Biochemical Principles and Functional Aspects of Pipecolic Acid Biosynthesis in Plant Immunity.

The nonprotein amino acid pipecolic acid (Pip) regulates plant systemic acquired resistance and basal immunity to bacterial pathogen infection. In Arabidopsis (Arabidopsis thaliana), the lysine (Lys) aminotransferase AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1) mediates the pathogen-induced accumulation of Pip in inoculated and distal leaf tissue. Here, we show that ALD1 transfers the α-amino group of l-Lys to acceptor oxoacids. Combined mass spectrometric and infrared spectroscopic analyses of in vitro assays and plant extracts indicate that the final product of the ALD1-catalyzed reaction is enaminic 2,3-dehydropipecolic acid (DP), whose formation involves consecutive transamination, cyclization, and isomerization steps. Besides l-Lys, recombinant ALD1 transaminates l-methionine, l-leucine, diaminopimelate, and several other amino acids to generate oxoacids or derived products in vitro. However, detailed in planta analyses suggest that the biosynthesis of 2,3-DP from l-Lys is the major in vivo function of ALD1. Since ald1 mutant plants are able to convert exogenous 2,3-DP into Pip, their Pip deficiency relies on the inability to form the 2,3-DP intermediate. The Arabidopsis reductase ornithine cyclodeaminase/µ-crystallin, alias SYSTEMIC ACQUIRED RESISTANCE-DEFICIENT4 (SARD4), converts ALD1-generated 2,3-DP into Pip in vitro. SARD4 significantly contributes to the production of Pip in pathogen-inoculated leaves but is not the exclusive reducing enzyme involved in Pip biosynthesis. Functional SARD4 is required for proper basal immunity to the bacterial pathogen Pseudomonas syringae Although SARD4 knockout plants show greatly reduced accumulation of Pip in leaves distal to P. syringae inoculation, they display a considerable systemic acquired resistance response. This suggests a triggering function of locally accumulating Pip for systemic resistance induction.

Deep, Quantitative Coverage of the Lysine Acetylome Using Novel Anti-acetyl-lysine Antibodies and an Optimized Proteomic Workflow.

Introduction of antibodies specific for acetylated lysine has significantly improved the detection of endogenous acetylation sites by mass spectrometry. Here, we describe a new, commercially available mixture of anti-lysine acetylation (Kac) antibodies and show its utility for in-depth profiling of the acetylome. Specifically, seven complementary monoclones with high specificity for Kac were combined into a final anti-Kac reagent which results in at least a twofold increase in identification of Kac peptides over a commonly used Kac antibody. We outline optimal antibody usage conditions, effective offline basic reversed phase separation, and use of state-of-the-art LC-MS technology for achieving unprecedented coverage of the acetylome. The methods were applied to quantify acetylation sites in suberoylanilide hydroxamic acid-treated Jurkat cells. Over 10,000 Kac peptides from over 3000 Kac proteins were quantified from a single stable isotope labeling by amino acids in cell culture labeled sample using 7.5 mg of peptide input per state. This constitutes the deepest coverage of acetylation sites in quantitative experiments obtained to-date. The approach was also applied to breast tumor xenograft samples using isobaric mass tag labeling of peptides (iTRAQ4, TMT6 and TMT10-plex reagents) for quantification. Greater than 6700 Kac peptides from over 2300 Kac proteins were quantified using 1 mg of tumor protein per iTRAQ 4-plex channel. The novel reagents and methods we describe here enable quantitative, global acetylome analyses with depth and sensitivity approaching that obtained for other well-studied post-translational modifications such as phosphorylation and ubiquitylation, and should have widespread application in biological and clinical studies employing mass spectrometry-based proteomics.

Study of the adjuvanticity of lysine lipopeptides; carbamate analogs elicit strong Th1 and Th2 response to ovalbumin in mice.

Bacterial lipoproteins and their synthetic analogs are strong immune modulators of the early host responses. In view of the strong adjuvanticity of bacterial lipopeptide mimics bearing lysine residues, a focused library of lipidated dipeptides and tripeptides has been synthesized with a view to understand the pattern of activity vis a vis the site and extent of lipidation. Compounds 4, 5 and 14 stimulate OVA specific IgG titer, neutralization of antibodies (IgG1 and IgG2a), T lymphocyte sub-sets (CD4/CD8) and its production of soluble mediators for Th1 (IFN-γ)/Th2 (IL-4) cytokines and costimulatory molecules (CD80/CD86) which are ideal traits of immune adjuvants. The results support lipidated lysine dipeptides as potent enhancers of humoral and cell mediated immune responses and thus might become promising immune-adjuvants for self adjuvanted vaccines.

Th-cytotoxic T-lymphocyte chimeric epitopes extended by Nepsilon-palmitoyl lysines induce herpes simplex virus type 1-specific effector CD8+ Tc1 responses and protect against ocular infection.

Molecularly defined vaccine formulations capable of inducing antiviral CD8+ T-cell-specific immunity in a manner compatible with human delivery are limited. Few molecules achieve this target without the support of an appropriate immunological adjuvant. In this study, we investigate the potential of totally synthetic palmitoyl-tailed helper-cytotoxic-T-lymphocyte chimeric epitopes (Th-CTL chimeric lipopeptides) to induce herpes simplex virus type 1 (HSV-1)-specific CD8+ T-cell responses. As a model antigen, the HSV-1 glycoprotein B498-505 (gB498-505) CD8+ CTL epitope was synthesized in line with the Pan DR peptide (PADRE), a universal CD4+ Th epitope. The peptide backbone, composed solely of both epitopes, was extended by N-terminal attachment of one (PAM-Th-CTL), two [(PAM)2-Th-CTL], or three [(PAM)3-Th-CTL] palmitoyl lysines and delivered to H2b mice in adjuvant-free saline. Potent HSV-1 gB498-505-specific antiviral CD8+ T-cell effector type 1 responses were induced by each of the palmitoyl-tailed Th-CTL chimeric epitopes, irrespective of the number of lipid moieties. The palmitoyl-tailed Th-CTL chimeric epitopes provoked cell surface expression of major histocompatibility complex and costimulatory molecules and production of interleukin-12 and tumor necrosis factor alpha proinflammatory cytokines by immature dendritic cells. Following ocular HSV-1 challenge, palmitoyl-tailed Th-CTL-immunized mice exhibited a decrease of virus replication in the eye and in the local trigeminal ganglion and reduced herpetic blepharitis and corneal scarring. The rational of the molecularly defined vaccine approach presented in this study may be applied to ocular herpes and other viral infections in humans, providing steps are taken to include appropriate Th and CTL epitopes and lipid groups.

Four different clones of mouse anti-acetyllysine monoclonal antibodies having different recognition properties share a common immunoglobulin framework structure.

By employing two different immunogens and two different antibody-screening strategies, we established four mouse hybridoma clones producing monoclonal antibodies against N epsilon -acetyllysine. Three different protocols were used in this study; i.e., mice were (1) immunized with an N epsilon -acetyllysine-containing peptide, Gly-Lys(Ac)- epsilon -aminocaproic acid (Aca)-Cys, conjugated to KLH, and the hybridoma clones were screened for their reactivity to a histone H3 peptide containing five acetyllysines; (2) immunized as in "1" and screened with chemically acetylated bovine serum albumin (BSA); (3) immunized with chemically acetylated keyhole limpet hemocyanin (KLH) and screened with chemically acetylated BSA. Antibodies produced by the four different hybridomas established here all reacted with acetyllysine residues, but their reactivity was not the same when evaluated with enzyme-linked immunosorbent assay (ELISA), Western blotting, and resonant mirror sensor analyses. Among the three protocols examined, protocol "3" was especially useful to obtain hybridomas producing anti-N epsilon -acetyllysine antibodies that could detect not only the acetylated histones but also other acetylated proteins. By cloning and sequencing the cDNAs encoding the variable regions of the antibodies, we found that their framework sequences were almost the same, which suggests that some framework amino acids in addition to their complementarity determining regions (CDRs) directly contribute to their recognition function.

The effect of amino acid spacers on the antigenicity of dimeric peptide--inducing cross-reacting antibodies to a cell surface protein antigen of Streptococcus mutans.

In the course of developing a synthetic peptide vaccine for dental caries, we identified a unique 13-mer peptide named PAc(365-377), TYEAALKQYEADL, as a minimum peptide inducing cross-inhibiting antibodies to a cell surface protein antigen (PAc) of Streptococcus mutans. However, the peptide could hardly induce the production of antibody in the absence of adjuvant. Thus using this peptide as a unit peptide, tandem constructs of dimeric unit peptide with or without spacer amino acid residues were synthesized, and their antigenicities were examined in B10.D2 mice. Significant augmentation of antigenicity was obtained in all of the dimeric unit peptides with spacers, especially for lysine spacers. In addition, analysis for cross-reactivity of anti-construct antibodies against a set of double valine-substituted analogues of the unit peptide revealed that the di-lysine spacer might be more effective in inducing the cross-reacting antibodies to rPAc.

Preparation of a monoclonal antibody to N(epsilon)-(Hexanonyl)lysine: application to the evaluation of protective effects of flavonoid supplementation against exercise-induced oxidative stress in rat skeletal muscle.

The monoclonal antibody to N(epsilon)-(hexanonyl)lysine (HEL), a novel adduct formed by the reaction of linoleic acid hydroperoxide and lysine, has been prepared and characterized. The obtained antibody specifically recognized the HEL moiety. Using the monoclonal antibody, we evaluated the protective effects of feeding eriocitrin, which is one of flavonoids in lemon fruit, on oxidative modification induced by exercise in rats. The supplementation of eriocitrin significantly suppressed the increase in HEL in the skeletal muscle by exercise. The result suggests that the determination of HEL may be a good method for evaluation of the protective effect of beneficial food factors against oxidative stress.

D-LysAsnProTyr tetrapeptide: a novel B-cell stimulant and stabilized bursin mimetic.

The tetrapeptide I (D-lysine-L-asparaginyl-L-prolyl-L-tyrosine or D-LysAsnProTyr), and analogue sequences, were synthesized and evaluated for the ability to stimulate immune cell subsets. These sequences were selected based on their perceived ability to readily adopt a beta-turn structure. In vitro immunological assays revealed a robust stimulation of mitogen activated B-cell proliferation and a modest to significant stimulation of cytotoxic T lymphocytes (CTLs). Further, this in vitro stimulation of B-cells was accompanied by an in vivo expansion of B-cells in C57BL/6 mice, as demonstrated by immunophenotyping experiments. Interestingly, a conformational analysis of the low energy conformers of I and the endogenous B-cell stimulant bursin (LysHisGlyNH2) shows that these molecules can be superimposed. However, I displayed significantly enhanced physiological stability. For a number of reasons, I may be a particularly useful vaccine adjuvant.

Camel single-domain antibody inhibits enzyme by mimicking carbohydrate substrate.

Whereas antibodies have demonstrated the ability to mimic various compounds, classic heavy/light-chain antibodies may be limited in their applications. First, they tend not to bind enzyme active site clefts. Second, their size and complexity present problems in identifying key elements for binding and in using these elements to produce clinically valuable compounds. We have previously shown how cAb-Lys3, a single variable domain fragment derived from a lysozyme-specific camel antibody naturally lacking light chains, overcomes the first limitation to become the first antibody structure observed penetrating an enzyme active site. We now demonstrate how cAb-Lys3 mimics the oligosaccharide substrate functionally (inhibition constant for lysozyme, 50 nM) and structurally (lysozyme buried surface areas, hydrogen bond partners, and hydrophobic contacts are similar to those seen in sugar-complexed structures). Most striking is the mimicry by the antibody complementary determining region 3 (CDR3) loop, especially Ala104, which mimics the subsite C sugar 2-acetamido group; this group has previously been identified as a key feature in binding lysozyme. Comparative simplicity, high affinity and specificity, potential to reach and interact with active sites, and ability to mimic substrate suggest that camel heavy-chain antibodies present advantages over classic antibodies in the design, production, and application of clinically valuable compounds.

Conjunctival mast cells and the allergic late phase reaction.

An active model of ocular anaphylaxis was developed in guinea pigs to evaluate the histopathology of the early (EPR) and late (LPR) phase reaction, focusing on the role of mast cells. Five groups (n = 6) of animals were actively immunized by first injecting into each of the axillary and inguinal lymph node areas, 0.25 ml of an emulsion containing 1 mg dinitrophenyl bovine gamma-globulin (DNP-BCG) with 0.5 ml complete Freund's adjuvant. After two weeks, an intramuscular injection of 0.5 ml of an emulsion containing 1 mg DNP-BGG with 0.5 ml of incomplete adjuvant was administered. One month after the first injection, animals were sacrificed after topical ocular challenge with 10 microliters of 1 mg/ml divalent hapten, di-DNP-lysine, in one eye and phosphate buffered saline (PBS) in the fellow eye as control. Clinical reactions were graded over time and histology evaluated at the endpoint (time 0, 0.5, 3, 9, and 24 h). Results showed that all animals clearly developed both an EPR and an LPR, as either a biphasic, multiphasic or prolonged clinical response. A small percentage of mast cells were degranulated at baseline, whereas, at 0.5 h, 95% of mast cells were degranulated in the eyes treated with specific hapten and 25% in the control eyes treated with PBS. At 3 h, 84% of the mast cells were degranulated. This value rose to 89% at 9 h, and remained unchanged at 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Early and late phases of ocular anaphylaxis in actively immunized guinea pigs.

A model of ocular anaphylaxis with distinct early- and late-phase components was studied in actively immunized guinea pigs. Twenty guinea pigs were injected with dinitrophenylated (DNP) bovine gamma globulin emulsified in Freund's complete adjuvant and challenged topically with di-DNP-lysine. Clinical signs were monitored over a 48 h period. An early-phase reaction (EPR) characterized by conjunctival edema, conjunctival erythema, lid swelling, and lid redness was observed. This reaction peaked at 0.5 h after challenge and subsided to a low point at 3-4 h. Subsequently, a second episode of lid swelling and lid redness was observed at 4-8 h. All animals in both groups exhibited an EPR. In addition, 75% of the animals underwent an EPR and an LPR. No animals exhibited an isolated LPR. Of the animals that underwent a dual response, 47% were biphasic, 6% were prolonged and 47% were multiphasic. The development of an active model of ocular anaphylaxis exhibiting both EPR and LPR components will enable studies of mechanisms which regulate the frequency and magnitude of these ocular allergic responses.

Cellular interactions in immune regulation. Hapten-specific suppression by non-T cells and T cell mediated reversal of suppression.

The ability of lymphoid cells from immunized animals to regulate the response of naive B ceils to the immunizing hapten was studied. Mice were immunized with trinitrophenylated (TNP) bovine gamma globulin (BGG) in complete Freund's adjuvant, and their spleen cells were examined in vivo and in vitro for the presence of specific inhibitory activity. This activity was found to peak 1 wk after immunization, was active against TNP on both T-dependent (BGG) and T-independent (Ficoll and polyacrylamide beads) carriers, and was demonstrable both by mixed cell transfers and mixed cell culture experiments. In in vitro studies, it was shown that the inhibition of the response to TNP- polyacrylamide beads by immune spleen cells was mediated by a non-T cell, possibly a B cell, because the suppressor activity was enriched in a purified B cell preparation. A role for macrophages was not formally ruled out. A specific suppressor factor was produced in vitro by immune spleen cells cultured in the absence of antigen. The suppressor activity was modulated by T .cells because elimination of T cells from the normal spleen cell population decreased suppression; elimination of T cells from the immune spleen cell population did not effect suppression, but elimination of T cells from both the normal and immune spleen cell populations allowed the expression of marked specific suppression. Thus, T cells present in the normal spleen cell population augment the degree of suppression, whereas T cells present in the immune spleen cell population decrease the degree of suppression; that is, T cells present in the immune spleen cell population had the ability to specifically abrogate suppression ("abrosuppression") in a T-independent immune response. It is proposed that the response to a T- independent antigen is regulated by specific suppressor activity generated by a non-T cell and augmented by the interaction of this cell with a T cell. The suppressor activity can be blocked by a specific abrosuppressor T cell. It is suggested that, because suppressor activity appears dominant in the naive state of the immune system, the induction of specific abrosuppressor activity may be essential if an immune response is to take place.

Anti-timothy IgE formation: suppression with antigen D-dGl conjugates.

Antigen D fragments (AgD1 and AgD2) and chemically synthesized quercetin-glutathione were conjugated to the synthetic polypeptide copolymer of D-glutamic acid and D-lysine (dGL). These conjugates were tested at varying epitope densities to determine their ability to suppress a secondary anti-antigen B IgE response. The data showed that all of the conjugates used with epitope densities of 5-20 groups per dGL produced significant dose-dependent suppression of a secondary IgE response. The duration of the observed suppression was short (about 30 days), but could be extended by additional treatment with the conjugate prior to the loss of unresponsiveness.

Ineffectiveness of lipopolysaccharide for preventing the tolerance induction in bone marrow-derived lymphoid cells with dinitrophenyl-poly-L-(glutamic acid, lysine).

The effect of endotoxin or lipopolysaccharide (LPS) on tolerance induction in bone marrow-derived lymphoid cells (B cells) was investigated. Dinitrophenylated amino acid copolymer-L-(glutamic acid, lysine) (DNP-GL) acts as a potent tolerogen on normal and DNP-primed B cells. LPS significantly enhanced the anti-sheep red blood cell plaque-forming cell (anti-SRBC PFC) response that occurred after the immunization with a low dose of SRBC. LPS did not induce the primary anti-DNP PFC response after the injection of DNP-GL, nor did it prevent the tolerance induction in normal and DNP-primed B cells that occured after the administration of DNP-GL.

The mechanism of tolerance induction in thymus-derived lymphocytes; I. intracellular inactivation of hapten-reactive helper T lymphocytes by hapten-nonimmunogenic copolymer of D-amino acids.

Treatment of a p-azobenzoate (PAB) derivative of a copolymer of D-glutamic acid and D-lysine (D-GL) induced a profound state of unresponsiveness to PAB-reactive helper T lymphocytes generated in PAB-mouse gamma globulin (MGG)-primed mice. This unresponsiveness in T lymphocytes was specific for PAB-reactive cells, since the bacterial alpha-amylase-, keyhole limpet hemocyanin-, or ovalbumin-primed helper T lymphocytes were not suppressed by PAB-D-GL treatment. Taking advantage of the relative ease with which PAB-D-GL can induce specific unresponsiveness to helper T lymphocytes in an animal previously primed with PAB-MGG, it was possible to approach certain questions concerning the mechanisms of tolerance-induction and the fate of tolerant helper T lymphocytes in the PAB-D-GL model by utilizing a classical adoptive cell transfer systemmelimination of the possibility of carry-over of the tolerogen with cells or of the generation of suppressor cells as the result of PAB-D-GL treatment as an explanation of the suppression of helper T-cell activity strongly inplicates the existence of a central intracellular mechanism of specific tolerance on the helper T-cell level. The possibility that suppression of the activity of PAB-reactive helper T lymphocytes by PAB-D-GL reflects simple blocking of surface receptor molecules on T lymphocytes was ruled out as it was found that the helper activity of PAB-reactive cells was minimally suppressed even when PAB-D-GL was directly exposed in vitro to helper T lymphocytesmmoreover, the most conclusive evidence on te the tolerant state induced by in vivo exposure of primed T cells to PAB-D-GL. It appears, therefore, that specific tolerance induced by PAB-D-GL' TO PAB-reactive helper T lymphocytes is an example of irreversible inhibition of T-cell reactivity to antigen, reflecting yet to be determined events at the intra- and subcellular levels.

Genetic control of immune response. The dose of antigen given in aqueous solution is critical in determining which mouse strain is high responder to poly(LTyr, LGlu)-poly(LPro)--poly(LLys).

Antibody response to different doses of (T,G)-Pro--L, given in aqueous solution, was investigated in the high responder SJL and low responder DBA/1 strains by measuring hemolytic plaque-forming cells (PFC) in the spleens as well as hemagglutination titers in the sera. The gene responsible for the difference between the two strains in the response to this antigen, given in complete Freund's adjuvant, has been previously denoted Ir-3. This gene is not linked to the major histocompatibility locus. In the response to the optimal dose (1 mug) of antigen, no difference could be shown between the strains. The peak of the response and the numbers of direct and indirect PFC were similar in both strains in the primary and secondary response. After injection of higher doses (10-100 mug) of antigen, both the direct and indirect PFC responses were lower in the low responder than in the high responder strain. Moreover, the peak of the response occurred earlier in the high responder strain in the primary response to the 10 mu dose of antigen. After administration of a suboptimal dose (0.02 mug) of antigen, the low responder strain produced in the primary response 4-20 times more indirect plaques than the high responder strain. Also the number of direct plaques was higher in the low responder than in the high responder strain. The serum antibody responses to the optimal and higher doses of antigen were parallel to the PFC responses. From inhibition of PFC with free antigen, it was concluded that a similar proportion of cells was producing high and low affinity antibodies to (T,G)-Pro--L in both strains. High and low zone tolerance could be induced in the two strains with (T,G)-Pro--L, but no difference could be shown between the strains. It is suggested that the Ir-3 gene plays a role in the regulation of the balance stimulation and suppression according to the dose of antigen given.