The first lipocalin with enzymatic activity.
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Acute-Phase Proteins↗
Biomedical subjects
M C Peitsch
Publications and source records attributed to M C Peitsch.
Localization and molecular modelling of the membrane-inserted domain of the ninth component of human complement and perforin.
Upon interaction with the membrane-bound C5b-8 complex, the ninth component of complement (C9) unfolds and inserts into the membrane of cells on which surface complement has been activated. Consequently C9 oligomerization occurs and transmembrane channels of varying sizes are formed. The domain of the unfolded protein interacting with the cell membrane has so far not been identified since, unlike many integral membrane proteins, the C9 sequence does not contain a continuous stretch of hydrophobic amino acids. We studied the interaction of C9 with the lipid bilayer using the membrane-restricted photoaffinity label 3-(trifluoromethyl)-3-(m[125I]iodophenyl)diazirine (125I-TID). C9 was assembled on liposomes and after photoactivation, several labeled and non-labeled peptides, obtained by chemical and enzymatic cleavage or the 125I-TID-labeled C9, were analyzed. The segment from 176 to 345 was identified as the region containing the membrane-interacting structure. By means of secondary structure predictions, we identified two amphipathic alpha-helices (292-308 and 313-333) separated by a turn (309-312). Based on these results, we constructed a molecular model for the membrane-spanning region of C9. By analogy, we also constructed a model for this domain in perforin/cytolysin, a pore-forming protein found in the cytoplasmic granules of cytotoxic T-lymphocytes.
Affinity Labels↗
Is apolipoprotein D a mammalian bilin-binding protein?
Human apolipoprotein D (APO-D) is a serum glycoprotein that has no sequence similarity with other apolipoproteins but rather belongs to the alpha 2-microglobulin superfamily whose other members transport small hydrophobic ligands in a wide variety of biological contexts. To investigate the ligand specificity of APO-D, we analyzed its relationship with the other members of this superfamily and constructed a detailed molecular model using the atomic coordinates of its most closely related homolog--insecticyanin from the tobacco hornworm, Manduca sexta. We studied the geometry of the binding pocket of APO-D and the topology of characteristic patches of both hydrophobic and polar side chains that also occur in crystal structures of insecticyanin and bilin-binding protein from the butterfly Pieris brassicae. From the data obtained we hypothesize that heme-related compounds may be more favorable ligands for APO-D than either cholesterol or cholesteryl ester. Preliminary experiments showed that purified human APO-D binds bilirubin in an approximately one-to-one molar ratio. These results suggest a new biological role for APO-D that is more congruent with its tissue distribution and evolutionary history.
Amino Acid Sequence↗
A purification method for apolipoprotein A-I and A-II.
Apolipoproteins A-I and A-II were isolated from precipitates obtained by cold ethanol fractionation of human plasma. The starting material used in this report was precipitate B of the Kistler and Nitschmann method which corresponds approximately to fraction III of the Cohn and Oncley procedure. Through the use of urea, chloroform, and ethanol in appropriate concentrations, apolipoproteins A-I and A-II were isolated by a simple extraction technique avoiding time-consuming ultracentrifugation. Starting from 10 g of centrifuged precipitate B, approximately 100 mg of apolipoprotein A-I and 10 mg of apolipoprotein A-II were obtained. When incubated with normal human or rabbit plasma, both apolipoproteins were readily incorporated into high-density lipoproteins. Apolipoprotein A-I obtained by the cold ethanol method activated lecithin-cholesterol acyltransferase to the same extent as apolipoprotein A-I prepared by the classical flotation method. Apolipoprotein A-II had no such properties by itself, but was capable of potentiating lecithin-cholesterol acyltransferase activity of apolipoprotein A-I.
Apolipoprotein A-I↗
Phosphorylcholine acts as a Ca2+-dependent receptor molecule for lymphocyte perforin.
Large granular lymphocytes and cytolytic T-lymphocytes (CTL) contain numerous cytoplasmic granules thought to be responsible, at least in part, for the cytolytic activity of these effector cells. Isolated granules are lytic for a variety of target cells and the granule proteins are specifically released upon target-cell interaction. Major proteins in mouse CTL granules are a family of seven serine proteases designated granzymes A to G, and a pore-forming protein called perforin (cytolysin). Purified perforin is cytolytic in the presence of Ca2+ and shows ultrastructural, immunological and amino-acid sequence similarities to complement component C9. Despite these similarities, perforin and C9 are clearly distinct in their mode of target-cell recognition. Whereas C9 insertion is absolutely dependent on a receptor moiety assembled from the complement proteins C5b, C6, C7, and C8 on the target-cell membrane, no requirement for a receptor molecule has been reported for perforin. Here, we demonstrate that phosphorylcholine acts as a specific, Ca2+-dependent receptor molecule for perforin.
Animals↗
A rapid and efficient method for the purification of the complement subcomponents C1r and C1s in zymogen form using fast protein chromatography.
The purification of the subcomponents C1r and C1s of the first component of complement involves multiple steps and is time-consuming. This accounts for the frequently observed partial activation of the subcomponents. In this report we propose a simplified procedure of purification using a batch method and fast protein chromatography avoiding a shift of pH. The method provides C1r and C1s in a yield of 35 and 60% respectively. In addition, this study provides a simple and sensitive test to assess functional purity of C1r and C1s with respect to the other C1 subcomponents.
Chromatography, High Pressure Liquid↗
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↗
Antibody-independent activation of C1. I. Differences in the mechanism of C1 activation by nonimmune activators and by immune complexes: C1r-independent activation of C1s by cardiolipin vesicles.
C1 activation is controlled by the regulatory protein C1-inhibitor (C1-INH). In contrast to immune-complex-induced activation, which is insensitive to C1-INH, antibody-independent activation of C1 is modulated by C1-INH. The mechanisms regulating nonimmune activation were studied with two phospholipids varying in their capacity to activate C1 in the presence of C1-INH: cardiolipin (CL) and phosphatidylglycerol (PG). Whereas C1-INH consistently suppressed activation by PG vesicles, a dose-dependent increase in C1 activation was measured with CL vesicles above 40 mole %. A similar dose-response binding of C1s requiring C1q, but not C1r, was detected only on CL vesicles, but neither on PG vesicles nor on immune complexes. This binding was Ca2+-dependent, suggesting that dimeric C1s is involved and was inhibited by spermine. The C1q-bound C1s was specifically cleaved at 37 degrees C into its active 58 kDa and 28 kDa chains, in the absence of C1r. On the addition of anti-CL antibodies, the C1q-mediated cleavage of C1s by CL vesicles was specifically inhibited. The cleavage of C1r on CL vesicles was also determined. When macromolecular C1 was offered in the presence of C1-INH, C1r cleavage was detected; however, the presence of C1s was a critical factor for C1r activation, because it was required on CL vesicles, but not on immune complexes. These results show that nonimmune activation of C1 presents specific features which distinguish it from immune complex-induced activation. These characteristics varied with the capacity of antibody-independent activators to activate C1 in the presence of C1-INH.
Antigen-Antibody Complex↗
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↗
Large-scale protein modelling and integration with the SWISS-PROT and SWISS-2DPAGE databases: the example of Escherichia coli.
Knowledge-based molecular modelling of proteins has proven useful in many instances, including the rational design of mutagenesis experiments, but it has generally been limited by the availability of expensive computer hardware and software. To overcome these limitations, we developed the SWISS-MODEL server for automated knowledge-based protein modelling. The SWISS-MODEL server uses the Brookhaven Protein Data Bank as a source of structural information and automatically generates protein models for sequences which share significant similarities with at least one protein of known three-dimensional structure. We have now used the software framework of the server to generate large collections of protein models, and established the SWISS-MODEL Repository, a new database for automatically generated and theoretical protein models. This repository is directly integrated with the SWISS-PROT and SWISS-2DPAGE databases through the ExPASy World Wide Web server (URL is http://expasy.hcuge.ch). Here we present an illustration of this process by an application to the Escherichia coli sequences.
Bacterial Proteins↗
Structural and functional characterization of complement C8 gamma, a member of the lipocalin protein family.
Human complement component C8 exhibits an unusual structure in that it contains three chains, two of which, alpha and beta, display high sequence homology to other complement and CTL pore-forming proteins. The third chain, C8 gamma, is covalently linked to C8 alpha by a disulfide linkage; it is demonstrated that Cys40 of C8 gamma is linked to Cys164 of C8 alpha, a unique cysteine located in a loop located between the cysteine-rich LDL-receptor class A module and the membrane-inserting region of C8 alpha. C8 gamma was recently identified as a member of the lipocalin protein family, in which all proteins were either shown to, or are believed to bind small hydrophobic ligands. The present results now demonstrate that C8 gamma incorporates retinol and retinoic acid in the presence of 2 M NaCl. Molecular modeling of C8 gamma, based on the crystal structure of the homologous beta-lactoglobulin, reveals a structure of eight antiparallel beta-strands, bearing a highly hydrophobic binding pocket. The residues participating in the pocket formation are highly conserved when compared with the structures of beta-lactoglobulin and retinol-binding protein, both of which are known to interact with retinol. It is therefore proposed that C8 gamma may act as a retinol transporting protein in plasma.
Alpha-Globulins↗
Human CD40 ligand: molecular cloning, cellular distribution and regulation of IgE synthesis.
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CD40 Ligand↗