Enhanced major histocompatibility complex class I-dependent presentation of antigens modified with cationic and fusogenic peptides.

Soluble extracellular protein antigens are notoriously poor stimulators of CD8+ cytotoxic T-lymphocyte (CTL) responses, largely because these antigens have inefficient access to an endogenous cytosolic pathway of the major histocompatibility complex (MHC) class I-dependent antigen presentation. Here, we present a strategy that facilitates antigen penetration into the cytosol of antigen-presenting cells (APC) by addition to the antigen of charge-modifying peptide sequences. As a result of this intervention, the charge modification enhances antigen uptake into APC by counteracting the repulsive cell surface charge, and then endosomal membranes are disrupted with a subsequent release of antigen into the cytosol. This technology significantly improves MHC class I-dependent antigen presentation to CTL, enabling a more efficient generation of specific CTL immunity in vivo. The strategy described here has potential for use in developing efficient vaccines for antigen-specific immunotherapy of human malignancies.

Designing proteins that work using recombinant technologies.

Therapeutic proteins have been engineered for a variety of purposes including reduced antigenicity, longer half-life, simplified process development, and increased affinity. Fusion proteins bring together functions from two different molecules creating therapeutics with completely novel activities. Protein engineering technologies have relied on rational design, directed evolution, DNA shuffling, RNA-peptide fusion, phage and ribosomal display methods to select out candidate protein forms with the desired therapeutic properties. Engineered site-specific pegylation and glycosylation strategies have improved circulation half-life, reduced immunogenicity and increased protein therapeutic stability. In this review we describe how protein engineering techniques have been used to select out, improve stability and clinical efficacy of protein therapeutics.

Controlled formation of model homo- and heterodimer coiled coil polypeptides.

Sequence-simplified coiled coil polypeptides were synthesized and their folding properties characterized in order to define the role of charged border residues at the coiled coil interface for the controlled formation of homodimer and heterodimer structures. Three peptides were designed to form parallel coiled coils with valine and leucine occupying the hydrophobic interface positions a and d, respectively, of the heptad repeat abcdefg. The polypeptide designated E/K42, with the heptad repeat sequence VSSLESK, contained glutamate and lysine in the interface border positions e and g, respectively, and was designed to form a coiled coil homodimer at neutral pH. Two other polypeptides, designated E/E35 and K/K35, have the heptad repeats VSSLESE and VSSLKSK, respectively. E/E35 contains only glutamic acid at both e and g positions; K/K35, only lysine, E/E35 and K/K35 were designed to form a stable coiled coil heterodimer when combined at neutral pH. All three polypeptides were prepared by solid-phase synthesis and purified by reverse-phase high-performance liquid chromatography followed by size-exclusion chromatography. E/K42 formed a stable dimeric coiled coil structure as determined by circular dichroism and size-exclusion chromatography. The alpha-helical content of E/K42 was highest at neutral pH and decreased at extremes of pH. The alpha-helical structure of E/K42 at micromolar concentrations had a Tm of 62-65 degrees C and exhibited a concentration dependence of thermal denaturation consistent with dimer formation. In contrast to results with E/K42, a mixture of E/E35 and K/K35, but neither alone, forms alpha-helix at neutral pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Formation of heterodimers between wild type and mutant trp aporepressor polypeptides of Escherichia coli.

Availability of the three-dimensional structure of the trp repressor of Escherichia coli and a large group of repressor mutants has permitted the identification and analysis of mutants with substitutions of the amino acid residues that form the tryptophan binding pocket. Mutant aporepressors selected for study were overproduced using a multicopy expression plasmid. Equilibrium dialysis with 14C-tryptophan and purified mutant and wild type aporepressors was employed to determine tryptophan binding constants. The results obtained indicate that replacement of threonine 44 by methionine (TM44) or arginine 84 by histidine (RH84) lowers the affinity for tryptophan approximately two- and four-fold, respectively. Replacement of arginine 54 by histidine (RH84) or glycine 85 by arginine (GR85) results in complete loss of tryptophan binding activity. Purified mutant and wild type aporepressors were used in in vitro heterodimer studies. The trp repressor of E. coli functions as a stable dimer. A large number of trp repressor mutants produces defective repressors that are transdominant to the wild type repressor in vivo. The transdominance presumably results from the formation of inactive or slightly active heterodimers between the mutant and wild type polypeptide subunits. An in vitro assay was developed to detect and measure heterodimer formation. Heterodimer formation was thermally induced, and heterodimers were separated on nondenaturing polyacrylamide gels. Aporepressors readily formed heterodimers upon treatment at 65 degrees C for 3 minutes. Heterodimer formation was significantly retarded by the presence of the corepressor, L-tryptophan. Indole-3-propionic acid, 5-methyl tryptophan, and other analogs of tryptophan, as well as indole, also inhibited heterodimer formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Major histocompatibility complex class I-restricted presentation of protein antigens without prior intracellular processing.

Proteins in their native form are incapable of stimulating antigen (Ag)-specific T cells, which can only recognize major histocompatibility complex (MHC)-bound peptides that have been generated by intracellular processing within antigen-presenting cells (APCs). Here, we show that APCs can trigger MHC class I-restricted T-cell responses after presenting proteins without conventional intracellular processing, provided the immunostimulatory MHC class I-binding peptide sequence is incorporated at the carboxy-terminal position. Such MHC-bound proteins do not stimulate T cells directly, because the contact between MHC/peptide complex and its cognate ligand is sterically hindered by the amino-terminal bulk of the protein. Removal of the latter via an extracellular Ag proteolysis by the T-cell- and/or APC-derived enzymes is required for effective T-cell stimulation. Our data challenge the established concept that only small peptides can bind to the MHC class I molecules.

Nucleotide sequence and genetic characterization reveal six essential genes for the LIV-I and LS transport systems of Escherichia coli.

The nucleotide sequence of the genes encoding the high affinity, branched-chain amino acid transport systems LIV-I and LS has been determined. Seven genes are present on a 7568-base pair DNA fragment, six of which participate directly in branched-chain amino acid transport. Two periplasmic amino acid-binding proteins are encoded by the livJ (LIV-BP) and livK (LS-BP) genes. These two proteins confer specificity on the LIV-I and LS transport systems. livK is the first gene in a polycistronic message that includes four genes encoding membrane components, livHMGF. The protein products of the livHMGF genes are shared by the two systems. An analysis of the livH and livM DNA sequences suggests that they encode hydrophobic proteins capable of spanning the membrane several times. The LivG and LivF proteins are less hydrophobic, but are also tightly associated with the membrane. Both LivG and LivF contain the consensus sequence for adenine nucleotide binding observed in many other transport proteins. A deletion strain that does not express any of the liv genes was constructed. This strain was used to show that each of the membrane component genes is required for high affinity leucine transport, including two genes, livM and livF, for which no previous genetic evidence had been obtained.

Structure-function analysis of FLT3 ligand-FLT3 receptor interactions using a rapid functional screen.

FLT3 ligand (FLT3L) stimulates primitive hematopoietic cells by binding to and activating the FLT3 receptor (FLT3R). We carried out a structure-activity study of human FLT3L in order to define the residues involved in receptor binding. We developed a rapid method to screen randomly mutagenized FLT3L using a FLT3R-Fc fusion protein to probe the relative binding activities of mutated ligand. Approximately 60,000 potential mutants were screened, and the DNA from 59 clones was sequenced. Thirty-one single amino acid substitutions at 24 positions of FLT3L either enhanced or reduced activity in receptor binding and cell proliferation assays. Eleven representative proteins were purified and analyzed for receptor affinity, specific activity, and physical properties. Receptor affinity and bioactivity were highly correlated. FLT3L affinity for receptor improved when four individual mutations that enhance FLT3L receptor affinity were combined in a single molecule. A model of FLT3L three-dimensional structure was generated based on sequence alignment and x-ray structure of macrophage colony-stimulating factor. Most residues implicated in receptor binding are widely dispersed in the primary structure of FLT3L, yet they localize to a surface patch in the tertiary model. A mutation that maps to and is predicted to disrupt the proposed dimerization interface between FLT3L monomers exhibits a Stokes radius that is concentration-dependent, suggesting that this mutation disrupts the FLT3L dimer.