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T E Hugli

Publications and source records attributed to T E Hugli.

At least 19 recordsLinked to original sources
PubMed1992

Influence of free thiol group(s) on autoantibody-defined epitope of proliferating cell nuclear antigen.

Proliferating cell nuclear Ag (PCNA) is an intranuclear protein involved in DNA replication directed by DNA polymerase delta and is the target Ag of autoantibodies in some patients with SLE. There is evidence that the epitope on PCNA recognized by human autoantibodies is conformation-dependent and is not a continuous peptide sequence. Thimerosal, a mercury-containing sulfhydryl blocking compound, markedly reduced or abolished the reactivity of this autoantibody-defined PCNA epitope. The thimerosal effect was observed in various Ag-detecting systems including indirect immunofluorescence, immunodiffusion, immunoprecipitation, and flow cytometry. The mechanism of the thimerosal effect appeared to be mediated through free but not readily accessible sulfhydryl group or groups. The sulfhydryl-modification by thimerosal could be reversed by competition with thiol-containing compounds. Experimentally induced mAb to PCNA generated by immunization with purified PCNA have been shown to recognize epitopes that are continuous peptide sequences and these epitopes were not affected by thimerosal. It has been shown that the human autoantibody-defined epitope is related to the function of PCNA because autoantibodies are able to inhibit DNA polymerase delta directed DNA replication, whereas experimentally induced antibodies are not. These studies show that certain sulfhydryl groups in PCNA have a role in determining the antigenicity of the epitope recognized by autoantibody and raise the possibility that certain sulfhydryl groups might also be associated with its function.

Autoantibodies↗
PubMed1992

Activation of complement components C3 and C5 by a cysteine proteinase (gingipain-1) from Porphyromonas (Bacteroides) gingivalis.

Complement components C3 and C5 are susceptible to limited proteolysis by an arginine-specific cysteine proteinase isolated from Porphyromonas gingivalis. This bacterium is an anaerobe commonly associated with severe periodontal disease. Infection by P. gingivalis is accompanied by an acute inflammatory response, complete with extensive neutrophil involvement. This prompted us to investigate a possible direct role for complement in periodontitis evoked by P. gingivalis. Exposure of C3 and C5 to the cysteine proteinase at molar ratios between 1:25 and 1:100 (enzyme to substrate ratios) resulted in a time-dependent, limited degradation of each component. C3 was converted in a stepwise manner to C3a-like and C3b-like fragments with evidence of extensive further degradation of the C3a-like portion of the molecule. We were unable to demonstrate C3a activity in the C3 digestion mixtures. C3 degradation appears to involve primarily the alpha-chain. Proteolysis of C5 also progresses in a stepwise manner producing an initial internal cleavage of the alpha-chain to generate 30- and 86-kDa fragments. Further digestion of the 86-kDa amino-terminal fragment of the alpha-chain leads to the release of C5a or a C5a-like fragment that is biologically active for neutrophil activation. The fact that a potent chemotactic factor, i.e. C5a, can be generated from C5 by a proteinase derived from P. gingivalis suggests a recruiting mechanism for attracting neutrophils to the gingival lesion site in periodontal disease.

Adhesins, Bacterial↗
PubMed1992

Identification and characterization of the effector region within human C5a responsible for stimulation of IL-6 synthesis.

Human C fragment C5a is known to be a proinflammatory mediator and more recently shown to be a potent modulator of both humoral and cell-mediated immunity. We recently reported that natural and recombinant C5a induces the synthesis of IL-6-specific mRNA and secreted protein from human monocytes. Our studies using analogue peptides that are homologous to the carboxyl-terminal sequence of human C5a, indicate that the "effector" site for inducing IL-6 synthesis resides within the C-terminal region (C5a (70-74)) of the C5a molecule. C5a peptides containing the exact sequence of the natural factor were found to retain full agonist activity but exhibited low potency (0.01-0.1% of intact C5a). It was also shown that amino acid substitutions in the C5a peptides by aromatic/hydrophobic residues, outside the immediate effector site, resulted in analogue peptides with a substantial increase in potency relative to the most active natural peptide (C5a (56-74)). Moreover, these peptides approach the potency of natural C5a for induction of IL-6. Taken together, these results suggest that the inflammatory and immunoregulatory activities associated with C5a may, in part, be due to the synthesis of IL-6.

Amino Acid Sequence↗
PubMed1992

Biologic activity of synthetic analogues of C5a anaphylatoxin.

The C activation fragment C5a is the most potent plasma-derived chemotactic factor known. This humoral factor induces both neutrophil and macrophage activation at low nanomolar concentrations. We have synthesized a series of C-terminal C5a analogues that exhibit all of the characteristic biologic activities of C5a. These peptides apparently contain the effector site for C5a receptor-mediated cellular activation, but express only a fraction of the potency of intact C5a. We have demonstrated the following in vitro activities for these C5a peptides: 1) ileal (guinea pig) contraction; 2) platelet (guinea pig) activation; and 3) neutrophil (human) polarization and chemotaxis. The effect of C5a peptides in vivo was evaluated by measuring enhancement in vascular permeability. Although potencies of the most effective synthetic C5a analogues were on the order of 0.01 to 0.1% that of the natural factor, our biologic data confirm that the C5a peptides are full agonists of the intact factor and may be useful substitutes for intact C5a. Furthermore, our results indicate that elongation of the C5a analogues from 10 to 19 residues in length contributes little toward enhancing or decreasing potency of the synthetic C5a analogues. Replacement of residues in the effector region by D-amino acids or by introduction of a cyclic group to reduce flexibility of the backbone decreased potency of the analogues. Substitution of His 67 by Phe in the decapeptide C5a 65-74 resulted in a significant increase in potency of the C5a analogue. The marked enhancement in potency from replacing His 67 by Phe in analogue C5a peptides identifies an important hydrophobic subsite. We conclude that site-specific amino acid modifications in or near the C-terminal effector site sequence can diminish or optimize potency of the model C5a peptides. However, there apparently are subsites on folded C5a, from regions other than the C-terminal portion of the molecule, that contribute significant receptor interactions. These subsites must be identified and incorporated into C5a model peptide designs before expression of full potency by synthetic analogues of this factor will be realized.

Amino Acid Sequence↗
PubMed1992

A mechanism of action for anaphylatoxin C3a stimulation of mast cells.

Incubation of either C3a, C3ades Arg, or synthetic analogues of the C-terminal sequence of C3a with purified rat peritoneal mast cells resulted in a rapid and dose-dependent histamine release. The natural factors C3a and C3ades Arg were the most active of the factors tested exhibiting EC50 values of 3.3 and 2.2 microM, respectively. The corresponding 21- and 22-residue C-terminal analogues of C3a (Y21R and Y21) were less potent than intact factor exhibiting EC50 values of 10.9 and 25.1 microM, respectively. Histamine was released in a nonlytic manner and the mast cell stimulation by both natural and synthetic factors was sensitive to pertussis toxin, neuraminidase, benzalkonium chloride, and to an excess of calcium. C3a stimulated the generation of inositol polyphosphates that was inhibited by either pertussis toxin or benzalkonium chloride. The C3a anaphylatoxin also directly stimulates purified G proteins (i.e., GTPase activity) in a dose-dependent manner. The evident correlation between efficiency of C3a and C3a analogues to stimulate purified G proteins and their capacity to induce cellular histamine release led us to conclude that C3a fails to activate mast cells via a mechanism involving specific receptors on the cell. Instead, we propose that C3a either causes direct activation of G proteins of the Gi subtype, with a subsequent activation of phospholipase C, or interacts with a binding site of the cell surface specific for cationic molecules that is coupled to the G protein cascade.

Amino Acid Sequence↗
PubMed1992

Neutrophil chemotactic factors promote leukocytosis. A common mechanism for cellular recruitment from bone marrow.

We investigated cellular responses in a rabbit to i.v. administration of five established chemotactic factors (leukotriene B4 (LTB4), platelet-activating factor (PAF), C5a, N-Formyl-Met-Leu-Phe (F-MLF), and IL-8), and each exerted a characteristic effect on circulating white blood cell levels. All five factors induced a rapid and transient leukopenia. The blood was nearly devoid of circulating neutrophils 5 min after administration of each chemotactic factor. Other leukocytes were also variably depleted during the leukopenic phase, including eosinophils, basophils, monocytes, and lymphocytes. The lymphocyte numbers remained significantly depressed (approximately 30%) for as long as 3 h after administration of PAF or f-MLF. Each chemotactic factor produced a marked neutrophilia (i.e., 250-400% of baseline levels) after the initial leukopenia. Eosinophil numbers were elevated along with the neutrophil response in the C5a- and LTB4-treated animals. Basophil levels were significantly elevated only in LTB4-treated animals. The cellular response to PAF, f-MLF, and IL-8 appeared to be specific for the neutrophils. The kinetic profiles of the neutrophilia induced by PAF (10 micrograms/kg) or f-MLF (2.5 micrograms/kg) were similar, with maximal responses occurring 3 to 4 h after administration. In contrast, LTB4 (10 micrograms/kg), IL-8 (2.5 micrograms/kg), and C5a (5 micrograms/kg) induced a more rapid neutrophilia, with peak responses occurring 1 to 1.5 h after injection, and remaining elevated for 3 to 4 h. In all animals the neutrophilia was accompanied by a relative increase in the number of nonsegmented neutrophils (bands), suggesting that a major component of leukocytosis is caused by the release of bone marrow reserves. Phenidone (10 mg/kg), a dual cyclooxygenase/5-lipoxygenase inhibitor, affected neither the neutropenia nor the neutrophilia induced by C5a, f-MLF, or PAF. The protein synthesis inhibitor actinomycin D also failed to suppress neutrophil responses induced by either C5a or PAF. These results suggest that leukocytosis is a common response induced by all neutrophil chemotactic factors. Leukocytosis appears to be a direct result of the dynamic adaptive response of neutrophils to chemotactic factor stimulation without involvement of a secondary mediator system.

Animals↗
PubMed1992

A protease-sensitive site in the proposed Ca(2+)-binding region of human serum amyloid P component and other pentraxins.

Serum amyloid P component (SAP) is a decamer of 10 identical 25.5-kDa subunits. Limited proteolysis of SAP with alpha-chymotrypsin cleaves the subunit into two fragments of 18 and 7.5 kDa, although the fragments stay together in the decamer under nondenaturing conditions. Proteolysis does not occur in the presence of Ca2+ (10 mM). Cleavage with alpha-chymotrypsin prevents the Ca(2+)-dependent binding of SAP to zymosan extract, nucleosomes, and DNA. The alpha-chymotrypsin cleavage site identified is in a region of SAP that is highly conserved in members of the human C-reactive protein (CRP) family of proteins (pentraxins) to which SAP belongs and is similar to the Ca(2+)-binding site in calmodulin and related Ca(2+)-binding proteins (Nguyen, N.Y., Suzuki, A., Boykins, R.A., & Liu, T.-Y., 1986, J. Biol. Chem. 261, 10456-10465). Treatment of SAP with other proteases (trypsin, Pronase, and Nagarse protease) yields fragmentation patterns upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that are similar to those obtained with alpha-chymotrypsin. Two other members of the pentraxin family of proteins, hamster female protein and rabbit CRP, also exhibit similar fragmentation patterns on SDS-PAGE when treated with the various proteases. Recently, it has been shown that the homologous protein, human CRP, is cleaved in the same homologous position as cleavage of SAP by alpha-chymotrypsin, resulting in the loss of Ca(2+)-binding (as shown by equilibrium dialysis) and Ca(2+)-dependent binding reactivities (Kinoshita, C.M., Ying, S.-C., Hugli, T.E., Siegel, J.N., Potempa, L.A., Jiang, H.J., Houghten, R.A., & Gewurz, H., 1989, Biochemistry 28, 9840-9848).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Cold-dependent activation of complement: recognition, assessment, and mechanism.

Cold-dependent activation of complement (CDAC) is a phenomenon characterized by low hemolytic complement activity in chilled serum. Complement component levels are normal when measured immunologically, and there is normal hemolytic activity in EDTA plasma or serum maintained at 37 degrees C. Little attention has been paid to CDAC except in Japan, and current unfamiliarity with it, even by clinical immunologists, can lead to confusion and unnecessary laboratory tests. A 66-year-old patient with a complex medical history is described whose complement tests showed abnormalities characteristic of CDAC. Evidence for classical complement pathway activation in the cold was obtained by CH50 measurements, by hemolytic C4 determinations, by C4a, C3a, and C4d generation, and by quantitating C1s-C1r-(C1 inhibitor)2 complexes. A good correlation was observed among these parameters. Cryoprecipitates were absent. CDAC activity has persisted for over 5 years and is greater at 13 than at 4 degrees C. Activation is ablated by heating at 56 degrees C and restored by the addition of C1 to the heated serum. Adsorption by streptococcal protein G-Sepharose and precipitation by 2.5% polyethylene glycol support the hypothesis that CDAC is caused by aggregated IgG. The CDAC factor(s) also induces complement activation in normal serum but has not interfered with Raji cell or C1q binding tests or with FACS analysis. More limited studies of a second individual experiencing CDAC yielded similar results.

Adult↗
PubMed1991

Designing synthetic superagonists of C3a anaphylatoxin.

An extensive structure-activity study of synthetic analogues of the C3a anaphylatoxin was conducted. Our goal was to map C3a-C3a receptor interactions by designing synthetic analogue molecules having maximal biologic potency. Nonspecific binding of the polycationic C3a to polyanionic molecules on cellular surfaces often obscures specific binding to the receptor. Less cationic synthetic C3a analogues would be useful tools in identifying and characterizing the various cell types having C3a receptors. These factors should also be useful as pharmacologic probes for mechanism studies, as high-affinity ligands for target cell identification, and for receptor isolation. Attachment of amino-terminal hydrophobic groups such as Fmoc to C3a analogues [as orginally introduced by Gerardy-Schahn et al. (1988) Biochem. J. 255, 209] markedly enhanced the potency of synthetic C3a peptides. The enhancement effect on potency from introducing hydrophobic groups to C3a analogues was interpreted as possibly being nonspecific. Our systematic search for an optimal peptide length, composition, and N-terminal hydrophobic unit resulted in several superpotent C3a analogues having 200-1500% the potency of natural C3a. One particularly potent C3a peptide was designed by incorporating two tryptophanyl residues at the N-terminal end of a 15-residue C3a analogue. The superpotent peptide W-W-G-K-K-Y-R-A-S-K-L-G-L-A-R has several residues differing (underlined) from the sequence corresponding to positions 63-77 in human C3a, a region that contains the essential functional site of the molecule. This 15-residue model peptide exhibited the greatest biological potency of all peptides tested, being 12-15 times more active than natural C3a. Since an optimal distance was found to exist between the N-terminal hydrophobic unit (W-W) and the C-terminal primary binding site (LGLAR), we concluded that the hydrophobic unit interacts specifically with a secondary binding site on the C3a receptor. The presence of both a primary (effector) and secondary (hydrophobic) binding site on these linear synthetic ligands, which can interact cooperatively with the C3a receptor, presumably accounts for the high relative potency of the analogues. Our design of superpotent analogues of C3a demonstrates the feasibility for constructing small synthetic peptides to mimic natural biologic factors that depend on secondary or tertiary structure for their activity. These synthetic peptide studies demonstrate that a linear array of amino acids (e.g., W-W) can successfully substitute for a conformation-dependent binding site on a bioactive factor.

Amino Acid Sequence↗
PubMed1991

Evidence for in vivo degradation of C3a anaphylatoxin by mast cell chymase. I. Nonspecific activation of rat peritoneal mast cells by C3ades Arg.

The anaphylatoxin C3a (125I-HC3a) is extensively degraded when exposed to isolated rat mast cells (RMC). Degradation occurs without prior stimulation of these cells. The protease responsible for C3a degradation has been identified as chymase. Mixed peritoneal cells, containing equal numbers of mast cells compared with incubates containing more highly purified RMC, promoted less degradation of the C3a molecule than did the purified RMC. These variable levels of spontaneous activation (ie, chymase release) of RMC in vitro were concluded to be a function of the cellular handling and isolation procedures. No degradation occurred when low levels of 125I-HC3ades Arg (10(-8) mol/l [molar]) were introduced in the peritoneum of a rat, unless the mast cells were stimulated by prior introduction of specific activators. Evidence that the enzyme being released in the peritoneal cavity of rats was chymase was provided both by its appearance after adding specific mast cell activators (ie, compound 48/80 and anti-IgE [gamma E immunoglobulin]) and by inhibition with chymostatin. Because 125I-HC3ades Arg at low levels (10(-8) mol/l) was not degraded in the rat peritoneum, expression of chymase by the rat mast cells could be monitored in situ during mast cell stimulation. 125I-HC3ades Arg was rapidly converted (in 2 to 5 minutes) to smaller fragments in the peritoneal cavity of the rat after either 10 micrograms of compound 48/80 or anti-IgE was injected. Introduction of higher levels of HC3a or HC3ades Arg (2.5 to 5.0 x 10(-6) mol/l) to the peritoneal cavity of the rat stimulated both chymase release and HC3a degradation without other mast cell activators being present. HC3ades Arg was equally as effective in vivo as HC3a in stimulating the rat peritoneal mast cell to release chymase. It was concluded that the mechanism of RMC activation by C3a (C3ades Arg) was nonspecific and similar to the process by which polyamines and polycations stimulate mast cells, as C3a is a highly cationic molecule. The fact that C3a may in turn be destroyed in minutes by the recruited protease (chymase) defines a potentially important physiologic control mechanism. This cellular control process, demonstrated here for rat mast cells, may function in other animals, including man, for regulating tissue levels of factors such as the anaphylatoxins that are potentially capable of mast cell activation through nonspecific (polycationic) mechanisms.

Animals↗
PubMed1990

Anaphylatoxin binding and degradation by rat peritoneal mast cells. Mechanisms of degranulation and control.

Incubation of radiolabeled human C3a with rat peritoneal mast cells resulted in high levels of uptake and extensive degradation of the ligand. Both cell-bound and free radiolabeled human C3a underwent extensive degradation by rat mast cells even at 0 degrees C. We examined several protease inhibitors for their ability to prevent degradation of radiolabeled human C3a by the rat mast cells. The inhibitors PMSF, chymostatin, and soybean trypsin inhibitor were most effective in preventing radiolabeled human C3a degradation. Degradation of the cell-bound ligand was totally inhibited only by PMSF. These compounds are effective inhibitors of a chymotrypsin-like enzyme (chymase) extracted from rat mast cells. Chemical cross-linking of radiolabeled human C3a to surface components on the rat mast cells, in the presence of PMSF, revealed one major and two minor bands. The mast cell component in both the major and minor bands proved to be chymase-associated based on a direct comparison with purified chymase isolated from rat mast cells. However, neither antichymase antibody nor chymase inhibitors influenced the degranulating activity of C3a on rat mast cells that occur independently of the C3a-chymase interactions. We conclude that there are neither specific C3a-binding sites on rat mast cells nor specific receptors whose occupancy leads to cellular activation. Although human C3ades Arg is inactive on guinea pig ileal and lung tissue, it binds to and induces degranulation of rat mast cells, as well as enhances vascular permeability in rat skin, at concentrations nearly identical to that of intact C3a. The fact that both C3a and C3ades Arg stimulated mast cell activation, at concentrations in excess of 10(-6) M, argues against specific binding sites for the anaphylatoxin on rat mast cells. It is proposed that the cationic C3a molecule activates rat mast cells in a secretory and nonlytic manner by a nonspecific mechanism similar to that of other polybasic compounds.

Anaphylatoxins↗
PubMed1990

C5a-mediated release of interleukin 6 by human monocytes.

Recombinant human C5a (rC5a) was assessed for its ability to induce interleukin 6 (IL-6) production in human peripheral blood-derived mononuclear cell (PBMC) cultures. rC5a was observed to induce IL-6 production as measured by hybridoma growth promotion (B9.9 assay) and human B cell differentiation (SKW6.4 assay). Optimal IL-6 production was obtained after 24 hr stimulation with 0.1-0.5 micrograms/ml rC5a. In addition, natural human C5ades Arg and natural porcine C5a were able to induce a similar level of IL-6. The observed IL-6 activity appeared not to be due to endotoxin contamination since heat treatment (100 degrees C/15 min) inhibited rC5a induction of IL-6. The rC5a stimulation also induced an increase in steady-state IL-6 mRNA as determined by Northern blot analysis. Pretreatment of PBMC with leucine-methyl ester to deplete monocytes reduced the rC5a-induced IL-6 production to background levels. In addition, stimulation of purified T cell preparations with rC5a produced little IL-6 activity, suggesting that monocytes are the major source of IL-6 in this system. These results suggest that the inflammatory and immunoregulatory activities of C5a may in part be due to the stimulation of IL-6 release, a cytokine which possesses potent pleiotropic functions.

Complement C5a↗
PubMed1990

Contractile effect of the chemotactic factors f-Met-Leu-Phe and C5a on the human isolated umbilical artery. Role of cyclooxygenase products and tissue macrophages.

Factors that are chemotactic for phagocytic leukocytes are known to elicit important acute circulatory changes, and the role of circulating leukocytes in these models is controversial. To evaluate the role of the blood vessel wall in the absence of circulating cells, spiral strips of human umbilical artery were exposed in vitro to the chemotactic peptides f-Met-Leu-Phe (1-100 nM) or C5a (2.5-25 nM). Contractile responses were observed for both peptides. Certain agonist analogues and a selective antagonist of the chemotactic action of f-Met-Leu-Phe behaved correspondingly as agonists and antagonist of the contractile effect on umbilical artery. The anaphylatoxin C3a also exerted a contractile effect on the tissue (25 nM and above), but this effect was highly tachyphylactic. Inhibitory drugs were used to examine the contributions of secondary mediators in eliciting the effects of C5a and f-Met-Leu-Phe. The contractile effect of both peptides was massively inhibited either by indomethacin or the thromboxane A2/prostaglandin H2 antagonist SQ 29548. Dazmegrel, a thromboxane A2 synthetase inhibitor, had partial inhibitory effects on contractions induced by either peptide. The contractile effect of C3a was prevented by indomethacin pretreatment. Vascular strips did not release measurable histamine in the bathing fluid after challenge with C5a or f-Met-Leu-Phe. The tissue apparently contains neither histamine nor mast cells. Autoradiography of 125I-labeled C5a or f-Met-Leu-Phe analogue showed specific binding of the peptides to cells dispersed in the vessel wall, but more frequently at the periphery. Cells stained positively for alpha-naphthyl acetate esterase showed a similar distribution. Pure cultures of smooth muscle cells derived from the umbilical artery failed to release prostanoids when exposed to f-Met-Leu-Phe or C5a, whereas fresh strips of this artery released more thromboxane B2 than the baseline in response to these peptides. We conclude that macrophagelike cells, present in the vessel wall, are the likely target cells for the chemotactic peptides. These cells trigger a contractile effect of the smooth muscle by generating cyclooxygenase products.

Anaphylatoxins↗
PubMed1990

C5a-induced neutrophilia. A primary humoral mechanism for recruitment of neutrophils.

The leukocytosis activity of C5a was studied in a rabbit model. Blood samples were drawn for cell counts through a catheter placed in an artery of one rabbit ear after injection of either porcine or human C5a into a vein in the opposite ear. These studies indicate the potential of C5a to mobilize bone marrow neutrophils following transient C5a-induced neutropenia, based on counts of nonsegmented neutrophils. The numbers of circulating neutrophils can be selectively elevated by 300% to 400%, within 1 to 2 hours, in rabbits given only microgram quantities (1 to 5 x 10(-9) mol/l) of C5a or C5adesArg. These quantities of C5a are equivalent to 1% to 2% of complement C5 activation. The time-course of the induced neutrophilia is characteristic of C5a, increasing rapidly in the first 10 to 20 minutes after injection and attaining a maximum level at 2 to 5 hours, then decreasing slowly to normal levels over the next 4 to 6 hours. After boiling at 100 degrees C for 10 minutes, C5a (C5ades Arg) lost its leukocytosis activity, indicating that the cellular effect was not caused by endotoxin. Other known leukocytosis factors, such as epinephrine, dexamethasone, lipopolysaccharide, and the prostanoid 15(S)-15-methyl PGF2 alpha, produced a distinctly different profile of leukocyte mobilization than that of C5a. The C5a-induced neutrophilia was not inhibited by pretreating these animals with indomethacin, suggesting that it is not a prostanoid-induced effect. One hypothesis is that no secondary cellular mediator system is involved in C5a-mediated leukocytosis, but rather than C5a alone is responsible for a rapid mobilization of neutrophils from bone marrow pools, and perhaps marginated pools, following neutropenia. Circulating neutrophils are activated by C5a and thereby become deformed and adherent, leading to a neutropenia, sequestration, and depletion of cells. After the neutropenic event an immediate neutrophilic response is required for replacement of this particular cell population and to re-establish homeostasis. Therefore the role of C5a may be just as important as other known leukocytosis factors (fragments from C3, for example) in promoting complement-dependent neutrophil mobilization in response to tissue injury, infections, or extracorporeal blood treatments.

Animals↗
PubMed1989

Elucidation of a protease-sensitive site involved in the binding of calcium to C-reactive protein.

C-reactive protein (CRP) is a Ca2+-binding protein composed of five identical 23-kDa subunits arranged as a cyclic pentamer, present in greatly elevated concentration in the blood during the acute phase of processes involving tissue injury and necrosis. In the present work, it was found that treatment of human CRP with Pronase or Nagarse protease produces two major fragments which remain associated in physiological buffers but are separable under denaturing conditions. To localize the cleavage site(s), the fragments were characterized according to molecular mass, amino acid composition, partial amino acid sequence, and reactivity with monoclonal antibodies specific for the fragments and for defined CRP epitopes including residues 147-152 and 199-206. Nagarse protease cleaves the CRP subunit between residues 145 and 146, producing two fragments, 16 and 6.5 kDa (calculated molecular mass). Pronase cleaves the CRP subunit between residues 146 and 147, producing a 16-kDa fragment (A1) and a 6.5-kDa fragment (B); an additional fragment (A2) approximately 1 kDa smaller than fragment A1 is also apparently produced due to a secondary cleavage site in fragment A1. Cleavage appears to be completely inhibited in the presence of 1 mM CaCl2. Ca2+ does not protect cleaved CRP from heat-induced aggregation (i.e., precipitation) as it does the intact protein. Protease-cleaved CRP loses the ability to bind to the Ca2+-dependent ligand phosphorylcholine but remains the ability to bind to the Ca2+-independent ligand arginine-rich histone. Equilibrium dialysis indicates that intact CRP binds 2 mol of Ca2+/mol of subunit with a Kd of 6 X 10(-5) M.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

The molecular architecture of human complement component C6.

The molecular architecture of human complement component C6 was elucidated at several levels of structural organization. The entire primary structure of C6 was determined by sequencing C6 cDNA that was cloned from a human liver lambda gt11 library. The polypeptide chain of C6 contains 913 amino acids. The protein is homologous with the other terminal components of complement, C7-C9. Specifically, C6 has 29% of its residues identical with C7, and 55 of the 56 cysteines found in C7 match those in C6. The C6 polypeptide chain is cross-linked by 32 disulfide bonds, and most of the cysteines are located in short (34-77 amino acids) discrete segments that exhibit homology with a wide variety of other proteins such as thrombospondin, the low density lipoprotein receptor, epidermal growth factor, and complement factors H and I. C6 is a glycoprotein, and it has two oligosaccharide groups attached to asparagines located near the amino and the carboxyl termini of the molecule. The organization of secondary structural elements in C6 was elucidated using circular dichroism spectroscopy and an empirical method based on sequence analysis. C6 has an estimated 12% alpha-helix, but is comparatively richer in beta-sheet (29%) and beta-turns (21%). Most of the predicted alpha-helical structure resides in a portion of the polypeptide chain that is free of cysteine and which shares homology with C9 and perforin. The tertiary structure of the C6 molecule was visualized by transmission electron microscopy; it has a sickle shape with dimensions of 144 x 66 A. The combined results are discussed and comparisons made with the other late acting components of complement and perforin.

Amino Acid Sequence↗