Paul Falmagne

Using two-dimensional electrophoresis, we have recently identified in human bronchoalveolar lavage fluid a novel protein, termed B166, with a molecular mass of 17 kDa. Here, we report the cloning of human and rat cDNAs encoding B166, which has been renamed AOEB166 for antioxidant enzyme B166. Indeed, the deduced amino acid sequence reveals that AOEB166 represents a new mammalian subfamily of AhpC/TSA peroxiredoxin antioxidant enzymes. Human AOEB166 shares 63% similarity with Escherichia coli AhpC22 alkyl hydroperoxide reductase and 66% similarity with a recently identified Saccharomyces cerevisiae alkyl hydroperoxide reductase/thioredoxin peroxidase. Moreover, recombinant AOEB166 expressed in E. coli exhibits a peroxidase activity, and an antioxidant activity comparable with that of catalase was demonstrated with the glutamine synthetase protection assay against dithiothreitol/Fe3+/O(2) oxidation. The analysis of AOEB166 mRNA distribution in 30 different human tissues and in 10 cell lines shows that the gene is widely expressed in the body. Of interest, the analysis of N- and C-terminal domains of both human and rat AOEB166 reveals amino acid sequences presenting features of mitochondrial and peroxisomal targeting sequences. Furthermore, human AOEB166 expressed as a fusion protein with GFP in HepG2 cell line is sorted to these organelles. Finally, acute inflammation induced in rat lung by lipopolysaccharide is associated with an increase of AOEB166 mRNA levels in lung, suggesting a protective role for AOEB166 in oxidative and inflammatory processes.

A recombinant lambda bacteriophage has been isolated that carries DNA from Streptococcus pneumoniae and expresses a potent hemolysin that has been shown to be pneumolysin, the sulfhydryl-activated toxin of the pneumococcus. Hemolytic activity is inhibited by cholesterol and neutralized by serum against streptolysin O. The cloned gene expresses two polypeptides (Mrs, 56,000 and 53,000) in an Escherichia coli in vitro transcription-translation system, and both are precipitated by the addition of anti-alveolysin serum and anti-streptolysin O serum in the presence of Staphylococcus aureus cells. Expression of pneumolysin occurs when the gene is cloned in both possible orientations in pUC8. The DNA sequence of a 5-kilobase ClaI fragment that carries the pneumolysin gene has been determined. An open reading frame was identified that encodes a polypeptide of 471 amino acids that is hydrophobic in character and has an N-terminal amino acid sequence which is identical to that deduced from amino acid sequencing of the purified protein. The predicted amino acid sequence of the polypeptide reveals a single cysteine residue located 44 residues from the C terminus. Putative promoter and ribosome binding sites have been identified 5' to the pneumolysin coding sequence.

A 67000 Mr bovine pregnancy-associated glycoprotein (bPAG) has been isolated from fetal cotyledons and purified to homogeneity by HPLC. The purification was monitored by a double immunodiffusion test and by RIA in conjunction with an antiserum raised against a crude fraction of placenta-specific antigens. The molecular weight of bPAG was estimated to be 67000 by SDS-PAGE. The isoelectric points (pI) of the four isoforms, determined by high-resolution analytical electrofocusing in polyacrylamide gel, were 4.4, 4.6, 5.2, and 5.4. The carbohydrate content of the bPAG consisted of approximately 10.02 +/- 1.09% neutral sugar and variant amounts of sialic acid (from 0.29 +/- 0.06% in the most basic isoform to 2.1 +/- 0.31% in the most acidic isoform). A specific antiserum was raised against the purified bPAG. A specific RIA showed that the bPAG was antigenically unrelated to BSA, alphafetoprotein (AFP), and human schwangerschafts-spezifischen (pregnancy-specific) beta 1 glycoprotein (SP1). According to some characteristics (e.g. the molecular weight), the purified bPAG may correspond to a form of the pregnancy-specific protein B previously described by Sasser and colleagues (Biol Reprod 1986; 35:936-942).

We used infrared attenuated total reflection spectroscopy to study the structure of diphtheria toxin (DT) and its fragments A, B, CB1, and CB4 as a function of the pH in the absence and in the presence of phospholipid vesicles. Binding of DT to asolectin or DL-alpha-dipalmitoylphosphatidylcholine-DL-alpha-dipalmitoylphosphatidic acid liposomes at pH 7.3 results in a 10% increase in its alpha-helix content. At pH 4, in the presence of liposomes, the secondary structure of DT is characterized by the appearance of a beta-sheet structure with strengthened hydrogen bonds which did not exist before pH lowering. DT fragment B displays little conformational change upon pH lowering in the presence of liposomes. However, the alpha-helix content of CB1 increases by 10%, and polarization measurements indicate that the alpha-helices of CB1 at pH 4 are oriented parallel to the lipid acyl chains. On the other hand, the alpha-helix content of CB4 decreases dramatically while the low frequency beta-sheet content increases. Dichroism measurements demonstrate that this sheet lies close to a parallel to the bilayer surface. The fragment A of DT experiences a large conformational change upon pH lowering and binds to the liposome membrane even in the absence of DT fragment B. The conformational modification of DT fragment A is fully reversed when pH is brought back to 7.3.