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J Tschopp

Publications and source records attributed to J Tschopp.

At least 217 records · Page 12Linked to original sources
PubMed1989

Molecular structure and functional characterization of a human complement cytolysis inhibitor found in blood and seminal plasma: identity to sulfated glycoprotein 2, a constituent of rat testis fluid.

A component of soluble terminal complement complexes was identified and affinity-purified to homogeneity by using a monoclonal antibody previously developed against the soluble C5b-9 complex. The protein, which we have designated complement cytolysis inhibitor (CLI), has a molecular mass of 70 kDa and consists of two nonidentical, disulfide-linked subunits of 35 kDa. Partial amino acid sequences determined for the amino-termini of the two subunits were identical with those of a recently characterized serum protein called SP-40,40. An almost full-length cDNA clone of 1651 base pairs was isolated from a human liver cDNA library by using long synthetic oligonucleotides as probes. The encoded amino acid sequence of CLI consists of 427 amino acid residues preceded by a 21-residue-long typical signal peptide and shows an overall 75.6% amino acid sequence homology to sulfated glycoprotein 2 (SGP-2), a major Sertoli cell-derived protein of rat testis fluid. As in SGP-2, proteolytic processing between residues 206 and 207 yields the two disulfide-linked subunits of plasma CLI. CLI and SGP-2 were shown to be orthologous single-copy genes in humans and rats by Southern blotting experiments. In addition, CLI was immunologically identified in human seminal plasma. Functional studies with purified terminal complement components showed that CLI suppresses the cytolytic potential of nascent C5b-7 complexes at physiological blood plasma concentrations (approximately 50 micrograms/ml). Its presence on the surface of mature sperm cells and its relative abundance in seminal plasma (approximately 250 micrograms/ml) suggest that CLI protects sperm cells and epithelial tissues against complement attack in the male reproductive tract.

Amino Acid Sequence↗
PubMed1989

Activation-dependent epitopes in the terminal complement pathway.

Assembly of the terminal complement complex (TCC) during complement activation is associated with considerable antigenic changes in the individual C5, C6, C7, C8 and C9 components. Numerous neoepitopes specific for the TCC are exposed. Similarly, native-restricted epitopes specific for the nonactivated components are concealed in the TCC. The present paper reviews these antigenic changes with special reference to available monoclonal antibodies and their utilization in assays to detect and quantify the TCC. Application of such assays in clinical medicine is discussed. Finally, a recommended terminology for the terminal complement pathway is put forward.

Antibodies, Monoclonal↗
PubMed1988

Characterization of granzymes A and B isolated from granules of cloned human cytotoxic T lymphocytes.

A human CD8+ CTL clone with cytolytic potential was shown to express two serine proteases, a 50-kDa homodimer and a 27-kDa monomer, which were purified from cytoplasmic granules. N-terminal sequencing of the purified proteins revealed that the 50-kDa homodimer is the gene product of the human Hanukah factor cDNA clone and that it represents the human homologue to granzyme A. Similarly, the 27-kDa protein was shown to be the serine esterase encoded by the human lymphocyte protease cDNA clone and corresponds to granzyme B. There was no evidence for the presence of other granzymes, in particular for the human homologues to murine granzymes C, D, E, and F. The substrate best cleaved by granzyme A was Gly-Pro-Arg-amido-4-methyl-coumarin after the Arg residue and the pH optimum was 8 to 8.5. Upon triggering of the TCR-CD3 complex with an anti-CD3 mAb, granzyme A was released into the culture medium. Furthermore, a granule-associated hemolytic activity was detected after salt extraction and partial purification of granule proteins. This suggests that hemolytically active human perforin can be obtained from inactive granules.

Amino Acid Sequence↗
PubMed1988

Lack of DNA degradation in target cells lysed by granules derived from cytolytic T lymphocytes.

It has been shown previously that fragmentation of target cell DNA is an early event in lysis mediated by cytolytic T lymphocytes (CTL). In this study, we have investigated whether CTL-derived granules that exhibit lytic activity also induce DNA fragmentation in murine target cells. Cytolytic granules isolated from three different alloreactive CTL clones were tested for the induction of DNA fragmentation in P815 and EL4 target cells, by using a Triton X-100-facilitated, radiolabeled DNA release assay. In contrast to the CTL clones from which they were derived, the cytolytic granules did not induce DNA fragmentation. Agarose gel electrophoretic analysis of DNA confirmed the lack of discrete DNA fragments in target cells lysed by CTL-derived granules. Possible explanations for the difference in the ability of CTL and CTL-derived granules to trigger DNA fragmentation are discussed.

Animals↗
PubMed1988

The heparin binding domain of S-protein/vitronectin binds to complement components C7, C8, and C9 and perforin from cytolytic T-cells and inhibits their lytic activities.

S-Protein/vitronectin is a serum glycoprotein that inhibits the lytic activity of the membrane attack complex of complement, i.e., of the complex including the proteins C5b, C6, C7, C8, and C9n. We show that intact S-protein/vitronectin or its cyanogen bromide generated fragments also inhibit the hemolysis mediated by perforin from cytotoxic T-cells at 45 and 11 microM, respectively. The glycosaminoglycan binding site of S-protein/vitronectin is responsible for the inhibition, since a synthetic peptide corresponding to a part of this highly basic domain (amino acid residues 348-360) inhibits complement- as well as perforin-mediated cytolysis. In the case of C9, the synthetic peptide binds to the acidic residues occurring in its N-terminal cysteine-rich domain (residues 101-111). Antibodies raised against this particular segment react 25-fold better with the polymerized form of C9 as compared with its monomeric form, indicating that this site becomes exposed only upon the hydrophilic-amphiphilic transition of C9. Since the cysteine-rich domain of C9 has been shown to be highly conserved in C6, C7, and C8 as well as in perforin, the inhibition of the lytic activities of these molecules by S-protein/vitronectin or by peptides corresponding to its heparin binding site may be explained by a similar mechanism.

Animals↗
PubMed1988

Antibody-independent activation of the complement system by mitochondria is mediated by cardiolipin.

Non-immune activation of the first component of complement (C1) by the heart mitochondrial inner membrane has been investigated. Cardiolipin, the only strong activator of C1 among phospholipids, is present in large amounts in the heart mitochondrial inner membrane. We therefore studied its contribution to C1 activation by mitochondria. The proteins of the mitochondrial inner membrane were found to activate C1 only weakly, in contrast with the phospholipid fraction which induces strong C1 activation. Furthermore, the digestion of mitochondrial inner membranes with proteolytic enzymes did not affect C1 activation. Additional support in favour of cardiolipin being the responsible activator came from competition experiments with mitochondrial creatine kinase (mt-CPK) and adriamycin, known to bind to cardiolipin. Both mt-CPK and adriamycin displaced C1q from the mitochondrial inner membrane. In addition, C1q displaced mt-CPK bound to mitoplasts.

Animals↗
PubMed1988

cDNA cloning of granzyme C, a granule-associated serine protease of cytolytic T lymphocytes.

A cDNA clone corresponding to the complete amino acid sequence of a putative protease CCP2 of murine cytotoxic T lymphocytes was isolated and sequenced. The clone encodes a 248-residue long serine esterase. The deduced N-terminal amino acid sequence is identical over 40 residues to that of granzyme C, a protease of unknown function present in granules of cytotoxic lymphocytes. Analysis of the sequence of granzyme C/CCP2 reveals high homology to other granzyme proteases, i.e. granzyme A (40%) and granzyme B (67%) and to rat mast cell protease II (46%). The amino acids lining the specificity pocket are well conserved between granzyme B, C, and rat mast cell protease II, but not granzyme A, suggesting a similar general specificity of these three proteases.

Amino Acid Sequence↗
PubMed1988

Inhibition of lymphocyte protease granzyme A by antithrombin III.

T-lymphocytes contain a cytoplasmic granule associated homo-dimeric protease designated granzyme A. Upon T-cell target cell interaction, the granules undergo exocytosis and granzyme A, and other granule constituents, are released. Here we show that granzyme A secreted into plasma is immediately inactivated by antithrombin III. The rate of complex formation is enhanced 400-fold in the presence of heparin. Two different complexes are generated: granzyme A-antithrombin III and granzyme A-(antithrombin III)2, respectively, indicating that both active centers of granzyme A are functional. Thus, the proteolytic activity of lymphocyte protease granzyme A, whose physiologically relevant function is unknown, is well regulated in plasma.

Animals↗
PubMed1988

Identification and sequencing of cDNA clones encoding the granule-associated serine proteases granzymes D, E, and F of cytolytic T lymphocytes.

Cytoplasmic granules of cytolytic T lymphocytes contain at least six related serine esterases (granzymes) that are released together with perforin, a pore-forming protein related to complement component C9, during target-cell lysis. Polyclonal antibodies were used to isolate a large number of cDNA clones from an expression library derived from cytolytic-T-cell mRNA. Three distinct full-length cDNA clones coding for granzymes D, E, and F were identified by restriction site mapping and nucleotide sequencing. The three deduced amino acid sequences are highly similar to one another (between 72% and 90% amino acid identities) and to the sequences of granzymes B and C, cathepsin G, and rat mast-cell proteases I and II (between 43% and 57% amino acid identities). Cysteine residues capable of forming intramolecular disulfide bonds are conserved, as are the catalytic-site residues characteristic of serine proteases. Comparison of the cDNA-derived protein sequences with the amino termini of the isolated granzymes provides evidence that they are stored in a fully processed, activated form after removal of the signal peptide and two additional residues (propeptide) at the amino terminus. Immunoelectron microscopic studies demonstrated that granzymes D, E, and F are present in the same morphologically distinct cytoplasmic granules in which perforin has been found previously.

Amino Acid Sequence↗
PubMed1988

Identification of a human C5 beta-chain epitope exposed in the native complement component but concealed in the SC5b-9 complex.

Monoclonal antibodies obtained after immunization of mice with human C5 were screened for activity against native C5 and SC5b-9. Clone 568 reacted with the C5 beta-chain in western blotting and with purified native C5 in double antibody enzyme immunoassays. The reactivity of clone 568 against serum decreased markedly when serum was activated with zymosan. The residual activity was found to be against free C5, whereas the antibody did not react at all with the SC5b-9 complex. Thus, the antibody 568 reacts with an activation-dependent C5 epitope, which is concealed when C5 is incorporated into the terminal complement complex.

Animals↗
PubMed1988

Granzymes, a family of serine proteases released from granules of cytolytic T lymphocytes upon T cell receptor stimulation.

The cytolytic potential of T effector cells appears to be intimately related to the presence of proteins stored in specialized cytoplasmic granules. A striking biological property of isolated granules is their lytic activity for a variety of target cells in a nonrestricted manner. Proteins contained within these granules of CTLs are specifically released upon target cell recognition. We have isolated and characterized six granule-associated proteins in two murine CTL lines in addition to the pore-forming and target membrane-disrupting perforin. Six full length cDNA clones have been identified in a CTL-specific cDNA expression library which code for the granule-associated serine esterases, designated as granzymes A to F. Granzymes A and B represent the genuine proteins encoded by the H factor/CTLA-3 cDNA and the CTLA-1/CCPI cDNA, respectively. The covalent amino acid structures of all six granzymes show the hallmarks for serine proteases and are highly related to that of rat mast cell protease I and II and cathepsin G, which have been found in granules of mast cells and neutrophilic granulocytes, respectively. The primary translation products are processed by removal of a hydrophobic signal peptide and a two residue-long propeptide at the amino-terminus. Immuno-electron microscopy shows that granzymes and perforin are stored together within secretory granules of CTLs. Simultaneous release of at least two of these granzymes has been observed during degranulation of a murine CTL line by anti-T3 antibodies. The biological role, particularly the proteolytic events elicited by granzyme A and other granzymes in the context of target cell recognition, are not known at present. It is unlikely that they form a proteolytic activation cascade together with pore-forming proteins analogous to the complement system. The strictly regulated secretion of granzymes and the lack of measurable enzymatic activity in the case of granzymes B, C, E and F towards a variety of synthetic substrates suggest a highly specific function for each of them.

Amino Acid Sequence↗
PubMed1988

Appearance of granule-associated molecules during activation of cytolytic T-lymphocyte precursors by defined stimuli.

Lysis of target cells by cytolytic T lymphocytes (CTL) is associated with the exocytosis of cytoplasmic granules. Purified granules from CTL cell lines contain a pore-forming protein (perforin), tree serine esterases, granzyme A (60,000 MW), granzyme B (29,000 MW), and granzyme C (27,000 MW). We have compared the kinetics of appearance of cytolytic activity with that of perforin and granzyme A activity during activation of lymphocytes from normal animals with leukoagglutinin (LA) and recombinant interleukin-2 (rIL-2). Unstimulated lymph node cells do not express any of these activities, which appear between Day 3 and Day 4 of stimulation and increase rapidly to reach a pronounced peak on Day 6. On Day 7 all the activities are considerably lower, even though the cells still proliferate exponentially. There is a good correlation between the kinetics of appearance of all of these activities. Using antisera against perforin and against granzyme C, one can detect positive cytoplasmic granules in a small fraction of cells on Day 3; by Day 5, 80-90% of the cells are stained. This proportion decreases again on Day 7.

Animals↗
PubMed1987

Structural homology of human complement component C8 gamma and plasma protein HC: identity of the cysteine bond pattern.

Anti-C8 alpha-gamma specific antibodies were used to isolate cDNA clones from a human liver expression library. Antibodies affinity-purified on the expressed hybrid protein of one clone bound exclusively to the gamma-chain of reduced C8 alpha-gamma. This clone, as well as a second full length cDNA clone obtained by hybridization screening, were sequenced and the complete primary structure for C8 gamma was established. Cyanogen bromide cleavage of C8 alpha-gamma released a 12 kDa carboxy-terminal C8 gamma fragment under both reducing and nonreducing conditions which was identified by fragment-specific, affinity-purified antibodies. Our data clearly show that C8 gamma has one internal disulfide bridge between cys-76 and cys-168 within the carboxy-terminal 12 kDa fragment, whereas the remaining cysteine residue 40 forms the disulfide bridge with C8 alpha. The overall sequence homology to plasma protein HC (23% amino acid identities) and the conservation of one internal cysteine bond and one free, surface-located cysteine residue suggests a highly conserved three-dimensional structure of C8 gamma and protein HC and also a possible functional relationship between these proteins.

Alpha-Globulins↗
PubMed1987

A unique epitope exposed in native complement component C9 and hidden in the terminal SC5b-9 complex enables selective detection and quantification of non-activated C9.

Recently, monoclonal antibodies recognizing epitopes exposed in activation products of complement but hidden in the native components have been characterized and used for selective quantification of the activated protein. We now demonstrate that an epitope in the native component C9 is hidden in the terminal SC5b-9 complex. A monoclonal antibody against this epitope enabled selective detection of C9 without influence of the amount of SC5b-9 present. This antibody recognizing native soluble C9 was used to construct a quantitative double-antibody ELISA with unique sensitivity and specificity. Combination of this assay with an assay previously described for selective quantification of the SC5b-9 complex provides an important tool for evaluating terminal pathway activation of complement.

Animals↗
PubMed1987

Complementary DNA cloning of complement C8 beta and its sequence homology to C9.

The complete amino acid sequence of mature C8 beta has been derived from the DNA sequence of a cDNA clone identified by expression screening of a human liver cDNA library. Comparison with the amino acid sequence of C9 shows an overall homology with few deletions and insertions. In particular, the cysteine-rich domains and membrane-inserting regions of C9 are well conserved. These findings are discussed in relation to a possible mechanism of membrane attack complex formation.

Amino Acid Sequence↗
PubMed1987

A family of serine esterases in lytic granules of cytolytic T lymphocytes.

Cytoplasmic granules of cytolytic T lymphocytes (CTLs) contain, in addition to the pore-forming protein perforin, a family of highly homologous serine esterases, granzymes A-H. The serine esterase affinity label diisopropyl fluorophosphate reacts strongly with granzymes A and D, to a lesser extent with B, E, F, G, and H, and not at all with C and F. For granzymes A and D, synthetic substrates have been found. Antibodies raised against granzyme B strongly cross-react with A, G, and H, and antibodies to granzyme D recognize C, E, and F. These antigenic relationships correlate with similarities in the N-terminal amino acid sequences. At least 60% homology is observed between the eight proteins, and all are similar to rat mast cell protease 2. Sequence analysis suggests the identity of granzyme A with a protease predicted from a CTL-specific cDNA clone (H factor) and of granzyme B, G, or H with a protein encoded by the CTL-specific cDNA clone CTLA 1/CCP 1.

Amino Acid Sequence↗
PubMed1987

Antibody-independent activation of C1. II. Evidence for two classes of nonimmune activators of the classical pathway of complement.

Nonimmune activation of the first component of complement (C1) by cardiolipin (CL) vesicles present specific features which were not demonstrated on immune complexes. CL vesicles which activate C1 in the presence of C1-inhibitor (C1-INH) were found to bind C1s in the absence of C1r, and to induce a specific C1r-independent cleavage of C1q-bound C1s. Therefore, several known natural nonimmune activators were analyzed by comparing their ability to activate C1 in the presence of C1-INH and to mediate a C1r-independent cleavage of C1s. Freshly isolated human heart mitochondria (HHM) activated C1 only in the absence of C1-INH. However, mitoplasts derived from HHM (HHMP) activated C1 regardless of the presence of C1-INH, and induced a specific cleavage of C1q-bound C1s. The same pattern was observed in the case of smooth E. coli and a semi-rough E. coli strain. DNA, known to activate C1 only in the absence of C1-INH, does not induce C1s cleavage in the absence of C1r. Thus, nonimmune activators can be classified into two distinct categories. "Strong" activators, such as CL vesicles, HHMP, or the semi-rough E. coli strain J5 can activate C1 in the presence of C1-INH. By using C1qs2 as a probe, they exhibit a specific, C1r-independent cleavage of C1s. C1s-binding to C1q is a critical factor for the activation process in this group. In the case of "weak" activators, such as E. coli smooth strains, DNA, or HHM, no C1s-binding to activator-bound C1q was detected, and C1r-independent C1s cleavage and C1 activation in the presence of C1-INH were not observed. As in the case of immune complexes, C1r activation appears to play a key role in the C1 activation by "weak" activators.

Calcium↗