Françoise Schoentgen

From structure to function: possible biological roles of a new widespread protein family binding hydrophobic ligands and displaying a nucleotide binding site

A cytosolic 21–23 kDa protein isolated from bovine brain was demonstrated to bind hydrophobic ligands, particularly phosphatidylethanolamine. The protein was encountered in numerous tissues of several species. High expression of the mRNA encoding the 21–23 kDa protein was found in rat testes. Immunohistochemical studies showed the presence of the 21–23 kDa protein in the elongated spermatids and epididymal fluid of rat testis and in brain oligodendrocytes of developing rats. As the bovine, human and rat brain 21–23 kDa proteins had only few sequence homologies with already known proteins, it was concluded that they belong to a new protein family. In order to get additional information on the structural features of the 21–23 kDa protein, we built a molecular model which displayed a nucleotide binding site. The affinity of the bovine brain 21–23 kDa protein towards nucleotides as well as its association with cytosolic proteins and small GTP‐binding proteins were demonstrated. Recently, significant sequence homologies were found with an antigen from Onchocerca volvulus, a fruit fly odorant‐binding protein and the yeast protein TFS1 which is a dosage‐dependant suppressor of CDC25 mutations. A positive regulation of RAS is carried out by CDC25 product which facilitates the GDP/GTP exchange on RAS proteins. These results imply that 21–23 kDa proteins function in oxidoreduction reactions and signal mechanisms during cell growth and maturation.

Insect lysozymes from three species of lepidoptera: Their structural relatedness to the c (chicken) type lysozyme

SummarySequence studies of the N-terminal halves of the lysozymes isolated fromBombyx mori, Galleria mellonella andSpodoptera littoralis (Lepidoptera) allow us to classify these enzymes among the c (chicken) type lysozymes.

Amino acid sequence and immunological properties of chachalaca egg white lysozyme

SummaryThe amino acid sequence of lysozyme c from chachalaca egg white was determined. Like other bird lysozymes c, that of the chachalaca has 129 amino acid residues. It differs from other avian lysozymes c by 27 to 31 amino acid substitutions as well as by being devoid of phenylalanine. It contains substitutions at 9 positions which are invariant in the other 7 bird lysozymes of known sequence. Although the chachalaca is classified zoologically in the order Galliformes, which includes chickens and other pheasant-like birds, its lysozyme differs more from those of pheasant-like birds than do the lysozymes c of ducks. Phylogenetic analysis of the sequence comparisons confirms that the lineage leading to chachalaca lysozyme c separated from that leading to other galliform lysozymes c before the duck lysozyme c lineage did. This indicates a contrast between protein evolution and evolution at the organismal level. Immunological comparison of chachalacalysozyme c with other lysozymes of known sequence provides further support for the proposal that immunological cross-reactivity is strongly dependent on degree of sequence resemblance among bird lysozymes.

Sheep κ-casein peptides inhibit platelet aggregation

The C-terminal part (residues 106-171) of sheep kappa-casein, called caseinoglycopeptide (CGP), inhibits thrombin- and collagen-induced platelet aggregation in a dose-dependent manner (mean inhibitory concentration (IC50) 215 microM and 100 microM, respectively). An enzymatic hydrolysate of CGP was fractionated by reverse phase high performance liquid chromatography: three peptides KDQDK (residues 112-116), TAQVTSTEV (residues 163-171) and QVTSTEV (residues 165-171) completely inhibited thrombin-induced platelet aggregation. CGP at a concentration near its IC50 had a very long life when incubated in human or guinea-pig plasma. An ex vivo experiment showed that 17% of CGP was found 60 min after its i.v. bolus injection in guinea-pig. By hydrophobic cluster analysis, human fibrinogen and sheep kappa-casein peptides, inhibitors of platelet aggregation, were compared and we observed similarities for their C-terminal parts and for their short peptides (RGDF and KDQDK).

Amino acid sequence of the Homo sapiens brain 21-23-kDa protein (Neuropolypeptide h3), Comparison with its counterparts from Rattus norvegicus and Bos taurus species, and expression of its mRNA in different tissues

The amino acid sequence of neuropolypeptide h3 from Homo sapiens brain has been determined. It revealed that h3 is the exact counterpart of the 21-kDa protein from Bos taurus brain and the 23-kDa protein from Rattus norvegicus brain: The three proteins belong to the same 21-23-kDa protein family. Multiple tissue Northern blots showed that the mRNA encoding the 21-23-kDa protein is expressed in different amounts according to tissues and species; it is particularly abundant in Rattus norvegicus testis.

Immunological Investigation of a 21-Kilodalton Cytosolic Basic Protein in Rat Brain

A gamma-globulin fraction was isolated from the antiserum raised against a 21-kilodalton (kDa) basic protein which was purified from bovine brain cytosol. This fraction was employed to study the immunocytochemical localization of the 21-kDa protein during the development of rat brain. Immunostaining was observed on oligodendrocytes and their processes at all stages of development investigated. This immunostaining was less prominent in very young and adult brains. Myelin fibers were always moderately stained; neurons and astrocytes were not immunolabelled. The electron microscopic study revealed that the labelling covers the entire cytoplasm of the oligodendrocytes, being more dense along the membranes of the rough endoplasmic reticulum and the plasma membrane. Other cytoplasmic organelles were unstained. The present report emphasizes that 21-kDa protein may serve as a specific marker for oligodendroglial cells in the central nervous system despite its presence in peripheral organs.

Structural data concerning the major rat brain myelin proteolipid P7 apoprotein.

In a previous paper, we described the isolation of the major rat brain myelin proteolipid W apoprotein by preparative sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis; the establishment of the N-terminal sequence up to the twentieth amino acid was thus possible [ 11. Identical sequences were found by studying the P7 apoprotein of human myelin [2] and corresponding apoproteins of bovine white matter [3]. Proteolipid preparations have been described to be resistant to the action of endopeptidases [4]. However Lees et al. [5] concluded that proteolytic digestion was possible in the presence of non-ionic detergent. In the present paper, we report for the first time the isolation and the sequence of several tryptic peptides. The apoprotein N-terminal sequence could be lengthened and the C-terminal sequence precised. Furthermore, the previously reported arginine content of P7 apoprotein calculated as 6 residues/m01 on a molecuIar weight basis of 23 500 [l] could be confirmed. The six arginine containing peptides were characterized.

Homology between the human vitamin D-binding protein (group specific component), α-fetoprotein and serum albumin

41 Amino acid long N‐terminal sequences of the three major human vitamin D‐binding proteins (group‐specific components Gc1F, Gc1S and Gc2) were characterized: they were identical. By computer analyses, the alignment of this N‐terminal sequence with several sequences of human serum pre‐proalbumin and human pre‐α‐fetoprotein was established

Improved Accuracy of Low Affinity Protein–Ligand Equilibrium Dissociation Constants Directly Determined by Electrospray Ionization Mass Spectrometry

There is continued interest in the determination by ESI-MS of equilibrium dissociation constants (KD) that accurately reflect the affinity of a protein–ligand complex in solution. Issues in the measurement of KD are compounded in the case of low affinity complexes. Here we present a KD measurement method and corresponding mathematical model dealing with both gas-phase dissociation (GPD) and aggregation. To this end, a rational mathematical correction of GPD (fsat) is combined with the development of an experimental protocol to deal with gas-phase aggregation. A guide to apply the method to noncovalent protein–ligand systems according to their kinetic behavior is provided. The approach is validated by comparing the KD values determined by this method with in-solution KD literature values. The influence of the type of molecular interactions and instrumental setup on fsat is examined as a first step towards a fine dissection of factors affecting GPD. The method can be reliably applied to a wide array of low affinity systems without the need for a reference ligand or protein.

Two cytosolic protein families implicated in lipid-binding: main structural and functional features.

1. According to the important biological role of fatty acids and phospholipids in cell membranes, two cytosolic proteins implicated in their binding and transport in brain were considered, namely: Fatty Acid-Binding Protein and basic 21 kDa protein. 2. They were reviewed as well as their related protein families. 3. Although the two protein groups do not present significant sequence homologies, they share several similar properties and might thus be implicated in common physiological functions.