[Enzyme optimization, mutagenesis and directed evolution]. / Optimiser les enzymes, mutagenèse et évolution dirigées.

Among the different areas of biotechnology, enzyme engineering represents a growing field where major progress has been recently made. Indeed, chemical, pharmaceutical or food industries have increased needs for enzymes. This increase requires enzyme optimization in order to achieve, together or separately, greater operational stability, better specificity, increased solubility or preferential enantioselectivity. Directed and random mutagenesis, the classical methods of enzymatic engineering, have proved to be efficient in some cases, but are quite tricky. Directed evolution is a hybrid method recently developed in order to reproduce the random mechanisms of evolution in vitro. This method has now been used to optimise an increasing number of enzymes. In our research group, a directed evolution project has been initiated on a bacterial phosphotriesterase, a promising enzyme, capable of efficiently detoxifying organophosphorus nerve agents.

[Discovery and crystallographic structure of human apolipoprotein]. / Découverte et structure cristallographique d'une apolipoprotéine humaine.

We report the serendipitous discovery of a human plasma phosphate binding protein (HPBP). This 38 kDa protein is co-purified with paraoxonase (PON1). The association between HPON1 and HPBP is modulated by phosphate and calcium concentrations. The HPBP X-ray structure solved at 1.9 A resolution is similar to the prokaryotic phosphate solute-binding proteins (SBPs) associated with ATP binding cassette transmembrane transporters, though phosphate-SBPs have never been characterized or predicted from nucleic acid databases in eukaryotes. However, HPBP belongs to the family of ubiquitous eukaryotic proteins named DING, meaning that phosphate-SBPs are also widespread in eukaryotes. The absence of complete genes for eukaryotic phosphate-SBP from databases is intriguing, but the astonishing 90% sequence conservation of genes between evolutionary distant species suggests that the corresponding proteins play an important function. HPBP is the first identified transporter capable of binding phosphate ions in human plasma. Thus it is thought to become a new predictor and a potential therapeutic agent for phosphate-related diseases such as atherosclerosis.

Purification, molecular characterization and catalytic properties of a Pseudomonas fluorescens enzyme having cholinesterase-like activity.

An enzyme with a cholinesterase (ChE) activity, produced by Pseudomonas fluorescens, was purified to homogeneity in a three-step procedure. Analysis by non-denaturing and SDS-PAGE, and by isoelectric focusing, indicated that the enzyme was a monomer of 43 kDa, with a pI of 6.1. The N-terminal sequence, AEPLKAVGAGEGQLDIVAWPGYIEA, showed some similarities with proteins of the ChE family and a strong similarity with a protein from Escherichia coli with unknown structure and function. Cholinesterase activity at pH 7.0 and 25 degreesC was maximum with propionylthiocholine as substrate (kcat,app=670 min-1), followed by acetylthiocholine, and significantly lower with butyrylthiocholine. Catalytic specificity (kcat/Km) was the same for propionylthiocholine and acetylthiocholine, but was two orders of magnitude lower for butyrylthiocholine. Kinetics of thiocholine ester hydrolysis showed inhibition by excess substrate which was ascribed to binding of a second substrate molecule, leading to non-productive ternary complex (Km=35 microM, KSS=0.49 mM with propionylthiocholine). There was low or no reactivity with organophosphates and carbamates. The enzyme inhibited by echothiophate (kII=0.44x102 M-1 min-1) was not reactivated by pralidoxime methiodide. However, the P. fluorescens enzyme had affinity for procainamide and decamethonium, two reversible ChE inhibitors used as affinity chromatography ligand and eluant, respectively. Although similarity of the N-terminal amino acid sequence of the enzyme with an internal sequence of ChEs is weak, its catalytic activity towards thiocholine esters, and its affinity for positively charged ligands supports the contention that this enzyme may belong to the ChE family. However, we cannot rule out that the enzyme belongs to another structural family of proteins having cholinesterase-like properties. The reaction of the enzyme with organophosphates suggests that it is a serine esterase, and currently this enzyme may be termed as having a cholinesterase-like activity.

Two alloalbumins with identical electrophoretic mobility are produced by differently charged amino acid substitutions.

We describe the amino acid substitutions of albumins Sondrio and Paris 2, two slow moving variants of human serum albumin, which show an identical electrophoretic mobility on cellulose acetate at three different pH values. These variants have been found in several instances in a wide geographic area including Northern Italy and France. Both alloalbumins were isolated from the sera of heterozygous subjects. Isoelectric focusing analysis of CNBr fragments from the purified variants allowed us to localize the mutation of albumin Sondrio in fragment CNBr V (residues 330-446) and that of albumin Paris 2 in CNBr VII (residues 549-585). Sequential analysis of the variant CNBr VII established the molecular defect of albumin Paris 2 as 563 Asp----Asn. Fragments CNBr V from normal and Sondrio albumins were isolated on a preparative scale and subjected to tryptic and V8 proteinase digestion. Sequence determination of the abnormal tryptic and V8 peptides revealed that the variant arises from the substitution of glutamic acid 333 by lysine. Thus, a +1 change in the C-terminal region of the albumin molecule produces a variant with the same electrophoretic mobility as an alloalbumin with a +2 substitution in the central domain, suggesting a higher degree of exposure to the solvent of the C-terminal tailpiece. Both amino acid substitutions are consistent with a G----A transition in the first position of the corresponding codon in the structural gene.

Thermal stability of acetylcholinesterase from Bungarus fasciatus venom as investigated by capillary electrophoresis.

Previous studies on the conformation of the monomeric acetylcholinesterase (AChE) from the krait (Bungarus fasciatus) venom showed that the protein possesses a large permanent dipole moment. These studies predicted that thermal irreversible denaturation must occur via partially unfolded states. The thermal stability of Bungarus AChE was determined using capillary electrophoresis (CE) with optimized conditions. Runs performed at convenient temperature scanning rates provided evidence for an irreversible denaturation process according to the Lumry and Eyring model. The mid-transition temperature, T(m), and the effective enthalpy change, DeltaH(m) were determined at different pH. The temperature dependence of the free energy, DeltaG, of Bungarus AChE unfolding was drawn using values of T(m), DeltaH(m) and DeltaC(p) determined by CE. The thermodynamic parameters for the thermal denaturation of the monomeric snake enzyme were compared with those of different dimeric and tetrameric ChEs. It was shown that the changes in the ratio of DeltaH(cal/)DeltaH(vH) and DeltaC(p) reflect the oligomerization state of these proteins. All these results indicate that wild-type monomeric Bungarus AChE is a stable enzyme under standard conditions. However, designed mutants of this enzyme capable of degrading organophosphates have to be engineered to enhance their thermostability.

Albumin Paris 2: a new genetic variant distinguished by isoelectric focusing.

Until recently, the characterization of genetic variants of human serum albumin was performed by electrophoretic typing prior to the determination of their amino acid substitutions. We describe a procedure using isoelectric focusing in the presence of urea for the analysis of the genetic variation of albumin. This procedure allowed a clear distinction of a new variant, previously found to be identical with albumin Sondrio according to its relative electrophoretic mobilities at 3 pHs. This new variant, the third rare albumin allotype identified in the Ile-de-France region, was called albumin Paris 2.

A sensitive double-diffusion microassay suitable for the detection of idiotype-antiidiotype precipitates.

Using agarose gel coated on GelBond film sheets and using Coomassie blue stain followed by silver stain, a sensitive double-diffusion microassay has been developed for detecting small amounts of precipitate forming during idiotype-antiidiotype reactions. The sensitivity of the method is 10-100 times greater than classical immunodiffusion tests. Other advantages include the need for minimal amounts of unconcentrated sample, the absence of radioactive or toxic substrates, no interference due to a second or third antibody coat such as are used in immunoenzymatic techniques, and the possibility of a direct evaluation of qualitative data such as identity, cross-reactivity or non-identity. As little as 40 ng antibody could be detected, corresponding to an antibody concentration of 8 micrograms/ml, making the microassay useful for rapid screening of idiotype-antiidiotype precipitates during routine analysis of hybridoma supernatants.

Measuring conformational stability of proteins using an optimized temperature-controlled capillary electrophoresis approach.

The thermal denaturation process of a model protein, bovine beta-lactoglobulin, was analyzed using capillary zone electrophoresis (CZE). For this purpose, a commercial CE apparatus was improved, allowing efficient control and accurate measurement of the temperature up to 95 degrees C. Under various pH conditions, transition temperature (Tm), enthalpy change (delta H) and entropy change (delta S) associated with the thermal denaturation were determined. Moreover, the technique is unique in its ability to estimate the heat capacity change (delta Cp). This work shows that CZE, performed even when electroosmotic flow occurs, is an innovative approach for determining the stability curves of proteins. Accordingly, CZE is a powerful tool to study protein unfolding/folding quickly and with minimal sample requirements.

Dual effect of high electric field in capillary electrophoresis study of the conformational stability of Bungarus fasciatus acetylcholinesterase.

The effect of high electric field in capillary zone electrophoresis (CZE) was evaluated for the study of the thermally induced unfolding of Bungarus fasciatus acetylcholinesterase. This monomer enzyme is characterised by two interdependent uncommon structural features, the asymmetrical distribution of charged residues and a relatively low thermal denaturation temperature. Both traits were presumed to interfere in the thermal unfolding of this enzyme as investigated by CZE. This paper analyses the effect of high electric field on the behaviour of the enzyme native state. It is shown that increasing the applied field causes denaturation-like transition of the enzyme at a current power which does not induce excessive Joule heating in the capillary. The susceptibility to electric field of proteins like cholinesterases, with charge distribution anisotropy, large permanent dipole moment and notable molecular flexibility associated with moderate thermal stability, was subsequently discussed.

[Capillary electrophoresis for monitoring stability of pharmaceutical proteins]. / Application de l'électrophorèse capillaire au contrôle de la stabilité des protéines pharmaceutiques.

Like chemical drugs, pharmaceutical proteins have to be of strictly controlled purity to become biopharmaceuticals. Detection, identification and characterization of impurities and compounds present in purified biopharmaceuticals are of central interest. Simultaneous development of structural biology, biotechnologies and of highly resolutive analytical tools, allow significant progress in this field to be achieved. Thus, capillary electrophoresis can be advantageously used for monitoring protein stability. As illustrated by the examples of potential enzymes for detoxification of organophosphates, prophylaxis and treatment of poisoning by pesticides and nerve agents or for skin decontamination, the present feature article shows that this new methodological approach allows: to determine purity and homogeneity of proteins;to analyze their resistance to denaturing conditions such as heat or high electric fields;to detect the presence of unwanted hidden protein-bound ligands capable of altering functional conformation and stability of enzymes, and moreover susceptible to be released and to induce side effects, such as immunologic response; and to establish the role of parameters controlling the "good compromise" between conformational stability and plasticity for allowing optimal functional efficiency of enzymes. Finally, capillary electrophoresis has proved to be a pertinent tool to validate the conformity of purified enzymes to a status of biopharmaceutical.

Stabilization of the active form(s) of human paraoxonase by human phosphate-binding protein.

While there is a consensus that human PON1 (paraoxonase-1) has a protective role, its primary biological function remains unclear. A protective role against poisoning by organophosphates [OPs (organophosphorus compounds)] drove earlier works. Clinical interest has recently focused on a protective role of PON1 against vascular diseases. PON1 resides mainly on HDL (high-density lipoprotein) particles, and converging recent works show that both its activities and stability dramatically depend on this versatile and dynamic molecular environment. The discovery that HPBP (human phosphate-binding protein) has a firm tendency to associate with PON1 has steered new directions for characterizing PON1 functional state(s). Storage stability studies provided evidence that HPBP is involved in maintaining physiologically active PON1 conformation(s). Thermal stability studies showed that human PON1 is remarkably thermostable and that its association with HPBP strongly contributes to slowing down the denaturation rate. A hybrid PON1, displaying mutations that stabilized recombinant enzyme expressed in Escherichia coli, was shown to be more thermostable than natural human PON1. Predictably, its stability was unaffected by the presence of HPBP. Synergistic efforts on characterizing natural PON1 and rPON1 (recombinant PON1) provide information for the design of future stable mutants of PON1-based bioscavengers to be used as safe and effective countermeasures to challenge OPs. Maintaining a stable environment for such administrable human rPON1 should, at least, preserve the anti-atherogenic activity of the enzyme.

[Polyclonal DNase abzyme produced by anti-idiotypic internal image method]. / Un abzyme DNase polyclonal produit par la méthode de l'image interne anti-idiotypique.

The concept of antigen internal image was applied to the production of catalytic antibodies. An antibody raised in rabbits to DNase (Ab1), acted as a competitive inhibitor of the catalysis, and thus was assumed to contain anti-active site Ab. This Ab1 was used to elicit a polyclonal anti-idiotypic antibody (Ab2). This later exhibited a DNA recognition specificity, suggesting the existence of structural internal images mimicking the conformation of the active site. Moreover, Ab2 were able to hydrolyse DNA, indicating the existence of internal images mimicking the enzymatic activity of DNase. Consequently, a strategy can thus be considered using the idiotypic way, for the production of abzymes in the form of internal images of enzyme active sites.

[Characterization of genetic variants of human albumin by isoelectric focusing]. / Caractérisation des variants génétiques de l'albumine humaine par focalisation isoélectrique.

Normal human serum albumin and bisalbuminic fractions from genetic variants of european origin have been studied by ultrathin-layer isoelectric focusing performed on whole sera or after purification of albumin fractions by affinity chromatography on Blue-Trisacryl. Narrow range ampholytes giving pH gradient between 5 and 8, together with the use of 8 M urea, provided suitable patterns allowing to discriminate the various allotypes. In these conditions, both normal albumin and heterozygous variants were microheterogeneous with several main bands; in the case of normal albumin, four major bands were found, while variants exhibited additional bands differing in number and pI. The position of additional bands comparatively to that of normal albumin was consistent with the electrophoretic behavior of variants. Fast moving allotypes exhibited additional bands with more anodal pIs, whereas slow moving variants were characterized by cathodal additional bands. The number and position of these bands allowed to characterize some variants, when they failed to distinguish between some others, identical patterns being correlated to identical mutations arising at different locations on the albumin molecule. These observations indicate that isoelectric focusing could allow a clear distinction of albumin variants, provided that their mutation were different, or could confirm the occurrence of identical mutation when IEF patterns are indistinguishable. In addition to electrophoretic mobilities at various pH, analytical isoelectric focusing could be a useful technique employed as a second step in the identification of allotypes prior to the determination of the structural change characterizing the variant.

Human albumin genetic variants: an attempt at a classification of European allotypes.

The relative mobility of albumin and proalbumin genetic variants was estimated by means of cellulose acetate electrophoresis performed with three buffer systems at different pH (8.6, 5.0, and 6.9) after addition of a reference protein and dilution of sera. Numerous experiments using samples of reference variants corroborated the accuracy and reproducibility of this technique. The estimation of the variants' relative mobility at three pH allowed us to distinguish three fast-moving variants (Gent, Vanves, and Reading) and five slow-moving variants (Sondrio, Roma, Christchurch, Lille, and B) in the French population. The frequency of alloalbuminemia in this population is .0004 and is characterized by the high occurrence of albumin B and of the two proalbumin variants, Christchurch and Lille. In order to classify the variants of European origin, the methodology that we developed, owing to its more resolutive possibilities, should be employed as a first step in their identification until establishment of a structural nomenclature making mention of the amino acid substitution characterizing each variant.

[Genetic variants of human albumin: structural characterization of allotypes used as references for electrophoretic classification]. / Variants génétiques de l'albumine humaine: caractérisation structurale des allotypes utilisés comme références dans la classification électrophorétique.

Eight different types of genetic variants of albumin are observed in the French population. The analysis of electrophoretic patterns of sera containing these variants, performed a three different pHs (8.6, 5.0 and 6.9) after addition of a reference protein (transferrin), allows the identification each variant by a quantitative estimation of its relative mobilities. The accuracy and reproducibility of the technique make it a useful reference method, commonly employed for studying European variants. The samples used as references for five genetic variant types, proalbumins Christchurch and Lille, albumins Vanves, B and Reading, were subjected to sequence analysis to determine the nature and localization of their structural change. Together with the mutations of albumins Gent and Roma previously described, the data presented here make available seven reference specimens for which the structural changes are characterized out of the eight variants known to exist in France.

Comparative study of sturgeon oocyte soluble proteins by isoelectric focusing.

1. Soluble caviar (oocyte) proteins of four sturgeon species from the Caspian sea, Acipenser stellatus (Sevrouga), Acipenser guldenstadti (Ossietre), Acipenser nudiventris (Chipe) and Huso huso (Belouga) were studied by isoelectric focusing. 2. Isoelectric focusing patterns of these proteins differ from one species to another and allow the identification of the specific origin of caviar. 3. Moreover, this technique allowed the discrimination of a subspecies, Acipenser guldenstadti persicus from the species. 4. This biochemical characterization of caviar proteins allowing identification of sturgeon species producing caviar could be added to data used in fraud tests.

[High molecular-weight polymers in human albumin solutions. Quantitative determination by polyacrylamide gradient electrophoresis]. / Les hauts polymères dans les solutions d'albumine humaine. Leur évaluation quantitative par électrophorèse en gradient de polyacrylamide.

Isolated albumin almost always contains polymerized forms which appear during preparation and storage of the protein. The proportion of polymerized forms reflects the degree of stability of the solution. The quantitative estimation of the polymers is usually performed by gel chromatography. In this work, the high resolution power of polyacrylamide gradient gel electrophoresis (Gradient PAGE) was used to separate the polymers present in the preparations of human serum albumin. The analysis of the different peaks obtained by gel chromatography allows to conclude that peak 1 contains aggregates and high polymers, peak 2 trimer and dimer and peak 3 the monomer of albumin. The aggregates of the peak 1 can be dissociated by SDS and correspond, in gradient PAGE, to the high polymers. By using gradient PAGE in the presence of SDS under the conditions described in this paper, it is possible to estimate the proportion of high polymers and aggregates present in albumin preparations. These results are similar to those obtained by chromatography followed by a protein assay but noticeably inferior to those resulting from measurements performed by absorbance at 280 nm.

[Diversity and relative inaccuracy of internal images of antigen group A revealed by IEF analysis]. / Diversité et infidélité relative des images internes de l'antigène de groupe A révélées par l'analyse par IEF.

The diversity of a polyclonal anti-idiotypic response (Ab2) to a murine monoclonal anti-A (Ab1) was investigated after purification of two Ab2 populations. One was eluted from human polyclonal anti-A column and the other from Ab1. Analysis of the Ab specificity, as well as screening of the clonotypic distribution, were achieved after splitting Ab by IEF; this was followed by immunoblotting and probing with various anti-ABH mAb. The first population reacted with almost all the murine anti-ABH mAb, as well as with four human anti-A mAb, and consequently consisted of Ab2 beta. The second was composed of "true" Ab2 directed against Ab1. In the first population internal images mimicked either A Ag, or H Ag, or some epitopes common to both. This study demonstrates the plurality of internal images-bearing Ig molecules, some mimicking completely, and some only partially or even unfaithfully the nominal A determinant. The analysis of this idiotypic cascade proves the existence of a degeneracy of the initial restricted antigenic specificity. The consequences of such a process are discussed.

Pst I polymorphism of the antithrombin III gene in a French population.

Pst I endonuclease fragment length polymorphism of the antithrombin III probe was studied in a French population. Among 25 subjects tested, 15 were found heterozygous at the Pst I site, so that this polymorphism might serve as a useful markers of antithrombin III gene in families with antithrombin III deficiency.

[Rare phenotypes of alpha-1 antitrypsin: study of a case of the M1X variant]. / Phénotypes rares de l'alpha-1 antitrypsine: étude d'un cas de variant M1X.

Alpha-1 antitrypsin is the major component responsible for the normal alpha 1 band in human serum. Some genetic variants giving double alpha-1 band, may be associated with pathological process. In the course of a systematic screening of blood donors a double-band alpha-1 pattern was observed in a serum, due to the heterozygous expression of a genetic variant of the PI system. A possible clinical significance of the variant was investigated by characterizing it. The very rare allotype PI*X was identified and its frequency in the population of french blood donors was estimated around to one for 10,000.