Sequential Comparison of Peptides Containing Half-cystine Residues from Ovalbumins of Six Avian Species

Hen ovalbumin contains one cystine disulfide (Cys⁷³-Cys¹²⁰) and four cysteine sulfhydryl groups (Cys¹¹,Cys³⁰,Cys³⁶⁷, and Cys³⁸²) in a single polypeptide chain of 385 amino acid residues. To investigate whether or not such a structure is shared by related avian species, the contents of disulfide-involved half-cystine residues and their positions in the primary structure of ovalbumins from five species were compared with those of hen ovalbumin. Ovalbumins were alkylated with a fluorescent dye, IAEDANS, under disulfide-reduced and disulfide-intact conditions and digested with a number of proteolytic enzymes. The sequences were deduced from peptides containing half-cystine residues labeled with the fluorescent dye. The results showed that the number of free cysteine sulfhydryl groups of ovalbumins was different among the species, three for guinea fowl and turkey (Cys¹¹, Cys³⁶⁷, and Cys³⁸²); and two for Pekin duck, mallard duck, and Emden goose (Cys¹¹ and Cys³³¹). On the other hand, a single intrachain disulfide bond could be identified from ovalbumins of five species using a combination of peptide mapping and N-terminal amino acid sequencing analysis under reduced and non-reduced conditions, in which the intrachain disulfide bond was like that of hen ovalbumin (Cys⁷³-Cys¹²⁰). The results also indicated that the variations in amino acid sequences on these peptides containing half-cystine residues bear a close relationship with the phylogeny of the six species.     

Refolding mechanism of ovalbumin: investigation by using a starting urea-denatured disulfide isomer with mispaired CYS367-CYS382

Ovalbumin, a member of the serpin superfamily, contains one cystine disulfide (Cys73-Cys120) and four cysteine sulfhydryls (Cys11, Cys30, Cys367, and Cys382) in the native state. To investigate the folding mechanism of ovalbumin, a urea-denatured disulfide isomer with a mispaired disulfide Cys367-Cys382 (D[367-382]) and its derivative (D[367-382/CM-73]) in which a native cystine counterpart of Cys73 is blocked by carboxymethylation were produced. Both the denatured isomers refolded within an instrumental dead time of 4 ms into an initial burst intermediate IN with partially folded conformation. After the initial burst phase, most of the D[367-382] molecules further refolded into the native form. In contrast, upon dilution of D[367-382/CM-73] with the refolding buffer, the protein stayed in the IN state as a stable form, which displayed a partial regain of the native secondary structure and a compact conformation with a similar Stokes radius to the native form. The structural characteristics of IN were clearly differentiated from those of an equilibrium intermediate IA that was produced by dilution with an acidic buffer of urea-denatured ovalbumin; IA showed much more hydrophobic dye binding and a larger Stokes radius than the IN state, despite their indistinguishable far-UV circular dichroic spectra. The non-productive nature of IA highlighted the importance of a compact conformation of the IN state for subsequent native refolding. These observations were consistent with a refolding model of ovalbumin that includes the regain of the partial secondary structure and of the compactness of overall conformation in an initial burst phase before the subsequent native refolding.     

Profile of the disulfide bonds in acetylcholinesterase

The inter- and intrasubunit disulfide bridges for the 11 S form of acetylcholinesterase isolated from Torpedo californica have been identified. Localized within the basal lamina of the synapse, the dimensionally asymmetric forms of acetylcholinesterase contain either two (13 S) or three (17 S) sets of catalytic subunits linked to collagenous and noncollagenous structural subunits. Limited proteolysis of these molecules yields a tetramer of catalytic subunits that sediments at 11 S. Each catalytic subunit contains 8 cysteine residues which were identified following tryptic digestion of the reduced, 14C-carboxymethylated protein. The tryptic peptides were purified by gel filtration followed by reverse-phase high performance liquid chromatography (HPLC) and then sequenced. The disulfide bonding profile was determined by treating the native, nonreduced 11 S form of acetylcholinesterase with a fluorescent, sulfhydryl-specific reagent, monobromobimane, prior to tryptic digestion. Peptides again were resolved by gel filtration and reverse-phase HPLC. One fluorescent cysteine-containing peptide was identified, indicating that a single sulfhydryl residue, Cys231, was present in its reduced form. Three pairs of disulfide-bonded peptides were identified. These were localized in the polypeptide chain based on the cDNA-deduced sequence of the protein and were identified as Cys67-Cys94, Cys254-Cys265, and Cys402-Cys521. Hence, three loops are found in the secondary structure. Cys572, located in the carboxyl-terminal tryptic peptide, was disulfide-bonded to an identical peptide and most likely forms an intersubunit cross-link. Since the 6 cysteine residues in acetylcholinesterase that are involved in intrachain disulfide bonds are conserved in the sequence of the homologous protein thyroglobulin, it is likely that both proteins share a common folding pattern in their respective tertiary structures. Cys231 and the carboxyl-terminal cysteine residue Cys572 are not conserved in thyroglobulin.     

Structural determinants of substrate access to the disulfide oxidase Erv2p

Erv2p is a small, dimeric FAD-dependent sulfhydryl oxidase that generates disulfide bonds in the lumen of the endoplasmic reticulum. Mutagenic and structural studies suggest that Erv2p uses an internal thiol-transfer relay between the FAD-proximal active site cysteine pair (Cys121-Cys124) and a second cysteine pair (Cys176-Cys178) located in a flexible, substrate-accessible C-terminal tail of the adjacent dimer subunit. Here, we demonstrate that Cys176 and Cys178 are the only amino acids in the tail region required for disulfide transfer and that their relative positioning within the tail peptide is important for activity. However, intragenic suppressor mutations could be isolated that bypass the requirement for Cys176 and Cys178. These mutants were found to disrupt Erv2p dimerization and to increase the activity of Erv2p for thiol substrates such as glutathione. We propose that the two Erv2p subunits act together to direct the disulfide transfer to specific substrates. One subunit provides the catalytic domain composed of the active site cysteine residues and the FAD cofactor, while the second subunit appears to have two functions: it facilitates disulfide transfer to substrates via the tail cysteine residues, while simultaneously shielding the active site cysteine residues from non-specific reactions.     

Structure of an antifreeze polypeptide from the sea raven. Disulfide bonds and similarity to lectin-binding proteins.

The antifreeze polypeptide (AFP) from the sea raven, Hemitripterus americanus, is a member of the cystine-rich class of blood antifreeze proteins which enable survival of certain fishes at sub-zero temperatures. Sea raven AFP contains 129 residues with 10 half-cystine residues. We have analyzed these half-cystine residues and established that all 10 of the half-cystine residues appeared to be involved in disulfide bond formation and that disulfide bonds linked Cys7 to Cys18, Cys35 to Cys125, and Cys89 to Cys117. These assignments were established by extensive proteolytic digestions of native AFP using pepsin and thermolysin and purification of the peptides by Sephadex G-15 gel filtration chromatography, anion exchange chromatography, and C18 reverse-phase high performance liquid chromatography. Cystine-containing peptides were detected by a colorimetric assay using nitrothiosulfobenzoate. Disulfide-containing peptides were reduced and alkylated, purified, and analyzed by amino acid analysis. The unreduced disulfide-linked peptides were sequenced directly by automated Edman degradations to confirm the disulfide assignments. Possible arrangements of the two remaining disulfide bonds include linkages Cys69/111 to Cys100/101. The sea raven AFP shares structural similarity with pancreatic stone protein and several lectin-binding proteins, especially with respect to half-cystines, glycines, and bulky aromatic residues. Two of the disulfide linkages we determined for sea raven AFP: Cys7-Cys18 and Cys35-Cys125, are conserved in these proteins. These similarities in covalent structure suggest that the sea raven AFP, pancreatic stone protein, and several lectin-binding proteins comprise a family of proteins which may possess a common fold.     

Localization of disulfide bonds in the cystine knot domain of human von Willebrand factor

von Willebrand factor (VWF) is a multimeric glycoprotein that is required for normal hemostasis. After translocation into the endoplasmic reticulum, proVWF subunits dimerize through disulfide bonds between their C-terminal cystine knot-like (CK) domains. CK domains are characterized by six conserved cysteines. Disulfide bonds between cysteines 2 and 5 and between cysteines 3 and 6 define a ring that is penetrated by a disulfide bond between cysteines 1 and 4. Dimerization often is mediated by additional cysteines that differ among CK domain subfamilies. When expressed in a baculovirus system, recombinant VWF CK domains (residues 1957-2050) were secreted as dimers that were converted to monomers by selective reduction and alkylation of three unconserved cysteine residues: Cys(2008), Cys(2010), and Cys(2048). By partial reduction and alkylation, chemical and proteolytic digestion, mass spectrometry, and amino acid sequencing, the remaining intrachain disulfide bonds were characterized: Cys(1961)-Cys(2011) (), Cys(1987)-Cys(2041) (), Cys(1991)-Cys(2043) (), and Cys(1976)-Cys(2025). The mutation C2008A or C2010A prevented dimerization, whereas the mutation C2048A did not. Symmetry considerations and molecular modeling based on the structure of transforming growth factor-beta suggest that one or three of residues Cys(2008), Cys(2010), and Cys(2048) in each subunit mediate the covalent dimerization of proVWF.     

Structural properties of recombinant ovalbumin and its transformation into a thermostabilized form by alkaline treatment.

The recombinant ovalbumin produced in Escherichia coli was purified from the cytoplasmic fraction and analyzed for its chemical and conformational properties. The recombinant ovalbumin displayed almost exactly the same circular dichroism and intrinsic tryptophan fluorescence spectra as egg white ovalbumin. As in the egg white protein, four cysteine sulfhydryls and one cystine disulfide were contained in the recombinant protein, according to the results of amino acid analyses; the disulfide bond was found by a peptide mapping analysis to correspond to the native cystine, Cys73-Cys120. According to a gel electrophoresis analysis, the presence of the disulfide bond was accounted for by specific oxidation of the corresponding cysteine residues during purification of the cytoplasmic protein. Unlike the identity in the conformational and peptide structures, none of the post-translational modifications (N-terminal acetylation, phosphorylation, and glycosylation) that are known with egg white ovalbumin were detected in the recombinant protein. The recombinant ovalbumin was transformed into a thermostabilized form in a similar manner to the transformation of egg white protein into S-ovalbumin; alkaline treatment increased the temperature for thermostability by 8.7 degrees C. These data strongly suggest that the post-translational modifications of ovalbumin are not related to the formation mechanism for S-ovalbumin.     

Isolation and characterization of disulfide-bonded peptides from the three globular domains of aggregating cartilage proteoglycan

The aggregating cartilage proteoglycan core protein contains two globular domains near the N terminus (G1 and G2) and one near the C terminus (G3). The G1-G3 domains contain 10, 8, and 10 cysteine residues, respectively. The disulfide assignments of the G1 domain have previously been deduced (Neame, P. J., Christner, J. E., and Baker, J. R. (1987) J. Biol. Chem. 262, 17768-17778) as Cys1-Cys2, Cys3-Cys6, Cys4-Cys5, Cys7-Cys10, and Cys8-Cys9, in which the numbers cited after the half-cystine residues are their relative positions from the N terminus. Here we describe a method for the isolation of disulfide-bonded peptides from tryptic digests of bovine nasal cartilage monomer. Sequence analysis of these peptides has allowed us to confirm the pairings previously determined for the G1 domain and to assign a disulfide pattern for the G2 domain of Cys11-Cys14, Cys12-Cys13, Cys15-Cys18, and Cys16-Cys17, in which the Cys15-Cys18 pairing was deduced indirectly. Similarly, for the G3 domain, a pattern of Cys19-Cys20, Cys21-Cys24, Cys22-Cys23, Cys25-Cys27, and Cys26-Cys28 was assigned, in which the Cys22-Cys23 pair was deduced indirectly. The G2 domain therefore contains disulfide bonding which is characteristic of the tandem repeat structures found in the G1 domain and link protein, and the G3 domain contains the three disulfide linkages previously assigned to the family of C-type animal lectins. The method described here, which combines anion-exchange, cation-exchange, and reversed-phase chromatography, should have broad application to the isolation of disulfide-bonded peptides from other heavily glycosylated proteins and proteoglycans.     

Partial assignment of disulfide pairs in neurophysins

The original report assigning the pairing of neurophysin's 14 half-cystine residues (Schlesinger et al. (1972), Proc. Natl. Acad. Sci., U.S.A., 69,3350-3353) was based on an incorrect amino acid sequence. In the present study, re-investigation of the results of proteolytic fragmentation of bovine neurophysins indicates that the majority of the original assignments were incorrect. Three disulfide pairs are now assigned as Cys21-Cys44, Cys67-Cys85 and Cys74-Cys79. The pairing pattern indicates that neurophysin's variable carboxyl terminal region, separately encoded by the third gene exon, does not form a self-contained domain.     

The status of half-cystine residues and locations of N-glycosylated asparagine residues in human eosinophil peroxidase

The determination by protein chemistry methods of the half-cystine status in human eosinophil peroxidase (EPO) is reported. EPO is two-chained and has a total of 14 half-cystine residues. Cys141 and Cys152 form an intrachain bridge in the light chain of EPO. Disulfide bridges connect Cys253 and Cys263, Cys257 and Cys287, Cys359 and Cys370, Cys570 and Cys635, and Cys676 and Cys701, forming five intrachain disulfide bridges in the heavy chain of EPO. Cys291 and Cys455 are found to be unpaired, containing free sulfhydryl groups. The pattern of disulfide bridges is in agreement with that predicted from the X-ray crystallographic structure of canine myeloperoxidase (MPO) (Zeng, J., and Fenna, R. E. (1992) J. Mol. Biol. 226, 185-207) to be general for the class of mammalian peroxidases, including EPO, MPO, lactoperoxidase (LPO), and thyroid peroxidase (TPO). Of four candidate sites in EPO for attachment of glucosamine-based carbohydrate, Asn327 and Asn363 are occupied, whereas Asn700 and Asn708 are unsubstituted. Furthermore, a discrepancy in the literature regarding the sequence of residues 645-659 is resolved.     

Amino acid sequence and S-S bonds of Penicillium brevicompactum guanyl-specific ribonuclease

The primary structure of Penicillium brevicompactum guanyl-specific RNase was determined. The enzyme consists of 102 amino acid residues, Mr 10801. The 4 cysteine residues of the RNase are linked in pairs by disulfide bonds: Cys2-Cys10, Cys6-Cys101. P. brevicompactum RNase structure is similar to RNase T1; the degree of homology is 66%.     

Human tissue factor contains thioester-linked palmitate and stearate on the cytoplasmic half-cystine

The state of the five half-cystine residues in human tissue factor (TF) has been characterized. The results indicate that the four half-cystines in the extracellular domain of TF form two disulfide bonds and the half-cystine in the cytoplasmic region is acylated by palmitic acid and stearic acid. The extracellular disulfide cross-links, Cys49-Cys57 and Cys186-Cys209, were deduced from the analysis of tryptic peptides. Acylation of the cytoplasmic half-cystine was demonstrated by purifying and characterizing fibroblast TF from cells labeled with [3H]palmitic acid. Radiolabeled fibroblast TF was observed by autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The tritiated material covalently bound to the protein was identified as [3H]palmitate and [3H]stearate by reverse-phase high-pressure liquid chromatography. Deacylation of TF with hydroxylamine resulted in the spontaneous generation of disulfide-linked TF dimers. This result suggests that the disulfide-linked TF dimer, a minor component of most TF preparations, and the recently described heterodimeric form of TF are artifacts produced by deacylation of Cys245 and subsequent interchain disulfide bond formation.     

Disulfide isoforms of recombinant glia maturation factor beta

Recombinant human glia maturation factor beta (r-hGMF-beta) is a single-chain polypeptide (141 amino acid residues) containing three cysteines, at positions 7, 86 and 95. Nascent r-hGMF-beta exists in the reduced state and has no biological activity. The protein can be activated through oxidative refolding by incubation with a mixture of reduced and oxidized glutathione. Reverse-phase HPLC analysis of the refolded r-hGMF-beta shows the presence of four peaks, corresponding to the reduced form plus three newly generated intrachain disulfide-containing isoforms predicted from the number of cysteine residues. Only one isoform shows biological activity when tested for growth suppression on C6 glioma cells. We infer from the HPLC elution pattern that the active form contains the disulfide bridge Cys86-Cys95.     

Disulfide arrangement of human insulin-like growth factor I derived from yeast and plasma.

The disulfide arrangement of yeast derived human insulin-like growth factor I (yIGF-I) was determined using a combination of Staphylococcus aureus V8 protease mapping, fast-atom-bombardment mass spectrometry as well as amino acid sequence and composition analysis. Three disulfide bridges were found between the following cysteine residues: Cys6-Cys48, Cys47-Cys52 and Cys18-Cys61. IGF-I isolated from human plasma (pIGF-I) was found to have an identical disulfide configuration. A yeast-derived isomeric form of IGF-I (yisoIGF-I) exhibited an altered disulfide arrangement: Cys6-Cys47, Cys48-Cys52 and Cys18-Cys61. Radioreceptor analysis of pIGF-I and yIGF-I showed high specific activity, 20,000 U/mg. However, yisoIGF-I demonstrated a severely reduced ability to bind to the IGF-I receptor (19%) and was less potent in provoking a mitogenic response in Balb/C 3T3 fibroblasts (50% at doses 10-100 ng/ml). The data demonstrate the importance of correct disulfide arrangement in IGF-I for full biological activity.     

The structure of hepadnaviral core antigens. Identification of free thiols and determination of the disulfide bonding pattern.

A set of wild-type and mutant human, woodchuck, and duck hepatitis viral core proteins have been prepared and used to study the free thiol groups and the disulfide bonding pattern present within the core particle. Human (HBcAg) and woodchuck (WHcAg) core proteins contain 4 cysteine residues, whereas duck (DHcAg) core protein contains a single cysteine residue. Each of the cysteines of HBcAg has been eliminated, either singly or in combinations, by a two-step mutagenesis procedure. All of the proteins were shown to have very similar physical and immunochemical properties. All assemble into essentially identical core particle structures. Therefore disulfide bonds are not essential for core particle formation. No intra-chain disulfide bonds occur. Cys107 is a free thiol buried within the particle structure, whereas Cys48 is present partly as a free sulfhydryl which is exposed at the surface of the particle. Cys61 is always and Cys48 is partly involved in interchain disulfide bonds with the identical residues of another monomer, whereas Cys183 is always involved in a disulfide bond with the Cys183 of a different monomer. WHcAg has the same pattern of bonding, whereas DHcAg lacks any disulfide bonds, and the single free sulfhydryl, Cys153 which is equivalent to Cys107 of HBcAg, is buried.     

Studies in serum support rapid formation of disulfide bond between unpaired cysteine residues in the VH domain of an immunoglobulin G1 molecule

Recombinant monoclonal antibodies undergo extensive posttranslational modifications. In this article, we characterize major modifications, separated by cation exchange chromatography, on an immunoglobulin G1 (IgG1) monoclonal antibody (mAb). We found that N-terminal cyclization of glutamine residues to pyroglutamate on the light and heavy chains are the major isoforms resolved during cation exchange chromatography. However, using CEX, we also separated and identified isoforms with unpaired cysteine residues in the V_H domain of the molecule (Cys22-Cys96). Omalizumab, a therapeutic anti-IgE antibody, has unpaired cysteine residues in the V_H domain between Cys22 and Cys96, and the Fab fragment, containing the unpaired cysteine residues, is reported to have reduced potency. Dynamic interchain disulfide rearrangement, with slow kinetics, was recently reported to take place in serum for an IgG2 molecule and resulted in predictable mature isoforms. Analytical evaluation of our mAb, after recovery from serum, revealed that the unpaired intrachain cysteine residues (Cys22-Cys96) reformed their disulfide bond. The significance of this study is that correct pairing occurred rapidly, and we speculate that thiol molecules such as cysteine, homocysteine, and glutathione in serum provide an environment, outside the endoplasmic reticulum, for correct linkage.     

Analyses of Native Disulfide Bond Formations in the Early Stage of the Folding Process in Mutant Lysozymes Where the Long-Range Interactions in the Denatured State Were Modulated

In order to clarify whether modulation of long-range interactions in the denatured state affect native disulfide bond (SS bond) formations of hen egg white lysozyme (HEL) containing a pair of cysteine residues, we examined the extent of SS bond formation among 12 variants containing a pair of cysteines. The loss of clusters 5 and 6 in the denatured state affected the formation of Cys30-Cys115 and Cys6-Cys127 respectively.     

Analyses of native disulfide bond formations in the early stage of the folding process in mutant lysozymes where the long-range interactions in the denatured state were modulated

In order to clarify whether modulation of long-range interactions in the denatured state affect native disulfide bond (SS bond) formations of hen egg white lysozyme (HEL) containing a pair of cysteine residues, we examined the extent of SS bond formation among 12 variants containing a pair of cysteines. The loss of clusters 5 and 6 in the denatured state affected the formation of Cys30-Cys115 and Cys6-Cys127 respectively.     

Role of an intrachain disulfide bond in the conformation and stability of ovalbumin

Ovalbumin, which contains one intrachain disulfide bond and four cysteine sulfhydryls, was reduced with dithiothreitol under non-denaturing conditions, and its conformation and stability were compared with those of the disulfide-bonded form. The CD spectrum in the far-UV region revealed that the overall conformation of the reduced form is similar to that of the disulfide-bonded one. Likewise, the inaccessibility to trypsin and the non-reactivity of the four cysteine sulfhydryls, exhibited by the native disulfide-bonded ovalbumin, were still retained in the disulfide-reduced form. Thus, the reduced ovalbumin appeared to substantially take the native-like conformation. However, the near-UV CD spectrum slightly differed between the native and disulfide-reduced forms. Protein alkylation with a fluorescent dye and subsequent sequence analysis showed that the two sulfhydryls (Cys73 and Cys120) originating from the disulfide bond are highly reactive in the reduced form. Furthermore, upon proteolysis with subtilisin, the N-terminal side of Cys73 was cleaved in the reduced form, but not in the disulfide-bonded one. Upon heat denaturation, the transition temperature of the reduced form was lower, by 6.8 degrees C, than that of the disulfide-bonded one. Thus, we concluded that ovalbumin has a native-like conformation in its disulfide-reduced form, but that the local conformation of the reduced form fluctuates more than that of the disulfide-bonded one. Such local destabilization may be related to the decreased stability against heat denaturation.     

Location of disulfide bonds within the sequence of human serum cholinesterase

Human serum cholinesterase was digested with pepsin under conditions which left disulfide bonds intact. Peptides were isolated by high pressure liquid chromatography, and those containing disulfide bonds were identified by a color assay. Peptides were characterized by amino acid sequencing and composition analysis. Human serum cholinesterase contains 8 half-cystines in each subunit of 574 amino acids. Six of these form three internal disulfide bridges: between Cys65-Cys92, Cys252-Cys263, and Cys400-Cys519. A disulfide bond with Cys65 rather than Cys66 was inferred by homology with Torpedo acetylcholinesterase. Cys571 forms a disulfide bridge with Cys571 of an identical subunit. This interchain disulfide bridge is four amino acids from the carboxyl terminus. A peptide containing the interchain disulfide is readily cleaved from cholinesterase by trypsin (Lockridge, O., and La Du, B. N. (1982) J. Biol. Chem. 257, 12012-12018), suggesting that the carboxyl terminus is near the surface of the globular tetrameric protein. The disulfide bridges in human cholinesterase have exactly the same location as in Torpedo californica acetylcholinesterase. There is one potential free sulfhydryl in human cholinesterase at Cys66, but this sulfhydryl could not be alkylated. Comparison of human cholinesterase, and Torpedo and Drosophila acetylcholinesterases to the serine proteases suggests that the cholinesterases constitute a separate family of serine esterases, distinct from the trypsin family and from subtilisin.