Sites of iodination in recombinant human brain-derived neurotrophic factor and its effect on neurotrophic activity.

Recombinant human brain-derived neurotrophic factor (BDNF) is now under extensive investigation because of its potential clinical applications. Radioactively labeled proteins are usually required to study receptor binding and pharmacokinetic properties of proteins. This study was undertaken to see if iodination affects the biological and conformational properties of a recombinant BDNF. BDNF was iodinated using a stoichiometric amount of nonradioactive cold NaI to minimize multiple iodinations. Of the four tyrosines present in BDNF--Tyr-52, Tyr-54, Tyr-63, and Tyr-86--only Tyr-63 and Tyr-86 were iodinated under the experimental conditions used. Iodination of Tyr-63 resulted in modification without alteration of the biological activity, whereas iodination of Tyr-86 resulted in a molecule with highly compromised biological activity. Similar inactivation was observed if both Tyr-63 and Tyr-86 were iodinated. These modified proteins exhibited conformation and dimerization apparently identical to those of the native protein, as demonstrated by analytical ultracentrifugation, gel filtration, light scattering, and circular dichroism. From these results, we concluded that Tyr-52 and Tyr-54 are not accessible to the reagent and are probably buried in the hydrophobic core, whereas Tyr-63 and Tyr-86 are exposed on the surface of the molecule; of the two exposed residues, only Tyr-86 contributes to the biological activity.

Identification of the residues involved in homodimer formation of recombinant human erythropoietin.

Recombinant human erythropoietin (rHuEPO) is biologically functional when in a monomeric state; upon extensive heating, rHuEPO forms a dimer. The nature of this dimeric linkage was investigated after isolation of the dimer by gel filtration. The dimer fraction was subjected to tryptic digestion, and the peptides were separated by reversed-phase HPLC. SDS-PAGE, N-terminal sequencing, capillary electrophoresis and mass spectrometry (both liquid-chromatographic electrospray and matrix-assisted laser desorption ionization) were employed to compare the tryptic peptides from heat-treated rHuEPO and untreated rHuEPO. Results demonstrated that elevated heat broke the intramolecular disulfide bond between Cys-7 and Cys-161 and an intermolecular disulfide bond then formed from these residues, producing a covalently linked rHuEPO homodimer. Dimer formation was also mathematically modeled and shown to fit a simple equilibrium.

Covalent dimerization of recombinant human interferon-gamma.

An apparently nonreducible covalent dimer has been consistently observed as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These studies endeavor to understand the nature of this covalent dimerization. Amino acid sequence analysis of a recombinant human interferon-gamma stored for 2 years showed a major sequence starting with the intact N-terminal methionine and a minor sequence corresponding to the C-terminal seven amino acids of the intact protein. Moreover, when the same material was analyzed by gel filtration in the presence of 8 M urea, a minor peak corresponding to the dimer was observed prior to the monomer peak. Reducing and nonreducing SDS-PAGE also showed a minor band corresponding to a dimer. These results suggest that the reactions of C-terminal processing and dimerization have occurred during storage. Reverse-phase chromatography of stored, unfractionated material showed three peaks. Mass spectral analysis of the first, second, and third peaks gave molecular weights of 16,900, 16,100, and 33,000. Since no major cleavage was observed in the N-terminal region of the protein, the observed masses suggest that the first peak corresponds to residues 1 to 144 (a full-length molecule), the second peak to residues 1 to 137 (des 7 interferon-gamma), and the third peak to a dimer. Calculation of theoretical molecular weight from the amino acid sequence suggests that this dimer corresponds to some combination of the intact protein and des 7 protein. Tryptic peptide maps in conjunction with sequence and mass analyses identified a new tryptic peptide in the map of the dimer corresponding to residue 133 to 137 followed by residues 1 to 7. The conclusion is recombinant methionyl human interferon-gamma undergoes a specific cleavage at the C-terminal side of residue 137 phenylalanine, and a conventional peptide bond was formed between residues 137 and 1 methionine.

Recombinant human brain-derived neurotrophic factor (rHuBDNF). Disulfide structure and characterization of BDNF expressed in CHO cells.

Three disulfide linkages of recombinant human brain-derived neurotrophic factor (BDNF) were determined by peptide sequence analysis and characterized by mass spectrometry. The three disulfide bonds for BDNF expressed in Chinese hamster ovary cells include Cys-13-Cys-80, Cys-58-Cys-109 and Cys-68-Cys-111, and the disulfide structure was homologous to that of nerve growth factor.

Site-specific mutagenesis of the catalytic subunit of cholera toxin: substituting lysine for arginine 7 causes loss of activity.

Cholera and pertussis toxins each contain a subunit with ADP-ribosyltransferase activity, sharing a region of nearly identical amino acid sequence near the NH2 terminus. Previous investigations have shown that substitution of a lysine residue for Arg-9 in the catalytic A subunit of pertussis toxin substantially eliminates its enzyme activity. We now report that substitution of lysine for the position-equivalent Arg-7 of cholera toxin subunit A leads to a similar loss of catalytic activity. This result suggests a correlation of function with structure between the sequence-related cholera and pertussis toxin A subunits and may contribute to the design of a vaccine containing an enzymatically inert analog of cholera toxin.

Oxidative refolding of insulin-like growth factor 1 yields two products of similar thermodynamic stability: a bifurcating protein-folding pathway.

Can one protein sequence encode two structures? Oxidative folding of human insulin-like growth factor 1 (IGF-1), a globular protein of 70 residues, is shown to yield two products of similar thermodynamic stability. This observation is of particular interest in light of the recent demonstration that two of the three disulfide bonds in native IGF-1 rearrange in the presence of dithiothreitol [Hober, S., et al. (1992) Biochemistry 31, 1749-1756]. Kinetics of the IGF-1 folding pathway were monitored by high-performance liquid chromatography (rp-HPLC). Disulfide-pairing schemes of intermediates and products were established by peptide mapping. Two disulfide isomers were obtained as products: one with native insulin-like pairing [6-48; 18-61; 47-52] (designated native IGF-1; 60% yield) and the other with alternative pairing [6-47; 18-61; 48-52] (designated IGF-swap; 40% yield). The predominant early intermediate contains the single disulfide 18-61, which is shared in common by the two products. Relative yields of native IGF-1 and IGF-swap are independent of protein concentration under dilute conditions. In the absence of an added thiol reagent, each isomer is stable indefinitely at neutral pH; in the presence of an added thiol reagent, the two isomers interconvert with an Arrhenius activation barrier of 12 kcal/mol. Interconversion does not require complete reduction and yields the same ratio of products as initial folding, demonstrating thermodynamic control. Spectroscopic studies using circular dichroism (CD), infrared spectroscopy (FTIR), two-dimensional 1H-NMR (2D-NMR), and photochemical dynamic nuclear polarization (photo-CIDNP) suggest that IGF-1 and IGF-swap adopt similar secondary structures but distinct tertiary folds. Implications of these observations for understanding the topology of protein-folding pathways are discussed.

Structural requirements for addition of O-linked carbohydrate to recombinant erythropoietin.

To define the structural requirements for addition of O-linked glycosylation in vivo, recombinant erythropoietin (rEPO) variants were constructed. Thirty-three independent Ser or Thr substitutions were constructed and examined to see which were subject to O-linked carbohydrate addition. Variants with Thr mutations at positions 123 and 125, but not elsewhere, contained additional carbohydrate, which suggests that several positions around the existing O-linked glycosylation site (Ser126), but not elsewhere, contain the necessary information for O-linked carbohydrate addition. Two forms of the Thr125 variant were identified. One form was glycosylated only at residue 125, and a second form was glycosylated at both Thr125 and Ser126, the normal O-glycosylation site. We have also found that glycosylation is less efficient when rEPO is improperly folded and that prolines at -1 and +1 relative to the O-glycosylation site enhance glycosylation.

Isolation and characterization of three recombinant human granulocyte colony stimulating factor His-->Gln isoforms produced in Escherichia coli.

This report demonstrates that three variant isoforms of recombinant methionyl human granulocyte colony stimulating factor are present in small quantities in the crude preparation solubilized from Escherichia coli inclusion bodies. These isoforms were separated from the main form of the factor during purification and further isolated by a series of cationic exchange chromatographic separations. They exhibit full in vitro biological activity and have slightly lower pI's. Structural characterization of the intact proteins and their isolated peptides by sequence determination and mass spectrometric analysis revealed that they are methionyl granulocyte colony stimulating factors having a His-->Gln replacement at sequence position 53, 157, or 171, respectively. The specific His-->Gln change suggests the occurrence of mistranslation during protein synthesis. These variant forms are chromatographically separable during purification and are not detectable in the final purified form of the factor.