of this important molecule.

Infection-Dependent Nuclear Localization of US17, a Member of the US12 Family of Human Cytomegalovirus-Encoded Seven-Transmembrane Proteins

ABSTRACT The human cytomegalovirus (HCMV) US12 gene family is a group of predicted seven-transmembrane, G-protein-coupled receptor-related proteins, about which little is known. Specific rabbit polyclonal antibodies detected US17 and US18 beginning 54 and 36 h after infection, respectively, with expression of both proteins dependent on viral DNA synthesis. While US14 and US18 are expressed exclusively in the cytoplasm, we unexpectedly found abundant expression of US17 in both the cytoplasm and nucleoplasm. N- and C-terminally tagged versions of US17 were readily detected in the cytoplasm of transfected mammalian cells, but not in nuclei, suggesting that nuclear localization involves other viral proteins or an infection-triggered cellular process. There was no specific colocalization between US17 and other nuclear expressed HCMV-encoded proteins (IE-2, DNA polymerase processivity factor, and pp28/UL99). To determine whether the observed nuclear localization might be the product of a process by which a soluble C-terminal segment of the full-length protein is expressed, we constructed a recombinant virus that incorporates a synthetic epitope at its N terminus, which in conjunction with the antipeptide antibody that targets its predicted cytoplasmic C-terminal segment, enables simultaneous independent detection of both termini. In cells infected with the recombinant, the US17 N and C termini had limited colocalization, with the N-terminal segment not detected in nuclei, supporting the segmentation hypothesis. Consistent with this, a fragment with an apparent molecular size of 10 kDa was detected by immunoblotting. We have identified the first viral example of a seven-transmembrane protein that is either segmented or expressed in nuclei. Further study will be required to learn the mechanism by which this occurs and the function of the nuclear localizing segment. This likely represents yet another mechanism by which a virus has hijacked or modified cellular regulatory pathways for its benefit.

Antipeptide Antibody Responses following Intranasal Immunization: Effectiveness of Mucosal Adjuvants

ABSTRACT Toxicity is a major factor limiting the development and use of potent adjuvants for human mucosally delivered vaccines. Novel adjuvant formulations have recently become available, and in the present study two have been used for intranasal immunization with a synthetic peptide immunogen (MAP-M2). This peptide represents a multiple antigenic peptide containing multiple copies of a mimotope M2, a peptide mimic of a conformational epitope of the fusion protein of measles virus. MAP-M2 was administered intranasally to experimental animals together with synthetic oligodeoxynucleotides containing unmethylated CpG motifs with or without a mutant of wild-type enterotoxin of Escherichia coli (LTR72). The combination of the mutant toxin LTR72 and the CpG repeats, codelivered with a peptide immunogen, induced both local and systemic peptide- and pathogen-specific humoral and cellular immune responses comparable to those obtained after intranasal immunization with the wild-type toxin LT. In addition, this combination of adjuvants induced a predominantly immunoglobulin G2a antibody response. If both the LTR72 and CpG adjuvants are shown to be safe for use in humans, this particular combination would appear to have potential as an adjuvant for mucosally delivered vaccines in humans.

Cryptic and Exposed Invariable Regions of VlsE, the Variable Surface Antigen of Borrelia burgdorferisl

ABSTRACT Borrelia burgdorferi, the Lyme disease spirochete, possesses a surface protein, VlsE, which undergoes antigenic variation. VlsE contains two invariable domains and a variable one that includes six variable and six invariable regions (IRs). Five of the IRs are conserved among strains and genospecies of B. burgdorferisensu lato. IR6 is conserved, immunodominant, and exposed at the VlsE surface but not at the spirochete surface, as assessed in vitro. In the present study, the remaining conserved IRs (IR2 to IR5) were investigated. Antisera to synthetic peptides based on each of the IR2 to IR5 sequences were produced in rabbits. Antipeptide antibody titers were similarly high in all antisera. Native VlsE was immunoprecipitable with antibodies to IR2, IR4, and IR5 but not to IR3, indicating that the first three sequences were exposed at the VlsE surface. However, negative surface immunofluorescence and in vitro antibody-mediated killing results indicated that none of the IRs were accessible to antibody at the spirochetal surface in vitro.

DDM-PGE2-mediated cytoprotection in renal epithelial cells by a thromboxane A2 receptor coupled to NF-κB

The present studies were conducted to determine the pharmacological nature of a cytoprotective 11-deoxy-16,16-dimethyl-PGE2(DDM-PGE2) receptor in LLC-PK1 cells. DDM-PGE2-mediated cytoprotection against 2,3,5-(trisglutathion- S-yl)hydroquinone (TGHQ)-mediated cytotoxicity can be reproduced using thromboxane A2(TXA2) receptor (TP) agonists (U46619 and IBOP), and the cytoprotective response to DDM-PGE2 and TP agonists is inhibited by TP antagonists (SQ-29,548 and ISAP). Western blot analysis using an antipeptide antibody against the human platelet TP receptor (55 kDa) identified a particulate associated 54-kDa protein. DDM-PGE2-mediated 12- O-tetradecanoyl phorbol-13-acetate (TPA) responsive element (TRE) binding activity is not inhibited by cyclooxygenase inhibitors (aspirin and indomethacin) or a TXA2 synthase inhibitor (sulfasalazine), suggesting that the biological response to DDM-PGE2 is not dependent on de novo TXA2biosynthesis. Peak DDM-PGE2- and U46619-mediated TRE binding activity and nuclear factor-κB (NF-κB) binding activity are inhibited by SQ-29,548. The full cytoprotective response to DDM-PGE2 requires an 8-h pulse with agonist. DDM-PGE2-mediated TRE and NF-κB binding activity remain elevated in the presence of agonist and rapidly decay following agonist washout, suggesting a direct correlation between DDM-PGE2-mediated cytoprotection and persistent DNA binding activities. TPA, a protein kinase C activator, induces cytoprotection and a persistent increase of NF-κB binding activity. DDM-PGE2-mediated cytoprotection and NF-κB binding activity but not TRE binding activity are inhibited by sulfasalazine. We conclude that the DDM-PGE2 receptor is a TP receptor and that the cytoprotective response may be mediated in part by NF-κB.