Séverine Frutiger

Human α and β parvalbumins

alpha and beta parvalbumins are Ca(2+)-binding proteins of the EF-hand type. We determined the protein sequence of human brain alpha parvalbumin by mass spectrometry and cloned human beta parvalbumin (or oncomodulin) from genomic DNA and preterm placental cDNA. beta parvalbumin differs in 54 positions from alpha parvalbumin and lacks the C-terminal amino acid 109. From MS analyses of alpha and beta parvalbumins we conclude that parvalbumins generally lack posttranslational modifications. alpha and beta parvalbumins were differently expressed in human tissues when analyzed by immunoblotting and polymerase-chain-reaction techniques. Whereas alpha parvalbumin was found in a number of adult human tissues, beta parvalbumin was restricted to preterm placenta. The pattern of alpha parvalbumin expression also differs in man compared to other vertebrates. For example, in rat, alpha parvalbumin was found in extrafusal and intrafusal skeletal-muscle fibres whereas, in man, alpha parvalbumin was restricted to the muscle spindles. Different functions for alpha and beta parvalbumins are discussed.

A human de‐ubiquitinating enzyme with both isopeptidase and peptidase activities in vitro

Some enzymatic and physicochemical properties of a human ubiquitin‐specific isopeptidase are reported. The enzyme was purified to homogeneity from red blood cells and its specificity towards polymeric ubiquitin substrates suggests a de‐ubiquitinating activity capable of cleaving ‘head‐to‐tail’ polyUb chains as well as isoamide ‘branched’ Ub dimers. K M values show a 10 fold preference for the cleavage of branched Ub dimers over head‐to‐tail Ub dimers. The enzymatic activity can be strongly inhibited by various peptides containing either of the cleavage site sequences found in Ub polymers, but not by unrelated peptides. The enzyme is monomeric under reducing conditions and exhibits a globular shape with an average diameter of 9 nm, an S20,w value of 5.2 S and a molar mass of 110 kDa ± 10%. Because the enzyme cleaves both peptide‐linked and isopeptide‐linked Ub moieties from substrates, we propose to name it de‐ubiquitinase rather than isopeptidase.

Increased stability upon heptamerization of the pore-forming toxin aerolysin

Aerolysin is a bacterial pore-forming toxin that is secreted as an inactive precursor, which is then processed at its COOH terminus and finally forms a circular heptameric ring which inserts into membranes to form a pore. We have analyzed the stability of the precursor proaerolysin and the heptameric complex. Equilibrium unfolding induced by urea and guanidinium hydrochloride was monitored by measuring the intrinsic tryptophan fluorescence of the protein. Proaerolysin was found to unfold in two steps corresponding to the unfolding of the large COOH-terminal lobe followed by the unfolding of the small NH2-terminal domain. We show that proaerolysin contains two disulfide bridges which strongly contribute to the stability of the toxin and protect it from proteolytic attack. The stability of aerolysin was greatly enhanced by polymerization into a heptamer. Two regions of the protein, corresponding to amino acids 180–307 and 401–427, were identified, by limited proteolysis, NH2-terminal sequencing and matrix-assisted laser desorption ionization-time of flight, as being responsible for stability and maintenance of the heptamer. These regions are presumably involved in monomer/monomer interactions in the heptameric protein and are exclusively composed of β structure. The stability of the aerolysin heptamer is reminiscent of that of pathogenic, fimbrial protein aggregates found in a variety of neurodegenerative diseases.

Human free secretory component is composed of the first 585 amino acid residues of the polymeric immunoglobulin receptor

The main objective of this work was to unequivocally determine the C‐terminal sequence of human milk free secretory component (SC). It was found to end at arginine‐585, i.e. 33 amino acids downstream from the major heterogeneous C‐terminal residue previously identified for colostrum SC. In contrast, our data showed that the C‐terminal end of SC was found to be homogeneous. Conflicting assignments, Asp/Gln, a missing Asn‐211, Asp/Asn, Glu/Gln were corrected and found to agree with the cDNA sequence. An Ala/Val substitution at position 562 (domain VI) was identified. Its genetic significance is uncertain at present.

cDNA cloning of a human 100 kDa de-ubiquitinating enzyme: the 100 kDa human de-ubiquitinase belongs to the ubiquitin C-terminal hydrolase family 2 (UCH2)

The full length cDNA encoding a 100 kDa human de‐ubiquitinating enzyme, referred to as de‐ubiquitinase was obtained using one clone selected from a randomly sequenced human brain cDNA library and specific primers. The sequence of 18 peptides generated from the de‐ubiquitinase isolated from outdated human erythrocytes matched perfectly with the predicted amino acid sequence, which would encode a protein containing 858 amino acids (calculated M r = 95,743 Da). Homology search disclosed that the protein is a member of a large family of ubiquitin C‐terminal hydrolases (UCH2), that was defined on the basis of the presence of two specific patterns, ‘the Cys‐ and His‐domains’, which are likely to be involved in the de‐ubiquitinating activity [7]. An additional conserved region, ‘the aspartic acid domain’, was also identified, the functional role of which is unknown.

The yeast protein encoded by PUB1 binds T-rich single stranded DNA.

We have characterized binding activities in yeast which recognise the T-rich strand of the yeast ARS consensus element and have purified two of these to homogeneity. One (ACBP-60) is detectable in both nuclear and whole cell extracts, while the other (ACBP-67) is apparent only after fractionation of extracts by heparin-sepharose chromatography. The major binding activity detected in nuclear extracts was purified on a sequence-specific DNA affinity column as a single polypeptide with apparent mobility of 60kDa (ACBP-60). This protein co-fractionates with nuclei, is present at several thousand copies per cell and has a Kd for the T-rich single strand of the ARS consensus between 10(-9) and 10(-10) M. Competition studies with simple nucleic acid polymers show that ACBP-60 has marginally higher affinity for poly dT30 than for a 30 nt oligomer containing the T-rich strand of ARS 307, and approximately 10 fold higher affinity for poly rU. Internal sequence information of purified p60 reveals identity with the open reading frames of genes PUB1 and RNP1 which encode polyuridylate binding protein(s). The second binding activity, ACBP-67, also binds specifically to the T-rich single strand of the ARS consensus, but with considerably lower affinity than ACBP-60. Peptide sequence reveals that the 67kDa protein is identical to the major polyA binding protein in yeast, PAB1.

Thiol–disulfide redox buffers maintain a structure of immunoglobulin A that is essential for optimal in vitro binding to secretory component

We have shown that human secretory component (SC) binds in vitro to different samples of human and murine dimeric immunoglobulin A (IgA). The binding ratio in the IgA/SC complex is 1:1. IgA which is stably bound to SC is separated from unreacted IgA by anion exchange chromatography. A part of IgA/SC complexes formed in vitro is unstable to this elution; the proportion varies between different samples of IgA; it increases following prolonged incubation of IgA at 37 degrees C. Incubation of IgA with glutathione/glutathione disulfide (GSH/GSSG) redox buffers increases the proportion able to form a stable complex with SC to approximately 90%. The presence of bound SC is not essential for this process but does allow it to occur at a lower GSH/GSSG concentration. The stable IgA/SC complex consists of a structure with a disulfide bond between IgA and SC apparently in equilibrium with a structure in which this bond is absent. The proportion bound covalently is similar for different samples of IgA and is insensitive to incubation with GSH/GSSG. It is significantly greater for secretory IgA (sIgA) and for IgA and SC incubated together with a starting mixture of cysteine/cystine. Monoclonal, antigen-specific IgA, all of which is optimally bound to SC in essentially the same way as in native sIgA, can be isolated in high yield. Our results support a mechanism for optimal binding of IgA to SC, that can occur both in vitro and in vivo, in which a thiol disulfide interchange occurs between a free IgA thiol and a sensitive SC disulfide following the initial non-covalent interaction.

Amino Acid Analysis at High Sensitivity: A Critical Review of the Communications

Some 13 oral and poster presentations (ignoring repeated communications) were given specifically on methodology for amino acid analysis. These concerned the well documented methods of precolumn derivatisation with FM0C-C1, PITC, OPA, DABS-Cl and Dns-Cl. Unfortunately, though all these precolumn methods have been applied for several years, there still appears to be no consensus as to a high sensitivity replacement for the ninhydrin postcolumn detection system.