To examine the role of germinal centers (GCs) in the generation and selection of high affinity antibody-forming cells (AFCs), we have analyzed the average affinity of (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific AFCs and serum antibodies both during and after the GC phase of the immune response. In addition, the genetics of NP-binding AFCs were followed to monitor the generation and selection of high affinity AFCs at the clonal level. NP-binding AFCs gradually accumulate in bone marrow (BM) after immunization and BM becomes the predominant locale of specific AFCs in the late primary response. Although the average affinity of NP-specific BM AFCs rapidly increased while GCs were present (GC phase), the affinity of both BM AFCs and serum antibodies continued to increase even after GCs waned (post-GC phase). Affinity maturation in the post-GC phase was also reflected in a shift in the distribution of somatic mutations as well as in the CDR3 sequences of BM AFC antibody heavy chain genes. Disruption of GCs by injection of antibody specific for CD154 (CD40 ligand) decreased the average affinity of subsequent BM AFCs, suggesting that GCs generate the precursors of high affinity BM AFCs; inhibition of CD154-dependent cellular interactions after the GC reaction was complete had no effect on high affinity BM AFCs. Interestingly, limited affinity maturation in the BM AFC compartment still occurs during the late primary response even after treatment with anti-CD154 antibody. Thus, GCs are necessary for the generation of high affinity AFC precursors but are not the only sites for the affinity-driven clonal selection responsible for the maturation of humoral immune responses.

Organophosphorus compounds include many synthetic, neurotoxic substances that are commonly used as insecticides. The toxicity of these compounds is due to their ability to inhibit the enzyme acetylcholine esterase. Some of the most toxic organophosphates have been adapted for use as chemical warfare agents; the most well known are GA, GB, GD, GF, VX and VR. All of these compounds contain a chiral phosphorus center with the S P -enantiomers being significantly more toxic than the R P -enantiomers. Phosphotriesterase (PTE) is an enzyme capable of detoxifying these agents, but the stereochemical preference of the wild-type enzyme is for the R P -enantiomers. A series of enantiomerically pure chiral nerve agent analogues has been developed containing the relevant phosphoryl centers found in GB, GD, GF, VX and VR. Wild-type and mutant forms of PTE have been tested for their ability to hydrolyze this series of compounds. Mutant forms of PTE with significantly enhanced, as well as relaxed or reversed stereoselectivity, have been identified. A number of variants showed dramatically improved kinetic constants for the catalytic hydrolysis of the more toxic S P -enantiomers. Improvements of up to three orders of magnitude relative to the wild type enzyme were observed. Some of these mutants were tested against racemic mixtures of GB and GD. The kinetic constants obtained with the chiral nerve agent analogues accurately predict the improved activity and stereoselectivity against the authentic nerve agents used in this study.Organophosphorus compounds have been utilized for more than 50 years as insecticides for the protection of agricultural crops (1) and similar compounds have been developed as chemical warfare agents (2). The structures of these latter compounds are presented in Scheme 1 and include tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), VX and VR. GA has a cyanide leaving group, the three remaining G-agents (GB, GD, and GF) have a fluoride leaving group, and the two versions of VX have a thiolate leaving group. The toxicity of these organophosphonates is due to the inactivation of acetylcholinesterase (AChE), an enzyme that catalyzes the hydrolysis of acetylcholine at neural synapses, through the phosphonylation of an active site serine residue (3). GA, GB, GF, VX, and VR contain a chiral phosphorus center and thus each of these nerve agents has two stereoisomers, while soman has four stereoisomers because of an additional chiral center within the pinacolyl substituent. The enantiomers are differentially toxic; the S Pstereoisomer of sarin reacts with AChE approximately ~10 4 times faster than the R Pstereoisomer and the two S P -stereoisomers of soman react ~10 5 times faster than the two † This work was supported by the NIH (GM 68550).

Dangerous organophosphorus (OP) compounds have been used as insecticides in agriculture and in chemical warfare. Because exposure to OP could create a danger for humans in the future, butyrylcholinesterase (BChE) has been developed for prophylaxis to these chemicals. Because it is impractical to obtain sufficient quantities of plasma BChE to treat humans exposed to OP agents, the production of recombinant BChE (rBChE) in milk of transgenic animals was investigated. Transgenic mice and goats were generated with human BChE cDNA under control of the goat ␤-casein promoter. Milk from transgenic animals contained 0.1-5 g/liter of active rBChE. The plasma half-life of PEGylated, goat-derived, purified rBChE in guinea pigs was 7-fold longer than non-PEGylated dimers. The rBChE from transgenic mice was inhibited by nerve agents at a 1:1 molar ratio. Transgenic goats produced active rBChE in milk sufficient for prophylaxis of humans at risk for exposure to OP agents.organophosphorus nerve agent ͉ recombinant protein expression ͉ transgenic production H uman plasma butyrylcholinesterase (huBChE) (EC 3.1.1.8) is a globular, tetrameric serine esterase with a molecular mass of Ϸ340 kDa that is stable in plasma with a half-life of Ϸ12 days (1, 2). Although the physiological function of huBChE is unclear, the enzyme prevents intoxication of animals exposed to organophosphorus (OP) compounds (3, 4). The huBChE enzyme also hydrolyzes many ester-containing drugs, such as cocaine and succinylcholine (5). The toxicity of OP agents is due to irreversible inhibition of acetylcholinesterase and the subsequent continuous stimulation of neurons by acetylcholine (6). Administration of exogenous huBChE, which irreversibly binds OP agents to prevent inactivation of acetylcholinesterase and continuous cholinergic stimulation, is a potential strategy for preventing toxicity from OP agents (4). Although huBChE has been obtained from human plasma by a large scale purification technique, this procedure is severely limited by the volume of human plasma needed (7). It is unlikely that a sufficient amount of enzyme could be purified commercially by this technique. Because of the 1:1 stoichiometry required for protection against exposure to OP agents (8), large quantities of huBChE are needed for effective prophylaxis and treatment of exposure. Compared with other potential enzymatic bioscavengers of OP agents, huBChE has a broad spectrum of activity, a relatively long half-life, and limited, if any, physiological side effects (9). Producing recombinant BChE (rBChE) is an alternative to purification of the enzyme from human plasma. Recombinant huBChE has been expressed in Escherichia coli (10), albeit in a nonfunctional form; mammalian 293T (11); and CHO (12) cells. However, these expression systems cannot economically produce sufficient quantities of rBChE with a residence time similar to native huBChE that would allow development of the enzyme as an agent for prophylaxis against OP poisoning.The production of recombinant proteins by the mammary g...

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The role of apoptosis in affinity maturation was investigated by determining the affinity of (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific antibody-forming cells (AFCs) and serum antibody in transgenic mice that overexpress a suppressor of apoptosis, Bcl-xL, in the B cell compartment. Although transgenic animals briefly expressed higher numbers of splenic AFCs after immunization, the bcl-x L transgene did not increase the number or size of germinal centers (GCs), alter the levels of serum antibody, or change the frequency of NP-specific, long-lived AFCs. Nonetheless, the bcl-x L transgene product, in addition to endogenous Bcl-xL, reduced apoptosis in GC B cells and resulted in the expansion of B lymphocytes bearing VDJ rearrangements that are usually rare in primary anti-NP responses. Long-lived AFCs bearing these noncanonical rearrangements were frequent in the bone marrow and secreted immunoglobulin G1 antibodies with low affinity for NP. The abundance of noncanonical cells lowered the average affinity of long-lived AFCs and serum antibody, demonstrating that Bcl-xL and apoptosis influence clonal selection/maintenance for affinity maturation.

The concept of using cholinesterase bioscavengers for prophylaxis against organophosphorous nerve agents and pesticides has progressed from the bench to clinical trial. However, the supply of the native human proteins is either limited (e.g., plasma-derived butyrylcholinesterase and erythrocytic acetylcholinesterase) or nonexisting (synaptic acetylcholinesterase). Here we identify a unique form of recombinant human butyrylcholinesterase that mimics the native enzyme assembly into tetramers; this form provides extended effective pharmacokinetics that is significantly enhanced by polyethylene glycol conjugation. We further demonstrate that this enzyme (but not a G117H/E197Q organophosphorus acid anhydride hydrolase catalytic variant) can prevent morbidity and mortality associated with organophosphorous nerve agent and pesticide exposure of animal subjects of two model species.countermeasures | nonconventional warfare agents | organophosphorous pesticides | protein engineering | transgenic plants B utyrylcholinesterase (BChE) is the major cholinesterase (ChE) in the serum of humans (1, 2). Although the closely related enzyme acetylcholinesterase (AChE) is well described as the primary synaptic regulator of cholinergic transmission, a definitive physiological role for BChE has not yet been demonstrated (3). BChE is catalytically promiscuous and hydrolyzes not only acetylcholine (ACh), but also longer-chain choline esters (e.g., butyrylcholine, its preferred substrate, and succinylcholine) and a variety of non-choline esters, such as acetylsalicylic acid (aspirin) and cocaine (4, 5). Moreover, BChE binds most environmentally occurring ChE inhibitors as well as man-made organophosphorous (OP) pesticides and nerve agents (NAs) (6, 7-10).The systemic biodistribution and affinity for ChE inhibitors allow endogenous BChE to provide broad-spectrum protection against various toxicants by their sequestration before they reach cholinergic synapses. However, under realistic high-dose exposure scenarios, BChE serum levels are too low to afford adequate protection, resulting in persistent cholinergic excitation due to irreversible inhibition of AChE and subsequent accumulation of ACh. Sublethal manifestations of this state include unregulated exocrine secretion and gastrointestinal hypermotility. Death usually results from unregulated stimulation at neuromuscular junction leading to hemodynamic instability and tetanic contraction of the respiratory muscles (11,12).Current OP poisoning therapy consists of atropine for muscarinic ACh receptor blockade and diazepam for symptomatic management of convulsions (12). Additionally, oxime therapy with 2-pralidoxime (2-PAM) can effectively reactivate some but not all OP-AChE adducts (13)(14)(15). This standard therapeutic approach can reduce mortality, but insufficiently prevents the incapacitation associated with OP toxicity (12, 16).Prophylaxis by administration of exogenous ChEs has proven successful in reducing OP-associated morbidity and mortality, but requires the availability of rel...

and Cerasoli, Douglas M., "Structure/function analyses of human serum paraoxonase (HuPON1) mutants designed from a DFPaselike homology model" (2004 AbstractHuman serum paraoxonase (HuPON1) is a calcium-dependent enzyme that hydrolyzes esters, including organophosphates and lactones, and exhibits anti-atherogenic properties. A few amino acids have been shown to be essential for the enzyme's arylesterase and organophosphatase activities. Until very recently, a three-dimensional model was not available for HuPON1, so functional roles have not been assigned to those residues. Based on sequence-structure alignment studies, we have folded the amino acid sequence of HuPON1 onto the sixfold h-propeller structure of squid diisopropylfluorophosphatase (DFPase). We tested the validity of this homology model by circular dichroism (CD) spectroscopy and site-directed mutagenesis. Consistent with predictions from the homology model, CD data indicated that the structural composition of purified HuPON1 consists mainly of h-sheets. Mutants of HuPON1 were assayed for enzymatic activity against phenyl acetate and paraoxon. Substitution of residues predicted to be important for substrate binding (L69, H134, F222, and C284), calcium ion coordination (D54, N168, N224, and D269), and catalytic mechanism of HuPON1 (H285) led to enzyme inactivation. Mutants F222Y and H115W exhibited substrate-binding selectivity towards phenyl acetate and paraoxon, respectively. The homology model presented here is very similar to the recently obtained PON1 crystal structure, and has allowed identification of several residues within the enzyme active site. D

We have used hydrogen-exchange labeling detected by 2D NMR to study antibody-protein interactions for two monoclonal antibodies raised against horse cytochrome c. The data show that these antibodies bind mainly to the large 37-59 omega-loop of the cytochrome c molecule. In addition, the results provide some suggestive evidence concerning units of local structural flexibility in cytochrome c.