Biomedical subjects

Wilhelm Schwaeble

Publications and source records attributed to Wilhelm Schwaeble.

10 recordsLinked to original sources

PubMed2005

An in vivo role for Trypanosoma cruzi calreticulin in antiangiogenesis.

Angiogenesis leads to neovascularization from existing blood vessels. It is associated with tumor growth and metastasis and is regulated by pro- and antiangiogenic molecules, some of them currently under clinical trials for cancer treatment. During the last few years we have cloned, sequenced and expressed a Trypanosoma cruzi calreticulin gene (TcCRT). Its product, TcCRT, a 45 kDa protein, is more than 50% identical to human CRT (HuCRT). TcCRT, present on the surface of trypomastigotes, binds both C1q and mannan binding lectin and inhibits the classical activation pathway of human complement. Since TcCRT is highly homologous to a functional antiangiogenic fragment from HuCRT (aa 120-180), recombinant (r) and native (n) TcCRT were tested in their antiangiogenic effects, in the chick embryonic chorioallantoid membrane (CAM) assay. Both proteins mediated highly significant antiangiogenic effects in the in vivo CAM assay. This effect was further substantiated in experiments showing that the plasmid construct pSecTag/TcCRT also displayed significant antiangiogenic properties, as compared to the empty vector. Most likely, the fact that antiangiogenic substances act preferentially on growing neoplasic tissues, but not on already established tumors, is due to their effects on emerging blood vessels. The results shown here indicate that TcCRT, like its human counterpart, has antiangiogenic properties. These properties may explain, at least partly, the reported antineoplasic effect of experimental T. cruzi infection.

Angiogenesis Inhibitors↗

PubMed2005

Does Trypanosoma cruzi calreticulin modulate the complement system and angiogenesis?

Calreticulin, a calcium-binding protein that is highly conserved in its multiple functions, is present in a wide spectrum of subcellular compartments in virtually every cell of higher organisms. In this article, we propose a dual role for parasite calreticulin, with emphasis on the Trypanosoma cruzi model. By modulating the vertebrate complement system, calreticulin might provide the parasite with an effective immune-escape mechanism. Alternatively, by inhibiting angiogenesis, the parasite molecule might protect the host from ongoing neoplasic aggressions. Many questions are still unanswered, particularly those regarding the consequences that these interactions could have in vivo for both the parasite and the host.

Angiogenesis Modulating Agents↗

PubMed2005

F(ab')2 antibody fragments against Trypanosoma cruzi calreticulin inhibit its interaction with the first component of human complement.

Trypanosoma cruzi calreticulin (TcCRT), described in our laboratory, retains several important functional features from its vertebrate homologues. We have shown that recombinant TcCRT inhibits the human complement system when it binds to the collagenous portion of C1q. The generation of classical pathway convertases and membrane attack complexes is thus strongly inhibited. In most T. cruzi-infected individuals, TcCRT is immunogenic and mediates the generation of specific antibodies. By reverting the C1q / TcCRT interaction, a parasite immune evasion strategy, these antibodies contribute to the host/parasite equilibrium. In an in vitro correlate of this situation, we show that the Clq/TcCRT interaction is inhibited by F(ab')2 polyclonal anti-TcCRT IgG fragments. It is therefore feasible that in infected humans anti-TcCRT antibodies participate in reverting an important parasite strategy aimed at inhibiting the classical complement pathway. Thus, membrane-bound TcCRT interacts with the collagenous portion Clq, and this Clq is recognized by the CD91-bound host cell CRT, thus facilitating parasite internalization. Based on our in vitro results, it could be proposed that the in vivo interaction between TcCRT and vertebrate Clq could be inhibited by F(ab')2 fragments anti-rTcCRT or against its S functional domain, thus interfering with the internalization process.

Animals↗

PubMed2004

The classical activation pathway of the human complement system is specifically inhibited by calreticulin from Trypanosoma cruzi.

The high resistance of Trypanosoma cruzi trypomastigotes, the causal agent of Chagas' disease, to complement involves several parasite strategies. In these in vitro studies, we show that T. cruzi calreticulin (TcCRT) and two subfragments thereof (TcCRT S and TcCRT R domains) bind specifically to recognition subcomponents of the classical and lectin activation pathways (i.e., to collagenous tails of C1q and to mannan-binding lectin) of the human complement system. As a consequence of this binding, specific functional inhibition of the classical pathway and impaired mannan-binding lectin to mannose were observed. By flow cytometry, TcCRT was detected on the surface of viable trypomastigotes and, by confocal microscopy, colocalization of human C1q with surface TcCRT of infective trypomastigotes was visualized. Taken together, these findings imply that TcCRT may be a critical factor contributing to the ability of trypomastigotes to interfere at the earliest stages of complement activation.

Animals↗

PubMed2004

Role of calreticulin from parasites in its interaction with vertebrate hosts.

Although parasites range from protozoan to complex, evolutionary advanced arthropods, in general, a hallmark of parasite life cycles is their ability to adapt to changes in temperature, pH and host defense strategies. Calreticulin, a calcium-binding protein, highly conserved and multifunctional, is present in every cell of higher organisms, except erythrocytes. The surprising array of calreticulin-associated functions include lectin-like chaperoning, calcium storage and signaling, modulation of gene expression, cell adhesion, enhancement of phagocytosis of C1q or collectin opsonized apoptotic cells, inhibition of angiogenesis and tumoral growth, inhibition of perforin pore formation in T and NK cells, and inhibition of C1q-dependent complement activation. Likewise, calreticulin is present in a wide spectrum of sub cellular compartments. Parasite calreticulin shows a surprisingly high degree of conservation within the framework of its functional domains. Its role within the parasite/host relationship needs to be assessed further, in particular with regard to its impact on parasite infectivity, by helping to evade from its hosts' immune response. With special emphasis on calreticulin from Trypanosoma cruzi, the intracellular protozoan agent of American trypanosomiasis (Chagas' disease), we wish to exemplify and highlight the various implications of parasite calreticulin, within the pathophysiology of parasite-mediated human and animal disease.

Animals↗

PubMed2004

Involvement of the lectin pathway of complement activation in antimicrobial immune defense during experimental septic peritonitis.

A critical first line of defense against infection is constituted by the binding of natural antibodies to microbial surfaces, activating the complement system via the classical complement activation pathway. In this function, the classical activation pathway is supported and amplified by two antibody-independent complement activation routes, i.e., the lectin pathway and the alternative pathway. We studied the contribution of the different complement activation pathways in the host defense against experimental polymicrobial peritonitis induced by cecal ligation and puncture by using mice deficient in either C1q or factors B and C2. The C1q-deficient mice lack the classical complement activation pathway. While infection-induced mortality of wild-type mice was 27%, mortality of C1q-deficient mice was increased to 60%. Mice with a deficiency of both factors B and C2 lack complement activation via the classical, the alternative, and the lectin pathways and exhibit a mortality of 92%, indicating a significant contribution of the lectin and alternative pathways of complement activation to survival. For 14 days after infection, mannan-binding lectin (MBL)-dependent activation of C4 was compromised. Serum MBL-A and MBL-C levels were significantly reduced for 1 week, possibly due to consumption. mRNA expression profiles did not lend support for either of the two MBL genes to respond as typical acute-phase genes. Our results demonstrate a long-lasting depletion of MBL-A and MBL-C from serum during microbial infection and underline the importance of both the lectin and the alternative pathways for antimicrobial immune defense.

Animals↗

PubMed2003

Natural substrates and inhibitors of mannan-binding lectin-associated serine protease-1 and -2: a study on recombinant catalytic fragments.

Mannan-binding lectin-associated serine protease (SP) (MASP)-1 and MASP-2 are modular SP and form complexes with mannan-binding lectin, the recognition molecule of the lectin pathway of the complement system. To characterize the enzymatic properties of these proteases we expressed their catalytic region, the C-terminal three domains, in Escherichia coli. Both enzymes autoactivated and cleaved synthetic oligopeptide substrates. In a competing oligopeptide substrate library assay, MASP-1 showed extreme Arg selectivity, whereas MASP-2 exhibited a less restricted, trypsin-like specificity. The enzymatic assays with complement components showed that cleavage of intact C3 by MASP-1 and MASP-2 was detectable, but was only approximately 0.1% of the previously reported efficiency of C3bBb, the alternative pathway C3-convertase. Both enzymes cleaved C3i 10- to 20-fold faster, but still at only approximately 1% of the efficiency of MASP-2 cleavage of C2. We believe that C3 is not the natural substrate of either enzyme. MASP-2 cleaved C2 and C4 at high rates. To determine the role of the individual domains in the catalytic region of MASP-2, the second complement control protein module together with the SP module and the SP module were also expressed and characterized. We demonstrated that the SP domain alone can autoactivate and cleave C2 as efficiently as the entire catalytic region, while the second complement control protein module is necessary for efficient C4 cleavage. This behavior strongly resembles C1s. Each MASP-1 and MASP-2 fragment reacted with C1-inhibitor, which completely blocked the enzymatic action of the enzymes. Nevertheless, relative rates of reaction with alpha-2-macroglobulin and C1-inhibitor suggest that alpha-2-macroglobulin may be a significant physiological inhibitor of MASP-1.

Binding, Competitive↗

PubMed2003

Molecular comparison of apocrine released and cytoplasmic resident carbonic anhydrase II.

We have previously shown that carbonic anhydrase II usually described as a cytoplasmic resident isoform (cCAH II) is secreted by the rat coagulating gland (sCAH II) via the apocrine secretion mode. To get more detailed information why CAH II is cytoplasmic resident in some organs and secreted in others we cloned and sequenced the cDNA of rat coagulating gland sCAH II. The sequence of the secretory form was found to be completely identical with the cCAH II. Therefore, a signal peptide targeting sCAH II for apocrine secretion can be excluded. Considering the fact that other apocrine secreted proteins are glycosylated, cCAH II and sCAH II were analyzed for carbohydrate substitutions. As expected for a cytoplasmic protein, no glycan modification could be identified in cCAH II. In contrast, sCAH II carried exclusively Gal, GlcNAc and Fuc residues in a molar ratio of 1:0.8:0.5. Carbohydrate linkage analyses demonstrated the presence of terminal Fuc, terminal, 3-substituted and 3,6-disubstituted Gal as well as 4-substituted and 3,4-disubstituted GlcNAc. The composition of the glycan constituents as well as deglycosylation experiments clearly proved that sCAH II carries neither conventional mammalian-type N-glycans nor mucin-type O-linked sugar chains. Lacking a signal peptide for ER translocation, glycosylation of sCAH II must occur within the cytoplasmic compartment. Further studies have to elucidate whether or not glycosylation of sCAH II is essential for the apocrine release of the protein.

Amino Acid Sequence↗

PubMed2002

In vivo biosynthesis of endogenous and of human C1 inhibitor in transgenic mice: tissue distribution and colocalization of their expression.

We have produced transgenic mice expressing human C1 inhibitor mRNA and protein under the control of the human promoter and regulatory elements. The transgene was generated using a minigene construct in which most of the human C1 inhibitor gene (C1NH) was replaced by C1 inhibitor cDNA. The construct retained the promoter region extending 1.18 kb upstream of the transcription start site, introns 1 and 2 as well as a stretch of 2.5 kb downstream of the polyadenylation site, and therefore carried all known elements involved in transcriptional regulation of the C1NH gene. Mice with high serum levels of human C1 inhibitor, resulting from multiple tandem integrations of the C1 inhibitor transgene, were selected. Immunohistochemistry in combination with in situ hybridization was applied to localize the sites of C1 inhibitor biosynthesis and to demonstrate its local production in brain, spleen, liver, heart, kidney, and lung. The distribution of human C1 inhibitor-expressing cells was qualitatively indistinguishable from that of its mouse counterpart, but expression levels of the transgene were significantly higher. In the spleen, production of C1 inhibitor was colocalized with that of a specific marker for white pulp follicular dendritic cells. This study demonstrates a stringently regulated expression of both the endogenous and the transgenic human C1 inhibitor gene and reveals local biosynthesis of C1 inhibitor at multiple sites in which the components of the macromolecular C1 complex are also produced.

Animals↗

PubMed2002

The mannan-binding lectin-associated serine proteases (MASPs) and MAp19: four components of the lectin pathway activation complex encoded by two genes.

Mannan-binding lectin (MBL) and ficolins (L-ficolin and H-ficolin) initiate the lectin pathway of complement activation upon binding to microbial carbohydrates. The activation is mediated by associated serine proteases, termed MASPs, since they were discovered as MBL-associated serine proteases. The MASP family comprises three serine proteases, MASP-1, MASP-2 and MASP-3 and a non-enzymatic protein, MAp19. The MASPs show identical domain structure, shared also with C1r and C1s. MASP-1 and MASP-3 are alternative splice products of a single gene, MASP1/3, and have identical A chains, whereas they have individual B chains, encompassing the serine protease domain. MASP2 and MAp19 are alternative splice products of the MASP-2 gene, with MAp19 consisting of the first two domains of MASP-2 plus additional four amino acid residues. MASP-2 is the protease responsible for activating C4 and C2 to generate the C3 convertase, C4bC2b. The biological function of the remaining three proteins has not yet been resolved.

Animals↗