Glycoprotein hormone assembly in the endoplasmic reticulum: II. Multiple roles of a redox sensitive beta-subunit disulfide switch

All three human glycoprotein hormone heterodimers are assembled in the endoplasmic reticulum by threading the glycosylated end of alpha-subunit loop two (alpha2) beneath a disulfide "latched" strand of the beta-subunit known as the "seatbelt." This remarkable event occurs efficiently even though the seatbelt effectively blocks the reverse process, thereby stabilizing each heterodimer. Studies described here show that assembly is facilitated by the formation, disruption, and reformation of a loop within the seatbelt that is stabilized by the most easily reduced disulfide in the free beta-subunit. We refer to this disulfide as the "tensor" because it shortens the seatbelt, thereby securing the heterodimer. Formation of the tensor disulfide appears to precede and facilitate seatbelt latching in most human choriogonadotropin beta-subunit molecules. Subsequent disruption of the tensor disulfide elongates the seatbelt, thereby increasing the space beneath the seatbelt and the beta-subunit core. This permits the formation of hydrogen bonds between backbone atoms of the beta-subunit cystine knot and the tensor loop with backbone atoms in loop alpha2, a process that causes the glycosylated end of loop alpha2 to be threaded between the seatbelt and the beta-subunit core. Contacts between the tensor loop and loop alpha2 promote reformation of the tensor disulfide, which explains why it is more stable in the heterodimer than in the uncombined beta-subunit. These findings unravel the puzzling nature of how a threading mechanism can be used in the endoplasmic reticulum to assemble glycoprotein hormones that have essential roles in vertebrate reproduction and thyroid function.     

蛋白质中二硫键附近肽段识别规律研究

本文对蛋白质中二硫键附近的残基进行了计算机统计分析,结果发现平行和反平行残基间存在着特异的配对规律。这种残基间的相互作用或识别,可能与蛋白质折叠过程中正确地形成二硫键有关。该结果有助于蛋白质工程设计。     

Identification and characterization of disulfide bonds in proteins and peptides from tandem MS data by use of the MassMatrix MS/MS search engine

A new database search algorithm has been developed to identify disulfide-linked peptides in tandem MS data sets. The algorithm is included in the newly developed tandem MS database search program, MassMatrix. The algorithm exploits the probabilistic scoring model in MassMatrix to achieve identification of disulfide bonds in proteins and peptides. Proteins and peptides with disulfide bonds can be identified with high confidence without chemical reduction or other derivatization. The approach was tested on peptide and protein standards with known disulfide bonds. All disulfide bonds in the standard set were identified by MassMatrix. The algorithm was further tested on bovine pancreatic ribonuclease A (RNaseA). The 4 native disulfide bonds in RNaseA were detected by MassMatrix with multiple validated peptide matches for each disulfide bond with high statistical scores. Fifteen nonnative disulfide bonds were also observed in the protein digest under basic conditions (pH = 8.0) due to disulfide bond interchange. After minimizing the disulfide bond interchange (pH = 6.0) during digestion, only one nonnative disulfide bond was observed. The MassMatrix algorithm offers an additional approach for the discovery of disulfide bond from tandem mass spectrometry data.     

Reduction and S-nitrosation of the neuropeptide oxytocin: implications for its biological function.

Oxytocin (OT; Cys-Tyr-Ile-Gln-Asn-Cys-Pro-leu-Gly), a posterior pituitary peptide hormone, is characterized by a Cys1-Cys6 disulfide bond in its stable, isolated state. This paper describes a simple, one-step method for the production of OT in its reduced, dithiol form (OT dithiol), free of reducing agent. The effects of temperature, pH, and metal-ion chelators on the autoxidation of OT dithiol were examined to establish if this form is likely to persist under biological conditions. It was found that OT dithiol has a half-life of 1.8h with respect to reformation of OT disulfide at 37 degrees C and pH 6.9 in the presence of the copper chelators, DTPA and neocuproine. S-Nitrosation of OT dithiol by acidified nitrite at pH 3.0 was examined by absorption spectroscopy and HPLC-UV-MS, which revealed that both singly and doubly S-nitrosated OT are formed. These results suggest novel chemical aspects to OT signaling, the biological implications of which are discussed here.     

Evidence for the presence of disulfide bridges in opioid receptors essential for ligand binding. Possible role in receptor activation

The mobility of purified mu opioid binding protein in SDS-polyacrylamide gek electrophoresis is sensitive to the presence of reducing agents. In the presence of increasing concentrations of DTT the apparent molecular weight increases in a stepwise fashion from 53 kDa to 65 kDa. This reduction in mobility is attributed to the successive breakage of disulfide bridges, resulting in an increasingly asymmetric molecule. Treatment of cell membranes from various brain areas with reducing agents, such as DTT, produced a concentration-dependent inhibition of opioid binding. Sensitivity to DTT inhibition varied between receptor types, mu greater than delta much greater than kappa. For mu receptors, agonist binding was considerably more sensitive to DTT than antagonist binding. Inhibition by DTT is readily reversible and is unaffected by Na+ and/or Mg2+ ions. Reversibility may be partially prevented by the inclusion of a low concentration of a reducing reagent such as glutathione which does not inhibit binding but blocks reformation of disulfide bonds. Scatchard analysis of saturation data shows that DTT causes a pronounced decrease in binding affinity with little effect on receptor number. It is suggested that disulfide bonds are essential for ligand binding and that cleavage of one or more of these bonds may play a role in opioid receptor activation by agonists.     

Glutathione as an essential factor for chaperon-mediated activation of lactonizing lipase (LipL) from Pseudomonas sp. 109

Pseudomonas sp. 109 produces a unique lipase (LipL) which efficiently catalyzes intramolecular transesterification of omega-hydroxyesters to form macrocyclic lactones. The production of the enzymatically active LipL requires a specific molecular chaperon (LimL protein) together with a low-M(r) lipase-activation-factor (LAF) of unknown structure. From 50 g of Pseudomonas cells, 2.15 mg of LAF was purified as a sulfobenzofurazanyl derivative after methanol extraction, derivatization, and C(18) reverse-phase HPLC. One-dimensional and two-dimensional 600 MHz (1)H-NMR and fast atom bombardment mass spectrometry (FAB-MS) revealed that LAF is glutathione. Because several SH compounds (L-cysteine and mercaptoethanol) were similarly effective to native LAF in the activation of LipL, and because only LipL contains two cysteinyl residues forming an intramolecular disulfide bond, it is concluded that the reduction of and reformation of the intramolecular disulfide bond of LipL is essential to liberate free and fully active LipL.     

What can disulfide bonds tell us about protein energetics, function and folding: simulations and bioninformatics analysis

We study the impact of disulfide bonds on protein stability and folding. Using lattice model simulations, we show that formation of a disulfide bond stabilizes a protein to an extent that depends on the distance along the chain between linked cysteine residues. However, the impact of disulfide bonds on folding kinetics varies broadly, from acceleration when disulfides are introduced in or close to the folding nucleus, to slowing when disulfides are introduced outside the nucleus. Having established the effect of disulfide bonds on stability, we study the correlation between the number of disulfide bonds and the composition of certain amino acid classes with the goal to use it as a statistical probe into factors that contribute to stability of proteins. We find that the number of disulfides is negatively correlated with aliphatic hydrophobic but not aromatic content. It is surprising that we observe a strong correlation of disulfide content with polar (Q,S,T,N) amino acid content and a strong negative correlation with charged (E,D,K,R) content. These findings provide insights into factors that determine protein stability and principles of protein design as well as possible relations of disulfide bonds and protein function.     

Count data in biology—Data transformation or model reformation?

Statistical analyses are an integral component of scientific research, and for decades, biologists have applied transformations to data to meet the normal error assumptions for F and t tests. Over the years, there has been a movement from data transformation toward model reformation—the use of non‐normal error structures within the framework of the generalized linear model (GLM). The principal advantage of model reformation is that parameters are estimated on the original, rather than the transformed scale. However, data transformation has been shown to give better control over type I error, for simulated data with known error structures. We conducted a literature review of statistical textbooks directed toward biologists and of journal articles published in the primary literature to determine temporal trends in both the text recommendations and the practice in the refereed literature over the past 35 years. In this review, a trend of increasing use of reformation in the primary literature was evident, moving from no use of reformation before 1996 to >50% of the articles reviewed applying GLM after 2006. However, no such trend was observed in the recommendations in statistical textbooks. We then undertook 12 analyses based on published datasets in which we compared the type I error estimates, residual plot diagnostics, and coefficients yielded by analyses using square root transformations, log transformations, and the GLM. All analyses yielded acceptable residual versus fit plots and had similar p‐values within each analysis, but as expected, the coefficient estimates differed substantially. Furthermore, no consensus could be found in the literature regarding a procedure to back‐transform the coefficient estimates obtained from linear models performed on transformed datasets. This lack of consistency among coefficient estimates constitutes a major argument for model reformation over data transformation in biology.     

Characterization and analysis of scFv-IgG bispecific antibody size variants

ABSTRACT

Bispecific antibodies are an emergent class of biologics that is of increasing interest for therapeutic applications. In one bispecific antibody format, single-chain variable fragments (scFv) are linked to or inserted in different locations of an intact immunoglobulin G (IgG) molecule to confer dual epitope binding. To improve biochemical stability, cysteine residues are often engineered on the heavy- and light-chain regions of the scFv to form an intrachain disulfide bond. Although this disulfide bond often improves stability, it can also introduce unexpected challenges to manufacturing or development. We report size variants that were observed for an appended scFv-IgG bispecific antibody. Structural characterization studies showed that the size variants resulted from the engineered disulfide bond on the scFv, whereby the engineered disulfide was found to be either open or unable to form an intrachain disulfide bond due to cysteinylation or glutathionylation of the cysteines. Furthermore, the scFv engineered cysteines also formed intermolecular disulfide bonds, leading to the formation of highly stable dimers and aggregates. Because both the monomer variants and dimers showed lower bioactivity, they were considered to be product-related impurities that must be monitored and controlled. To this end, we developed and optimized a robust, precise, and accurate high-resolution size-exclusion chromatographic method, using a statistical design-of-experiments methodology.     

Limited tendency of alpha-helical residues to form disulfide bridges: a structural explanation.

Disulfide bridges have an enormous impact on the structure of a large number of proteins and polypeptides. Understanding the structural basis that regulates their formation may be important for the design of novel peptide-based molecules with a specific fold and stability. Here we report a statistical analysis of the relationships between secondary structure and disulfide bond formation, carried out using a large database of protein structures. Our analyses confirm the observation sporadically reported in previous investigations that cysteine residues located in alpha-helices display a limited tendency to form disulfide bridges. The very low occurrence of the disulfide bond in all alpha-chains compared to all beta-chains indicates that this property is also evident when proteins with different topologies are investigated. Taking advantage of the large database that endorsed the analysis on relatively rare motifs, we demonstrate that cysteine residues embedded in 3(10) helices present a good tendency to form disulfide bonds. This result is somewhat surprising since 3(10) helices are commonly assimilated into alpha-helices. A plausible structural explanation for the observed data has been derived combining analyses of disulfide bond sequence separation and of the length of the different secondary structure elements.     

The mapping of an antibody-binding region on the Mycobacterium tuberculosis 19 kilodalton antigen.

To localize the epitopes of four independently derived murine mAb IT-10, IT-12, IT-16, and IT-19 on the 19-kDa Ag protein of Mycobacterium tuberculosis, expression plasmids were constructed containing deletions of the gene encoding the 19-kDa protein. Reaction of the 4 mAb with Western blots of the truncated recombinant proteins revealed two epitope specificities in the recognition of the 19-kDa protein. IT-10 was found to be dependent only on the presence of amino acids surrounding the first cysteine residue, whereas IT-12, IT-16, and IT-19 all required the presence of both the first and third cysteine residues. These two cysteine residues are separated by 135 amino acids, and are considered to be brought together by tertiary folding of the protein to form an assembled epitope for IT-12, IT-16, and IT-19. These three mAb demonstrated differing sensitivities to the modification of reduced 19-kDa protein using iodoacetamide: a treatment that should have prevented the reformation of disulfide bonds within the protein. This result suggests that, although IT-12, IT-16, and IT-19 appear to be specific for the same epitope, there are probably fine-specificity differences in this recognition. IT-10 was not sensitive to the absence of disulfide bonds within the 19-kDa protein, suggesting that the epitope is not conformationally sensitive, and is likely to be linear in nature.     

Role of the amino- and carboxy-terminal regions in the folding and oligomerization of wheat high molecular weight glutenin subunits

The high molecular weight glutenin subunits are considered one of the most important components of wheat (Triticum aestivum) gluten, but their structure and interactions with other gluten proteins are still unknown. Understanding the role of these proteins in gluten formation may be aided by analyses of the conformation and interactions of individual wild-type and modified subunits expressed in heterologous systems. In the present report, the bacterium Escherichia coli was used to synthesize four naturally occurring X- and Y-type wheat high molecular weight glutenin subunits of the Glu-1D locus, as well as four bipartite chimeras of these proteins. Naturally occurring subunits synthesized in the bacteria exhibited sodium dodecyl sulfate-polyacrylamide gel electrophoresis migration properties identical to those of high molecular weight glutenin subunits extracted from wheat grains. Wild-type and chimeric subunits migrated in sodium dodecyl sulfate gels differently than expected based on their molecular weights due to conformational properties of their N- and C-terminal regions. Results from cycles of reductive cleavage and oxidative reformation were consistent with the formation of both inter- and intramolecular disulfide bonds in patterns and proportions that differed among specific high molecular weight glutenin species. Comparison of the chimeric and wild-type proteins indicated that the two C-terminal cysteines of the Y-type subunits are linked by intramolecular disulfide bonds, suggesting that the role of these cysteines in glutenin polymerization may be limited.     

Protein engineering of subtilisin BPN': enhanced stabilization through the introduction of two cysteines to form a disulfide bond

Introduction of a disulfide bond by site-directed mutagenesis was found to enhance the stability of subtilisin BPN' (EC 3.4.21.14) under a variety of conditions. The location of the new disulfide bond was selected with the aid of a computer program, which scored various sites according to the amount of distortion that an introduced disulfide linkage would create in a 1.3-A X-ray model of native subtilisin BPN'. Of the several amino acid pairs identified by this program as suitable candidates, Thr-22 and Ser-87 were selected by using the additional requirement that the individual cysteine substitutions occur at positions that exhibit some degree of variability in related subtilisin amino acid sequences. A subtilisin variant containing cysteine residues at positions 22 and 87 was created by site-directed mutagenesis and was shown to have an activity essentially equivalent to that of the wild-type enzyme. Differential scanning calorimetry experiments demonstrated the variant protein to have a melting temperature 3.1 degrees C higher than that of the wild-type protein and 5.8 degrees C higher than that of the reduced form (-SH HS-) of the variant protein. Kinetic experiments performed under a variety of conditions, including 8 M urea, showed that the Cys-22/Cys-87 disulfide variant undergoes thermal inactivation at half the rate of that of the wild-type enzyme. The increased thermal stability of this disulfide variant is consistent with a decrease in entropy for the unfolded state relative to the unfolded state that contains no cross-link, as would be predicted from the statistical thermodynamics of polymers.     

重组二硫键异构酶在大肠杆菌中表达条件的统计优化

二硫键形成蛋白A (disulfidebondformationproteinA ,DsbA)是大肠杆菌周质胞腔中辅助多种含有二硫键的蛋白质正确折叠并具有生物学活性的一种二硫键异构酶.通过统计实验设计的方法将生产重组DsbA的发酵过程进行了优化.首先通过Plackett Burman设计挑选出了对DsbA表达量影响较大的四个因素,然后再利用杂合设计进行实验,并通过拟合得到了响应曲面函数,但该函数的驻点是鞍点,因此不具有全局的极值.最后通过约束优化得到了较佳的实验点,在该实验点下DsbA的表达量比基本培养条件下提高了5 0 .14 % .     

Conformational characterization of disulfide bonds: A tool for protein classification

Background

Throughout evolution, mutations in particular regions of some protein structures have resulted in extra covalent bonds that increase the overall robustness of the fold: disulfide bonds. The two strategically placed cysteines can also have a more direct role in protein function, either by assisting thiol or disulfide exchange, or through allosteric effects. In this work, we verified how the structural similarities between disulfides can reflect functional and evolutionary relationships between different proteins. We analyzed the conformational patterns of the disulfide bonds in a set of disulfide-rich proteins that included twelve SCOP superfamilies: thioredoxin-like and eleven superfamilies containing small disulfide-rich proteins (SDP).

Results

The twenty conformations considered in the present study were characterized by both structural and energetic parameters. The corresponding frequencies present diverse patterns for the different superfamilies. The least-strained conformations are more abundant for the SDP superfamilies, while the “catalytic” +/−RHook is dominant for the thioredoxin-like superfamily. The “allosteric” -RHSaple is moderately abundant for BBI, Crisp and Thioredoxin-like superfamilies and less frequent for the remaining superfamilies. Using a hierarchical clustering analysis we found that the twelve superfamilies were grouped in biologically significant clusters.

Conclusions

In this work, we carried out an extensive statistical analysis of the conformational motifs for the disulfide bonds present in a set of disulfide-rich proteins. We show that the conformational patterns observed in disulfide bonds are sufficient to group proteins that share both functional and structural patterns and can therefore be used as a criterion for protein classification.     

Engineering of a staphylokinase-based fibrinolytic agent with antithrombotic activity and targeting capability toward thrombin-rich fibrin and plasma clots

Current clinically approved thrombolytic agents have significant drawbacks including reocclusion and bleeding complications. To address these problems, a staphylokinase-based thrombolytic agent equipped with antithrombotic activity from hirudin was engineered. Because the N termini for both staphylokinase and hirudin are required for their activities, a Y-shaped molecule is generated using engineered coiled-coil sequences as the heterodimerization domain. This agent, designated HE-SAKK, was produced and assembled from Bacillus subtilis via secretion using an optimized co-cultivation approach. After a simple in vitro treatment to reshuffle the disulfide bonds of hirudin, both staphylokinase and hirudin in HE-SAKK showed biological activities comparable with their parent molecules. This agent was capable of targeting thrombin-rich fibrin clots and inhibiting clot-bound thrombin activity. The time required for lysing 50% of fibrin clot in the absence or presence of fibrinogen was shortened 21 and 30%, respectively, with HE-SAKK in comparison with staphylokinase. In plasma clot studies, the HE-SAKK concentration required to achieve a comparable 50% clot lysis time was at least 12 times less than that of staphylokinase. Therefore, HE-SAKK is a promising thrombolytic agent with the capability to target thrombin-rich fibrin clots and to minimize clot reformation during fibrinolysis.     

Conservation of amino acids into multiple alignments involved in pairwise interactions in three-dimensional protein structures

We present an original strategy, that involves a bioinformatic software structure, in order to perform an exhaustive and objective statistical analysis of three-dimensional structures of proteins. We establish the relationship between multiple sequences alignments and various structural features of proteins. We show that amino acids implied in disulfide bonds, salt bridges and hydrophobic interactions have been studied. Furthermore, we point out that the more variable the sequences within a multiple alignment, the more informative the multiple alignment. The results support multiple alignments usefulness for predictions of structural features.     

Molecular cloning, expression and characterization of protein disulfide isomerase from Conus marmoreus

The oxidative folding of disulfide-rich conotoxins is essential for their biological functions. In vivo, disulfide bond formation is mainly catalyzed by protein disulfide isomerase. To elucidate the physiologic roles of protein disulfide isomerase in the folding of conotoxins, we have cloned a novel full-length protein disulfide isomerase from Conus marmoreus. Its ORF encodes a 500 amino acid protein that shares sequence homology with protein disulfide isomerases from other species, and 70% homology with human protein disulfide isomerase. Enzymatic analyses of recombinant C. marmoreus protein disulfide isomerase showed that it shared functional similarities with human protein disulfide isomerase. Using conotoxins tx3a and sTx3.1 as substrate, we analyzed the oxidase and isomerase activities of the C. marmoreus protein disulfide isomerase and found that it was much more efficient than glutathione in catalyzing oxidative folding and disulfide isomerization of conotoxins. We further demonstrated that macromolecular crowding had little effect on the protein disulfide isomerase-catalyzed oxidative folding and disulfide isomerization of conotoxins. On the basis of these data, we propose that the C. marmoreus protein disulfide isomerase plays a key role during in vivo folding of conotoxins.     

Synthesis of radioiodine labeled dibenzyl disulfide for evaluation of tumor cell uptake

Benzyl 4-halobenzyl and ally benzyl disulfide were synthesized as diallyl disulfide analogues and their tumor growth inhibitory effects on the cancer cells (SNU C5 and MCF-7) were comparable to that of diallyl disulfide, indicating that the disulfide functional group was responsible for the tumor growth inhibitory effects. Cu(I)-assisted radioiodination of benzyl 4-bromobenzyl disulfide gave benzyl 4-[123I/125I]iodobenzyl disulfide in 30-40% radiochemical yield. The radiolabeled disulfide was taken up by the cancer cells in a time-dependent manner, and the uptake was inhibited by the pretreatment of S-methyl methanethiosulfonate (MMTS), phorone and diallyl disulfide. This study suggested that the radiolabeled dibenzyl disulfide was taken up by the cancer cells via thiol-disulfide exchange and retained inside the cells.