Nonconserved Active Site Residues Modulate CheY Autophosphorylation Kinetics and Phosphodonor Preference

In two-component signal transduction, response regulator proteins contain the catalytic machinery for their own covalent phosphorylation and can catalyze phosphotransfer from a partner sensor kinase or autophosphorylate using various small molecule phosphodonors. Although response regulator autophosphorylation is physiologically relevant and a powerful experimental tool, the kinetic determinants of the autophosphorylation reaction and how those determinants might vary for different response regulators and phosphodonors are largely unknown. We characterized the autophosphorylation kinetics of 21 variants of the model response regulator <i>Escherichia coli</i> CheY that contained substitutions primarily at nonconserved active site positions D + 2 (CheY residue 59) and T + 2 (CheY residue 89), two residues C-terminal to conserved D57 and T87, respectively. Overall, the CheY variants exhibited a >10⁵-fold range of rate constants (<i>k</i>_phos/<i>K</i>_S) for reaction with phosphoramidate, acetyl phosphate, or monophosphoimidazole, with the great majority of rates enhanced versus that of wild-type CheY. Although phosphodonor preference varied substantially, nearly all the CheY variants reacted faster with phosphoramidate than acetyl phosphate. Correlation between the increased positive charge of the D + 2 and T + 2 side chains and faster rates indicated electrostatic interactions are a kinetic determinant. Moreover, sensitivities of rate constants to ionic strength indicated that both long-range and localized electrostatic interactions influence autophosphorylation kinetics. The increased nonpolar surface area of the D + 2 and T + 2 side chains also correlated with an enhanced autophosphorylation rate, especially for reaction with phosphoramidate and monophosphoimidazole. Computer docking suggested that highly accelerated monophosphoimidazole autophosphorylation rates for CheY variants with a tyrosine at position T + 2 likely reflect structural mimicry of phosphotransfer from the sensor kinase histidyl phosphate

Datasheet1_Peanut butter feeding induces oral tolerance in genetically diverse collaborative cross mice.docx

BackgroundEarly dietary introduction of peanut has shown efficacy in clinical trials and driven pediatric recommendations for early introduction of peanut to children with heightened allergy risk worldwide. Unfortunately, tolerance is not induced in every case, and a subset of patients are allergic prior to introduction. Here we assess peanut allergic sensitization and oral tolerance in genetically diverse mouse strains.ObjectiveWe aimed to determine whether environmental adjuvant-driven airway sensitization and oral tolerance to peanut could be induced in various genetically diverse mouse strains.MethodsC57BL/6J and 12 Collaborative Cross (CC) mouse strains were fed regular chow or ad libitum peanut butter to induce tolerance. Tolerance was tested by attempting to sensitize mice via intratracheal exposure to peanut and lipopolysaccharide (LPS), followed by intraperitoneal peanut challenge. Peanut-specific immunoglobulins and peanut-induced anaphylaxis were assessed.ResultsWithout oral peanut feeding, most CC strains (11/12) and C57BL/6J induced peanut-specific IgE and IgG1 following airway exposure to peanut and LPS. With oral peanut feeding none of the CC strains nor C57BL/6J mice became sensitized to peanut or experienced anaphylaxis following peanut challenge.ConclusionAllergic sensitization and oral tolerance to peanut can be achieved across a range of genetically diverse mice. Notably, the same strains that became allergic via airway sensitization were tolerized by feeding high doses of peanut butter before sensitization, suggesting that the order and route of peanut exposure are critical for determining the allergic fate.</p

Image1_Peanut butter feeding induces oral tolerance in genetically diverse collaborative cross mice.tif

BackgroundEarly dietary introduction of peanut has shown efficacy in clinical trials and driven pediatric recommendations for early introduction of peanut to children with heightened allergy risk worldwide. Unfortunately, tolerance is not induced in every case, and a subset of patients are allergic prior to introduction. Here we assess peanut allergic sensitization and oral tolerance in genetically diverse mouse strains.ObjectiveWe aimed to determine whether environmental adjuvant-driven airway sensitization and oral tolerance to peanut could be induced in various genetically diverse mouse strains.MethodsC57BL/6J and 12 Collaborative Cross (CC) mouse strains were fed regular chow or ad libitum peanut butter to induce tolerance. Tolerance was tested by attempting to sensitize mice via intratracheal exposure to peanut and lipopolysaccharide (LPS), followed by intraperitoneal peanut challenge. Peanut-specific immunoglobulins and peanut-induced anaphylaxis were assessed.ResultsWithout oral peanut feeding, most CC strains (11/12) and C57BL/6J induced peanut-specific IgE and IgG1 following airway exposure to peanut and LPS. With oral peanut feeding none of the CC strains nor C57BL/6J mice became sensitized to peanut or experienced anaphylaxis following peanut challenge.ConclusionAllergic sensitization and oral tolerance to peanut can be achieved across a range of genetically diverse mice. Notably, the same strains that became allergic via airway sensitization were tolerized by feeding high doses of peanut butter before sensitization, suggesting that the order and route of peanut exposure are critical for determining the allergic fate.</p

Table1_Peanut butter feeding induces oral tolerance in genetically diverse collaborative cross mice.docx

BackgroundEarly dietary introduction of peanut has shown efficacy in clinical trials and driven pediatric recommendations for early introduction of peanut to children with heightened allergy risk worldwide. Unfortunately, tolerance is not induced in every case, and a subset of patients are allergic prior to introduction. Here we assess peanut allergic sensitization and oral tolerance in genetically diverse mouse strains.ObjectiveWe aimed to determine whether environmental adjuvant-driven airway sensitization and oral tolerance to peanut could be induced in various genetically diverse mouse strains.MethodsC57BL/6J and 12 Collaborative Cross (CC) mouse strains were fed regular chow or ad libitum peanut butter to induce tolerance. Tolerance was tested by attempting to sensitize mice via intratracheal exposure to peanut and lipopolysaccharide (LPS), followed by intraperitoneal peanut challenge. Peanut-specific immunoglobulins and peanut-induced anaphylaxis were assessed.ResultsWithout oral peanut feeding, most CC strains (11/12) and C57BL/6J induced peanut-specific IgE and IgG1 following airway exposure to peanut and LPS. With oral peanut feeding none of the CC strains nor C57BL/6J mice became sensitized to peanut or experienced anaphylaxis following peanut challenge.ConclusionAllergic sensitization and oral tolerance to peanut can be achieved across a range of genetically diverse mice. Notably, the same strains that became allergic via airway sensitization were tolerized by feeding high doses of peanut butter before sensitization, suggesting that the order and route of peanut exposure are critical for determining the allergic fate.</p

Table2_Peanut butter feeding induces oral tolerance in genetically diverse collaborative cross mice.docx

BackgroundEarly dietary introduction of peanut has shown efficacy in clinical trials and driven pediatric recommendations for early introduction of peanut to children with heightened allergy risk worldwide. Unfortunately, tolerance is not induced in every case, and a subset of patients are allergic prior to introduction. Here we assess peanut allergic sensitization and oral tolerance in genetically diverse mouse strains.ObjectiveWe aimed to determine whether environmental adjuvant-driven airway sensitization and oral tolerance to peanut could be induced in various genetically diverse mouse strains.MethodsC57BL/6J and 12 Collaborative Cross (CC) mouse strains were fed regular chow or ad libitum peanut butter to induce tolerance. Tolerance was tested by attempting to sensitize mice via intratracheal exposure to peanut and lipopolysaccharide (LPS), followed by intraperitoneal peanut challenge. Peanut-specific immunoglobulins and peanut-induced anaphylaxis were assessed.ResultsWithout oral peanut feeding, most CC strains (11/12) and C57BL/6J induced peanut-specific IgE and IgG1 following airway exposure to peanut and LPS. With oral peanut feeding none of the CC strains nor C57BL/6J mice became sensitized to peanut or experienced anaphylaxis following peanut challenge.ConclusionAllergic sensitization and oral tolerance to peanut can be achieved across a range of genetically diverse mice. Notably, the same strains that became allergic via airway sensitization were tolerized by feeding high doses of peanut butter before sensitization, suggesting that the order and route of peanut exposure are critical for determining the allergic fate.</p

High affinity mAbs selected from DTLacO cells.

<p>(A) Above, binding profiles of successive DTLacO LacI-HP1 populations selected for recognition of cell surface receptors, VEGFR2, TIE2 and TROP2. Rounds of selection designated above peaks (S0–S8). Below, saturation binding kinetics, indicating apparent k_D. (B) Specificity of selected DTLacO populations. FACS analysis of binding of cell populations selected for high affinity recognition of VEGFR2, TIE2 or TROP2 to recombinant VEGFR2, TIE2, TROP2, SAv or ovalbumin (OVA). Solid peaks represent the negative reference control (secondary antibody alone), and green lines represent staining with antigen. (C) Schematic alignment of V_H and V_λ regions of mAbs selected for binding to VEGFR2, TIE2 and TROP2. Thin horizontal blue lines represent chicken framework regions, thicker horizontal lavender lines against background shading identify CDRs, vertical bars indicate single residue differences relative to the most common DTLacO sequence, and triangle indicates insertion.</p

Selection and humanization of anti-FN14 and anti-FZD10 mAbs.

<p>(A) Schematic of time course of selection of anti-FN14 and anti-FZD10 mAbs, with selection steps indicated by S, and apparent affinities (k_D) of recombinant chimeric mAbs shown below. (B) Schematic alignment of V_H and V_λ regions of mAbs selected for binding to FN14 and FZD10. Thin horizontal lines represent chicken framework regions, thicker horizontal lines against background shading identify CDRs, vertical bars indicate single residue differences relative to the most common DTLacO sequence, and triangle indicates insertion. (C) Antibody humanization. V_H and V_λ regions of humanized mAbs hFS24 and hFZ2 schematically aligned to the human V_H-III or V_λ-III consensus (top lines). Thin horizontal lines represent human framework regions; asterisks denote the two residues eliminated from the N-terminal of the light chain; vertical lines outside background shading identify Vernier zone residues preserved in humanized mAbs; other notations as in Panel B. (D) Apparent affinities (k_D) of humanized and progenitor mAbs.</p

Rapid evolution of anti-SAv antibodies in DTLacO cells.

<p>(A) SAv binding profile of successive selected cell populations of DTLacO (left) or DTLacO E47-LacI (right) cells. Selection was carried out on average weekly. Cell numbers plotted relative to SAv-PE fluorescent signal. Populations at successive rounds of selection designated above peaks (S0–S7). Pre, populations prior to any sorting (gray fill). (B) Saturation binding kinetics of DTLacO E47-LacI S7 population. (C) Sequences of high affinity selected anti-SAv mAb compared to the germline <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036032#pone.0036032-Reynaud1" target="_blank">[16]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036032#pone.0036032-Reynaud2" target="_blank">[17]</a>. CDRs are identified by background shading. The D6 sequence was chosen as the germline D element for comparison <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036032#pone.0036032-Reynaud2" target="_blank">[17]</a>. Note the 18-residue insertion/duplication in CDR1 of V_λ of the anti-SAv mAb, recapitulating an insertion in light-chain CDR1 reported by others selecting anti-SAv mAbs from DT40 cells that had not undergone any genetic engineering <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036032#pone.0036032-Seo1" target="_blank">[8]</a>.</p