Molecular identification of Knops blood group polymorphisms found in long homologous region D of complement receptor 1.

Complement receptor 1 (CR1) has been implicated in rosetting of uninfected red blood cells to Plasmodium falciparum-infected cells, and rosette formation is associated with severe malaria. The Knops blood group (KN) is located on CR1 and some of these antigens, ie, McCoy (McC) and Swain-Langley (Sl(a)), show marked frequency differences between Caucasians and Africans. Thus, defining the molecular basis of these antigens may provide new insight into the mechanisms of P falciparum malaria. Monoclonal antibody epitope mapping and serologic inhibition studies using CR1 deletion constructs localized McC and Sl(a) to long homologous repeat D of CR1. Direct DNA sequencing of selected donors identified several single nucleotide polymorphisms in exon 29 coding for complement control protein modules 24 and 25. Two of these appeared to be blood group specific: McC associated with K1590E and Sl(a) with R1601G. These associations were confirmed by inhibition studies using allele-specific mutants. A sequence-specific oligonucleotide probe hybridization assay was developed to genotype several African populations and perform family inheritance studies. Concordance between the 1590 mutation and McC was 94%; that between Sl(a) and 1601 was 88%. All but 2 samples exhibiting discrepancies between the genotype and phenotype were found to be due to low red cell CR1 copy numbers, low or absent expression of some alleles, or heterozygosity combined with low normal levels of CR1. These data further explain the variability observed in previous serologic studies of CR1 and show that DNA and protein-based genetic studies will be needed to clarify the role of the KN antigens in malaria.

Decay accelerating activity of complement receptor type 1 (CD35). Two active sites are required for dissociating C5 convertases.

The goal of this study was to identify the site(s) in CR1 that mediate the dissociation of the C3 and C5 convertases. To that end, truncated derivatives of CR1 whose extracellular part is composed of 30 tandem repeating modules, termed complement control protein repeats (CCPs), were generated. Site 1 (CCPs 1-3) alone mediated the decay acceleration of the classical and alternative pathway C3 convertases. Site 2 (CCPs 8-10 or the nearly identical CCPs 15-17) had one-fifth the activity of site 1. In contrast, for the C5 convertase, site 1 had only 0.5% of the decay accelerating activity, while site 2 had no detectable activity. Efficient C5 decay accelerating activity was detected in recombinants that carried both site 1 and site 2. The activity was reduced if the intervening repeats between site 1 and site 2 were deleted. The results indicate that, for the C5 convertases, decay accelerating activity is mediated primarily by site 1. A properly spaced site 2 has an important auxiliary role, which may involve its C3b binding capacity. Moreover, using homologous substitution mutagenesis, residues important in site 1 for dissociating activity were identified. Based on these results, we generated proteins one-fourth the size of CR1 but with enhanced decay accelerating activity for the C3 convertases.

Independently melting modules and highly structured intermodular junctions within complement receptor type 1.

A segment of complement receptor type 1 (CR1) corresponding to modules 15-17 was overexpressed as a functionally active recombinant protein with N-glycosylation sites ablated by mutagenesis (referred to as CR1 approximately 15-17(-)). A protein consisting of modules 15 and 16 and another corresponding to module 16 were also overexpressed. Comparison of heteronuclear nuclear magnetic resonance (NMR) spectra for the single, double, and triple module fragments indicated that module 16 makes more extensive contacts with module 15 than with module 17. A combination of NMR, differential scanning calorimetry, circular dichroism, and tryptophan-derived fluorescence indicated a complex unfolding pathway for CR1 approximately 15-17(-). As temperature or denaturant concentration was increased, the 16-17 junction appeared to melt first, followed by the 15-16 junction, and module 17 itself; finally, modules 15 and 16 became denatured. Modules 15 and 16 adopted an intermediate state prior to total denaturation. These results are compared with a previously published study [Clark, N. S., Dodd, I, Mossakowska, D. E., Smith, R. A. G., and Gore, M. G. (1996) Protein Eng. 9, 877-884] on a fragment consisting of the N-terminal three CR1 modules which appeared to melt as a single unit.

Mapping epitopes for 20 monoclonal antibodies to CR1.

Complement receptor type one (CR1; CD35) binds and processes C3b and C4b opsonized immune complexes and regulates complement activation. We have characterized the epitopes of 13 previously reported and seven new MoAbs to human CR1. The MoAbs formed seven groups based on their reactivity with a panel of deletion forms of CR1. Seventeen of the MoAbs reacted with CR1 at more than one site, a consequence of its repetitive sequence. All five of the MoAbs recognizing epitopes in the nearly identical repeats 3, 10, and 17, as well as one MoAb which reacted with repeats 8 or 1/2 of 9 and 15 or 1/2 of 16, blocked cofactor activity for C3b. Knowledge of the repeats bearing the epitopes for these MoAbs should facilitate the further characterization of CR1.

Structure-function analysis of the active sites of complement receptor type 1.

Two functionally distinct but homologous sites in complement receptor type 1 (CR1) (CD35) were further characterized by homologous substitution mutagenesis of two CR1 derivatives, each containing one site. In both sites, reducing negative and/or increasing positive charge augmented interaction with iC3/C3b and C4b, supporting a role of ionic forces in the binding reaction. In one case, substitution of Asp at the end of complement control protein repeat (CCP) 2 with an Asn transformed the protein, with negligible cofactor activity and iC3 binding, into a mutant with activities similar to native CR1. Consequently, this protein, one-fourth the size of CR1, is a therapeutic candidate for a complement inhibitor. Another important observation is that the residues between two CCPs contribute to activity, probably because they influence positioning of one CCP relative to the next. The initial characterization of the third CCP of an active site led to identification of three peptides necessary for binding. In line with earlier findings for the first two CCPs, interactions with iC3/C3b are similar but not identical to those with C4b, implying overlapping but distinct binding domains. Moreover, changes in cofactor activity usually, but not always, parallel alterations in binding, indicating that these two activities are separable. We also mapped epitopes for a blocking and a function enhancing monoclonal antibody. Their effects can be explained by epitope location. The first antibody binds near functionally important residues. The second may shield inhibitory (negatively charged) residues. These results represent a comprehensive analysis of the active sites of CR1, which is built of modules found in more than 50 mammalian proteins.

Comparative effects of valproate and the new experimental anticonvulsant drug alpha-ethyl-alpha-methyl-thiobutyrolactone on selected metabolite levels in the plasma, livers, and brains of infant mice.

The newly discovered anticonvulsant drug, alpha-ethyl-alpha-methylthio butyrolactone (alpha-EMTBL),is as effective as valproate (VPA) in blocking seizures induced by convulsant compounds and maximal electric shock in adult mice. Other data have suggested that alpha-EMTBL might be less toxic than VPA and longer acting. We compared the effects of a single therapeutic dose of the two drugs on levels of key metabolites in the blood, liver, and brain of normal suckling infant mice. Unlike VPA, alpha-EMTBL had no adverse effects on selected aspects of liver carbohydrate, fat (ketogenesis), coenzyme A (CoA), carnitine, or amino acid metabolism. In contrast to VPA, alpha-EMTBL did not alter concentrations of neurotransmitter anticonvulsant activity of alpha-EMTBL in adult mice and its lack of acute hepatotoxicty in infant mice, our findings support further study of alpha-EMTBL as a potential adjunct to the relatively limited armamentarium of effective antiepileptic drugs (AEDs) for clinical use.

Effects of acute, subacute, and chronic diabetes on carbohydrate and energy metabolism in rat sciatic nerve. Relation to mechanisms of peripheral neuropathy.

To address the problem of the pathogenesis of diabetic neuropathy, rats were made diabetic by alloxan administration, and sciatic nerves were sampled for electrolyte and water content and levels of selected carbohydrates and intermediates in energy metabolism at 3, 6, and 26 weeks. Significant increases were seen in the nerve content of glucose, sorbitol, and fructose. Decreases of myo-inositol were not statistically significant. Glucose-6-phosphate was increased at all times; fructose-1,6-bisphosphate was elevated at 6 and 26 weeks. Nerve ATP and phosphocreatine levels were both increased concomitantly, as was the energy charge. Nerve lactate levels increased only at 26 weeks when plasma lactate levels were also high. Plasma ketone bodies were elevated throughout the 26-week experimental interval. It is postulated that ketone bodies were being used as alternative metabolic fuels in diabetic nerve, thereby causing inhibition of pyruvate oxidation and increased aerobic production of lactate. Increased plasma ketone body levels could also inhibit hepatic lactate uptake. There was no other evidence for hypoxia/ischemia. Lactate:pyruvate ratios did not differ from control values at any time in these ketotic hypoinsulinemic animals. Five major hypotheses have been proposed to explain the pathogenesis of diabetic neuropathy: 1) hypoxia/ischemia, 2) hyperglycemic pseudohypoxia, 3) myo-inositol deficiency, 4) fructose and polyol accumulation and osmotic disequilibrium, and 5) nonenzymatic glycation of macromolecules by fructose and glucose. The data obtained in this study seem to fit best with hypotheses 4 and perhaps 5.

Analysis of the functional domains of complement receptor type 1 (C3b/C4b receptor; CD35) by substitution mutagenesis.

The complement receptor type 1 (CR1; CD35), carrying 30 short consensus repeats (SCRs), has two sites. Site 1 contains SCR-1 and SCR-2 and binds C4b. Site 2 contains SCR-8 and SCR-9 and was reported to bind mainly C3b (Klickstein, L. B., Bartow, T. J., Miletic, V., Rabson, L. D., Smith, J. A., and Fearon, D. T. (1988) J. Exp. Med. 168, 1699-1717). For the functional analysis we used two constructs, each with one site. CR1-4, composed of eight and one-half initial SCRs, carries site 1, binds C4b, and is cofactor for C4b cleavage. CR1-4(8,9), obtained from CR1-4 by converting site 1 to site 2, binds iC3/C3b and, unexpectedly, C4b. It is a cofactor for cleavage of both ligands. Its cofactor activity for C4b cleavage is greater than that of site 1. Analysis of the mutants constructed by interchanging homologous peptides between the two sites identified no sequences necessary for cofactor activity other than those required for binding. In site 2, peptides important for both ligands were found. Some modifications of either site led to higher activity for both ligands. Thus the activity of complement regulators can be increased by changing a few amino acids within SCRs, an important step toward the generation of more effective inhibitors of complement activation. Knowledge of the active sites of CR1 should be applicable to other SCR-containing proteins and should provide insights into the evolution of these proteins.

Reversal of the adverse chronic effects of the unsaturated derivative of valproic acid--2-n-propyl-4-pentenoic acid--on ketogenesis and liver coenzyme A metabolism by a single injection of pantothenate, carnitine, and acetylcysteine in developing mice.

Like treatment with the parent compound valproic acid (VPA), acute and/or chronic treatment with the unsaturated derivative, 2-n-propyl-4-pentenoic acid (4-en-VPA), decreased ketogenesis and lowered free CoA, acetyl CoA, and free carnitine levels in the livers of normal developing mice. Concomitantly, there were manifold increases in the content of medium-chain acyl CoA esters (4-en-VPA CoA and 4-en-VPA CoA metabolites). Acute cotreatment of 4-en-VPA-treated animals with pantothenate, carnitine, and acetylcysteine caused significant amelioration of these metabolic aberrations. In animals chronically treated with 4-en-VPA, a single injection of pantothenate, carnitine, and acetylcysteine returned the 4-en-VPA-depressed levels of beta-hydroxybutyrate in plasma and free CoA and acetyl CoA in liver to normal. These findings support the hypothesis that VPA- and 4-en-VPA-induced hepatic dysfunction is produced by CoA sequestration rather than by irreversible inhibition by alkylation of the enzymes of fatty acid beta-oxidation by reactive intermediates. The findings also support the important but little-known role of carnitine in CoA metabolism--carnitine relieves the inhibition of pantothenate kinase, the rate-controlling first enzyme in the pathway of CoA synthesis by its product, free CoA, and by CoA esters.

Amelioration of adverse effects of valproic acid on ketogenesis and liver coenzyme A metabolism by cotreatment with pantothenate and carnitine in developing mice: possible clinical significance.

Very young children with organic brain damage, intractable seizures, and developmental retardation are at particular risk of developing fatal hepatic dysfunction coincident with valproate therapy, especially if the children are also receiving other anticonvulsant drugs. The mechanism of valproate-associated hepatic failure in these children is unclear. There are two major theories of etiology. The first concerns the manyfold consequences of depletion of CoA due to sequestration into poorly metabolized valproyl CoA and valproyl CoA metabolites. The other theory proposes that the unsaturated valproate derivative 2-n-propyl-4-pentenoic acid and/or metabolically activated intermediates are toxic and directly cause irreversible inhibition of enzymes of beta-oxidation. The present study shows for the first time that in developing mice, when panthothenic acid and carnitine are administered with valproate, at least some of the effects of valproate are mitigated. Perhaps most importantly, the beta-hydroxybutyrate concentration in plasma and the free CoA and acetyl CoA levels in liver do not fall so low. Cotreatment with carnitine alone was without effect. Findings support the CoA depletion mechanism of valproate inhibition of beta-oxidation and other CoA- and acetyl CoA-requiring enzymic reactions and stress the role of carnitine in the regulation of CoA synthesis at the site of action of pantothenate kinase.

myo-inositol: a newly identified nonnitrogenous osmoregulatory molecule in mammalian brain.

Sugar alcohols have been found to play an important osmoregulatory role both in unicellular organisms and, more recently, in multicellular organisms, including mammals. This study shows that myo-inositol accumulates in the brains of chronically hypernatremic mice, as had been earlier found in rats, and demonstrates for the first time a profound decrease of myo-inositol in the brains of chronically hyponatremic mice. Together with decreases in better known cerebral osmoles (amino acids and related nitrogenous compounds), the decrease in myo-inositol apparently allows the brain to balance its intracellular osmolality with that of the plasma, permitting a normal brain water content (no edema) despite profound hyponatremia.

Effect of momentary stress on brain energy metabolism in weanling mice: apparent use of lactate as cerebral metabolic fuel concomitant with a decrease in brain glucose utilization.

The hypothesis that the anxiety induced by repeated injections affects brain energy metabolism was tested. Normal 19- to 21-day-old mice were stressed by two sham intraperitoneal injections within 4 min, at which time they were decapitated. Noninjected, control littermates were quickly decapitated. Momentary stress increased plasma glucose (12%), glycerol (85%), beta-hydroxybutyrate (108%), and lactate (153%)--a reflection of elevated plasma cortisol (25%) and glucagon (45%). In brain, stress increased levels of glucose-6-P (15%) and fructose-6-P (17%). The brain pyruvate concentration increased 74%; lactate 76%. Citrate, alpha-ketoglutarate, and malate increased 15, 95, and 37%, respectively. Levels of glycogen, glucose, phosphocreatine, ATP, ADP, and AMP were unchanged. The brain lactate/pyruvate ratio was normal but the brain/plasma lactate ratio fell 32%. Metabolite changes in the stressed animals were compatible with a decrease in the glycolytic flux at the phosphofructokinase step and a paradoxical increased flux in the Krebs citric acid cycle. The decreased brain/plasma lactate ratio supported increased uptake of lactate from plasma and increased brain lactate oxidation. Metabolite changes similar to those described above occurred in unstressed mice injected with lactate. Findings confirm a positive effect of stress on brain metabolism, support a role for lactate as an oxidative fuel for brain, and caution that the rate of cerebral glucose utilization may not always reflect brain energy (oxidative) metabolism accurately.

Valproate doubles the anoxic survival time of normal developing mice: possible relevance to valproate-induced decreases in cerebral levels of glutamate and aspartate, and increases in taurine.

We have previously reported that chronic administration of valproate in developing mice decreased brain aspartic and glutamic acid levels and increased the brain taurine content. The direction of the valproate-induced changes in the cerebral levels of these neurotransmitter amino acids - excitatory in the case of aspartate and glutamate, inhibitory in the case of taurine - appeared relevant to the mechanism of its anticonvulsant action. Since the neuropathology of hypoxia-ischemia also appears to be mediated by release of glutamate/aspartate at the synapse, the valproate-induced reduction of the levels of these neuroexcitatory/neurotoxic amino acids suggested that valproate might increase the tolerance of young mice to anoxia. A doubling of the length of survival of the intact animal in an atmosphere of pure nitrogen gas and a three-fold increase in the duration of respiratory activity (gasping) of the isolated head after chronic administration of valproate support the speculation.

Adaptive decreases in amino acids (taurine in particular), creatine, and electrolytes prevent cerebral edema in chronically hyponatremic mice: rapid correction (experimental model of central pontine myelinolysis) causes dehydration and shrinkage of brain.

The experimental model of central pontine myelinolysis--chronic (4-day) hyponatremia induced by daily injections of hypotonic dextrose solutions and vasopressin followed by rapid correction with saline--was used in young fasted and thirsted mice. In normal controls chronic fasting and thirsting lowered plasma and brain glucose levels and cerebral glycolytic and tricarboxylic acid cyclic metabolic fluxes. The fasting state had little effect on brain amino acids. Clinically, the animals became semistuporous; about one-third died. Chronic hyponatremia in fasted mice almost tripled the plasma glucose concentrations and increased the brain carbohydrate reserve. Levels of other brain glycolytic and Krebs citric acid cycle intermediates were similar to those of controls. Severe hyponatremia and hypoosmolality induced profound decreases in levels of brain electrolytes, amino acids (especially taurine), and creatine. These changes permitted a new osmotic balance between blood and brain and a normal brain water content. The behavior and mortality of the hyponatremic animals were not different from those of the fasted control mice. Correction of hyponatremia to normonatremic levels over a 9-hr period returned brain Na+ and K+ levels to normal but the contents of the measured amino acids and creatine were still reduced one-third or more. As a result, treatment produced a significant degree of dehydration and shrinkage of the brain. The findings stress the importance of amino acids (taurine in particular) and creatine levels, as well as electrolytes, in brain osmoregulation and suggest a role for an osmotic disequilibrium--blood osmolality higher than brain--in the production of brain lesions following rapid correction of chronic hyponatremia in animals and possibly in humans. Replenishment of depleted brain K+ and amino acid levels, as well as slow elevation of the chronically depressed level of plasma Na+, is recommended.

Delayed neurotoxicity of excitatory amino acids in vitro.

The acute neurotoxicity produced by glutamate and related excitatory amino acids is probably caused by depolarization leading to excessive anionic and cationic fluxes and osmotic lysis. Recently, a more delayed form of glutamate neurotoxicity, which is critically dependent upon calcium influx, has been described in cultured neocortex. We investigated this phenomenon in cultures of dispersed rat hippocampal neurons. When these cultures were briefly incubated with various excitatory amino acids in low extracellular chloride, there was no acute toxicity, but a gradual drop-out of neurons occurred over the next day. When calcium was removed from the extracellular medium during amino acid incubation, this late neuronal loss was not seen. Interestingly, blocking excitatory amino acid receptors in cultures after the amino acid exposure also prevented this delayed neuronal death. In addition, these treated cultures contained neurons with normal physiological properties, and had concentrations of adenosine triphosphate that were close to control values. The findings suggest an amino acid-induced calcium influx may elevate the release of endogenous excitatory transmitter, likely glutamate, and/or increase the sensitivity of these neurons to glutamate. These in vitro observations may partially explain the delayed neuronal loss seen in some pathological conditions affecting man.

Brain amino acids decrease in chronic hyponatremia and rapid correction causes brain dehydration: possible clinical significance.

In animals, rapid correction of chronic hyponatremia produces brain lesions similar to those seen in central pontine myelinolysis. This is the first study of the effects of rapid correction (9 h) of chronic hyponatremia (3 d) on brain electrolyte, water, and amino acid contents in young mice. Despite profound hyponatremia, decreases in brain electrolytes and amino acids permitted an apparent osmotic balance between blood and brain with a normal brain water content. Rapid elevation of the depressed plasma sodium concentration to normonatremic levels caused dehydration of the brain. Although brain Na+ and K+ levels were returned to normal, the relatively brief interval of treatment was insufficient to allow complete recovery of brain amino acid levels. Findings support an osmotic disequilibrium--plasma osmolality higher than brain--in the pathogenesis of the brain lesions following rapid correction of chronic hyponatremia and suggest caution in the rate of elevation of the depressed plasma Na+ levels.

Ketamine protects hippocampal neurons from anoxia in vitro.

Ketamine, a dissociative, general anesthetic, blocks the excitation produced by activating one class of excitatory amino acid receptors, the N-methyl-D-aspartate receptor in the rat. We have found that ketamine can protect hippocampal neurons in culture and slice from anoxia. When added to cultures immediately prior to anoxic exposure, ketamine prevented the neuronal destruction seen after a day of anoxia. Neurons appeared undamaged and had normal resting and action potentials. Adenosine triphosphate levels in ketamine-protected anoxic cultures were approximately two-thirds of normal controls. Ketamine also prevented the irreversible loss of the population spike seen in hippocampal slices after prolonged perfusion with anoxic buffer. These results suggest that ketamine may have therapeutic potential in preventing anoxic damage from stroke in man.