Intrachain Disulfide Bond Formation Research Articles

Mapping time dependent disulfide bond formation during in-vitro refolding of recombinant peptibody: A Fc-fusion protein

Disulfide bonds are commonly found in covalent interactions, which play a vital role in establishing the three-dimensional structure of proteins and maintaining their biological activity. This investigation is focused on time dependant mapping of intra and inter-disulfide bonds during in-vitro refolding of recombinant peptibody using LC-ESI-MS/MS. The selected recombinant peptibody is a homodimeric, aglycosylated Fc-fusion protein expressed in E. coli. The amino acid sequence in the disulfide bond containing peptides was confirmed at MS/MS level using the CID-based fragmentation approach. During in-vitro refolding of peptibody, a peptide with inter-chain disulfide bond is observed post 4 h, with low area intensity, and it reached a maximum in 96 h. However, maximum area intensity was reached for the intra-chain disulfide bond in 72 h. Since cysteine residues responsible for intra-chain disulfide bonds are present in complex part of the Fc domain, formation of intra-chain disulfide bonds may be an overall rate-limiting step in recombinant Romiplostim refolding. Proper folding and contributions by non-covalent interactions will be required to form intra-chain disulfide bond. Monitoring time-based formation of such non-covalent interactions was carried out using intrinsic fluorescence during protein refolding. Observations from intrinsic fluorescence show that 4–6 h is most crucial time for domain formation. Observations from this study will help characterize structural integrity and quality during process and product development for consistent product quality.

Effect of high temperature high pressure-acidic solution treated Auricularia auricula on the rheological property and structure of wheat flour dough

Incorporation of A. auricula into wheat flour dough could improve their nutritional properties, but utilization of A. auricula is limited as it causes a weakened dough and deterioration in the quality of product. In this study, A. auricula was treated using high temperature high pressure processing combined with acidic solution treatment, and the effect of A. auricula before and after the treatment on the rheological and structural properties of dough was investigated. Results showed that the high temperature high pressure-acidic solution treated A. auricula (HTHPA) did not block the formation of intrachain disulfide bonds in glutenins. On the contrary, it led to a higher aggregation extent of glutenin macropolymer by contributing to the crosslinking of proteins via noncovalent interactions. Rheological tests showed that the doughs with 2–8% HTHPA exhibited higher elastic and viscous moduli and shear-thinning behavior compared to the doughs with 2–8% untreated A. auricula (UA). This will contribute to the volumetric expansion of dough during proofing. Temperature sweep test revealed that addition of 2–8% HTHPA could reinforce the intermolecular interactions in the dough structure. This study will provide a new method to design and produce A. auricula-enriched staple foods with desirable rheological and structural properties.

Assembly and Folding Properties of Cytosolic IgG Intrabodies

Intrabodies, antibodies expressed within cells, offer an interesting way to target intracellular molecules, making them potentially useful for biotechnology and medicine. However, it remains controversial whether full-size IgG intrabodies expressed in the reducing environment of the cytosol of mammalian cells are workable and structurally sound. Herein, we settle this issue with a systematic investigation of the structure and functionality of four chimeric IgG1s with distinct variable (V) domains but identical constant (C) domains. Full-size IgGs expressed in the cytosol of HEK293 cells were either assembly-competent or -incompetent, depending on the intrinsic properties of the V regions. Structural integrity of the C region is required for H:L association and the formation of a functional antigen-binding site. Partial intrachain disulfide bond formation occurs in both H and L chains of cytosolic IgG intrabodies, whereas interchain disulfide bond formation was absent and dispensable for functional assembly. IgG1s expressed in the cytosol and via the ER were shown to assemble differently. Our findings provide insight into the features and possible utilization of full-size IgGs as cytosolic antibodies in biotechnological and medical applications.

Thiolated polymers: Bioinspired polymers utilizing one of the most important bridging structures in nature.

Thiolated polymers designated "thiomers" are obtained by covalent attachment of thiol functionalities on the polymeric backbone of polymers. In 1998 these polymers were first described as mucoadhesive and in situ gelling compounds forming disulfide bonds with cysteine-rich substructures of mucus glycoproteins and crosslinking through inter- and intrachain disulfide bond formation. In the following, it was shown that thiomers are able to form disulfides with keratins and membrane-associated proteins exhibiting also cysteine-rich substructures. Furthermore, permeation enhancing, enzyme inhibiting and efflux pump inhibiting properties were demonstrated. Because of these capabilities thiomers are promising tools for drug delivery guaranteeing a strongly prolonged residence time as well as sustained release on mucosal membranes. Apart from that, thiomers are used as drugs per se. In particular, for treatment of dry eye syndrome various thiolated polymers are in development and a first product has already reached the market. Within this review an overview about the thiomer-technology and its potential for different applications is provided discussing especially the outcome of studies in non-rodent animal models and that of numerous clinical trials. Moreover, an overview on product developments is given.

Disulfide Bond Formation and ToxR Activity in Vibrio cholerae

Virulence factor production in Vibrio cholerae is complex, with ToxRS being an important part of the regulatory cascade. Additionally, ToxR is the transcriptional regulator for the genes encoding the major outer membrane porins OmpU and OmpT. ToxR is a transmembrane protein and contains two cysteine residues in the periplasmic domain. This study addresses the influence of the thiol-disulfide oxidoreductase system DsbAB, ToxR cysteine residues and ToxR/ToxS interaction on ToxR activity. The results show that porin production correlates with ToxR intrachain disulfide bond formation, which depends on DsbAB. In contrast, formation of ToxR intrachain or interchain disulfide bonds is dispensable for virulence factor production and in vivo colonization. This study further reveals that in the absence of ToxS, ToxR interchain disulfide bond formation is facilitated, whereat cysteinyl dependent homo- and oligomerization of ToxR is suppressed if ToxS is coexpressed. In summary, new insights into gene regulation by ToxR are presented, demonstrating a mechanism by which ToxR activity is linked to a DsbAB dependent intrachain disulfide bond formation.

Electron Transfer Dissociation (ETD) of Peptides Containing Intrachain Disulfide Bonds

The fragmentation chemistry of peptides containing intrachain disulfide bonds was investigated under electron transfer dissociation (ETD) conditions. Fragments within the cyclic region of the peptide backbone due to intrachain disulfide bond formation were observed, including: c (odd electron), z (even electron), c-33 Da, z+33 Da, c+32 Da, and z-32 Da types of ions. The presence of these ions indicated cleavages both at the disulfide bond and the N-Cα backbone from a single electron transfer event. Mechanistic studies supported a mechanism whereby the N-Cα bond was cleaved first, and radical-driven reactions caused cleavage at either an S-S bond or an S-C bond within cysteinyl residues. Direct ETD at the disulfide linkage was also observed, correlating with signature loss of 33 Da (SH) from the charge-reduced peptide ions. Initial ETD cleavage at the disulfide bond was found to be promoted amongst peptides ions of lower charge states, while backbone fragmentation was more abundant for higher charge states. The capability of inducing both backbone and disulfide bond cleavages from ETD could be particularly useful for sequencing peptides containing intact intrachain disulfide bonds. ETD of the 13 peptides studied herein all showed substantial sequence coverage, accounting for 75%-100% of possible backbone fragmentation.

SNAP-25 Contains Non-Acylated Thiol Pairs that can Form Intrachain Disulfide Bonds: Possible Sites for Redox Modulation of Neurotransmission

Intrachain disulfide bond formation among the cysteine thiols of SNAP-25, a component of the SNARE protein complex required for neurotransmitter release, has been hypothesized to link oxidative stress and inhibition of synaptic transmission. However, neither the availability in vivo of SNAP-25 thiols, which are known targets of S-palmitoylation, nor the tendency of these thiols to form intrachain disulfide bonds is known. We have examined, in rat brain extracts, both the availability of closely spaced, or vicinal, thiol pairs in SNAP-25 and the propensity of these dithiols toward disulfide bond formation using a method improved by us recently that exploits the high chemoselectivity of phenylarsine oxide (PAO) for vicinal thiols. The results show for the first time that a substantial fraction of soluble and, to a lesser extent, particulate SNAP-25 contain non-acylated PAO-binding thiol pairs and that these thiols in soluble SNAP-25 in particular have a high propensity toward disulfide bond formation. Indeed, disulfide bonds were detected in a small fraction of soluble SNAP-25 even under conditions designed to prevent or greatly limit protein thiol oxidation during experimental procedures. These results provide direct experimental support for the availability, in a subpopulation of SNAP-25, of vicinal thiols that may confer on one or more isoforms of this family of proteins a sensitivity to oxidative stress.

Disulfide Bridge Disruption in the α2 Domain of the HLA Class I Molecule Leads to Low Expression of the Corresponding Antigen

Using sequence-based typing, we have identified a novel human leukocyte antigen (HLA)-A*30 allele, HLA-A*3014L, with a low expression pattern. The sequence of HLA-A*3014L is identical to that of HLA-A*3001 except for a G to C substitution in exon 3 at nucleotide position 563, resulting in an amino acid difference at position 164 (Cys to Ser). Due to the cysteine substitution, a disulfide bridge in the α2 domain of the HLA class I heavy chain cannot be formed. By using the standard microlymphocytotoxicity test, the HLA-A30 antigen cannot be detected. By flow cytometric analysis of the cell-surface expression at either 37°C or 30°C, a temperature-sensitive expression pattern of the HLA-A*3014L antigen was observed. Only by incubating the cells at 30°C, which increases the stability of HLA class I heavy chains, was a weak but clearly detectable HLA-A*3014L expression found. The mRNA expression level of the HLA-A*3014L allele was not affected by the nucleotide substitution. The intrachain disulfide bond formation in the α2 domain is essential for the normal expression of the HLA molecules. Reduced protein expression is probably caused by incorrect HLA class I heavy chain folding and HLA class I complex assembly.

A novel DFNA9 mutation in the vWFA2 domain of COCH alters a conserved cysteine residue and intrachain disulfide bond formation resulting in progressive hearing loss and site‐specific vestibular and central oculomotor dysfunction

Mutations within the COCH gene (encoding the cochlin protein) lead to auditory and vestibular impairment in the DFNA9 disorder. In this study, we describe the genetic mapping of progressive autosomal dominant sensorineural hearing loss first affecting high-frequency auditory thresholds within a human pedigree to the long arm of chromosome 14 in band q12. A maximal pairwise LOD score of 7.08 was obtained with marker D14S1021. We identified a c.1625G > T mutation in exon 12 of COCH that co-segregates with auditory dysfunction in the pedigree. The mutation results in a predicted p.C542F substitution at an evolutionarily conserved cysteine residue in the C-terminus of cochlin. The c.1625G > T transversion in COCH exon 12 represents the first reported mutation outside of the LCCL domain which is encoded by exons 4 and 5. The 542F mutant cochlin is translated and secreted by transfected mammalian cells. Western blot analysis under non-reducing and reducing conditions suggests that the 542F mutation alters intramolecular cochlin disulfide bond formation. In the vestibular system, a progressive horizontal canal hypofunction and a probable saccular otolith challenge were detected in family members with the c.1625G > T COCH alteration. Abnormal central oculomotor test results in family members with the c.1625G > T COCH alteration imply a possible central nervous system change not previously noted in DFNA9 pedigrees harboring mutations within the LCCL domain.

Identification and functional mapping of the Mycoplasma fermentans P29 adhesin.

Initial adherence interactions between mycoplasmas and mammalian cells are important for host colonization and may contribute to subsequent pathogenic processes. Despite significant progress toward understanding the role of specialized, complex tip structures in the adherence of some mycoplasmas, particularly those that infect humans, less is known about adhesins through which other mycoplasmas of this host bind to diverse cell types, even though simpler surface components are likely to be involved. We show by flow cytometric analysis that a soluble recombinant fusion protein (FP29), representing the abundant P29 surface lipoprotein of Mycoplasma fermentans, binds human HeLa cells and inhibits M. fermentans binding to these cells, in both a quantitative and a saturable manner, whereas analogous fusion proteins representing other mycoplasma surface proteins did not. Constructs representing nested N- or C-terminal truncations of FP29 allowed initial mapping of this specific adherence function to a central region of the P29 sequence containing a 36-amino-acid disulfide loop. A derivative of FP29 containing a mutation converting one participating Cys to Ser, precluding intrachain disulfide bond formation, retained full activity. Together these results suggest that the direct interaction of M. fermentans with a ligand on the HeLa cell surface involves a limited segment of the P29 surface lipoprotein and requires neither the disulfide bond nor the contribution of adjacent portions of the protein. Earlier results indicating phase-variable display of monoclonal antibody surface epitopes on P29, now recognized to be outside this ligand binding region, raise the possibility that variation of mycoplasma surface architecture might alter the presentation of the binding region and the adherence phenotype. Preliminary results further indicated that FP29 could inhibit binding to HeLa cells by Mycoplasma hominis, a distinct human mycoplasma species displaying the phase-variable adhesin Vaa, but not that by Mycoplasma capricolum, an organism infecting caprine species. This result raises the additional, testable possibility that a common host cell ligand for two human mycoplasma species may be recognized through structurally dissimilar adhesins that undergo phase variation by two distinct mechanisms, governing protein expression (Vaa) or surface masking (P29).

Heterogeneity in Human Cardiac Troponin I Standards

The LC–MS analysis of recombinant cardiac troponin I (cTnI) and cTnI extracted from human hearts showed a high degree of structural heterogeneity among all samples. The examined recombinant cTnI samples indicated posttranslational modifications, presumably due to their purification (i.e., 2-mercaptoethanol adducts and carbamylation) and related to their expression (i.e., an N-terminal expression tag). The extracted cTnI samples, while having a higher degree of structural heterogeneity, showed less structural variance between samples than the recombinant proteins. The LC–MS analysis of the extracted cTnI samples provided evidence of posttranslational modification by phosphorylation, acetylation, proteolytic cleavage, and intrachain disulfide bond formation.

ERO1-L, a Human Protein That Favors Disulfide Bond Formation in the Endoplasmic Reticulum

Oxidizing conditions must be maintained in the endoplasmic reticulum (ER) to allow the formation of disulfide bonds in secretory proteins. Here we report the cloning and characterization of a mammalian gene (ERO1-L) that shares extensive homology with the Saccharomyces cerevisiae ERO1 gene, required in yeast for oxidative protein folding. When expressed in mammalian cells, the product of the human ERO1-L gene co-localizes with ER markers and displays Endo-H-sensitive glycans. In isolated microsomes, ERO1-L behaves as a type II integral membrane protein. ERO1-L is able to complement several phenotypic traits of the yeast thermosensitive mutant ero1-1, including temperature and dithiothreitol sensitivity, and intrachain disulfide bond formation in carboxypeptidase Y. ERO1-L is no longer functional when either one of the highly conserved Cys-394 or Cys-397 is mutated. These results strongly suggest that ERO1-L is involved in oxidative ER protein folding in mammalian cells.

Oxidation Regulates the Inflammatory Properties of the Murine S100 Protein S100A8

The myeloid cell-derived calcium-binding murine protein, S100A8, is secreted to act as a chemotactic factor at picomolar concentrations, stimulating recruitment of myeloid cells to inflammatory sites. S100A8 may be exposed to oxygen metabolites, particularly hypochlorite, the major oxidant generated by activated neutrophils at inflammatory sites. Here we show that hypochlorite oxidizes the single Cys residue (Cys41) of S100A8. Electrospray mass spectrometry and SDS-polyacrylamide gel electrophoresis analysis indicated that low concentrations of hypochlorite (40 microM) converted 70-80% of S100A8 to the disulfide-linked homodimer. The mass was 20,707 Da, 92 Da more than expected, indicating additional oxidation of susceptible amino acids (possibly methionine). Phorbol 12-myristate 13-acetate activation of differentiated HL-60 granulocytic cells generated an oxidative burst that was sufficient to efficiently oxidize exogenous S100A8 within 10 min, and results implicate involvement of the myeloperoxidase system. Moreover, disulfide-linked dimer was identified in lung lavage fluid of mice with endotoxin-induced pulmonary injury. S100A8 dimer was inactive in chemotaxis and failed to recruit leukocytes in vivo. Positive chemotactic activity of recombinant Ala41S100A8 indicated that Cys41 was not essential for function and suggested that covalent dimerization may structurally modify accessibility of the chemotactic hinge domain. Disulfide-dependent dimerization may be a physiologically significant regulatory mechanism controlling S100A8-provoked leukocyte recruitment.

Close Association of the First and Fourth Extracellular Domains of the Duffy Antigen/Receptor for Chemokines by a Disulfide Bond Is Required for Ligand Binding

It has been demonstrated that the promiscuous chemokine binding profile of the Duffy antigen/receptor for chemokines (DARC) is given by its extracellular NH2-terminal region. However, the relationship among the Fy6, Fya/b, and Fy3 epitopes, localized in the first and fourth extracellular domains of DARC, respectively, and the chemokine binding sites remained a matter of controversy. Here, we performed cross-displacement and cross-inhibition experiments indicating that all anti-Fy6, anti-Fya, and anti-Fy3 monoclonal antibodies and interleukin 8 are antagonists for binding to red cells. Biopanning of phage peptide libraries with an anti-Fy6 monoclonal antibody led to the identification of the motif Phe22-Glu23, the mutation of which altered the binding of both anti-Fy6 and chemokines (interleukin 8, MGSA, RANTES (regulated on activation normal T cell expressed)) to DARC transfectants. These results characterized the core of the Fy6 epitope and provided definitive proof of the tight relationship between Fy6 and the chemokine receptor site. Analysis of red cells treated by sulfhydryl group-modifying reagents suggested that the chemokine receptor function of DARC required the integrity of disulfide bond(s) but not that of free sulfhydryl group(s). Accordingly, mutation of cysteines 51 and 276 abolished chemokine binding to DARC transfectants. Altogether, our results suggested that the chemokine binding pocket of DARC included sequences located in the first and fourth extracellular domains which are brought into close vicinity by a disulfide bridge.

Characterization of class I MHC folding intermediates and their disparate interactions with peptide and β2-microglobulin

Newly synthesized class I heavy chains achieve domain structure using disulfide bonds, assemble with beta-2 microglobulin ( β 2m), and bind peptide ligand to complete the trimeric complex. Although each of these initial events is thought to be critical for class I folding, their sequential order and effect on class I structure are unknown. Using mAb specific for distinct conformations of H-2L d and L q, we have defined folding intermediates of class I molecules. We show here that non-peptide-associated forms of L d or L q, detected by mAb 64-3-7 and designated L alt, lack numerous conformational epitopes surrounding their ligand binding sites. These results support the notion that L alt molecules have an open conformation. Interestingly, a significant proportion of L alt molecules were detected in association with β 2m and these L alt β 2m heterodimers were preferentially folded by peptide in cell lysates. These findings indicate that class I heavy chain β 2m association can precede ligand binding and that peptide is probably the limiting factor for completion of the L d/β 2m/peptide trimeric complex in vivo. The characteristics of L alt molecules were investigated further by ascertaining the disulfide bond status of these molecules and their association with β 2m and peptide. Treatment of cells with dithiothreitol (DTT), a membrane-permeable reducing agent, demonstrated that L alt molecules constitute a heterogeneous population including reduced, partially reduced, partially reduced and native class I molecules. Furthermore, partially reduced L d alt molecules, in a cell line expressing a mutant L d molecule lacking the α2 domain disulfide bond, accumulated intracellularly, were not β 2m-associated and displayed marginal peptide-induced folding in vitro. In accordance with this latter finding, peptide was found to preferentially convert fully disulfide-bonded forms of L d alt to conformed L d. Thus, we propose that intrachain disulfide bond formation precedes the association of class I heavy chain with β 2m and peptide, and that disulfide bond formation is required for efficient assembly, ligand binding and folding of the class I heavy chain.

Nucleotide sequence of a wheat cDNA encoding protein disulfide isomerase.

PDI (EC 5.3.4.1) is an ER-resident protein involved in the rearrangement of disulfide bonds of secretory proteins. This protein also appears as one of the two subunits of another ER resident protein, prolyl 4-hydroxylase (EC 1.14.11.2), and may be involved in various cellular processes (Noiva and Lennarz, 1992). PDI appears to be very abundant in developing wheat grains (Roden et al., 1982) and was also demonstrated to catalyze intrachain disulfide bond formation in a wheat y-gliadin storage protein (Bulleid and Freedman, 1988). To analyze the expression of PDI in developing grains and to study further its role in the folding and assembly of wheat storage proteins, we have cloned a full-length wheat PDI cDNA by screening a root-tip cDNA library using maize PDI as a DNA probe (Table I). Sequence analysis of the wheat cDNA revealed an open reading frame encoding a protein of 515 amino acids, which also exhibited 96 and 63% amino acid sequence identity with full-length PDI cDNAs from barley (accession No. L33250) and alfalfa (Shorrosh and Dixon, 1991), respectively. The deduced amino acid sequence of the wheat PDI also contained an N-terminal signal peptide of 25 amino acids, two conserved thioredoxin-like active sites, a potential N-linked glycosylation site, as well as a C-terminal KDEL sequence generally present in ER-resident proteins.

Ab initio association with beta 2-microglobulin during biosynthesis of the H-2Ld class I major histocompatibility complex heavy chain promotes proper disulfide bond formation and stable peptide binding.

In vitro translation studies indicate that the beta 2-microglobulin (beta 2-m) light chain influences the formation of intrachain disulfide bonds in class I major histocompatibility complex (MHC) molecules during their biosynthesis. We now have examined the influence of beta 2-m on class I MHC intrachain disulfide bond formation in vivo. Using beta 2-m+ and beta 2-m- derivatives of a cell line transfected with the mouse H-2Ld gene, we show that all of the H-2Ld molecules from beta 2-m+ cells have both the alpha 2 and alpha 3 intrachain disulfide bonds, whereas about 50% of the H-2Ld molecules from beta 2-m- cells have only one of these bonds. All of the free H-2Ld heavy chains from beta 2-m+ cells can undergo a peptide-induced conformational change and can bind exogenous peptide and beta 2-m stably in vitro. Only those H-2Ld molecules from beta 2-m- cells, which have both intrachain disulfide bonds, undergo a peptide- and beta 2-m-induced conformational change in vitro. These H-2Ld molecules do not bind beta 2-m and peptide stably in vitro. From these results emerges a greater understanding of the role of beta 2-m at the time of class I MHC molecule synthesis: beta 2-m promotes intrachain disulfide bond formation in the class I MHC molecule and additionally affects class I MHC structure to render it competent to form stable trimolecular complexes with peptide and beta 2-m.

The role of cysteine residues in the folding and association of the COOH-terminal propeptide of types I and III procollagen.

Procollagen chains assemble in a type-specific manner forming either homo- or heterotrimers. The molecular mechanisms underlying procollagen chain selectivity are unknown, although it is thought that the C-propeptide (COOH-terminal propeptide) is responsible for directing chain recognition and assembly. To define the processes involved in chain selection we reconstituted the initial stages of procollagen folding and assembly in a cell-free system. Using human pro-alpha 1(III) and pro-alpha 2(I) chains as prototypes of chains that are either capable or incapable of forming homotrimeric molecules, respectively, we constructed two minigenes (p alpha 1(III) delta 1 and p alpha 2(I) delta 1) that lacked most of the triple helical domains. The minigenes were transcribed in vitro and translated in a rabbit reticulocyte lysate supplemented with microsomal membranes under conditions that favored disulfide bond formation. Both pro-alpha 1(III) delta 1 and pro-alpha 2(I) delta 1 chains formed intrachain disulfide bonds within the C-propeptide. However, only pro-alpha 1(III) delta 1 chains were able to self-associate forming homotrimers stabilized by interchain disulfide bonds. The C-propeptide of the pro-alpha 1(III) chain contains 8 cysteine residues (Cys-1-8). We used a site-directed mutagenesis to investigate the role of specific cysteine residues in trimer formation and found that substitution of serine for Cys-1, Cys-2, Cys-3, and Cys-4 prevented interchain disulfide bonding and trimerization. Furthermore, mutations in Cys-1 and Cys-4 also prevented intrachain disulfide bond formation within the C-propeptide. The C-propeptide of the pro-alpha 2(I) chain contains only 7 cysteine residues, lacking cysteine at position 2. Substitution of the existing Ser residue with Cys did not produce a homotrimeric phenotype, indicating that additional recognition signals are required to determine chain selection.

Recombinant pulmonary surfactant protein D. Post-translational modification and molecular assembly.

Pulmonary surfactant protein D (SP-D) is a member of a family of collagenous C-type lectins that includes the serum mannose binding proteins and surfactant protein A. Recent studies have shown that rat SP-D (rSP-D) molecules are assembled as tetramers of trimeric subunits (12 mers) and that dodecamers can participate in higher orders of molecular assembly involving interactions of the amino-terminal peptide domains. In order to further study the assembly of SP-D in vitro, Chinese hamster ovary K1 cells were transfected with a full-length rat SP-D cDNA, and stable transfectants with high levels of SP-D production (approximately 6 x 10(6) dodecamers/cell/24 h) were obtained using a glutamine synthetase selection system. The secreted molecules (RrSP-D), which were purified by affinity chromatography on maltosyl-agarose, comigrated with rSP-D on SDS-polyacrylamide gel electrophoresis in the presence and absence of reduction, and coeluted with rSP-D dodecamers from 4% agarose. The major bacterial collagenase-resistant peptide showed a decreased mobility on reduction consistent with the formation of intrachain disulfide bonds. A 17-kDa pepsin-resistant fragment was isolated following overnight digestion with pepsin at 27 degrees C, confirming the formation of a triple helical domain comparable in size and thermal stability to that of natural SP-D. The expressed protein contained sialylated endoglycosidase F-sensitive carbohydrate; amino acid analysis of acid and alkaline hydrolysates demonstrated essentially normal levels of hydroxyproline, hydroxylysine, and hydroxylysine-glycosides. Electron microscopic studies showed a molecular structure indistinguishable from lung SP-D, with a similar small subpopulation of molecules showing higher orders of multimerization. Solid-phase neoglycoprotein binding assays gave the same saccharide inhibition profile as natural rat SP-D, and both proteins showed efficient saccharide-dependent agglutination of Escherichia coli. These studies demonstrate that a single genetically distinct chain type can account for the various and complex molecular assemblies of SP-D, and further verify the potential physiologic significance of the disulfide-bonded multimers and higher aggregates isolated from rat, bovine, and human lung lavage.

Formation of mitogenically active PDGF-B dimer does not require interchain disulfide bonds.

Platelet-derived growth factor (PDGF), a major mitogen for mesenchymal cells, is a disulfide-bonded dimer of two subunit polypeptides named A and B. All of the three possible dimeric forms, i.e. AA, BB, and AB, exist in nature. The dimeric structure has been presumed to be necessary for biological activity, since reduction of the dimer results in loss of activity and simultaneous conversion to monomeric form as determined by SDS-gel electrophoresis. However, reduction of the native molecule destroys intrachain, as well as interchain, disulfide bonds, and it is possible that the former rather than the latter are critical for proper conformation of the active protein. We show here that PDGF-B polypeptides in which all 8 cysteines or the 2nd, 4th, 5th, and 8th cysteines have been mutated to serines fail to form covalent dimers and possess dramatically less mitogenic activity than native PDGF-BB. Another mutant, PDGF-B(C2,4S), in which just the 2 cysteines involved in interchain disulfides were converted to serine, ran as a monomer on SDS-polyacrylamide gels as expected. Somewhat unexpectedly, however, the mitogenic activity of the PDGF-B(C2,4S) analog was similar to the activity of wild-type PDGF-BB disulfide-bonded dimer under physiological conditions. The activity of the analog was more sensitive to the effect of low pH than was the activity of wild-type PDGF-BB. Molecular weight analysis utilizing light scattering and sedimentation equilibrium demonstrated that the PDGF-B(C2,4S) analog exists as a noncovalent dimer at pH 4-7 but dissociates to a monomer at pH 2.5. Disulfide analysis of the mutant protein demonstrated that the intrachain disulfide bonds are the same as those formed in wild-type PDGF-BB homodimers. We conclude that proper formation of intrachain disulfide bonds is critical to maintaining the correct conformation of PDGF monomers, but that appropriately folded monomers can associate into active noncovalent dimers in the absence of interchain disulfide bonds. Interchain disulfide bonds thus appear to increase the stability of the PDGF dimer rather than being crucial to its existence.