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PubMed1982

Disulfide bond reduction inhibits norepinephrine accumulation in postganglionic sympathetic nerve endings.

Accumulation of l-[3H]norepinephrine by sympathetic nerves in mouse atria was inhibited by disulfide bond reducing agents such as dithiothreitol (IC50 approximately 0.4 mM) and beta-mercaptoethanol (IC50 approximately 1.1 mM). Dithiothreitol-induced inhibition of l-[3H]norepinephrine accumulation was reversed by exposing tissues to oxygen or to oxidizing agents such as potassium ferricyanide (0.03 mM) or 5,5'dithiobis-(2-nitrobenzoic acid) (1.0 mM). The cycle of reduction-induced inhibition and oxidation-induced reactivation could be repeated within tissues by sequential exposure to dithiothreitol and potassium ferricyanide. Exposing rat atrial to concentrations of dithiothreitol (1.0 mM) that produced substantial inhibition of l-[3H]norepinephrine accumulation (approximately 50%) did not affect spontaneous atrial rate or l-isoproterenol-induced increases in rate. In addition to disulfide bond reducing agents, sulfhydryl blocking agents such as N-ethylmaleimide (IC50 approximately 0.3 mM) and p-chloromercuriphenylsulfonic acid (IC50 approximately 0.2 mM) inhibited l-[3H]norepinephrine accumulation by mouse atria. Inhibition induced by sulfhydryl blockers could not be reversed by oxygen or by oxidizing agents. Furthermore, tissues exposed to dithiothreitol and subsequently exposed to N-ethylmaleimide could not be reactivated by oxygen or by oxidizing agent. The data suggest that a protein with a disulfide bond is associated with l-[3H]norepinephrine accumulation and that reduction of the disulfide bond inhibits amine accumulation. Reduced disulfide bonds that have not been alkylated can be reoxidized, but reduced disulfide bonds that are alkylated are irreversibly inactivated.

Animals↗
PubMed1993

Disulfide bond requirements for assembly of the platelet glycoprotein Ib-binding domain of von Willebrand factor.

von Willebrand factor (vWF) is a multimeric glycoprotein of plasma and the subendothelial matrix that interacts with specific platelet receptors to establish platelet adhesion at a site of vascular injury. The vWF domain containing the platelet receptor glycoprotein Ib-binding site can be expressed in heterologous cells as a recombinant homodimeric fragment that mediates platelet-platelet interaction, analogous to multimeric vWF. The recombinant domain, r116, contains 7 Cys residues within its 290-residue monomeric subunit paired in an unidentified intra- and intermolecular disulfide bond arrangement. In this report we define the disulfide bond-dependent framework of r116 that provides the domain with its essential structural features that support dimer formation and the generation of a disulfide bond-dependent epitope. The results demonstrate that a triplet of Cys residues at positions 459, 462, and 464 are essential for efficient dimer formation. An intramolecular Cys509/Cys695 disulfide loop is required for generating a functional dimeric molecule, and monomeric molecules containing a Cys509/Cys695 intramolecular disulfide bond are unable to support ristocetin-mediated platelet aggregation. The disulfide arrangement in r116 is similar, if not identical, to the proposed arrangement within the corresponding region of plasma vWF, and these studies document the inherent Cys-dependent maturation of an isolated vWF domain.

Disulfides↗
PubMed1994

Unfolding of colicin A during its translocation through the Escherichia coli envelope as demonstrated by disulfide bond engineering.

Three double cysteine mutants, each possessing a disulfide bond in its pore-forming domain, were used to study the translocation of colicin A through the Escherichia coli envelope. These mutated colicins were able to exert their in vivo channel activity only after their disulfide bonds had been reduced by dithiothreitol. In solution, the reduction of the disulfide bonds by dithiothreitol was a slow process whose kinetics depended on the position of the disulfide bond (t1/2 varying between 35 and 100 s). This t1/2 was strongly decreased (t1/2 = 8-9 s) upon predenaturation of the mutated colicins with urea. The t1/2 values of reduction of the mutants bound to E. coli-sensitive cells were similar to those of predenatured colicins. This suggested that the interaction of the oxidized double cysteine mutants with the E. coli envelope triggered their unfolding. The disulfide bonds did not prevent but delayed the translocation of the colicins. The amplitude of the delay and the time at which it occurred during translocation depended on the position of the disulfide bond. We could discriminate between the delays accumulated during binding to the receptor and those during the translocation via OmpF and the Tol proteins.

Bacterial Outer Membrane Proteins↗
PubMed1995

Characterization of fibroblasts with a unique defect in processing antigens with disulfide bonds.

A chinese hamster ovary (CHO) fibroblast, transfected with murine MHC class II genes, inefficiently stimulated CD4+ Th cells specific for OVA, hen egg lysozyme (HEL), and pork insulin which contain disulfide bonds. However, the fibroblasts elicited a T cell response to lambda repressor, which lacks disulfide bonds, and efficiently presented synthetic peptides. A somatic cell hybrid WALC, generated by fusing the hamster fibroblast with a murine L cell fibroblast, very efficiently processed OVA and HEL, suggesting that impaired processing was genetically complemented and was a recessive trait. The hamster fibroblasts were capable of processing two distinct denatured forms of OVA and carboxymethylated HEL, either as effectively or more efficiently than the B lymphoma cell. The CHO cells also displayed diminished disulfide reduction of an endocytosed [125I]tyramine linked to poly-(D-lysine) through a disulfide spacer compared with that of the cell hybrid, providing direct evidence for defective reductive cleavage by the CHO cells. Diminished aspartic acid-mediated proteolysis of Ag could not account for the phenotype, because cell lysates and separated organelles from the fibroblast possessed higher acidic aspartyl proteolytic activity than lysates and organelles from a B lymphoma cell. Thus, CHO cells exhibit a defect in processing Ag with disulfide bonds which is consistent with the impaired intracellular reduction of the disulfide bonds in endocytosed macromolecules.

Animals↗
PubMed1994

Mutational analysis of disulfide bridges in the type C atrial natriuretic peptide receptor.

The type C receptor (ANP-C or NPR-C) for the natriuretic peptides was demonstrated, by site-directed mutagenesis, to have an immunoglobulin-like disulfide bonding pattern that is very similar to that of the cytokine receptor superfamily. The mature form of ANP-C has a disulfide-linked homodimeric structure and contains 5 conserved cysteine residues per subunit, all in the extracellular domain. To identify the cysteine residue involved in the dimerization and further to determine the intramolecular disulfide bridges and their functional roles, cysteine to serine mutations of the 5 cysteine residues were constructed. An analysis of the mutant receptors expressed in COS-1 cells by 125I-ANP binding assay and by measuring difference in their electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels indicated that 1) the first 4 cysteine residues are joined sequentially, forming the Cys104-Cys132 and Cys209-Cys257 loops of 29 and 49 residues, respectively; 2) the two disulfide-linked loops are essential for the ligand binding activity; 3) the 5th cysteine residue Cys469 is used in the formation of covalently linked dimers; and 4) the covalent association of the subunit through the disulfide bond involving Cys469 has no apparent influence on ligand-receptor interactions. The intramolecular disulfide bond Cys104-Cys132 was also confirmed by direct protein sequencing of tryptic fragments of purified ANP-C receptor. The secondary structural features revealed here will be useful in understanding the structure and function relationships of not only the dimeric ANP-C receptor, which has only a short cytoplasmic tail, but also the ANP-A (GC-A) and ANP-B (GC-B) receptor subtypes, which have a guanylate cyclase domain in their long cytoplasmic tail and have recently been shown to possess an oligomeric structure, since they have similarly spaced cysteine residues in their extracellular domains.

Amino Acid Sequence↗
PubMed1993

Cysteine mutants of light chain-2 form disulfide bonds in skeletal muscle myosin.

Multiple disulfide bonds form in recombinant myosin light chain-2 mutants that contain an engineered cysteine at positions 2, 73, or 94, in addition to the endogenous cysteines at residues 126 and 155 (Saraswat, L.D., and Lowey, S. (1991) J. Biol. Chem. 266, 1977). By replacing one of the native cysteines with an alanine, mutants with a single pair of thiols were created: Cys2/Cys155, Cys2/Cys126, Cys73/Cys155 and Cys94/Cys155. Oxidation of these mutants resulted in a fast migrating band on nonreducing SDS gels, which was attributed to an intramolecular disulfide bond. To determine if disulfide formation could also occur when the light chains (LC) are bound to the myosin heavy chains, LCs were added to myosin which had been depleted of its native LC2 by an immunoadsorbent. When the reconstituted myosin was reacted with 5,5'-dithiobis(2-nitrobenzoic acid) in the absence of divalent cations, intramolecular disulfide bonds formed in the mutant and wild type LCs, but the LCs did not remain bound to the myosin heavy chains. Addition of magnesium ions prevented LC dissociation, but intramolecular disulfide bonds no longer formed. Instead, mutants containing cysteines in the NH2-terminal domain formed intermolecular disulfide bonds between the two heads of myosin. The ability to cross-link the heads demonstrates the existence of close head/head interactions in the myosin molecule, a feature that may be essential for regulation.

Animals↗
PubMed1994

Role of interchain disulfide bonds on the assembly and secretion of human fibrinogen.

The cysteines involved in joining the 2 half-molecules of fibrinogen and also those located on either side of the alpha-helical coiled-coil region, were substituted, by site-directed mutagenesis, with serine. Fibrinogen assembly and secretion were determined in transiently transfected COS cells. Our studies indicate that in order to assemble the 2 half-molecules into a dimer, it is not sufficient to only have the disulfide linkages which keep the 2 half-molecules intact. The disulfide rings which flank the coil-coiled region also play important roles in dimer assembly. Intact interchain disulfide linkages at the NH2-terminal end of the coiled-coil region are essential for assembly of the 2 half-molecules. Disruption of these disulfide rings leads to the formation and secretion of half-molecules. Disruption of the interchain disulfide rings at the COOH-terminal end of the coiled-coil region allows dimer formation, but the 6-chain molecule which is assembled is not secreted. Disruption of both disulfide rings at either end of the coiled-coil region disallows assembly of half-molecules and of dimeric fibrinogen.

Animals↗
PubMed1993

Peptide binding to protein disulfide isomerase occurs at a site distinct from the active sites.

Protein disulfide isomerase (PDI) is a multifunctional protein resident in the lumen of the rough endoplasmic reticulum that facilitates protein folding via disulfide bond isomerization. Previously we determined that PDI binds a variety of peptides that can be covalently attached to this protein via a photoreactive cross-linker. We have now investigated the relationship between the peptide binding site and the ability of PDI to catalyze disulfide bond isomerization. PDI has two identical sequences, -WCGHCK-, that have been demonstrated to be important in PDI-catalyzed disulfide isomerization. We have found that other proteins containing these thioredoxin-like active site sequences do not bind the photoreactive peptide probes. Moreover, although chemical modification of the 2 cysteines within the thioredoxin-like active site regions completely inhibits PDI-catalyzed disulfide isomerization, these modifications do not affect peptide binding by PDI. Both of these observations suggest that peptide binding occurs at a site other than the putative PDI active sites. To localize the site in PDI at which binding occurs, we used a radiolabeled peptide photoaffinity probe. Peptide fragments generated by cleavage of 125I-peptide-labeled PDI with cyanogen bromide yielded a single 8-kDa polypeptide fragment containing the 125I-labeled peptide site, but neither of the putative catalytic sites of PDI. An 125I-labeled tryptic peptide was generated from this cyanogen bromide fragment and determined by microsequencing to contain residues 451-476 of PDI; this 26-residue peptide is noteworthy because of its extremely high content of acidic amino acids. Based on these findings we conclude that the peptide binding site is located in the COOH-terminal domain of the protein, and it is distinct from the two active sites for PDI-catalyzed disulfide isomerization and from the region of PDI that has estrogen receptor sequence similarity.

Amino Acid Sequence↗
PubMed1993

Characterization of the disulfide motif in BNBD-12, an antimicrobial beta-defensin peptide from bovine neutrophils.

BNBD-12, a prototype beta-defensin peptide from bovine neutrophils, was chosen for determination of the disulfide motif in this family of tridisulfide antimicrobial peptides. Disulfide-containing fragments of BNBD-12 were generated by incubation with trypsin, and the amino acid composition of one tryptic fragment allowed for the assignment of one of the three disulfides. The remaining two disulfides, contained in a 16-residue tryptic oligopeptide, were characterized by amino acid analysis of fragments generated by a single round of Edman degradation which cleaved the Cys-Cys peptide bond present near the carboxyl terminus of BNBD-12. Cleavage of this bond produced two disulfide-containing oligopeptides, the compositions of which provided unambiguous assignments of the disulfides involved. The cystine motif in BNBD-12 differs from that of classical defensins, and indicates that the beta-defensins and defensins must have differently folded chains, though they share several functional properties.

Amino Acid Sequence↗
PubMed1996

Role of intramolecular disulfide bond formation in the assembly and secretion of apolipoprotein B-100-containing lipoproteins.

Apolipoprotein B-100 (apoB) is essential for the hepatic assembly and secretion of triglyceride-rich very low density lipoprotein (VLDL). The mechanism of VLDL assembly was explored by perturbing apoB folding in HepG2 cells with the thiol reducing agent dithiothreitol (DTT). Although apoB contains eight known disulfide bonds, seven of which are positioned in the amino-terminal 21% of the protein, its assembly and secretion was only partially blocked in cells treated with 2 mM DTT, a condition that fully blocks the secretion of other disulfide-bonded proteins. Nonreducing gel electrophoresis of an apoB-derived proteolytic peptide revealed that apoB escapes the secretory block normally caused by DTT because its amino-terminal disulfide bonds undergo maturation to a DTT-resistant form after completing synthesis of only the first approximately 20-25% of the protein. If, however, DTT was used under conditions that prevented the initial formation of amino-terminal disulfide bonds, lipoprotein secretion was blocked. Reduced forms of apoB were extremely labile and, unlike other disulfide-bonded proteins, incapable of achieving secretion competence posttranslationally. These results indicate that disulfide bond formation within the amino-terminus of apoB is essential for the proper folding and assembly of its downstream lipophilic sequences. The onset of DTT resistance while still a nascent polypeptide chain is consistent with a model in which the amino-terminal domain of apoB undergoes an independent folding and maturation process, the completion of which may represent an initiation phase of triglyceride-rich lipoprotein assembly.

Apolipoprotein B-100↗
PubMed1976

Time of occurrence of disulfide linking between procollagen chains.

The disulfide bonds that link together single collagen pro-alpha chains into trimers, prior to their secretion, were found to be synthesized after the pro-alpha chains were completed and released from polysomes and not while the pro-alpha chains were still nascent. After a 4- or 8-min incubation of cell cultures derived from embryonic chick sternum with [3H]proline, the nascent chains isolated from polysome preparations contained negligible amounts of disulfide-linked chains. After a 20-min incubation, some very large disulfide-linked material was found associated with the polysome preparation, but this material proved to be noncollagenous and also appeared to represent completed, and not nascent, chains. The presence of this proline-labeled species was confirmed in analyses of whole cell lysates, in which it is present as a mixture of monomers of approximately 290,000 molecular weight and disulfide-linked dimers. Pulse-chase experiments also gave results that were consistent with the conclusion that released single pro-alpha chains are the precursors of released disulfide-linked pro-alpha chains. Such experiments also showed that pro-alpha chains could continue to form disulfide-linked species in the presence of inhibitors of peptide chain elongation.

Animals↗
PubMed1995

Two resident ER-proteins, CaBP1 and CaBP2, with thioredoxin domains, are substrates for thioredoxin reductase: comparison with protein disulfide isomerase.

Protein disulfide-isomerase (PDI) is the best known representative of a growing family of enzymes with thioredoxin domains. Two such proteins with thioredoxin (Trx) domains, CaBP1 and CaBP2 (ERp72), have previously been isolated from rat liver microsomes. Here we report that they, like PDI are substrates for thioredoxin reductase and will catalyze NADPH-dependent insulin disulfide reduction. The activity of CaBP1 and CaBP2 in this assay was higher than that of PDI but lower than that of E. coli Trx. Furthermore, as isolated the thioredoxin domains of CaBP1 and CaBP2 were in disulfide form as judged by stoichiometric oxidation of 2 and 3 mol of NADPH in CaBP1 and CaBP2, respectively. The redox potential of the active site disulfide/dithiol was estimated from the equilibrium with a mutant E. coli Trx, P34H Trx, with a known redox potential (-235 mV). This showed that CaBP1 and CaBP2, like PDI, have a much higher redox potential than wild type thioredoxin (-270 mV) in agreement with a role in formation of protein disulfide bonds. In conclusion, in vitro CaBP1 and CaBP2 share catalytic properties in thiol disulfide-interchange reactions with PDI. Thus, the well known activity of PDI is not unique in the endoplasmic reticulum and CaBP1 and CaBP2 may be regarded as functional equivalents.

Animals↗
PubMed2006

A novel member of the protein disulfide oxidoreductase family from Aeropyrum pernix K1: structure, function and electrostatics.

The formation of disulfide bonds between cysteine residues is a rate-limiting step in protein folding. To control this oxidative process, different organisms have developed different systems. In bacteria, disulfide bond formation is assisted by the Dsb protein family; in eukarya, disulfide bond formation and rearrangement are catalyzed by PDI. In thermophilic organisms, a potential key role in disulfide bond formation has recently been ascribed to a new cytosolic Protein Disulphide Oxidoreductase family whose members have a molecular mass of about 26 kDa and are characterized by two thioredoxin folds comprising a CXXC active site motif each. Here we report on the functional and structural characterization of ApPDO, a new member of this family, which was isolated from the archaeon Aeropyrum pernix K1. Functional studies have revealed that ApPDO can catalyze the reduction, oxidation and isomerization of disulfide bridges. Structural studies have shown that this protein has two CXXC active sites with fairly similar geometrical parameters typical of a stable conformation. Finally, a theoretical calculation of the cysteine pK(a) values has suggested that the two active sites have similar functional properties and each of them can impart activity to the enzyme. Our results are evidence of functional similarity between the members of the Protein Disulphide Oxidoreductase family and the eukaryotic enzyme PDI. However, as the different three-dimensional features of these two biological systems strongly suggest significantly different mechanisms of action, further experimental studies will be needed to make clear how different three-dimensional structures can result in systems with similar functional behavior.

Aeropyrum↗
PubMed2004

Binding of phlorizin to the C-terminal loop 13 of the Na(+)/glucose cotransporter does not depend on the [560-608] disulfide bond.

The disulfide bonds of the Na(+)/glucose cotransporter (SGLT1) are believed to participate in the binding of the transport inhibitor phlorizin. Here, we investigated the role of the [560-608] disulfide bond on the phlorizin-binding function of the C-terminal loop 13 of SGLT1 using 3-iodoacetamidophlorizin (3-IAP) as a probe. The reactivity of 3-IAP to the fully reduced loop 13 was competitively inhibited by phlorizin, as evident from the MALDI mass spectra. It indicates that the disulfide bond is not mandatory for phlorizin binding. CD and equilibrium unfolding studies showed that the secondary structure and conformation stability of loop 13 were not affected by removing the disulfide bond. Furthermore, we generated a series of loop 13 mutants to assess the contribution of the disulfide bond to phlorizin binding. A positive correlation between the stability and phlorizin affinity of the mutant proteins was observed, implying that the protein stability, rather than the disulfide bond, is relevant to the phlorizin-binding function of loop 13.

Acrylamide↗
PubMed1981

Electrostatic influence of local cysteine environments on disulfide exchange kinetics.

The ionic strength dependence of the bimolecular rate constant for reaction of the negative disulfide 5,5'-dithiobis (2-nitrobenzoic acid) with cysteines in fragments of naturally occurring proteins was determined by stopped-flow spectroscopy. The Debye-Hückel relationship was applied to determine the effective charge at the cysteine and thereby determine the extent to which nearby neighbors in the primary sequence influence the kinetics. Corrections for the secondary salt effect on cysteine pKs were determined by direct spectrometric pH titration of sulfhydryl groups or by observation of the ionic strength dependence of kinetics of cysteine reaction with the neutral disulfide 2,2'-dithiodipyridine. Quantitative expressions was verified by model studies with N-acetyl-cystein. At ionic strengths equal to or greater than 20 mM, the net charge at the polypeptide cysteine site is the sum of the single negative charge of the thiolate anion and the charges of the amino acids immediately preceding and following the cysteine in the primary sequence. At lower ionic strengths, more distant residues influence kinetics. At pH 7.0, 23 degree C, and an ionic strength of 20 mM, rate constants for reaction of the negative disulfide with a cysteine having two positive neighbors, one positive and one neutral neighbor, or two neutral neighbors are 132000, 3350, and 367 s-1 M-1, respectively. This corresponds to a contribution to the activation energy of 0.65- 1.1 kcal/mol per ion pair involved in collision between the cysteine and disulfide regions. The results permit the estimation that cysteine local environments may provide a means of achieving a 10(6)-fold range in rate constants in disulfide exchange reactions in random-coil proteins. This range may prove useful in developing strategies for directing disulfide pairing in synthetic proteins.

Cyanogen Bromide↗
PubMed1978

Purification and characterization of cytoplasmic thioltransferase (glutathione:disulfide oxidoreductase) from rat liver.

An enzyme catalyzing thiol-disulfide interchange of glutathione and disulfides and the reaction between glutathione and thiosulfate esters has been purified 40 000-fold from rat liver cytosol. The enzyme, named thioltransferase (Askelöf, P., Axelsson, K., Eriksson, S., & Mannervik, B. (1974) FEBS Lett. 38, 263--267), was homogeneous in several electrophoretic systems, had an isoelectric point at pH 9.6, and contained 8.6% (w/w) carbohydrate. The catalytic activity had a distinct optimum at pH 7.5. A series of substrates was tested at a constant glutathione level; the kcat values (at 4mM glutathione) were all in the range of about 10(4) min-1. The substrates included mixed disulfides of glutathione, other low-molecular-weight disulfides, S-sulfocysteine and S-sulfoglutathione, and peptide disulfides such as insulin, oxytocin, ribonuclease, and the mixed disulfide of glutathione and egg-white lysozyme. The enzymatic reaction was inhibited by an excess of glutathione (greater than 4mM).

Animals↗
PubMed2002

Breaking and re-forming the disulfide bond at the high-potential, respiratory-type Rieske [2Fe-2S] center of thermus thermophilus: characterization of the sulfhydryl state by protein-film voltammetry.

A disulfide bond, adjacent to the [2Fe-2S] cluster, is conserved in all high-potential Rieske proteins from the respiratory and photosynthetic cytochrome bc(1) and b(6)f complexes but is absent from the low-potential, bacterial dioxygenase Rieske proteins. The role of the disulfide is unclear, since cysteine mutants have resulted in only apoprotein. The high stability of the soluble Thermus thermophilus Rieske protein permits chemical reduction of the disulfide bond and characterization of the sulfhydryl (dithiol) form by protein-film voltammetry. The effect of disulfide reduction on the cluster potential is small (DeltaE(0)' <or= -0.04 V) and attributed to relaxation of the disulfide tether between the protein loops ligating the cluster, including possible mechanical strain release and hydrogen-bonding modification. Above pH 6 an additional decrease in potential of the sulfhydryl form is assigned to the nearby negatively charged thiolates (DeltaE(0)' -0.16 to -0.12 V); the histidine-ligand nitrogen pKs are correspondingly increased. Entropies of reduction for the native and dithiolate forms are equal (-48 +/- 5 J K(-1) mol(-1), pH 7-8); thus changes in reduction potential are enthalpic in origin. Following sulfhydryl alkylation the cluster redox properties mirror those of the native protein (DeltaE(0)' approximately -0.1 V) over all pHs. While a sustained electrode potential of -0.85 V fails to reduce the disulfide, the free sulfhydryls recombine upon an oxidative excursion, at low pH, to restore the native redox properties. This unique behavior is attributed to preorganization of the two thiolate groups upon uptake of one or more protons by the sulfhydryl pair.

Bacterial Proteins↗
PubMed1997

Disulfide bond assignment in human interleukin-7 by matrix-assisted laser desorption/ionization mass spectroscopy and site-directed cysteine to serine mutational analysis.

Interleukin-7 (IL-7) is a proteinaceous biological response modifier that has a bioactive tertiary structure dependent on disulfide bond formation. Disulfide bond assignments in human (h)IL-7 are based upon the results of matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy and Cys to Ser mutational analyses. A gene encoding the hIL-7 was synthesized incorporating Escherichia coli codon usage bias and was used to express biologically active protein as determined by stimulation of precursor B-cell proliferation. MALDI mass spectroscopic analysis of trypsin-digested hIL-7 was performed and compared with the anticipated results of a simulated tryptic digestion. Many of the anticipated hIL-7 tryptic fragments were detected including one with a molecular mass equivalent to the sum of two polypeptides linked through a disulfide bond formed from Cys residues (Cys3 and Cys142). Subsequently, Cys to Ser substitution mutational analyses were performed. A hIL-7 variant with all six Cys substituted with Ser was found to be biologically inactive (EC50 > 1 x 10(-7) M). In contrast, a family of single disulfide bond-forming variants of hIL-7 were constructed by reintroducing Cys pairs (Cys3-Cys142, Cys35-Cys130, and Cys48-Cys93), and each could stimulate cell proliferation with an EC50 of 4 x 10(-9), 2 x 10(-8), and 2 x 10(-9) M, respectively. In single disulfide bond-forming mutants of hIL-7, the ability to stimulate cell proliferation was abolished in the presence of 2 mM dithiothreitol. The results presented strongly suggest that only a single disulfide bond is required for hIL-7 to form a tertiary structure capable of stimulating precursor B-cell proliferation.

Amino Acid Sequence↗