Design of yeast-secreted albumin derivatives for human therapy: biological and antiviral properties of a serum albumin-CD4 genetic conjugate.

Due to its remarkably long half-life, together with its wide in vivo distribution and its lack of enzymatic or immunological functions, human serum albumin (HSA) represents an optimal carrier for therapeutic peptides/proteins aimed at interacting with cellular or molecular components of the vascular and interstitial compartments. As an example, we designed a genetically engineered HSA-CD4 hybrid aimed at specifically blocking the entry of the human immunodeficiency virus into CD4+ cells. In contrast with CD4, HSA-CD4 is correctly processed and efficiently secreted by Kluyveromyces yeasts. In addition, its CD4 moiety exhibits binding and antiviral in vitro properties similar to those of soluble CD4. Finally, the elimination half-life of HSA-CD4 in a rabbit experimental model is comparable to that of control HSA and 140-fold higher than that of soluble CD4. These results indicate that the genetic fusion of bioactive peptides to HSA is a plausible approach toward the design and recovery of secreted therapeutic HSA derivatives with appropriate pharmacokinetic properties.

Functional characterization of human recombinant apolipoprotein AIV produced in Escherichia coli.

Apolipoprotein AIV (apoAIV), a protein which is known to activate the enzyme lecithin: cholesterol acyltransferase, to bind to apoAI/AII receptor sites and also to promote cholesterol efflux from adipose cells, may play an important role in reverse cholesterol transport. In this report, the high-level production of soluble recombinant mature human apoAIV (isoform 1) in Escherichia coli is described. The recombinant protein was purified by avoiding lipid extraction or denaturation. The apoAIV preparation was analysed by its reactivity with antibodies raised against human apoAIV, SDS-gel electrophoresis, isoelectric focusing and N-terminal sequencing. The purified recombinant protein retains an extra methionine at the N-terminus. Purified recombinant and natural apoAIV proteins were indistinguishable with regard to their denaturation properties, thermo-stability or their fluorescence emission properties in the presence of various quantities of a quenching agent. Complexes of ApoAIV with L-alpha-dimyristoyl-glycerophosphocholine (Myr2GroPCho), glycerophosphocholine (GroPCho), or L-alpha-1-palmitoyl-2-oleoylglycerophosphocholine (PamOleGroPCho) prepared from plasmatic and from recombinant apoAIV proteins have similar densities as revealed by analytical centrifugation. They also share the same cofactor properties for the lecithin:cholesterol acyltransferase reaction. Recombinant apoAIV complex with Myr2GroPCho was also able to bind to the same apoAI/AII receptor sites and to promote cholesterol efflux to an equal extent from adipose cells. It is concluded that the recombinant protein is functionally identical to the plasmatic apoAIV and may therefore be very useful in helping to elucidate the physiological role of apoAIV.

Mechanism of inactivation of the beta 2 subunit of Escherichia coli tryptophan synthase by monoclonal antibodies.

Monoclonal antibodies directed against the native form of the beta 2 subunit of Escherichia coli tryptophan synthase strongly inhibit both its tryptophan synthase and its serine deaminase activities. The mechanism of this inactivation is studied here, by monitoring quantitatively the absorption and fluorescence properties of different well-characterized successive intermediates in the catalytic cycle of tryptophan synthase. It is shown that the antibodies interfere specifically with the formation of one or the other of these intermediates. It is concluded that the antibodies either modify or block the molecular flexibility of the protein, thus preventing conformational changes that the protein has to undergo during the catalysis. At least two different stages of the catalytic process, each one sensitive to a different class of antibodies, are shown to involve molecular movements of the polypeptide chain. Indications are given on the regions of the molecule involved in these movements.

A physical-chemical and immunological comparison shows that native and renatured Escherichia coli tryptophan synthase beta 2 subunits are identical.

An acid-denaturation of the beta 2 subunit of Escherichia coli tryptophan synthase has been recently described. In the present study, renaturation yield of acid-denaturated beta 2, and the influence of temperature, protein concentration and presence of ligands are investigated. It is also demonstrated that 3 forms of the protein are obtained at the end of the renaturation process: one is fully active, and is identical to native beta 2, as indicated by some of its chemical and physical properties, as well as by its immunological reactivity towards monoclonal antibodies specific for the native protein. A second form is composed of high molecular weight insoluble and inactive aggregates. A third form consists of low molecular weight soluble and inactive aggregates. The results obtained for the immunochemical reactivity of these small aggregates indicate that they are formed with partly correctly folded beta monomers assembled by specific but incorrect quaternary interactions. The capacity of monoclonal antibodies to detect such incorrect structures and to characterize renatured proteins is particularly emphasized.

Alternate succession of steps can lead to the folding of a multidomain oligomeric protein.

The beta 2 subunit of Escherichia coli tryptophan synthase can be either unfolded in 6 M guanidine, or extensively denatured at acidic pH. These two denatured forms of beta 2 have different circular dichroism spectra and thus correspond to distinct physical states. Here we compare the folding pathways of these two different denatured forms of beta chains. We describe the kinetics of regain of a variety of physical, functional, and immunochemical signals characteristic of six successive steps previously identified on the folding pathway of guanidine unfolded beta 2. It is shown that whereas identical molecular events occur with the same kinetics, the two folding pathways are different, and involve different structural intermediates.

Kinetics of appearance of an early immunoreactive species during the refolding of acid-denatured Escherichia coli tryptophan synthase beta 2 subunit.

A reversible acid-denaturation process of the beta 2 subunit of Escherichia coli tryptophan synthase has been set up. The acid-denatured state has been physically characterized: though not in a random-coiled conformation, it is extensively denatured. The renaturation of this denatured state of beta 2 has been observed in a stopped-flow system, in the presence of a monoclonal antibody directed against native beta 2. It is shown that the association occurs very early in the folding of beta 2. The association rate constants of the antibody with the immunoreactive folding intermediate and with native beta 2 are the same (3 X 10(5) M-1.s-1). But at high antibody concentrations the formation of the antigen/antibody complex is rate limited by a rapid (5.4 X 10(-2) s-1) isomerization of refolding beta chains. This isomerization appears to reflect the formation of at least part of the epitope recognized by the antibody during the folding of beta 2. Further conformational adjustments occurring later in the folding pathway would then allow the ultimate structuring of the epitope.