A serine protease isolated from the bristles of the Amazonic caterpillar, Premolis semirufa, is a potent complement system activator.

BACKGROUND: The caterpillar of the moth Premolis semirufa, commonly named pararama, is found in the Brazilian Amazon region. Accidental contact with the caterpillar bristles causes an intense itching sensation, followed by symptoms of an acute inflammation, which last for three to seven days after the first incident. After multiple accidents a chronic inflammatory reaction, called "Pararamose", characterized by articular synovial membrane thickening with joint deformities common to chronic synovitis, frequently occurs. Although complement mediated inflammation may aid the host defense, inappropriate or excessive activation of the complement system and generation of anaphylatoxins can lead to inflammatory disorder and pathologies. The aim of the present study was to evaluate, in vitro, whether the Premolis semirufa's bristles extract could interfere with the human complement system. RESULTS: The bristles extract was able to inhibit the haemolytic activity of the alternative pathway, as well as the activation of the lectin pathway, but had no effect on the classical pathway, and this inhibition seemed to be caused by activation and consumption of complement components. The extract induced the production of significant amounts of all three anaphylatoxins, C3a, C4a and C5a, promoted direct cleavage of C3, C4 and C5 and induced a significant generation of terminal complement complexes in normal human serum. By using molecular exclusion chromatography, a serine protease of 82 kDa, which activates complement, was isolated from P. semirufa bristles extract. The protease, named here as Ps82, reduced the haemolytic activity of the alternative and classical pathways and inhibited the lectin pathway. In addition, Ps82 induced the cleavage of C3, C4 and C5 and the generation of C3a and C4a in normal human serum and it was capable to cleave human purified C5 and generate C5a. The use of Phenanthroline, metalloprotease inhibitor, in the reactions did not significantly interfere with the activity of the Ps82, whereas the presence of PMSF, serine protease inhibitor, totally blocked the activity. CONCLUSION: These data show that a serine protease present in the Premolis semirufa's bristles extract has the ability to activate the complement system, which may contribute to the inflammatory process presented in humans after envenomation.

A high-throughput protein refolding screen in 96-well format combined with design of experiments to optimize the refolding conditions.

Production of correctly folded and biologically active proteins in Escherichiacoli can be a challenging process. Frequently, proteins are recovered as insoluble inclusion bodies and need to be denatured and refolded into the correct structure. To address this, a refolding screening process based on a 96-well assay format supported by design of experiments (DOE) was developed for identification of optimal refolding conditions. After a first generic screen of 96 different refolding conditions the parameters that produced the best yield were further explored in a focused DOE-based screen. The refolding efficiency and the quality of the refolded protein were analyzed by RP-HPLC and SDS-PAGE. The results were analyzed by the DOE software to identify the optimal concentrations of the critical additives. The optimal refolding conditions suggested by DOE were verified in medium-scale refolding tests, which confirmed the reliability of the predictions. Finally, the refolded protein was purified and its biological activity was tested in vitro. The screen was applied for the refolding of Interleukin 17F (IL-17F), stromal-cell-derived factor-1 (SDF-1α/CXCL12), B cell-attracting chemokine 1 (BCA-1/CXCL13), granulocyte macrophage colony stimulating factor (GM-CSF) and the complement factor C5a. This procedure identified refolding conditions for all the tested proteins. For the proteins where refolding conditions were already available, the optimized conditions identified in the screening process increased the yields between 50% and 100%. Thus, the method described herein is a useful tool to determine the feasibility of refolding and to identify high-yield scalable refolding conditions optimized for each individual protein.

Isolation of the third component of complement and its derivative with anaphylatoxin-like activity from the plasma of the newt Cynops pyrrhogaster.

The third component of complement (C3) of a newt, Cynops pyrrhogaster, was purified using a fast protein liquid chromatography technique. The purified newt C3 consists of two polypeptide chains (the molecular masses of the alpha and beta-chains of C3 were 120,000 and 70,000, respectively) linked by disulfide bonds. The alpha-chain retained an internal thiolester bond that was cleaved with methylamine, and the N-terminal amino acid sequence of the alpha-chain was XVQLIDAKAGKAAKF. Digestion of newt C3 with trypsin yielded fragments that induced significant histamine release from newt peritoneal cells. These results indicate that newt C3 retains structural and functional properties shared with mammalian C3.

Bidder's organ extract induced anaphylaxis in experimental animals.

Bidder's organ (BO, a vestigeal organ), present in toad Bufo melanostictus (Schenider), is a characteristic feature of all male bufo. Its possible anaphylactic properties are investigated on experimental animals. BO extract produced both in vivo and in vitro anaphylactic reaction in guineapig. Dyspnoea and bronchoconstriction was a major cause of anaphylactic death. Blood histamine level was significantly increased in the anaphylactic animals. BO extract significantly released histamine from chopped lung preparation, an action antagonised by disodium chromoglycate. BO extract degranulated peritoneal mast cell in vitro. Passive cutaneous anaphylactic reactions were enhanced by BO extract and were significantly inhibited by disodium chromoglycate. Anaphylotoxin (identity not known) present in bidder's organ is probably involved in toad defence.

Adsorption of anaphylatoxins from the plasma of systemic lupus erythematosus patients using dextran sulfate cellulose columns.

Complement-derived anaphylatoxin may be one of the causes of vascular injury and an indicator of activity in systemic lupus erythematosus (SLE). The present study examines the effectiveness of dextran sulfate (DS) column immunoadsorption treatment to remove anaphylatoxins (C3a, C4a, and C5a) from the blood of patients with SLE. Seven SLE patients were subjected to immunoadsorption using DS-bound cellulose columns (Selesorb, Kaneka). Blood samples were taken both before and after the immunoadsorption session. Specimens were also obtained from both the inlets and outlets of the DS columns every 1,000 ml of treated plasma volume. The DS columns removed anaphylatoxins C3a and C4a from the separated plasma (from 775+/-334 ng/ml to 640+/-252 ng/ml, and from 1,303+/-847 ng/ml to 619+/-578 ng/ml, respectively) during the clinical anti-DNA apheresis procedure. In these study, the C5a levels in the circulating plasma of SLE patients were not elevated. To confirm whether DS-bound cellulose beads adsorbs anaphylatoxins in vitro, zymosan-activated plasma (ZAP) containing high levels of anaphylatoxins was incubated with DS-bound cellulose beads. The levels of C3a, C4a and C5a in the ZAP significantly decreased by mixing with DS-bound cellulose beads (P<0.05). Nevertheless, C3a and C4a in the peripheral blood were not significantly decreased after the immunoadsorption, suggesting that these anaphylatoxins bypass the DS columns in apheresis and return to the patient via the cell-rich fraction.

Biocompatibility evaluation of polyamide hemofiltration.

INTRODUCTION: Postdilution hemofiltration with a polyamide membrane is a renal replacement technique widely used, but very little information is available regarding the biocompatibility of this treatment. In this paper we report the results of an acute study of the biocompatibility of polyamide hemofiltration. PATIENTS AND METHODS: Complement activation such as C3a and C5a Des Arg (RIA), granulocyte degranulation like alpha 1 elastase intradialytic increase (ELISA) and the expression of high affinity membrane receptors for IL-2 (anti-TAC) were determined. Beta 2-microglobulin (RIA) intradialytic decrease, as well as its convective removal, was evaluated. The nature of protein layer adsorbed onto the polyamide membrane, at the end of the dialytic session was investigated with a new immunohistochemical technique. Cell-associated cytokine concentration (like IL-1 beta and IL-1Ra - ELISA) was determined on mononuclear cell lysates. RESULTS: A low degree of complement activation was detected with the polyamide membrane when data were adjusted for hemoconcentration and for 1 m2 of membrane surface area. An important convective removal not only of Beta 2-microglobulin (258+/-20 mg/session), but also of the activated anaphylatoxins (225+/-76 ng/ml for C3a and 22.5+/-4 ng/ml for C5a) was revealed. A marked deposition of all coagulation factors with no detectable amount of immunoglobulins and complement factors was revealed on the polyamide membrane at the end of the dialytic session. No intradialytic (for IL-1beta) (from 14. 1+/-3.0 to 13.5+/-2.9 pg/2.5 x 10(6) cell) and interdialytic (for IL-1Ra) (from 4572+/-1076 to 5408+/-615 pg/2.5 x 10(6) cell) cell-associated cytokine expression was induced by hemofiltration. DISCUSSION AND CONCLUSION: Polyamide hemofiltration is a highly biocompatible technique due to the use of a synthetic membrane with a sterile reinfusion fluid and the convective removal of the activated anaphylatoxins and Beta 2-microglobulin.

The possibility of improving biocompatibility by removing anaphylatoxins from the circuit of blood-perfused artificial organs.

The possibility of reducing bioreactivity during extracorporeal circulation by removing bioactive substances "turned on" in the devices is described in this paper. The bioreaction and bioactive products, as represented by neutrophil reaction and activated complement fragments generated during the use of a blood-perfused artificial organ, were studied as a model. Hemoperfusion with activated charcoal (AC) was used to remove the activated complement fragments. The adsorption of anaphylatoxins (C3a, C4a, and C5a) was evaluated following plasma perfusion through AC in vitro, all three of which were satisfactorily adsorbed. Six kinds of AC (three HEMA coated AC, two uncoated AC, and one cellulose coated AC) were compared with respect to adsorption of C5a in vitro. Uncoated AC adsorbed well and rapidly, but the coating with HEMA made adsorption take place slowly. The cellulose coating activated complement and did not decrease plasma C5a levels. After the adsorption of anaphylatoxins was confirmed, each of the AC columns was placed on-line after a cuprophan dialyzer in five chronic hemodialysis patients. C3a levels and neutrophil counts were compared with those obtained during usual hemodialysis (HD). By using heparin treated, uncoated AC, elevation of C3a levels and transient neutropenia were suppressed. Peak C3a with AC was reduced by 150% compared with prevalues, and the neutropenia nadir increased from 20% to 40% of baseline. The adsorption ability influenced the reduction in neutropenia. The heparinized uncoated AC and the column with large volumes of HEMA-coated AC had the best effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural characterization of the C4a anaphylatoxin from rat.

The C4a anaphylatoxin was purified from rat sera activated by heat-aggregated IgG. The anaphylatoxin was isolated by a three-step purification procedure and was judged to be homogeneous based on visualization of a single stained band after electrophoresis on both cellulose acetate membrane strips and on 9% SDS-polyacrylamide gels. Results from Ouchterlony and radioimmunoassay analysis indicated that neither rat C5A nor C3a contaminated the C4a preparation. Rat C4a is a glycoprotein estimated to be 11,000-12,000 mol. wt and contains 76 amino acid residues representing a mol. wt of 8577 and one oligosaccharide unit of 2000-3000 mol. wt. Rat C4a is weakly active in contracting guinea pig ileum at 0.1-1 microM, which is comparable with the activity of human C4a. Both human and bovine C4a are polypeptides free of carbohydrate while rat and presumably mouse C4a are glycoproteins. The complete primary structure of rat C4a anaphylatoxin has been elucidated as follows: (formula; see text)

Amino acid sequence of guinea pig C3a anaphylatoxin.

The anaphylatoxin peptide, C3a, was isolated in its inactive des-arginine form from complement-activated guinea pig serum and the complete amino acid sequence was determined. The peptide was isolated using a modification of previously described methods for anaphylatoxin purification and yielded 18 mg of guinea C3ades Arg judged homogeneous by cellulose acetate electrophoresis, high performance liquid chromatography, amino acid composition, and sequence analyses. The complete amino acid sequence of the molecule was determined by automated Edman degradation of intact C3ades Arg following performic acid oxidation, and of four peptides obtained from CNBr, endoproteinase Lys-C, or endoproteinase Glu-C (V8 protease) digests of reduced and alkylated C3ades Arg. The sequence is as follows: Guinea pig C3ades Arg has approximately 70% overall sequence identity with human, porcine, rat and mouse anaphylatoxins. As with C3a's from other species, the guinea pig molecule has an invariant C-terminal tetrapeptide sequence, LGLA, and six invariant cysteinyl residues at positions 23, 24, 37, 50, 57, and 58.

Inability of rat anaphylatoxin to induce histamine release in rats.

The role of rat anaphylatoxin in histamine release and increased vascular permeability during the first thirty minute period in zymosan-air-pouch inflammation, an experimental model of inflammation induced by zymosan in an air-pouch prepared on the back of rats, was investigated. Complement depletion by cobra venom factor did not affect the histamine release nor the increased vascular permeability in the inflammation of this type. In spite of apparent anaphylatoxin activity, zymosan activated serum (ZAS) failed to cause any significant release of histamine when infused in the air-pouch on the back. Anaphylatoxin purified from rat serum activated with zymosan in the presence of an inhibitor (epsilon-aminocaproic acid) of anaphylatoxin inactivator gave a single band in both polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE. The molecular weight estimated by SDS-PAGE was approx. 7,000. The purified rat anaphylatoxin failed to induce histamine release nor increased vascular permeability even at 50 micrograms/ml, although it caused contraction of guinea pig ileum at 0.8 micrograms/ml. These results suggest that rat anaphylatoxin does not participate in histamine release and increased vascular permeability in the zymosan-air-pouch inflammation.

Isolation of three separate anaphylatoxins from complement-activated human serum.

Recent methodologies used in preparing anaphylatoxins from complement-activated serum are described. Activation of the alternative pathway generates C3a and C5a; however, activation of the classical pathway is required to generate the anaphylatoxin from C4. This article describes an activation scheme that simultaneously generates all three of the anaphylatoxins (e.g., C3a, C4a and C5a) in human serum and outlines a procedure for isolating each as homogeneous products. Purification of intact anaphylatoxins directly from complement-activated serum takes place only if an exopeptidase in serum, known as carboxypeptidase N (SCPN), is properly inhibited. A new series of mercapto derivatives of arginine analogs are introduced as potent and effective inhibitors of SCPN. These inhibitors permit normal complement activation but prevent degradation of the released activation fragments C3a, C4a or C5a. The SCPN inhibitor previously used was 6-aminohexanoic acid (EACA), but it required a 1 M concentration for effective inhibition, the substituted mercapto-guanido compounds prove to be effective in the mM range.

Characterization of human C4a anaphylatoxin.

Human C4a anaphylatoxin was isolated from a Cls digest of the fourth component of complement. Isolation required a two-step procedure involving ion-exchange chromatography on CM-Sephadex C-50 and gel filtration on Sephadex G-50. Characterization of C4a indicated it is a highly cationic polypeptide (pI = 9.0-9.5) containing 77 residues with Mr = 8,759. C4a is devoid of tryptophan, histidine, and carbohydrate. Judged by the shape and magnitude of its circular dichroism spectrum, 54% of the polypeptide backbone of C4a assumes an alpha-helical conformation. Partial NH2-terminal sequence determination of C4a revealed a sequence identical with that published by Bolotin et al. (Bolotin, C., Morris, S., Tack, B., and Prahl, J. (1977) Biochemistry 16, 2008-2015) for the NH2 terminus of the alpha-subunit of human C4. Comparison of the NH2-terminal sequence of C4a with the sequences of complement activation fragments C3a (Hugli, T.E. (1975) J. Biol. Chem. 250, 8293-8301) and C5a (Fernandez, H.N., and Hugli, T.E. (1978) J. Biol. Chem, 253-6955-6962) showed that of the first 24 NH2-terminal residues of C4a, 6 were identical with those of C3a (25% homology) and 8 were identical with those of C5a (33% homology). These data represent the first chemical evidence for the existence of an evolutionary relationship among anaphylatoxins C3a, C4a, and C5a, and imply that a similar relationship exists among their precursor proteins.

Low molecular weight plasma proteins isolated from preparations of human immunoglobulin.

Low molecular weight proteins co-purified with IgG constitute 0.22% of the total protein purified from human plasma by ion-exchange chromatography on DEAE-cellulose. We have found that these low molecular weight proteins were obtained free of immunoglobulin by ultrafiltration in 5 M guanidinium chloride. Electrophoresis and isoelectric focusing in polyacrylamide gels demonstrated that this fraction of low molecular weight proteins is remarkably heterogeneous. Chromatography of an Mr 6000 to 12 000 fraction on hydroxyapatite resolved fourteen discrete protein peaks. Three of the peaks contained proteins which appeared to be homogeneous on acid-urea polyacrylamide gels. Two of these proteins were similar in composition to B2 globulin and may represent degradation products of some larger protein. The third protein was found to have an amino-terminal sequence identical to C3a. This population of low molecular weight plasma proteins has previously been shown to contain the cystic fibrosis mucociliary inhibitor and is here shown to contain two proteins similar to B2 globulin, C3a and many proteins remaining to be characterized. The presence of these low molecular weight proteins in measurable concentrations may be insufficiently appreciated in studies using 'purified' immunoglobulins as biological or chemical probes.

Mediators of immune complex-induced aggregation of polymorphonuclear neutrophils. I. C5a anaphylatoxin, neutrophil cationic proteins and their cleavage fragments.

This study reports the results of in vitro investigations on the aggregation of polymorphonuclear neutrophils (PMN) induced by the C5a anaphylatoxin complement component as well as the cationic proteins (CP), which are released by challenging PMN with immune complexes (IC). The carboxy-peptidase-derived des-Arg fragments of CP and C5a; CPi and C5ai, inactive in terms of anaphylactic and chemotactic activity, nevertheless showed a more potent ability to aggregate PMN than CP and C5a. The process of PMN aggregation required metabolic energy and divalent cations, Ca++ and Mg++. The microtubular system and the subplasmalemmal microfilaments appeared to be of critical importance. Electron microscopic studies on aggregates of PMN obtained on stimulation with CP, C5a, CPi and C5ai showed parallel tracts of variable length of cell membranes at the points where cells were in contact with each other.

Purification and partial amino acid sequence of classical anaphylatoxin from pig serum: identification with Des-Arg-C5a.

Complement was activated in hog serum and the biologically active peptide(s) derived from the fifth component of complement were purified. Treatment of the serum with yeast, without any precautions to inhibit serum arginine carboxypeptidase, allowed the recovery of only one active fraction, the classical anaphylatoxin; it was identified as des-Arg-C5a. When hog serum was activated with yeast in the presence of 1M epsilon-aminohexanoic acid (inhibitor of arginine carboxypeptidase), two active C5-derived fractions were obtained, C5a and des-Arg-C5a. A partial amino acid sequence of the classical anaphylatoxin (19 N-terminal and 12 C-terminal amino acids) has been elaborated. With one exception it is identical with hog C5a as far as that structure is known (12 N-, 3 C-terminal amino acids). The only difference is the C-terminal region: arginine, present in C5a, is absent from des-Arg-C5a which ends with ... Asn-Ile-Gln-Leu-Gly-OH. The finding that des-Arg-C5a, virtually free of any significant contamination with C5a, has considerable spasmogenic activity demonstrates that it is the classical anaphylatoxin, and that for spasmogenic activity arginine as the C-terminal amino acid is not absolutely essential.

Purification, characterization, and amino acid sequence of rat anaphylatoxin (C3a).

C3a anaphylatoxin derived from the third component of complement has been isolated from rat serum and its complete amino acid seuqence determined. A three-step purification procedure was employed that consisted of gel filtration on Sephadex G-100, followed by chromatography of the anaphylatoxin-containing pool on carboxymethylcellulose. A subsequent separation on DEAE-Sephadex resolved C3a from minor contaminating peptides. Biological studies have shown that purified rat anaphylatoxin is approximately twice as active as human or porcine C3a when tested for smooth-muscle contraction. In addition to the active form of rat anaphylatoxin, a serum carboxypeptidase B inactivated form of C3a (C3ades-Arg) was purified from rat serum and utilized in subsequent structural studies. Sequence analysis of rat C3a was facilitated by a long automated Edman degradation which established the first 55 residues of the anaphylatoxin. Overlapping peptides were generated by cyanogen bromide and trypsin, and the resultant fragments were sequenced by either automated or manual Edman procedures. The primary structure of rat C3a is 70% identical to the previously determined structures of human and porcine anaphylatoxin. Antisera raised to the purified rat peptide do not cross-react immunologically by Ouchterlony analysis with either human or porcine C3a.