Lacking Disulfide Bonds Research Articles

A pollen-specific RALF from tomato that regulates pollen tube elongation.

Rapid Alkalinization Factors (RALFs) are plant peptides that rapidly increase the pH of plant suspension cell culture medium and inhibit root growth. A pollen-specific tomato (Solanum lycopersicum) RALF (SlPRALF) has been identified. The SlPRALF gene encodes a preproprotein that appears to be processed and released from the pollen tube as an active peptide. A synthetic SlPRALF peptide based on the putative active peptide did not affect pollen hydration or viability but inhibited the elongation of normal pollen tubes in an in vitro growth system. Inhibitory effects of SlPRALF were detectable at concentrations as low as 10 nm, and complete inhibition was observed at 1 mum peptide. At least 10-fold higher levels of alkSlPRALF, which lacks disulfide bonds, were required to see similar effects. A greater effect of peptide was observed in low-pH-buffered medium. Inhibition of pollen tube elongation was reversible if peptide was removed within 15 min of exposure. Addition of 100 nm SlPRALF to actively growing pollen tubes inhibited further elongation until tubes were 40 to 60 mum in length, after which pollen tubes became resistant to the peptide. The onset of resistance correlated with the timing of the exit of the male germ unit from the pollen grain into the tube. Thus, exogenous SlPRALF acts as a negative regulator of pollen tube elongation within a specific developmental window.

In Vitro Activation of the Rhesus Macaque Myeloid α-Defensin Precursor proRMAD-4 by Neutrophil Serine Proteinases

Alpha-defensins are mammalian antimicrobial peptides expressed mainly by cells of myeloid lineage or small intestinal Paneth cells. The peptides are converted from inactive 8.5-kDa precursors to membrane-disruptive forms by post-translational proteolytic events. Because rhesus myeloid pro-alpha-defensin-4 (proRMAD-4((20-94))) lacks bactericidal peptide activity in vitro, we tested whether neutrophil azurophil granule serine proteinases, human neutrophil elastase (NE), cathepsin G (CG), and proteinase-3 (P3) have in vitro convertase activity. Only NE cleaved proRMAD-4((20-94)) at the native RMAD-4 N terminus to produce fully processed, bactericidal RMAD-4((62-94)). The final CG cleavage product was RMAD-4((55-94)), and P3 produced both RMAD-4((55-94)) and RMAD-4(57-94). Nevertheless, NE, CG, and P3 digests of proRMAD4 and purified RMAD-4((62-94)), RMAD-4((55-94)), and RMAD-4(57-94) peptides had equivalent in vitro bactericidal activities. Bactericidal peptide activity assays of proRMAD-4((20-94)) variants containing complete charge-neutralizing D/E to N/Q or D/E to A substitutions showed that (DE/NQ)-proRMAD-4((20-94)) and (DE/A)-proRMAD-4((20-94)) were as active as mature RMAD-4((62-94)). Therefore, proregion Asp and Glu side chains inhibit the RMAD-4 component of full-length proRMAD-4((20-94)), perhaps by a combination of charge-neutralizing and hydrogen-bonding interactions. Although native RMAD-4((62-94)) resists NE, CG, and P3 proteolysis completely, RMAD-4((62-94)) variants with disulfide pairing disruptions or lacking disulfide bonds were degraded extensively, evidence that the disulfide array protects the alpha-defensin moiety from degradation by the myeloid converting enzymes. These in vitro analyses support the conclusion that rhesus macaque myeloid pro-alpha-defensins are converted to active forms by serine proteinases that co-localize in azurophil granules.

Dimethylacetamide can be used as an alternative to glycerol for the successful cryopreservation of koala (Phascolarctos cinereus) spermatozoa

Swelling of koala sperm chromatin following cryopreservation has largely been attributed to the absence of intermolecular disulfide cross-linkages in the marsupial sperm nucleus. Fish spermatozoa also lack disulfide bonds within their chromatin, but have been successfully cryopreserved. The present study examined the hypothesis that the cryoprotectants used for fish sperm cryopreservation would confer a similar degree of protection on koala spermatozoa. Three concentrations each of five cryoprotectants (dimethyl sulfoxide, methanol, propylene glycol, ethylene glycol and dimethylacetamide (DMA)) were evaluated. Each treatment was compared against an established koala sperm cryopreservation protocol that uses 14% glycerol. Post-thaw assessment of progressive motility, plasma membrane integrity and mitochondrial membrane potential (MMP) revealed that protocols using 15% DMA achieved 62.2 +/- 3.6% (P < 0.05) sperm survival, of which 79% (P < 0.05) had high MMP, an improvement of 32% and 40%, respectively, over sperm frozen in 14% glycerol. The percentage of spermatozoa with swollen nuclei was also lowest when frozen in 15% DMA, both immediately after thawing (18.0 +/- 3.5%; P < 0.05) and after 2 h incubation at 35 degrees C (35.8 +/- 4.4%; P < 0.05). A second study was conducted to determine the optimal concentration of DMA for use in the cryopreservation of koala spermatozoa. High DMA concentrations (17.5% and 20%) resulted in significantly lower proportions of live spermatozoa showing high MMP immediately after thawing compared with spermatozoa frozen in the lower concentrations. The percentage of koala spermatozoa with swollen chromatin following cryopreservation was not affected by DMA concentration.

Roles of Salt and Conformation in the Biological and Physicochemical Behavior of Protegrin-1 and Designed Analogues: Correlation of Antimicrobial, Hemolytic, and Lipid Bilayer-Perturbing Activities

Protegrins are short (16-18 residues) cationic peptides from porcine leukocytes that display potent, broad-spectrum antimicrobial activity. Protegrin-1 (PG-1), one of five natural homologues, adopts a rigid beta-hairpin structure that is stabilized by two disulfide bonds. We have previously employed the principles of beta-hairpin design to develop PG-1 variants that lack disulfide bonds but nevertheless display potent antimicrobial activity [Lai, J. R., Huck, B. R., Weisblum, B., and Gellman, S. H. (2002) Biochemistry 41, 12835-12842.]. The activity of these disulfide-free variants, however, is attenuated in the presence of salt, and the activity of PG-1 itself is not. Salt-induced inactivation of host-defense peptides, such as human defensins, is thought to be important in some pathological situations (e.g., cystic fibrosis), and the variation in salt-sensitivity among our PG-1 analogues offers a model system with which to explore the origins of these salt effects. We find that the variations in antimicrobial activity among our peptides are correlated with the folding propensities of these molecules and with the extent to which the peptides induce leakage of contents from synthetic liposomes. Comparable correlations were observed between folding and hemolytic activity. The extent to which added salt reduces antimicrobial activity parallels salt effects on vesicle perturbation, which suggests that the biological effects of high salt concentrations arise from modulation of peptide-membrane interactions.

Functional properties of PDIA from Aspergillus niger in renaturation of proteins

Functional properties of protein disulfide isomerase A (PDIA) from Aspergillus niger were investigated using ribonuclease A, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and prochymosin as substrates. PDIA was shown to function as an isomerase catalyzing the refolding of denatured and reduced ribonuclease A. PDIA also exhibited trx-independent chaperone activity preventing the aggregation of reduced, denatured GAPDH, an enzyme lacking disulfide bonds. Both isomerase activity and chaperone function of PDIA were essential for the efficient refolding of the reduced, denatured prochymosin.

Interfacial Enzymology of Parvovirus Phospholipases A2

The capsid of parvoviruses proteins were recently shown to contain secreted phospholipase A(2) (sPLA(2))-like activity that is required during host cell entry. Parvoviral PLA(2) domains have little sequence identity with sPLA(2)s and lack disulfide bonds. In the present study, after bacterial expression and purification, the biochemical characterizations of these first PLA(2)s identified in viruses have been investigated, and a comparison has been made with other known PLA(2)s. The specific activities of three viral PLA(2)s differed by 3 orders of magnitude, with porcine parvovirus PLA(2) displaying a specific activity similar to that of the most active sPLA(2)s (e.g. human group IIA) and the human AAV2 and B19 parvoviral enzymes displaying approximately 10(3) lower specific activities (similar to human sPLA(2) groups IIE and XIIA). These differences were not caused by weaker Ca(2+) or interfacial binding. The specific activities of the viral PLA(2)s on zwitterionic or anionic phospholipid vesicles were comparable. The viral PLA(2)s did not display a preference for unsaturated versus saturated sn-2 fatty acyl chains and hydrolyzed all major classes of glycero-phospholipids except phosphatidylinositol. Incubation of mammalian cells with porcine parvovirus PLA(2) led to the release of arachidonic acid into the culture medium. Interestingly, among nine previously known sPLA(2) inhibitors, only a subset showed inhibition of the viral PLA(2)s and with weak potency, indicating that the active sites of these new enzymes are structurally distinct from those of sPLA(2)s. Based on these distinct enzymatic and structural properties, we propose to classify the parvovirus PLA(2)s within the PLA(2) superfamily as group XIII enzymes.

Microcin J25 has a threaded sidechain-to-backbone ring structure and not a head-to-tail cyclized backbone.

Microcin J25 is a 21 amino acid bacterial peptide that has potent antibacterial activity against Gram-negative bacteria, resulting from its interaction with RNA polymerase. The peptide was previously proposed to have a head-to-tail cyclized peptide backbone and a tight globular structure (Blond, A., Péduzzi, J., Goulard, C., Chiuchiolo, M. J., Barthélémy, M., Prigent, Y., Salomón, R. A., Farías, R. N., Moreno, F. & Rebuffat, S. Eur. J. Biochem. 1999, 259, 747-755). It exhibits remarkable thermal stability for a peptide of its size lacking disulfide bonds and in part this was previously proposed to derive from its macrocyclic structure. We show here that in fact the peptide does not have a head-to-tail cyclic structure but rather a side chain to backbone cyclization between Glu8 and the N-terminus. This creates an embedded ring that is threaded by the C-terminal tail of the molecule, forming a noose-like feature. The three-dimensional structure deduced from NMR data suggests that slippage of the noose is prevented by two aromatic residues flanking the embedded ring. Unthreading does not occur even when the molecule is enzymatically digested with thermolysin. The new structural interpretation fully accounts for previously reported NMR and biophysical data and is consistent with the remarkable stability of this potent antimicrobial peptide.

Overexpression and functional characterization of a serine carboxypeptidase inhibitor (I(C)) from Saccharomyces cerevisiae.

Carboxypeptidase Y (CPY) inhibitor, I(C), a cytoplasmic inhibitor of vacuolar proteinases in yeast, Saccharomyces cerevisiae, was purified by means of a high-level expression system using a proteinase-deficient strain, BJ2168, and an expression vector with the promoter GAL1. The purified I(C) exists as a monomeric beta-protein in solution with a mole-cular weight of 24,398.4 as determined by gel filtration chromatography, MALDI-TOF mass spectrometry, and far-UV CD spectroscopy. The acetylated N-terminal methionine residue is the sole posttranslational modification. I(C) specifically inhibits both the peptidase and anilidase activities of CPY with inhibitor constants (K(i)) of approximately 1.0 x 10(-9) M. The chemical modification of I(C) with sulfhydryl reagents indicated that it lacks disulfide bonds and has two free SH groups, which are responsible, not for the inhibitory function, but, apparently, for the folding of the overall structure. The formation of a complex of I(C) with CPY was highly specific, as evidenced by no detectable interaction with pro-CPY. Chemical modification studies of the CPY-I(C) complex with specific reagents demonstrated that the catalytic Ser146 and S1 substrate-binding site of CPY are covered in the complex.

Solution structure of the eukaryotic pore-forming cytolysin equinatoxin II: implications for pore formation

Sea anemones produce a family of 18–20 kDa proteins, the actinoporins, that lyse cells by forming pores in cell membranes. Sphingomyelin plays an important role in their lytic activity, with membranes lacking this lipid being largely refractory to these toxins. The structure of the actinoporin equinatoxin II in aqueous solution, determined from NMR data, consists of two short helices packed against opposite faces of a β-sandwich structure formed by two five-stranded β-sheets. The protein core has extensive hydrophobic interfaces formed by residues projecting from the internal faces of the two β-sheets. 15N relaxation data show uniform backbone dynamics, implying that equinatoxin II in solution is relatively rigid, except at the N terminus; its inferred rotational correlation time is consistent with values for monomeric proteins of similar mass. Backbone amide exchange rate data also support the view of a stable structure, even though equinatoxin II lacks disulfide bonds. As monitored by NMR, it unfolds at around 70°C at pH 5.5. At 25°C the structure is stable over the pH range 2.5–7.3 but below pH 2.5 it undergoes a slow transition to an incompletely unfolded structure resembling a molten globule. Equinatoxin II has two significant patches of positive electrostatic potential formed by surface-exposed Lys and Arg residues, which may assist its interaction with charged regions of the lipid head groups. Tyr and Trp residues on the surface may also contribute by interacting with the carbonyl groups of the acyl chains of target membranes. Data from mutational studies and truncated analogues identify two regions of the protein involved in membrane interactions, the N-terminal helix and the Trp-rich region. Once the protein is anchored, the N-terminal helix may penetrate the membrane, with up to four helices lining the pore, although other mechanisms of pore formation cannot be ruled out.

Sugarcane phytocystatins: Identification, classification and expression pattern analysis

The cystatins are tightly binding, but reversible, inhibitors of cysteine proteinases, which constitute a superfamily of evolutionary related proteins. They have been subdivided into three families: the cystatin family which contain two disulfide bonds, the stefin family which lack disulfide bonds, and the kininogen family composed of large glycoproteins containing three repeats similar to those found in the cystatin family. Members of the cystatin superfamily occurring in plants are currently known as phytocystatins, defined as proteins lacking disulfide bonds but possessing a conserved N-terminal amino acid sequence (L-A-R-[FY]-A-[VI]-X(3)-N). We have used the protein sequence deduced from seven phytocystatins (from the Arabidopsis thaliana genome project) and from the SUgarCane EST project (SUCEST) database to identify 25 possible sugarcane phytocystatins. Phylogenetic analysis has allowed us to cluster these phytocystatins into four distinct groups: (i) those with a characteristic N-terminal consensus, (ii) those with the same consensus plus a long C-terminal extension; (iii) those that lack the consensus but contain the highly conserved QxVxG motif found in all members of the superfamily and (iv) those that lack both the consensus and the QxVxG motif.

Studies on the function of yeast protein disulfide isomerase in renaturation of proteins

Renaturation of two enzymes lacking disulfide bonds, citrate synthase (CS), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and another protein containing disulfide bonds, lysozyme (LZM), were studied in order to dissect the possible chaperone function from the isomerase function of yeast protein disulfide isomerase (PDI). Our findings suggest no independent chaperone activity of yeast PDI with respect to the two enzymes lacking disulfide bonds, GAPDH and CS, since neither of these enzymes required PDI for renaturation. In contrast, a high level of renaturation of LZM was observed in the presence of PDI. Renaturation of LZM involved formation and rearrangement of disulfide bonds. Additional studies using LZM as a substrate were done to examine the role of cysteine residues in the two active sites of PDI. Studies with a series of cysteine to serine mutants and truncation mutants of yeast PDI revealed that the two active sites of PDI were not equal in activity. An intramolecular disulfide bond in at least one active site of PDI was required for the oxidation of reduced LZM. The first cysteine in each active site was necessary for disulfide bond rearrangement, i.e., isomerization, in LZM, while the second cysteine was not.

Distinct cysteine sulfhydryl environments detected by analysis of Raman S-H markers of Cys→Ser mutant proteins

Very little is known about the character or functional relevance of hydrogen-bonded cysteine sulfhydryl (S-H) groups in proteins. The Raman S-H band is a unique and sensitive probe of the local S-H environment. Here, we report the use of Raman spectroscopy combined with site-specific mutagenesis to document the existence of five distinguishable hydrogen-bonded states of buried cysteine sulfhydryl groups in a native protein. The 666 residue subunit of the Salmonella typhimurium bacteriophage P22 tailspike contains eight cysteine residues distributed through the elongated structure. The tailspike cysteine residues display an unusual Raman S-H band complex (2500-2600 cm−1 interval) indicative of diverse S-H hydrogen-bonding interactions in the native trimeric structure. To resolve specific Cys contributions to the complex Raman band we characterized a set of tailspike proteins with each cysteine replaced by a serine. The mutant proteins, once folded, were structurally and functionally indistinguishable from wild-type tailspikes, except for their Raman S-H signatures. Comparison of the Raman spectra of the mutant and wild-type proteins reveals the following hydrogen-bond classes for cysteine sulfhydryl groups. (i) Cys613 forms the strongest S-H…X bond of the tailspike, stronger than any heretofore observed for a protein. (ii) Cys267, Cys287 and Cys458 form robust S-H…X bonds. (iii) Moderate S-H…X bonding occurs for Cys169 and Cys635. (iv) Cys290 and Cys496 form weak hydrogen bonds. (v) It is remarkable that Cys287 contributes two Raman S-H markers, indicating the population of two distinct hydrogen-bonding states. The sum of the S-H Raman signatures of all eight mutants accurately reproduces the composite Raman band of the wild-type tailspike. The diverse cysteine states may be an outcome of the folding and assembly pathway of the tailspike, which though lacking disulfide bonds in the native state, utilizes transient disulfide bonds in the maturation pathway. This Raman study represents the first detailed assessment of local S-H hydrogen bonding in a native protein and provides information not obtainable directly by other structural probes. The method employed here should be applicable to a wide range of cysteine-containing proteins.

Oxidative refolding of Denatured/Reduced lysozyme utilizing the chaperone-like function of liposomes and immobilized liposome chromatography

Oxidative refolding of the denatured/reduced lysozyme was examined in the presence of small unilamellar vesicles (SUVs) essentially composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). SUVs facilitated the recovery of the enzymatic activity of lysozyme like molecular chaperones through the interaction with the refolding intermediate of lysozyme. The highest reactivation yield (87%) was obtained by delaying oxidation time (15-30 s) after dilution of the denaturant concentration was initiated in the presence of SUVs. SUVs supplemented with 1 mol % phosphatidylethanolamine were covalently coupled to gel beads, and then the interactions of SUVs with lysozyme at the various conformations were quantitatively examined with immobilized liposome chromatography (ILC). The reduced lysozyme lacking disulfide bonds was found to have a property similar to that of the molten globule state in terms of its local hydrophobicity, which was determined with the aqueous two-phase partitioning method. The reduced lysozyme was more clearly retarded on the ILC column than the native and 1 M guanidinium hydrochloride (GuHCl)-treated lysozymes with intact disulfide bonds. Similar results were obtained for alpha-lactalbumin, which has three-dimensional structure closely similar to that of lysozyme, regarding the membrane-protein interaction. These results can be interpreted as follows. In the early stage of the oxidative refolding, liposomes bound to the refolding intermediate of lysozyme lacking disulfide bonds. Then, liposomes assisted the formation of the tertiary structure of lysozyme by reducing protein intermolecular interactions, which usually cause the formation of the inactive aggregates. Consequently, the correct formation of the disulfide bonds was promoted. On the basis of the above results, the chromatographic oxidative refolding was also examined with ILC. The denatured/reduced lysozyme (9 mg/mL, 10 &mgr;L) was passed through the ILC column with a flow rate of 1 mL/min, which corresponds to the retention of lysozyme about 1 min in the ILC column. Subsequent oxidation of the eluted lysozyme resulted in the almost complete recovery (100%) of the original enzymatic activity of lysozyme.

Involvement of the C-terminal region of yeast proteinase B inhibitor 2 in its inhibitory action

Yeast proteinase B inhibitor 2 (YIB2), which is composed of 74 amino acid residues, is an unusual serine protease inhibitor, since it lacks disulfide bonds. To identify its reactive site for proteases, we constructed an expression system for a synthetic YIB2 gene and then attempted to change the inhibitory properties of YIB2 by amino acid replacements. The purified wild-type YIB2 inhibited the activity of subtilisin BPN′, a protein homologous to yeast proteinase B, although its binding ability was not strong, and a time-dependent decrease in its inhibitory activity was observed, demonstrating that wild-type YIB2 behaves as a temporary inhibitor when subtilisin BPN′ is the target protease. Since YIB2 exhibits sequence homology to the propeptide of subtilisin, which inhibits a cognate protease using its C-terminal region, we replaced the six C-termi nal residues of YIB2 with those of the propeptide of subtilisin BPN′ to make the mutant YIB2m1. This mutant exhibited markedly increased inhibitory activity toward subtilisin BPN′ without a time-dependent decrease in its inhibitory activity. Replacement of only the C-terminal Asn of YIB2 by Tyr, or deletion of the C-terminal Tyr of YIB2m1, inhibited subtilisin, but the ability of these mutants to bind subtilisin and their resistance to proteolytic attack were weaker than those of YIB2m1, indicating that the C-terminal residue contributes to the interaction with the protease to a greater extent than the preceding five residues and that the resistance of YIB2 to proteolyic attack is closely related to its ability to bind a protease. These results demonstrate that YIB2 is a unique protease inhibitor that involves its C-terminal region in the interaction with the protease.

Conformation and Antimicrobial Activity of Linear Derivatives of Tachyplesin Lacking Disulfide Bonds

Tachyplesin is a potent antimicrobial peptide isolated from the hemocytes of the horseshoe crab,Tachypleus tridentatus.Previous studies have shown that the 17-residue peptide has an intrinsic amphipathic structure conferred by two antiparallel β-sheets held rigidly by two disulfide bonds. Taking its short length into account and the potential of such a small polypeptide to take on multiple conformational states, one may assume that the disulfide bonds are relevant determinants of function. However, in order to gain a global perspective on the tolerance of cysteine residues in tachyplesin to amino acid substitutions, a series of linear peptides have been synthesized and their physicochemical properties analyzed. In these linear peptides, the cysteines have been replaced with amino acids possessing different side-chain properties, i.e., aliphatic hydrophobic (Ala, Leu, Ile, Val, and Met), aromatic hydrophobic (Phe and Tyr), and acidic (Asp). Activity assays using natural and synthetic membranes, and conformational measurements, highlight the subtle influence and variability of the amino acid side-chain properties on peptide structure. While an unequivocal interpretation of the results will have to await more refined structural measurements, our results indicate that a rigidly held disulfide-bonded β-pleated sheet structure may not be absolutely essential for antimicrobial activity. Furthermore, the results challenge the accepted dogma of structure–activity relationships among antimicrobial peptides and suggest that the maintenance of peptide hydrophobic–hydrophilic balance may be a critical parameter, in addition to structure, in the design of peptides with pharmaceutical relevance.

Manipulation of disulfide bonds differentially affects the intracellular transport, sorting, and processing of neuroendocrine secretory proteins.

To investigate if the prevention of disulfide bond formation affects the intracellular transport, sorting, and processing of a distinct set of neuroendocrine proteins in the regulated secretory pathway, we have treated Xenopus intermediate pituitaries with the thiol-reducing agent dithiothreitol. Pulse-chase incubations in combination with immunoprecipitation analysis were used to monitor the fates of the prohormone proopiomelanocortin (POMC), prohormone convertase PC2 and its helper protein 7B2, as well as secretogranin III. Manipulation of the disulfide bonds in POMC and proPC2 blocked their transport to the trans-Golgi network and strongly inhibited their processing. Reduction of the single disulfide bond in 7B2 did not disturb its transport and cleavage, but caused its missorting to the constitutive secretory pathway. Moreover, the liaison between proPC2 and 7B2 was prevented. Dithiothreitol did not affect transport, sorting, and cleavage of secretogranin III, which lacks disulfide bonds. When the reducing agent was washed away, POMC processing, proPC2 maturation, and the association between proPC2 and 7B2 were reestablished. Collectively, our findings indicate that manipulation of disulfide bonds differentially affects the fates of neuroendocrine proteins during their transit through the secretory pathway.

The ERO1Gene of Yeast Is Required for Oxidation of Protein Dithiols in the Endoplasmic Reticulum

We describe a conserved yeast gene, ERO1, that is induced by the unfolded protein response and encodes a novel glycoprotein required for oxidative protein folding in the ER. In a temperature-sensitive ero1-1 mutant, newly synthesized carboxypeptidase Y is retained in the ER and lacks disulfide bonds, as shown by thiol modification with AMS. ERO1 apparently determines cellular oxidizing capacity since mutation of ERO1 causes hypersensitivity to the reductant DTT, whereas overexpression of ERO1 confers resistance to DTT. Moreover, the oxidant diamide can restore growth and secretion in ero1 mutants. Genetic tests distinguish the essential function of ERO1 from that of PDI1. We show that glutathione is not required for CPY folding and conclude that Ero1p functions in a novel mechanism that sustains the ER oxidizing potential, supporting net formation of protein disulfide bonds.

Role of modified glutamic acid in the helical structure of conantokin-T

Circular dichroism (CD) and 2-dimensional NMR were used to study the solution conformation of conantokin-T (Con-T), a small peptide toxin found in the venom of fish-hunting cone snails, and its Glu-substituted analog. Con-T lacks disulfide bonds but contains many γ-carboxyglutamic acids (Gla), a post-translationally modified residue. Our results show that Con-T adopts an α-helical conformation in aqueous solution even in the absence of calcium. Glu replacements diminish both helicity and function of Con-T. The helical content of Con-T is higher than most natural helical peptides of this length in aqueous solution. The sequence of this small toxin incorporates several known elements that stabilize α-helical structure in peptides. Gla residues form several salt bridges that stabilize helical conformation of Con-T.

Defective antigen processing correlates with a low level of intracellular glutathione.

Previously, we reported that Chinese hamster ovary (CHO) cells transfected with murine mouse major histocompatibility complex class II genes, exhibit a unique antigen (Ag) processing defect whereby these cells are impaired in processing only Ag with disulfide bonds. Here, we examined various aspects of the intracellular reducing environment in the CHO cells to understand the underlying mechanism causing the defect. A cell hybrid generated by the fusion of CHO cells and L cell fibroblasts was used for comparison due to their competency in processing Ag. The transport pathway of cysteine within the CHO cells appeared normal. However, these cells had a significantly lower level of glutathione, a major physiological reducing thiol, compared to the cell hybrid. Treatment of the CHO cells with N-acetyl-L-cysteine did not augment their glutathione content nor their ability to process Ag. When the cell hybrid was treated with L-buthionine-(S,R)-sulfoximine (BSO), which significantly decreased their glutathione level, the hybrid poorly processed hen egg lysozyme (HEL) and ovalbumin, which have disulfide bonds. In contrast, BSO treatment did not affect the capacity of the hybrid to process pigeon cytochrome c and carboxymethylated HEL, which lack disulfide bonds. Therefore, low intracellular glutathione levels in antigen-presenting cells correlated with defective processing of Ag with disulfide bonds, indicating that this thiol may be a critical factor in regulating productive Ag processing.

Stability and folding of the SH3 domain of Bruton's tyrosine kinase.

Bruton's tyrosine kinase (BTK) plays an important role in B cell development. Deletion of C-terminal 14 amino acids of the SH3 domain of BTK results in X-linked agammaglobulinemia (XLA), an inherited disease. We report here on the stability and folding of SH3 domain of BTK. Peptides corresponding to residues 216-273 (58 residues) and 216-259 (44 residues) of BTK SH3 domain were synthesized by solid phase methods; the first peptide constitutes the entire SH3 domain of BTK while the latter peptide lacks 14 amino acid residues of the C-terminal. The 58 amino acid peptide forms mainly a beta-barrel type folding unit. Although small and lacking disulfide bonds, this peptide is extremely stable to thermal denaturation. Based on circular dichroism measurements, its melting temperature was found to be high, 82 degrees C at pH 6.0. However, the Gibbs free energy (delta GH2O) of the intrinsic stability and thermodynamic spontaneity of unfolding were found to be low, 2.6 kcal/mol by Gdn.HCl denaturation experiments, as compared to 12 kcal/mol obtained for larger single domain proteins, indicating poor stability of SH3 domain. Addition of 500 mM of Na2SO4 increased the free energy change delta GH2O to 4.0 kcal/mol, suggesting an ionic strength effect. The truncated peptide fails to fold correctly and adopts random coil conformation in contrast to 58 amino acid beta-barrel peptide, which exhibits high thermal stability but normal or low stability at ambient temperature. These results, to our knowledge the first to delineate the importance of C-terminal in structural integrity of SH3 domains, indicate also that improper folding and/or poor stability of mutant SH3 domain in BTK likely causes XLA.