PEGylation of native disulfide bonds in proteins

PEGylation has turned proteins into important new biopharmaceuticals. The fundamental problems with the existing approaches to PEGylation are inefficient conjugation and the formation of heterogeneous mixtures. This is because poly(ethylene glycol) (PEG) is usually conjugated to nucleophilic amine residues. Our PEGylation protocol solves these problems by exploiting the chemical reactivity of both of the sulfur atoms in the disulfide bond of many biologically relevant proteins. An accessible disulfide bond is mildly reduced to liberate the two cysteine sulfur atoms without disturbing the protein's tertiary structure. Site-specific PEGylation is achieved with a bis-thiol alkylating PEG reagent that sequentially undergoes conjugation to form a three-carbon bridge. The two sulfur atoms are re-linked with PEG selectively conjugated to the bridge. PEGylation of a protein can be completed in 24 h and purification of the PEG-protein conjugate in another 3 h. We have successfully applied this approach to PEGylation of cytokines, enzymes, antibody fragments and peptides, without destroying their tertiary structure or abolishing their biological activity.     

Glutathione is required to regulate the formation of native disulfide bonds within proteins entering the secretory pathway

The formation of native disulfide bonds is an essential event in the folding and maturation of proteins entering the secretory pathway. For native disulfides to form efficiently an oxidative pathway is required for disulfide bond formation and a reductive pathway is required to ensure isomerization of non-native disulfide bonds. The oxidative pathway involves the oxidation of substrate proteins by PDI, which in turn is oxidized by endoplasmic reticulum oxidase (Ero1). Here we demonstrate that overexpression of Ero1 results in the acceleration of disulfide bond formation and correct protein folding. In contrast, lowering the levels of glutathione within the cell resulted in acceleration of disulfide bond formation but did not lead to correct protein folding. These results demonstrate that lowering the level of glutathione in the cell compromises the reductive pathway and prevents disulfide bond isomerization from occurring efficiently, highlighting the crucial role played by glutathione in native disulfide bond formation within the mammalian endoplasmic reticulum.     

Identification of plasma proteins containing sulfite-reactive disulfide bonds

Plasma protein S-sulfonate compounds (RS-SO-3) have previously been shown to form, presumably by sulfitolysis of disulfide bonds, as a result of exposure to sulfite. In the investigations reported here, we identify two proteins in rabbit plasma, namely albumin and plasma fibronectin, which contain reactive sites for S-sulfonate formation. Separation and identification of these proteins following in vitro and in vivo exposure to sulfite was accomplished primarily by column chromatographic and electrophoretic techniques. In addition, the structure of presumed S-sulfonate groups was confirmed by the identification of cysteinyl-S-sulfonate residues in protein hydrolysates generated by enzymatic digestion. The molar ratio of RS-SO-3 in both albumin and plasma fibronectin was less than one. Data from our experiments suggest that the mixed disulfide site of non- mercaptalbumin is the reactive site for S-sulfonate formation. The site(s) of formation within the plasma fibronectin molecule was not investigated. The possible physiological significance of disulfide sulfitolysis of albumin and plasma fibronectin is discussed.     

Formation of disulfide bonds in proteins and peptides

For many proteins and peptides, disulfide bridges are prerequisite for their proper biological function. Many commercialized proteins are crosslinked by disulfide bridges that increase their resistance to destructive effects of extreme environment used in industrial processes or protect protein-based therapeutics from rapid proteolytic degradation. Manufacturing of these products must take into account oxidative refolding--a formation of native disulfide bonds by specific pairs of cysteines located throughout a sequence of linear protein. This review describes basic and practical aspects of oxidative folding that should be considered while designing and optimizing manufacturing of proteins using chemical synthesis, semi-synthesis and a recombinant expression.     

Intramolecular versus intermolecular disulfide bonds in prion proteins

Prion protein (PrP) is the major component of the partially protease-resistant aggregate that accumulates in mammals with transmissible spongiform encephalopathies. The two cysteines of the scrapie form, PrP(Sc), were found to be in their oxidized (i.e. disulfide) form (Turk, E., Teplow, D. B., Hood, L. E., and Prusiner, S. B. (1988) Eur. J. Biochem. 176, 21-30); however, uncertainty remains as to whether the disulfide bonds are intra- or intermolecular. It is demonstrated here that the monomers of PrP(Sc) are not linked by intermolecular disulfide bonds. Furthermore, evidence is provided that PrP(Sc) can induce the conversion of the oxidized, disulfide-intact form of the monomeric cellular prion protein to its protease-resistant form without the temporary breakage and subsequent re-formation of the disulfide bonds in cell-free reactions.     

Site-specific PEGylation of native disulfide bonds in therapeutic proteins

Native disulfide bonds in therapeutic proteins are crucial for tertiary structure and biological activity and are therefore considered unsuitable for chemical modification. We show that native disulfides in human interferon alpha-2b and in a fragment of an antibody to CD4(+) can be modified by site-specific bisalkylation of the two cysteine sulfur atoms to form a three-carbon PEGylated bridge. The yield of PEGylated protein is high, and tertiary structure and biological activity are retained.     

Sensitive quantitative analysis of disulfide bonds in polypeptides and proteins

A sensitive quantitative method has been developed to determine the number of disulfide bonds in peptides and proteins. The disulfide bonds of several peptides and proteins were cleaved quantitatively by excess sodium sulfite at pH 9.5 and room temperature. Guanidine thiocyanate (2 M) was added to the protein solutions in order to denature them and thereby make the disulfide bonds accessible. The reaction with sulfite leads to a thiosulfonate and a free sulfhydryl group; the concentration of the latter was determined by reaction with disodium 2-nitro-5-thiosulfobenzoate (NTSB) in the presence of excess sodium sulfite. The synthesis, purification, and characterization of NTSB are described. The assay is rapid, requiring 3-5 min for oligopeptides and 20 min for proteins, and is as sensitive and quantitative as the sulfhydryl group assay employing 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent). It can be used for the analysis of as little as 10(8) mol of disulfide bonds, with an error of +/- 3%.     

Differential trafficking of soluble and integral membrane secretory granule-associated proteins

The posttranslational processing enzyme peptidylglycine alpha-amidating monooxygenase (PAM) occurs naturally in integral membrane and soluble forms. With the goal of understanding the targeting of these proteins to secretory granules, we have compared the maturation, processing, secretion, and storage of PAM proteins in stably transfected AtT-20 cells. Integral membrane and soluble PAM proteins exit the ER and reach the Golgi apparatus with similar kinetics. Biosynthetic labeling experiments demonstrated that soluble PAM proteins were endoproteolytically processed to a greater extent than integral membrane PAM; this processing occurred in the regulated secretory pathway and was blocked by incubation of cells at 20 degrees C. 16 h after a biosynthetic pulse, a larger proportion of soluble PAM proteins remained cell-associated compared with integral membrane PAM, suggesting that soluble PAM proteins were more efficiently targeted to storage granules. The nonstimulated secretion of soluble PAM proteins peaked 1-2 h after a biosynthetic pulse, suggesting that release was from vesicles which bud from immature granules during the maturation process. In contrast, soluble PAM proteins derived through endoproteolytic cleavage of integral membrane PAM were secreted in highest amount during later times of chase. Furthermore, immunoprecipitation of cell surface-associated integral membrane PAM demonstrated that very little integral membrane PAM reached the cell surface during early times of chase. However, when a truncated PAM protein lacking the cytoplasmic tail was expressed in AtT-20 cells, > 50% of the truncated PAM-1 protein reached the cell surface within 3 h. We conclude that the trafficking of integral membrane and soluble secretory granule-associated enzymes differs, and that integral membrane PAM proteins are less efficiently retained in maturing secretory granules.     

The effects of disulfide bonds on the denatured state of barnase

The effects of engineered disulfide bonds on protein stability are poorly understood because they can influence the structure, dynamics, and energetics of both the native and denatured states. To explore the effects of two engineered disulfide bonds on the stability of barnase, we have conducted a combined molecular dynamics and NMR study of the denatured state of the two mutants. As expected, the disulfide bonds constrain the denatured state. However, specific extended beta-sheet structure can also be detected in one of the mutant proteins. This mutant is also more stable than would be predicted. Our study suggests a possible cause of the very high stability conferred by this disulfide bond: the wild-type denatured ensemble is stabilized by a nonnative hydrophobic cluster, which is constrained from occurring in the mutant due to the formation of secondary structure.     

Detecting native folds in mixtures of proteins that contain disulfide bonds

High-throughput in vitro refolding of proteins that contain disulfide bonds, for which soluble expression is particularly difficult, is severely impeded by the absence of effective methods for detecting their native forms. We demonstrate such a method, which combines mass spectrometry with mild reductions, requires no prior experimentation or knowledge of proteins' physicochemical characteristics, function or activity, and is amenable to automation. These are necessary criteria for structural genomics and proteomics applications.     

Barnacle cement proteins. Importance of disulfide bonds in their insolubility

Barnacles produce a cement that is a proteinaceous underwater adhesive for their secure attachment to the substratum. The biochemical properties of the cement have not previously been elucidated, because the insolubility of the cement proteins hampers their purification and characterization. We developed a non-hydrolytic method to render soluble most of the cement components, thereby allowing the proteins to be analyzed. Megabalanus rosa cement could be almost completely rendered soluble by its reduction with 0.5 m dithiothreitol at 60 degrees C in a 7 m guanidine hydrochloride solution, the high concentration of dithiothreitol being indispensable to achieve this. The effectiveness of this reduction treatment was confirmed by the detachment of the barnacle from the substratum. Three proteins comprising up to 94% of the whole cement were identified as the major cement components. The cDNA clone of one of these major proteins was isolated, and the site-specific expression of the gene in the basal portion of the adult barnacle, where the cement glands are located, was demonstrated. A sequence analysis revealed this cement component to be a novel protein of 993 amino acid residues, including a signal peptide. This is the first report of the major component of the barnacle cement protein complex.     

Disruption of disulfide bonds is responsible for impaired secretion in human complement factor H deficiency

Factor H, a secretory glycoprotein composed of 20 short consensus repeat modules, is an inhibitor of the complement system. Previous studies of inherited factor H deficiency revealed single amino acid substitutions at conserved cysteine residues, on one allele arginine for cysteine 518 (C518R) and on the other tyrosine for cysteine 941 (C941Y) (Ault, B. H., Schmidt, B. Z., Fowler, N. L., Kashtan, C. E., Ahmed, A. E., Vogt, B. A., and Colten, H. R. (1997) J. Biol. Chem. 272, 25168-25175). To ascertain if the phenotype, impaired secretion of factor H, is due to the C518R substitution or the C941Y substitution and to ascertain the mechanism by which secretion is impaired, we studied COS-1 and HepG2 cells transfected with wild type and several mutant factor H molecules. The results showed markedly impaired secretion of both C518R and C941Y factor H as well as that of factor H molecules bearing alanine or arginine substitutions at the Cys518-Cys546 disulfide bond (C518A, C546A, C546R, C518A-C546A). In each case, mutant factor H was retained in the endoplasmic reticulum and degraded relatively slowly as compared with most other mutant secretory and membrane proteins that are retained in the endoplasmic reticulum. These data indicate that impaired secretion of the naturally occurring C518R and C941Y mutant factor H proteins is due to disruption of framework-specific disulfide bonds in factor H short consensus repeat modules.     

RNase activity requires formation of disulfide bonds and is regulated by the redox state

The activity of many RNases requires the formation of one or more disulfide bonds which can contribute to their stability. In this study, we show that RNase activity and, to a much lesser extent, nuclease activity, are redox regulated. Intracellular RNase activity was altered in vitroby changes in the glutathione redox state. Moreover, RNase activity was abolished following exposure to reducing agents such as -ME or DTT. Following reduction with glutathione (GSH), RNase activity could be fully reactivated with oxidized glutathione (GSSG). In contrast, RNase activity could not be reactivated when reduced with DTT. Decreasing the level of glutathione in vivoin wheat increased RNase activity. Tobacco engineered to have an increased glutathione redox state exhibited substantially lower RNase activity during dark-induced senescence. These results suggest that RNase activity requires the presence of one or more disulfide bonds that are regulated by glutathione and demonstrate for the first time that RNase activity can be altered with an alteration in cellular redox state.     

Superoxide involvement in the reduction of disulfide bonds of wheat gel proteins

A soluble epoxide hydrase which catalyzes the hydration of 9,10-epoxypalmitic acid has been partially purified from cell-free preparations from $\text{Bacillus}\text{megaterium}$ ATCC 14581. The hydrase can be cleanly separated from a soluble cytochrome P-450-dependent monooxygenase complex, previously demonstrated in this bacterium, that can catalyze the epoxidation of palmitoleic acid.     

Ligation of oligonucleotides to nucleic acids or proteins via disulfide bonds.

We have developed general methods for joining together, via cleavable disulfide bonds, either two unprotected polynucleotides or a polynucleotide and a peptide or protein. To join two oligonucleotides, each is first converted to an adduct in which cystamine is joined to the 5'-terminal phosphate of the oligonucleotide by a phosphoramidate bond. The adducts are mixed and reduced with dithiothreitol. The dithiothreitol is then removed by dialysis. Oxidation by atmospheric oxygen occurs to yield the required dimer. To join an oligonucleotide to a cysteine-containing peptide or protein, the 5'-cystamine oligomer is first converted to a 2'-pyridyldisulfide adduct and then reacted with an excess of the peptide or protein. If the peptide does not contain a free cysteine residue, it is first treated with iminothiolane to introduce one or more sulfhydryl groups. We have used these procedures to join a 16 mer deoxynucleotide probe and MDV-1 RNA, a substrate of Q beta RNA polymerase. This adduct hybridizes with a complementary target DNA. We have also joined a 16mer probe to peroxidase and MDV-1 RNA to human IgG. The probe-peroxidase adduct maintains enzymatic activity and the MDV-1 RNA-IgG adduct binds to a complementary anti-IgG.     

Assignment of disulfide bonds in proteins by fast atom bombardment mass spectrometry

A rapid and sensitive method for assignment of disulfide bonds using fast atom bombardment mass spectrometry is described for hen egg white lysozyme and bovine ribonuclease A. The protein is initially digested to a mixture of peptides using chemical and enzymatic methods under conditions which minimize disulfide bond reduction and exchange. The digested sample is analyzed directly by fast atom bombardment mass spectrometry before and after chemical reduction of cystine residues. An important feature of the method is that it is not necessary to completely resolve the peptides in the digest chromatographically prior to analysis. The disulfide-containing peptides are also characterized directly by prolonged exposure of the sample to the high energy xenon atom beam which results in the reduction of cystine residues. Intra- as well as interchain disulfide bond assignments are made on the basis of the mass difference between the molecular ions (MH+) of the oxidized and reduced peptides. Confirmation of the mass assignments may be obtained from the mass spectra of the digests after one cycle of manual Edman degradation. Although the quantity of protein required to unambiguously assign all of the disulfide linkages will depend on the ease with which the appropriate peptide fragments can be formed, results from these studies indicate that approximately 1 nmol of protein is usually sufficient.     

Disruption of disulfide bond formation alters the trafficking of prothyrotropin releasing hormone (proTRH)-derived peptides

Rat prothyrotropin releasing hormone (proTRH) is processed in the regulated secretory pathway (RSP) of neuroendocrine cells yielding five TRH peptides and several non-TRH peptides. It is not understood how these peptides are targeted to the RSP. We show here that a disulfide bond in the carboxy-terminus of proTRH plays an important role in the trafficking of this prohormone. Recombinant proTRH was observed to migrate faster on a native gel when treated with dithiothreitol (DTT) suggesting the presence of a disulfide bond. In vitro disulfide bond formation was prevented either by DTT treatment or by mutating cysteines 213 and 219 to glycines. In both cases the peptides derived from these mutants exhibited increased constitutive release and processing defects when expressed in AtT20 cells, a neuroendocrine cell line used in our prior studies on proTRH processing. Immunocytochemistry revealed that wild-type proTRH and mutant proTRH localized in a punctate pattern typical of proteins sorted to the regulated secretory pathway. These data suggest that the proposed disulfide bond of proTRH is involved in sorting of proTRH-derived peptides and in their retention within maturing secretory granules. This is the first evidence of structural motifs being important for the sorting of proTRH.     

Effect of disulfide bonds on lysozyme dynamics

The effect of destruction of disulfide bonds on the dynamics of proteins was studied by an example of lysozyme by the methods of molecular dynamics. In lysozyme, in the absence of disulfide bonds, the characteristic times of motions of secondary structure devices increased 3-7 times, whereas the amplitudes of fluctuations of secondary structure devices practically did not vary. In the absence of S-S-bonds, the volume of the molecule decreased approximately by 2%, primarily due to a "cleft" between the major and the small domains of lysozyme. Thus, disulfide bonds not only "glue" the secondary structure devices of the protein but also play a role of "rods", maintaining a certain free volume of the molecule necessary for the realization of its functions.