Pathway of Oxidative Folding of a 3-Disulfide α-Lactalbumin May Resemble Either BPTI Model or Hirudin Model

Pathways of oxidative folding of disulfide proteins display a high degree of diversity and vary among two extreme models. The BPTI model is defined by limited species of folding intermediates adopting mainly native disulfide bonds. The hirudin model is characterized by highly heterogeneous folding intermediates containing mostly non-native disulfide bonds. αLA-IIIA is a 3-disulfide variant of α-lactalbumin (αLA) with a 3-D conformation essentially identical to that of intact αLA. αLA-IIIA contains 3 native disulfide bonds of αLA, two of them are located at the calcium binding β-subdomain (Cys⁶¹–Cys⁷⁷ and Cys⁷³–Cys⁹¹) and the third bridge is located within the α-helical domain of the molecule (Cys²⁸–Cys¹¹¹). We investigate here the pathway of oxidative folding of fully reduced αLA-IIIA with and without stabilization of its β-subdomain by calcium binding. In the absence of calcium, the folding pathway of αLA-IIIA was shown to resemble that of hirudin model. Upon stabilization of β-sheet domain by calcium binding, the folding pathway of αLA-IIIA exhibits a striking similarity to that of BPTI model. Three predominant folding intermediates of αLA-IIIA containing exclusively native disulfide bonds were isolated and structurally characterized. Our results further demonstrate that stabilization of subdomains in a protein may dictate its folding pathway and represent a major cause for the existing diversity in the folding pathways of the disulfide-containing proteins.     

Conformational analysis by quantitative NOE measurements of the β-proton pairs across individual disulfide bonds in proteins

NOEs between the β-protons of cysteine residues across disulfide bonds in proteins provide direct information on the connectivities and conformations of these important cross-links, which are otherwise difficult to investigate. With conventional [U-¹³C, ¹⁵N]-proteins, however, fast spin diffusion processes mediated by strong dipolar interactions between geminal β-protons prohibit the quantitative measurements and thus the analyses of long-range NOEs across disulfide bonds. We describe a robust approach for alleviating such difficulties, by using proteins selectively labeled with an equimolar mixture of (2R, 3S)-[β-¹³C; α,β-²H₂] Cys and (2R, 3R)-[β-¹³C; α,β-²H₂] Cys, but otherwise fully deuterated. Since either one of the prochiral methylene protons, namely β2 (proS) or β3 (proR), is always replaced with a deuteron and no other protons remain in proteins prepared by this labeling scheme, all four of the expected NOEs for the β-protons across disulfide bonds could be measured without any spin diffusion interference, even with long mixing times. Therefore, the NOEs for the β2 and β3 pairs across each of the disulfide bonds could be observed at high sensitivity, even though they are 25% of the theoretical maximum for each pair. With the NOE information, the disulfide bond connectivities can be unambiguously established for proteins with multiple disulfide bonds. In addition, the conformations around disulfide bonds, namely χ² and χ³, can be determined based on the precise proton distances of the four β-proton pairs, by quantitative measurements of the NOEs across the disulfide bonds. The feasibility of this method is demonstrated for bovine pancreatic trypsin inhibitor, which has three disulfide bonds.     

Androgen binding protein (ABP) in rabbit testis and epididymis

ABP has been measured in 105,000 g supernatants of testis and epididymls from rabbits of different ages and compared with a similar androgen binding protein (TeBG) in rabbit serum. Whereas the concentration of ABP in the caput epididymidis increased markedly from immaturity to adulthood, serum TeBG decreased, indicating that ABP and TeBG are regulated by different hormonal mechanisms.The concentration of ABP (pmoles/mg protein) in sexually mature rabbits was much higher in the epididymis than in the testis. Within the epididymis most of the ABP was concentrated within the caput, and very low amounts were found in the cauda, indicating that binding activity of ABP is destroyed as it passes through the epididymis.In addition to ABP (R_f ~0.7), rabbit epididymal supernatant contains a larger binding protein for 5α-dihydrotestosterone (DHT; 17β-hydroxy-5α-androstan-3-one) with slower electrophoretic mobility (R_f ~0.4) and a more rapid sedimentation rate on sucrose gradients (7S). This component is most probably the intracellular androgen receptor in the rabbit e pididymis.     

Immunohistochemical demonstration of androgen-binding protein in the rat prostatic gland.

Androgen-binding protein (ABP) is one of the best-characterized products of synthesis by the Sertoli cells in the rat. Although the exact physiological role of ABP remains to be determined, it has been widely used to study Sertoli cells and testicular function in this species. Since this protein is the principal carrier for testosterone in rat testis and epididymis, we decided to investigate ABP immunoreactivity (ABP-I) in androgen-dependent organs, including testicle, epididymides, prostate, and seminal vesicles. The location of ABP was investigated by immunohistochemistry using specific antisera against rat ABP. As previously described in the testis, rat ABP-I was identified in the seminiferous tubules within the cytoplasm of the Sertoli cells and the tubular luminae. The epididymis showed ABP-I only in epithelial cells of the proximal caput. We demonstrated ABP-I in the apical portions of epithelial cells of the rat prostate. Short-term castration and/or ligation of the efferent ducts did not suppress prostatic ABP-I. ABP-I was not present in seminal vesicles of control rats nor under any of the experimental conditions used throughout this study. The results also indicate the presence of ABP-I in prostatic epithelium, probably because of a mechanism similar to that described in epididymis. Our data support and enhance the concept that ABP may serve as a transmembrane carrier protein for androgens in androgen target organs in the male reproductive tract.     

Biochemical and molecular characterization of gliadins

Gliadins account for about 40–50% of the total proteins in wheat seeds and play an important role in the nutritional and processing quality of flour. Usually, gliadins can be divided into α-(α/β), γ-, and ω-groups, whereas the low-molecular-weight (LMW) gliadins are novel seed storage proteins. The low-molecular-weight glutenin subunits (LMW-GSs) are also designated as gliadins in a few publications. The genes encoding gliadins are mainly located on the short arms of group 6 and group 1 chromosomes, and not evenly distributed. Repetitive sequences cover most of the uncoding regions, which attributed greatly to the evolution of wheat genome. The primary structure of each gliadin is divided into several domains, and the long repetitive domains consist of peptide motifs. Conserved cysteine residues mainly form intramolecular disulfide bonds. The rare potential intermolecular disulfide bonds and the long repetitive domains play an important role in the quality of wheat flour. There is a general idea that gliadin genes, even prolamin genes, have a common origin and subsequent divergence leads to gene polymorphism. The γ-gliadins are considered to be the most ancient of the wheat prolamin family. Several elements in the 5′-flanking (e.g., CAAT and TATA box) and the 3′-flanking sequences have been detected, which has been shown to be necessary for the proper expression of gliadins. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 5, pp. 796–807. The text was submitted by the authors in English.     

Characterization of an androgen binding protein (ABP) in rat testis and epididymis

An androgen binding protein (ABP) was demonstrated in the 105,000 g supernatant of rat testis homogenate after charcoal extraction of endogenous steroids. Testis ABP proved to be identical to an ABP previously described in rat epididymis. It contained saturable high-affinity sites which exhibited binding specificity for dihydrotestosterone (6) and testosterone when measured by polyacrylamide gel electrophoresis or by competitive binding using charcoal adsorption. Binding to ABP was not affected by ribonuclease or neuraminidase but was decreased by the disulfide reducing agent, dithiothreitol and the sulfhydryl reagent, N-ethylmaleimide. Binding was abolished by treatment with proteolytic enzyme. The mean molecular radius of ABP was 2.92 nm as determined by the retardation of electrophoretic mobility in polyacrylamide gels of decreasing pore size. Assuming a partial specific volume of 0.66–0.74 the molecular weight was 86,000–91,000 for a spherical molecule. ABP binding was stable after treating at 45° C for 20 min. but was destroyed at 60° C. Binding was maximal between pH 7.5 and 9.0 and decreased at pH below 7.0.     

Estrogen in the adult male reproductive tract: A review

Testosterone and estrogen are no longer considered male only and female only hormones. Both hormones are important in both sexes. It was known as early as the 1930's that developmental exposure to a high dose of estrogen causes malformation of the male reproductive tract, but the early formative years of reproductive biology as a discipline did not recognize the importance of estrogen in regulating the normal function of the adult male reproductive tract. In the adult testis, estrogen is synthesized by Leydig cells and the germ cells, producing a relatively high concentration in rete testis fluid. Estrogen receptors are present in the testis, efferent ductules and epididymis of most species. However, estrogen receptor-α is reported absent in the testis of a few species, including man. Estrogen receptors are abundant in the efferent ductule epithelium, where their primary function is to regulate the expression of proteins involved in fluid reabsorption. Disruption of the α-receptor, either in the knockout (αERKO) or by treatment with a pure antiestrogen, results in dilution of cauda epididymal sperm, disruption of sperm morphology, inhibition of sodium transport and subsequent water reabsorption, increased secretion of Cl^-, and eventual decreased fertility. In addition to this primary regulation of luminal fluid and ion transport, estrogen is also responsible for maintaining a differentiated epithelial morphology. Thus, we conclude that estrogen or its α-receptor is an absolute necessity for fertility in the male.     

Solvent assistance in regiospecific disulfide formation in dimethylsulfoxide

Summary This paper describes a convenient oxidative refolding method that exploits the dual property of dimethylsulfoxide (DMSO) as an oxidant and a solvent ‘chaperon’ in assisting disulfide formation in peptides. Synthetic peptides with preferred secondary structures were used as models. For β-sheet peptides, an active fragment of fibroblast growth factor containing the tetrapeptide Arg-Ser-Arg-Arg at a reverse turn was used. A disulfide scan consisting of 16 analogs is designed in which different pairs of amino acids on the antiparallel β-sheets flanking the active determinants are replaced with cysteine. DMSO in aqueous buffer at pH 6 or 8 was found to minimize aggregation due to β-sheet formation in all 16 β-stranded peptides and provided monocyclic disulfide peptides within 7 h. In contrast, air oxidation required a significantly longer duration for completion under similar conditions, and only 8 of 16 peptides formed intramolecular disulfides. Rate accelerations at pH 8 were found in exo-disulfide formation involving peptides with amino terminal cysteine, irrespective of oxidation conditions. The exo-disulfide effect in accelerating disulfide formation may be useful for regiospecific disulfide formation. For α-helix peptides, the twodisulfide endothelin (ET) was used as a model. DMSO in combination with trifluoroethanol (TFE) was found to favor the desired bicyclic 1,4-disulfide bridged ET (1,4-ET) over the incorrectly folded 1,3-ET. Under aqueous conditions at pH 5–11, 1,4-ET to 1,3-ET was formed in the ratio of approximately 3:1, while the use of DMSO and TFE increased the ratio to 11:1. This solvent combination may stabilize an α-helical stretch found in ET and contribute to enhanced selectivity. Thus, our results show that DMSO in disulfide formation in an aqueous or helix-promoting solution may serve as an oxidant and a ‘chaperon’ solvent system to provide regiospecificity for oxidative disulfide formation.     

Role of ascorbic acid in metabolism of rat testis and epididymis in relation to the onset of puberty

The metabolism of ascorbic acid, cholesterol, serum testosterone level and activities of 3Β and 17Β hydroxysteroid dehydrogenases were studied in testis and Cauda epididymis of prepubertal, pubertal and postpubertal (5, 15, 30, 45, 55 and 60 day old) rats. The data showed that serum testosterone levels and 3Β and 17Β hydroxysteroid dehydrogenases were increased with the age. The ascorbic acid metabolism was found to be stabilized in testis at day 30 being comparable with the adult, whereas a spurt in its metabolism occurred by day 45 and a significant depletion in ascorbic acid content in relation to the passage of the first wave of spermatozoa through cauda epididymis. The results of this study clearly elucidate that ascorbic acid is involved in metabolism of testis and epididymis in developing postnatal rats, in relation to the increasing demands for attaining a stable hormonal milieu, and the onset of puberty and the passage of the first wave of spermatozoa,via the formation of its free radical monodehydroascorbic acid and charge transfer complex mechanism     

Characterization of the Fourth α Isoform of the Na,K-ATPase

The Na,K-ATPase is a major ion transport protein found in higher eukaryotic cells. The enzyme is composed of two subunits, α and β, and tissue-specific isoforms exist for each of these, α1, α2 and α3 and β1, β2 and β3. We have proposed that an additional α isoform, α4, exists based on genomic and cDNA cloning. The mRNA for this gene is expressed in rats and humans, exclusively in the testis, however the expression of a corresponding protein has not been demonstrated. In the current study, the putative α4 isoform has been functionally characterized as a novel isoform of the Na,K-ATPase in both rat testis and in α4 isoform cDNA transfected 3T3 cells. Using an α4 isoform-specific polyclonal antibody, the protein for this novel isoform is detected for the first time in both rat testis and in transfected cell lines. Ouabain binding competition assays reveal the presence of high affinity ouabain receptors in both rat testis and in transfected cell lines that have identical K _D values. Further studies of this high affinity ouabain receptor show that it also has high affinities for both Na⁺ and K⁺. The results from these experiments definitively demonstrate the presence of a novel isoform of the Na,K-ATPase in testis. Received: 4 December 1998/Revised: 1 February 1999     

Transformation of sperm histone during formation and maturation of rat spermatozoa.

Changes of chromosomal basic proteins of rats have been followed during transformation of spermatids into spermatozoa in the testis and during maturation of spermatozoa in the epididymis. Rat testis chromatin has been fractionated on the basis of differing sensitivity to shearing, yielding a soluble fraction and a condensed fraction. The sperm histone is found in the condense fraction. Somatic-type histones are found in both fractions. The somatic-type histones in the condensed fraction contains much more lysine-rich histone I, than does the somatic-type histones in the soluble fraction. This may suggest that the lysine-rich histone I is the last histone to be displaced during the replacement of somatic-type histones by sperm histone. After extensive shearing followed by sucrose centrifugation, the condensed portion of testis chromatin can be further fractionated into two morphologically distinctive fractions. One is a heavy fraction possessing an elongated shape typical of the head of late spermatids. The other is a light fraction which is presumably derived from spermatids at earlier stages of chromatin condensation and which is seen as a beaded structure in the light microscope. Sperm histone of testis chromatin can be extractable completely by guanidinium chloride without a thiol, wheras 2-mercaptoethanol is required for extraction of sperm histone from caput and cauda epididymal spermatozoa. The light fraction of the condensed testis chromatin contains unmodified and monophospho-sperm histone. The sperm histones of the heavy fraction is mainly of monophospho and diphospho species, whereas unmodified and monophosphosperm histones are found in caput and cauda epididymal spermatozoa. Labeling of cysteine sulfhydryl groups of sperm histone releases by 2-mercaptoethanol treatment shows that essentially all of the cysteine residues of sperm histone in testis chromatin are present as sulfhydryl groups, while those of sperm histone isolated from mature (cauda epididymal) spermatozoa are present as disulfide forms and approximately 50% of the cysteine residues of sperm histone obtained from caput epididymal spermatozoa are in disulfide forms. These results suggest that phosphorylation of sperm histone is involved in the process of chromatin condensation during transformation of spermatozoa in the epididymis.     

A disulfide linked model of the complement protein C8γ complexed with C8α indel peptide

In a recent study C8γ (complement protein) with Cys40Ala substitution and a C8α derived peptide with Cys164Ala substitution were co-crystallized and their binding mode was revealed. Computer modeling and molecular dynamics simulations were performed in order to check the hypothesis that the residues Ala164 of C8α and Ala40 of C8γ occupied the right position if cysteine residues were in their place for disulfide bonding. Substitution of these two alanine residues with cysteine along with disulfide bond creation via molecular modeling and subsequent molecular dynamics simulation of the complex corroborated the hypothesis, which was also confirmed from recent crystallographic data. Average RMSD between backbone atoms of the indel peptide during the MD trajectory in comparison with the corresponding sequence of crystal structure of the C8α/γ complex was found only 0.085 nm. Figure Modeling the C*y/α comlexation. Ribbon representation of the C8y complexed with C8α indel peptide initial (green/cyan) X-ray structure and the final MD conformation (magenta/orange) after imposing the disulfide link. Average RMSD between backbone atoms of the indel peptide during MD trajectory in comparison with the corresponding sequence of crystal structure of the C8α/y complex was found only 0.085nm. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Chemical construction of immunotoxins

Immunotoxins are chimeric proteins consisting of an antibody linked to a toxin. The antibodies most frequently used for the preparation of immunotoxins are murine monoclonal antibodies belonging to IgG isotype. The most used toxins for the chemical construction of immunotoxins are Ricin toxin A chain in its deglycosylated form and recombinant Pseudomonas endotoxin with the cell-binding domain deleted. The linkage of the antibody to the toxin can be accomplished by chemical methods using reagents that crosslink antibody to toxin. The usual crosslinkers attach disulfide groups into the antibody molecule to form a disulfide bond between the antibody and the toxin. Disulfide bonds are susceptible to reduction in the cytoplasm of the targeted cells thereby releasing the toxin so that it can exert its cytotoxic activity only into the cells (e.g., tumor cells) binding the antibody moiety. This article describes various methods to obtain antibodies and toxins and several procedures for their crosslinking as well as “in vitro” and “in vivo” testing of the immunotoxins efficacy.     

Domain <Emphasis Type="Italic">a</Emphasis>’ of protein disulfide isomerase plays key role in inhibiting α-synuclein fibril formation

α-Synuclein (αSyn) is the main component of Lewy bodies formed in midbrain dopaminergic neurons which is a pathological characteristic of Parkinson's disease. It has been recently showed to induce endoplasmic reticulum (ER) stress and impair ER functions. However, the mechanism of how ER responds to αSyn toxicity is poorly understood. In the present study, we found that protein disulfide isomerase (PDI), a stress protein abundant in ER, effectively inhibits αSyn fibril formation in vitro. In PDI molecule with a structure of abb’xa’c, domain a’ was found to be essential and sufficient for PDI to inhibit αSyn fibril formation. PDI was further found to be more avid for binding with intermediate species formed during αSyn fibril formation, and the binding was more intensive in the later lag phase. Our results provide new insight into the role of PDI in protecting ER from the deleterious effects of misfolded protein accumulation in many neurodegenerative diseases.     

Functional relationships of the mammalian testis and epididymis

The development of knowledge in four areas of research in male reproductive physiology of particular interest is reviewed. The concept of the blood-testis barrier (BTB), which arose following dye exclusion from the seminiferous tubules, has now been established as the differential transfer from interstitial fluid to tubular and rete testis fluids of molecules of physiological importance. The composition of fluid collected mostly from the rete testis of several species, not only reflects the nature of the barrier, but also the secretory capacity of the Sertoli cell. The functional significance of the transfer of molecules into testicular fluid and the composition of the fluid flowing into the epididymis are discussed. Sertoli cells establish the structural basis of the BTB during puberty and divide the seminiferous epitheliuym into basal and adluminal compartments. The Sertoli cell is the prime target for follicle stimulating hormone (FSH). The responses evoked by FSH are discussed, including special mention of androgen binding protein (ABP) and the protein hormone, 'inhibin', with FSH-suppressing properties. The control of FSH in the lamb is mentioned including new evidence to support a tubular source of a feedback agent with significance during the impuberal stage. Finally, some of the biochemical properties of the epididymis and its fluid contents are reviewed and the epididymal sperm are identified as the site of the antifertility action of the 6-chloro-6-deoxy sugars.     

Flow cytometric analysis of rodent epididymal spermatozoal chromatin condensation and loss of free sulfhydryl groups

Flow cytometric measurements were made on acridine orange (AO) and 7-diethylamino-3-(4'-maleimidylphenyl)-4-methyl-coumarin (CPM)-stained epididymal- and vas deferens-derived spermatozoal nuclei to follow the course of chromatin condensation and oxidation of free sulfhydryl groups, respectively, during passage through mouse and rat posttesticular reproductive tracts. Alterations of mouse and rat spermatozoal chromatin during transition from a testicular elongated spermatids to epididymal caput spermatozoa resulted in a threefold loss of DNA stainability with AO. Passage of spermatozoa from the caput to corpus epididymis was accompanied by an approximate 15% loss of DNA stainability, which was maintained at that level throughout passage into the vas deferens. AO stainability of epididymal spermatozoal nuclei was generally independent of -SH group stainability. CPM stainability of rat spermatozoal nuclei free -SH groups was 83%, 18%, and 11% of caput spermatozoal values for corpus, cauda epididymis, and vas deferens, respectively. Comparable values for mice were 69%, 20%, and 18%. CPM stainability was relatively homogeneous for these mouse and rat reproductive tract regions, except mouse corpus epididymis spermatozoal nuclei stained very heterogeneously. Rat spermatozoa detained by ligature up to 7 days in the caput, corpus, and cauda epididymi had CPM staining values equal to or below those of normal vas spermatozoa, indicating that disulfide (S-S) bonding is intrinsic to the spermatozoa and is independent of the epididymal environment. These data suggest that chromatin condensation and loss of spermatozoal DNA stainability during passage from the testis to the vas deferens are independent of S-S bonding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of disulfide bonds in α1-acid glycoprotein and their effect on the ability of the protein to interact with ethidium bromide

α1-Acid glycoprotein (orosomucoid) was purified from the human and murine blood sera using phenol deproteinization. As opposed to the murine protein, the human orosomucoid bound the fluorescent dye ethidium bromide but lost this ability after treatment with β-mercaptoethanol, which breaks disulfide bonds. Disulfide bonds between the Cys²³ and Cys¹⁶⁵ residues of the human orosomucoid and between the Cys⁹¹ and Cys¹⁸⁴ residues of the murine orosomucoid were identified.     

Production of disulfide-bonded proteins in Escherichia coli

Disulfide bonds are covalent bonds formed post-translationally by the oxidation of a pair of cysteines. A disulfide bond can serve structural, catalytic, and signaling roles. However, there is an inherent problem to the process of disulfide bond formation: mis-pairing of cysteines can cause misfolding, aggregation and ultimately result in low yields during protein production. Recent developments in the understanding of the mechanisms involved in the formation of disulfide bonds have allowed the research community to engineer and develop methods to produce multi-disulfide-bonded proteins to high yields. This review attempts to highlight the mechanisms responsible for disulfide bond formation in Escherichia coli, both in its native periplasmic compartment in wild-type strains and in the genetically modified cytoplasm of engineered strains. The purpose of this review is to familiarize the researcher with the biological principles involved in the formation of disulfide-bonded proteins with the hope of guiding the scientist in choosing the optimum expression system.