Intrachain Disulfide Research Articles

Replacement of Disulfides by Amide Bonds in the Relaxin-like Factor (RLF/INSL3) Reveals a Role for the A11–B10 Link in Transmembrane Signaling

The relaxin-like factor (RLF) also named insulin-like 3 (INSL3) consists of two polypeptide chains linked by two interchain and one intrachain disulfide bond. RLF binds to its receptor (LGR8 also named RXFP2) through the B chain and initiates transmembrane communication by activating the adenylate cyclase through the N-terminal region of both chains. Cystine A11-B10 occupies a unique position on the molecular surface just outside the binding region and between the two signaling ports. We have synthesized an RLF analogue in which the disulfide A11-B10 was replaced by a peptide bond and found that cAMP production ceased while receptor binding was not affected. In contrast, replacing the disulfide A24-B22 by a peptide bond reduced potency proportional to the binding affinity and lowered efficacy to 65%, while replacing disulfide A10-A15 by a peptide bond reduced binding affinity to 32% and lowered potency to 7% but maintained 100% efficacy. The exceptional properties of the derivative bearing an A11-B10 isopeptide cross-link suggests that the disulfide has a special role in signal transduction. We propose that disulfide A11-B10 serves as an insulator between the two ports, whereas the amide functionality disturbs the signal transmission complex likely due to changes in polarity. The clear separation between receptor binding and signal activation sites within this small protein permits one to study how the relaxin-like factor initiates the signal on the receptor that induces intracellular cAMP production.

Analyzing the genetic characteristics and function of the swine leukocyte antigen 2 gene in a Chinese inbreed of pigs

To study the genetic characteristics and function of swine leukocyte antigen (SLA) class I from the Hebao pig, a rare inbreed in China, a pair of primers was designed to amplify the SLA-2 gene (SLA-2-HB) and then the genetic characteristics of the gene were analyzed. The 3D homology modeling was used to analyze the structure and function of SLA-2-HB proteins. After cloning, sequencing and computer analysis, four SLA-2-HB alleles were found, all of 1119 bp. Sites 3-1097 were an open reading frame encoding 364 amino acids with two sets of intra-chain disulfide bonds comprising four cysteines situated in sites 125, 188, 227 and 283. By alignment of SLA-2-HB sequences with other SLA-2 alleles in the IPD database, 11 key variable amino acid sites were found in the extracellular domain of the SLA-2-HB alleles at sites 23(F), 24(I), 43(A), 44(K), 50(Q), 73(N), 95(I), 114(R), 155(G), 156(E) and 216(S), which could be used to differentiate other SLA-2 alleles. The 3D homology modeling demonstrated that the eight of 11 key variable amino acid sites were all in antigenic binding groove of SLA-2-HB proteins. The amino acid identities between SLA-2-HB and other SLA-2, SLA-1 and SLA-3 alleles were 86.2-97.0%, 85.0-93.9% and 83.3-88.6%, respectively. The phylogenetic tree of SLA-2-HB showed that it was relatively independent of the other SLA-2 genes. Furthermore, the SLA-2-HB alleles were similar to HLA-B15 and HLA-A2 functional domains and preserved some functional sites of HLA-A2. It was concluded that SLA-2-HB are novel alleles of SLA-2 and that the Hebao pig might have evolved independently in China.

Identification of in Vivo Disulfide Conformation of TRPA1 Ion Channel

TRPA1 (transient receptor potential ankyrin 1) is an ion channel expressed in the termini of sensory neurons and is activated in response to a broad array of noxious exogenous and endogenous thiol-reactive compounds, making it a crucial player in chemical nociception. A number of conserved cysteine residues on the N-terminal domain of the channel have been identified as critical for sensing these electrophilic pungent chemicals, and our recent EM structure with modeled domains predicts that these cysteines form a ligand-binding pocket, allowing for the possibility of disulfide bonding between the cysteine residues. Here, we present a comprehensive mass spectrometry investigation of the in vivo disulfide bonding conformation and in vitro reactivity of 30 of the 31 cysteine residues in the TRPA1 ion channel. Four disulfide bonds were detected in the in vivo TRPA1 structure: Cys-666-Cys-622, Cys-666-Cys-463, Cys-622-Cys-609, and Cys-666-Cys-193. All of the cysteines detected were reactive to N-methylmaleimide (NMM) in vitro, with varying degrees of labeling efficiency. Comparison of the ratio of the labeling efficiency at 300 μM versus 2 mM NMM identified a number of cysteine residues that were outliers from the mean labeling ratio, suggesting that protein conformation changes rendered these cysteines either more or less protected from labeling at the higher NMM concentrations. These results indicate that the activation mechanism of TRPA1 may involve N-terminal conformation changes and disulfide bonding between critical cysteine residues.

Molecular characteristics of the SLA-2 gene from Chinese Hebao pigs

To study the molecular characteristics of swine leukocyte antigen (SLA) class I from the Hebao pig, a rare inbreed in China, a pair of primers was designed to amplify the SLA-2 gene (SLA-2-HB) and then the molecular characteristics of the gene were analyzed by computer. After cloning, sequencing and computer analysis, four SLA-2-HB alleles were found, all of 1119 bp. Sites 3-1097 were an open reading frame encoding 364 amino acids with two sets of intra-chain disulfide bonds comprising four cysteines situated in sites 125, 188, 227 and 283. By alignment of SLA-2-HB sequences with other SLA-2 alleles in the DNA Data Bank of Japan/European Molecular Biology Laboratory/GenBank database, nine key variable amino acid sites were found in the extracellular domain of the SLA-2-HB alleles at sites 23(F), 24(I), 43(A), 44(K), 50(Q), 73(N), 95(I), 114(R) and 216(S), which could be used to differentiate other SLA-2 alleles. The amino acid identities between SLA-2-HB and other SLA-2, SLA-3 and SLA-1 alleles were 87.1-97.0%, 85.0-93.9% and 83.3- 88.6%, respectively. The phylogenetic tree of SLA-2-HB showed that it was relatively independent of the other SLA-2 genes. Furthermore, the SLA-2-HB alleles were similar to HLA-B15 and HLA-A2 functional do- mains and preserved some functional sites of HLA-A2. It was concluded that SLA-2-HB is an allele of SLA-2 and that the Hebao pig might have evolved independently in China.

Disulfide bonds are critical for tissue-nonspecific alkaline phosphatase function revealed by analysis of mutant proteins bearing a C201-Y or C489-S substitution associated with severe hypophosphatasia

Hypophosphatasia (HPP), a rare genetic disease characterized by reduced serum alkaline phosphatase (ALP) activity and failure in bone and tooth mineralization, is caused by mutations in tissue-nonspecific ALP (TNSALP) gene. Two missense mutations (C201Y and C489S, standardized nomenclature) of TNSALP, involved in intra-chain disulfide bonds, were reported in patients diagnosed with perinatal HPP (Taillandier A. et al. Hum. Mutat. 13 (1999) 171–172, Hum. Mutat. 15 (2000) 293). To investigate the role of the disulfide bond in TNSALP, we expressed TNSALP (C201Y) and TNSALP (C489S) in COS-1 cells transiently. Compared with the wild-type enzyme [TNSALP (W)], both the TNSALP mutants exhibited a diminished ALP activity in the cells, where a 66kDa immature form was predominant with a marginal amount of a 80kDa mature form of TNSALP. Detailed studies on Tet-On CHO established cell line expressing TNSALP (W) or TNSALP (C201Y) showed that the 66kDa form of TNSALP (C201Y) exists as a monomer in contrast to a dimer of TNSALP (W). Only a small fraction of the TNSALP (C201Y) reached cell surface as the 80kDa mature form, though most of the 66kDa form was found to be endo-β-N-acetylglucosaminidase H sensitive and rapidly degraded in proteasome following polyubiquitination. Collectively, these results indicate not only that the intra-subunit disulfide bonds are crucial for TNSALP to properly fold and assemble into the dimeric enzyme, but also that the development of HPP associated with TNSALP (C201Y) or TNSALP (C489S) is attributed to decreased cell surface appearance of the functional enzyme.

Disulfide-Driven Assembly and Aggregation of the P2X Receptor

P2X receptors are cation channels gated by extracellular ATP that are formed by the non-covalent assembly of three identical or homologous subunits. All P2X subunits contain ten strictly conserved cysteine residues (denoted here with sequential numbers, C1-C10) in the ectodomain that give rise to five intrachain disulfides per subunit. To examine the role of the cysteine residues in the folding and assembly of the P2X1 receptor, we took advantage of the reducing agent dithiothreitol (DTT) that inhibits formation of disulfides in living cells without perturbing most other functions (Braakman et al. EMBO J. 11, 1717-1722, 1992). in intact X. laevis oocytes, DTT kept newly synthesized P2X1 subunits in their less compact reduced form that aggregated, but did not trimerize or reach the plasma membrane. Upon DTT washout, the preformed P2X1 subunits oxidized into the more compact form and homotrimerized efficiently to form a fully functional ATP-gated receptor that appeared at the plasma membrane. in the mature homotrimeric state, the P2X1 receptor was DTT resistant. The impact of individual cysteine residues on homotrimerization was assessed by studying cysteine-to-serine single mutants. The resulting presence of an odd number of nine cysteine residues in the ectodomain significantly increased aggregate formation. Depending upon which residue was mutated, homotrimerization was impaired (C1S to C6S) or abolished (C7S to C10S), resulting in reduced or absent cell surface expression, respectively. in contrast to the wt rP2X1 receptor, the ectodomain of mutants C1S to C6S contained a maleimide-reactive thiol, indicating invariant accessibility of the unpaired cysteine residue. These results will be discussed in relation to the crystal structure of the zebrafish P2X4 receptor (Kawate et al. Nature 460, 592–599, 2009).

Biogenesis of Weibel-Palade bodies in von Willebrand's disease variants with impaired von Willebrand factor intrachain or interchain disulfide bond formation

Mutations of cysteine residues in von Willebrand factor are known to reduce the storage and secretion of this factor, thus leading to reduced antigen levels. However, one cysteine mutation, p.Cys2773Ser, has been found in patients with type 2A(IID) von Willebrand's disease who have normal plasma levels of von Willebrand factor. We hypothesize that disruption of either intra- or interchain disulfide bonds by cysteine mutations in von Willebrand factor has different effects on the biogenesis of Weibel-Palade bodies. The effect of specific cysteine mutations that either disrupt intrachain (p.Cys1130Phe and p.Cys2671Tyr) or interchain (p.Cys2773Ser) disulfide bonds on storage and secretion of von Willebrand factor was studied by transient transfection of human embryonic kidney cell line 293. Upon expression of von Willebrand factor these cells formed endothelial Weibel-Palade body-like organelles called pseudo-Weibel-Palade bodies. Storage of von Willebrand factor was analyzed with both confocal immunofluorescence and electron microscopy. Regulated secretion of von Willebrand factor was induced by phorbol 12-myristate 13-acetate. p.Cys1130Phe and p.Cys2671Tyr reduced the storage of von Willebrand factor into pseudo-Weibel-Palade bodies with notable retention of von Willebrand factor in the endoplasmic reticulum, whereas p.Cys2773Ser-von Willebrand factor was stored normally. As expected, wild-type von Willebrand factor formed proteinaceous tubules that were seen under electron microscopy as longitudinal striations in pseudo-Weibel-Palade bodies. p.Cys2773Ser caused severe defects in von Willebrand factor multimerization but the factor formed normal tubules. Furthermore, the basal and regulated secretion of von Willebrand factor was drastically impaired by p.Cys1130Phe and p.Cys2671Tyr, but not by p.Cys2773Ser. We postulate that natural mutations of cysteines involved in the formation of interchain disulfide bonds do not affect either the storage in Weibel-Palade bodies or secretion of von Willebrand factor, whereas mutations of cysteines forming intrachain disulfide bonds lead to reduced von Willebrand factor storage and secretion because the von Willebrand factor is retained in the endoplasmic reticulum.

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.

Molecular Analysis of Type 1 VWD Mutations: Effects of Mutant:Wild-Type Transfection Ratio and Protein Degradation

Abstract 1210 Background:Von Willebrand disease (VWD), the most frequently diagnosed bleeding disorder, is characterized by variable expressivity and incomplete penetrance. Bleeding severity in type 1 VWD does not always correlate with plasma VWF levels, except in cases of severe deficiency. It is possible that the phenotypic variability observed in type 1 VWD is related to the final ratio of mutant vs. wild-type (WT) subunits in the mature VWF multimeric structure. The aim of this study was to determine the role of mutant:WT transfection ratio on von Willebrand factor (VWF) expression, secretion and degradation in VWD type 1 mutations. Methods:Type 1 VWD mutations with reported normal multimer distribution were chosen from the D'-D3 region of VWF. Mutations of cysteine residues were eliminated to avoid interference with inter- and intra-chain disulfide linkages. Mutations were generated by performing site-directed mutagenesis on full-length human VWF cDNA within the pcDNA3.1(-)A vector, which appends VWF with a Myc-His tag (denoted mH). The following mutations were generated: M771I, R782Q, R924W, I1094T and T1156M.Mutant VWF was co-transfected with WT VWF contained within the pCIneo vector (mutant mH:WT pCIneo ratios investigated were 1:3, 2:2, 3:1, 4:0). Recombinant (r)VWF expression was measured using ELISA and concentrations were determined by comparison to a standard curve generated with pooled normal plasma. Multimer composition was analyzed using SDS-agarose gel electrophoresis followed by Western blot. Statistical significance was determined using one-way ANOVA with post-hoc Tukey test. Results:Homozygous expression of R924W or I1094T had no effect on rVWF expression or secretion compared to WT, while M771I, R782Q and T1156M significantly increased intracellular protein retention. Co-expression of M771I or R782Q at varying ratios with WT was able to partially correct rVWF secretion, although intracellular retention remained significantly higher than WT at all ratios (n=3, * p<0.05, Figure 1). Co-expression with WT cDNA was also able to correct T1156M retention in a dose-dependent manner (n=3, Figure 1), as described previously [Lethagen, Thromb and Haemost, 2002]. Multimer analysis of co-transfection supernatants exhibited normal and full distribution of multimers, as expected for type 1 VWD mutations. [Display omitted] Others have shown previously that heterodimers of WT and C1149R VWF, a type 1 VWD mutation, are degraded by the proteasome [Bodo et al, Blood, 2001], presumably via recognition of a folding defect within the mutant subunit. In order to determine the role of proteasomal degradation in the decreased secretion levels of our mutants, we performed experiments in the presence of the proteasome inhibitor MG-132. Treatment of co-transfected cells (mutant:WT 2:2) with 1 mM MG-132 for 16 hours prior to harvesting did not significantly affect secretion or overall expression of rVWF, suggesting that this pathway is not involved in the regulation of the expression of our mutants. Discussion:Our data demonstrate that M771I, T1156M and R782Q induce a significant increase in intracellular retention compared to WT protein, which could contribute to a quantitative deficiency in type 1 VWD, while R924W and I1094T do not appear to interfere with VWF production or secretion. Variable levels of intracellular retention have been observed in a previous study of VWF mutations identified in type 1 VWD patients [Eikenboom, et al, J Thromb Haemost, 2009]. While one interpretation of these results is that R924W or I1094T may not be causative mutations in type 1 VWD, other mechanisms including protein clearance and function remain to be explored. Although type 1 VWD mutations variably affect expression and secretion levels in vitro, studying platelet rolling on these mutants at a range of physiological shear stresses will provide valuable information regarding whether the degree of incorporation of mutant subunits into VWF multimers can affect supramolecular structure, and ultimately, hemostatic function. Disclosures:No relevant conflicts of interest to declare.

SPINK1: A New Therapeutic Target in Cancer?

The serine protease inhibitor Kazal type 1 (SPINK1)2 was initially isolated from bovine pancreas by Kazal and colleagues. A similar protease inhibitor was later identified in human pancreatic juice and called “pancreatic secretory trypsin inhibitor.” By means of immunologic techniques, SPINK1 was later identified as a tumor-associated peptide and isolated from the urine of a patient with ovarian cancer. Hence, it was called “tumor-associated trypsin inhibitor” (TATI) (1). This name has been used in most studies of its use as a tumor marker [reviewed in (2)]. TATI was shown to be a serum and urine marker for many types of cancer, but it was not found to be superior to earlier known markers that had been identified for diagnostic use. It was an independent prognostic marker for several types of cancer, however. Tumors producing SPINK1 have been shown to also produce trypsin, and because trypsin activates matrix metalloproteinases, which have been shown to mediate cancer invasion, an association between SPINK1 production and an adverse prognosis in cancer was ascribed to its coproduction with trypsin. SPINK1 was thought to have a role in cancer similar to the one it has in the pancreas, where it protects the pancreas by inhibiting prematurely activated trypsin (2). Mechanisms by which SPINK1 could exert alternative functions were suggested by its structural similarities with epidermal growth factor (EGF): a 50% sequence homology, a molecular size of approximately 6 kDa, and the presence of 3 intrachain disulfide bridges (2). This similarity with EGF prompted studies on the ability of SPINK1 to act as a growth factor. In 1985, SPINK1 was shown to bind to surface receptors and to stimulate DNA synthesis in …

Mutagenesis of Surfactant Protein D Informed by Evolution and X-ray Crystallography Enhances Defenses against Influenza A Virus in Vivo

The recognition of influenza A virus (IAV) by surfactant protein D (SP-D) is mediated by interactions between the SP-D carbohydrate recognition domains (CRD) and glycans displayed on envelope glycoproteins. Although native human SP-D shows potent antiviral and aggregating activity, trimeric recombinant neck+CRDs (NCRDs) show little or no capacity to influence IAV infection. A mutant trimeric NCRD, D325A/R343V, showed marked hemagglutination inhibition and viral neutralization, with viral aggregation and aggregation-dependent viral uptake by neutrophils. D325A/R343V exhibited glucose-sensitive binding to Phil82 hemagglutinin trimer (HA) by surface plasmon resonance. By contrast, there was very low binding to the HA trimer from another virus (PR8) that lacks glycans on the HA head. Mass spectrometry demonstrated the presence of high mannose glycans on the Phil82 HA at positions known to contribute to IAV binding. Molecular modeling predicted an enhanced capacity for bridging interactions between HA glycans and D325A/R343V. Finally, the trimeric D325A/R343V NCRD decreased morbidity and increased viral clearance in a murine model of IAV infection using a reassortant A/WSN/33 virus with a more heavily glycosylated HA. The combined data support a model in which altered binding by a truncated mutant SP-D to IAV HA glycans facilitates viral aggregation, leading to significant viral neutralization in vitro and in vivo. These studies demonstrate the potential utility of homology modeling and protein structure analysis for engineering effective collectin antivirals as in vivo therapeutics.

Identification of the endogenous cysteine-rich peptide trissin, a ligand for an orphan G protein-coupled receptor in Drosophila

There are many orphan G protein-coupled receptors (GPCRs), for which ligands have not yet been identified, in both vertebrates and invertebrates, such as Drosophila melanogaster. Identification of their cognate ligands is critical for understanding the function and regulation of such GPCRs. Indeed, the discovery of bioactive peptides that bind GPCRs has enhanced our understanding of mechanisms underlying many physiological processes. Here, we identified an endogenous ligand of the Drosophila orphan GPCR, CG34381. The purified ligand is a peptide comprised of 28 amino acids with three intrachain disulfide bonds. The preprotein is coded for by gene CG14871. We designated the cysteine-rich peptide “trissin” (it means for triple S–S bonds) and characterized the structure of intrachain disulfide bonds formation in a synthetic trissin peptide. Because the expression of trissin and its receptor is reported to predominantly localize to the brain and thoracicoabdominal ganglion, trissin is expected to behave as a neuropeptide. The discovery of trissin provides an important lead to aid our understanding of cysteine-rich peptides and their functional interaction with GPCRs.

ChemInform Abstract: The Relaxin Peptide Family — Structure, Function and Clinical Applications

AbstractReview: 89 refs.

An infectious clone of the highly pathogenic porcine reproductive and respiratory syndrome virus: Topology of glycoprotein 3 (GP3) addressing the intrachain disulfide bonds

The highly pathogenic porcine reproductive and respiratory virus (hpPRRSV) with discontinuous 30 amino acid (aa) deletion as a gene marker has caused great economic loss in pig industry and since 2007 has become the dominant strain prevalent in China and Vietnam since 2007. Reverse genetics method is a powerful tool urgently needed to be used on the hpPRRSV to study the intriguing molecular mechanism of transcription, replication and the virulence determinant factors. In our study, we successfully constructed a full-length infectious clone, prBJSY07, based on hpPRRSV isolate, BJSY07. The rescued virus, vrBJSY07, showed similar growth characters to those of the parental virus, BJSY07. We also found that a rescued virus vBJSY07 generated from pBJSY07 was viable but displayed decreased and delayed reproductive capacity, which might be caused by two amino acids mutations, S83C and S117C in glycoprotein 3 (GP3), acquired during the preparation of the infectious clone. The topology of the wild type GP3 and the mutant were further analyzed by bioinformatics and revealed that the mutated GP3 possessed slightly altered structure, most likely by forming a new disulfide bond between the two new cysteine residues. As GP3 is a cysteine-rich glycoprotein, common for viral glycoproteins, our results show that GP3 can accommodate even more cysteine mutations. Based on this, a topological model of GP3 is proposed by addressing the intrachain disulfides.

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.

Photolysis of Recombinant Human Insulin in the Solid State: Formation of a Dithiohemiacetal Product at the C-Terminal Disulfide Bond

Exposure of protein pharmaceuticals to light can result in chemical and physical modifications, potentially leading to loss of potency, aggregation, and/or immunogenicity. To correlate these potential consequences with molecular changes, the nature of photoproducts and their mechanisms of formation must be characterized. The present study focuses on the photochemical degradation of insulin in the solid state. Solid insulin was characterized by solid-state NMR, polarized optical microscopy and scanning electron microscopy; various insulin preparations were exposed to UV light prior to product analysis by mass spectrometry. UV-exposure of solid human insulin results in photodissociation of the C-terminal intrachain disulfide bond, leading to formation of a CysS(•) thiyl radical pair which ultimately disproportionates into thiol and thioaldehyde species. The high reactivity of the thioaldehyde and proximity to the thiol allow the formation of a dithiohemiacetal structure. Dithiohemiacetal is formed during the UV-exposure of both crystalline and amorphous insulin. Dithiohemiacetals represent novel structures generated through the photochemical modification of disulfide bonds. This is the first time that such structure is identified during the photolysis of a protein in the solid state.

Decreasing the configurational entropy and the hydrophobicity of EBV-derived peptide 11389 increased its antigenicity, immunogenicity and its ability of inducing IL-6

Peptide 11389 from CD21-binding region of EBV-gp350/220 protein binds to PBMCs inducing IL-6 expression and inhibiting EBV-binding to PBMCs. In addition, anti-peptide 11389 antibodies recognize EBV-infected cells and inhibit both EBV infection and IL-6 production in PBMCs. We have postulated that native structure stabilization of peptide 11389 sequence can increase its biological activity. The strategy was to modify its sequence to restrict the number of structures that peptide 11389 could acquire in solution (decreasing peptide's configurational entropy) and to weaken the non-relevant intermolecular interactions (decreasing its hydrophobicity), preserving CD21-interacting residues and structure as displayed in the native protein. Thirteen analog peptides were designed and synthesized; most of them were monomers containing an intra-chain disulfide bridge. Analog peptides 34058, 34060, 34061, 34296, 34298, 34299 and 34300 inhibited EBV invasion of PBMCs. Peptides 34059, 34060, 34295 and 34297 induced IL-6 levels in PBMCs (EC50=3.4, 3.3, 0.5, 0.5 μM, respectively) at higher potency than peptide 11389 (EC50=5.8 μM). Peptides 34057, 34059, 34060, 34301 and 34302 interacted with anti-EBV antibodies with affinities from 3 to 50 times higher than peptide 11389. Most of analog peptides were highly immunogenic and elicited antibodies that cross-react with EBV. In conclusion, we have designed peptides displaying higher biological activity than peptide 11389.

A novel lectin from fresh rhizome of Alisma orientale ( Sam.) Juzep

A novel lectin with a molecular mass of 22.5 kDa, designated as AOL, has been isolated from fresh rhizome of Alisma orientale ( Sam.) Juzep. The purification procedure involved ammonium sulfate precipitation, affinity chromatography on Affi-gel blue gel and gel filtration chromatography on Sephacryl S200. AOL was a monomer with intrachain disulfide bond; its isoelectric point was estimated to be 5.6. The lectin could be stained in red by periodic acid-Schiff (PAS) reagent, it consisted of 93.7% protein and 6.3% sugar. N-terminal amino acid sequence of AOL was determined to be EAVVDTNGDLIHTGSIH. AOL exhibited hemagglutinating activity in human erythrocytes. The hemagglutinating activity was mostly preserved up to 50 °C and was indiscernible when the temperature above 70 °C. It was stable over the pH range 2–11. Among the carbohydrates tested, l-arabinose, mannitol, d-xylose and l-rhamnose were capable of inhibiting the hemagglutinating activity. AOL was a metal dependent protein, metallic compounds including LiCl, FeCl 3, MnCl 2 and K 2Cr 2O 7 inhibited the activity whereas MgCl 2, ZnCl 2, BaCl 2, CuCl 2 and CaCl 2 did not affect its activity, which means all these metallic compounds cannot promote the activity of AOL. Proliferation of tumor cells HeLa cells was inhibited by AOL with an IC 50 of 7.3 μM, HepG2 cells and the normal cells MDCK was just slightly affected even at the concentration of 40 μM. AOL did not exhibit antifungal activity.

Deficiency in Ab response to insulin in gamma-IFN-inducible lysosomal thiol reductase (GILT) knock-out mice (100.29)

Abstract The γ-IFN-inducible lysosomal thiol reductase (GILT) is a unique thiol reductase with optimal activity at low pH, and is expressed in Ag presenting cells (APC). GILT is critical to processing protein Ag by catalyzing disulfide bond reduction, thus facilitating the unfolding of native Ag containing disulfide bonds for further processing. GILT-/- mice are defective in processing exogenous protein Ag. However the role of GILT in Ab response is not clear. Here we report that after immunization, GILT-/- mice failed to produce Ab to exogenous bovine insulin (bIns), which has A and B chains and contains both inter and intra-chain disulfide bonds, while Ab response to OVA was normal in both GILT-/- and wt B6 mice. GILT-/- APC was able to present bIns to an insulin B chain peptide-specific T hybridoma, although it produced less amounts of IL2, compared to when using B6 APC. IL2 production was MHC-II mediated (could be blocked by α-mouse, but not α-human, MHC-II Ab) and the reduced IL2 production of GILT-/- cells was bIns-specific since GILT-/- APC could not present HEL, another Ag containing disulfide bonds, to an HEL-specific T hybridoma to produce IL2. This data indicates that the T cell response to bIns is only partially affected in GILT-/- mice, which was further supported by the fact that CFSE labeled, in vivo bIns primed, GILT-/- CD4 cells proliferated equally well as wt B6 CD4 cells upon restimulation by bIns. Our data indicates a novel role of GILT in Ab response to insulin.

The natural occurrence of human fibrinogen variants disrupting inter-chain disulfide bonds (A Cys36Gly, A Cys36Arg and A Cys45Tyr) confirms the role of N-terminal A disulfide bonds in protein assembly and secretion

Analyses of site-directed fibrinogen mutants expressed in several recombinant models have previously shown that both inter- and intra-chain disulfide bonds are critical for fibrinogen assembly and secretion. Four naturally occurring mutations on AαCys36 and AαCys45 residues are reported here to be associated with decreased fibrinogen levels. This confirms the main role of the AαCys36-BβCys65 and AαCys45-γCys23 disulfide bonds in reaching a normal fibrinogen plasma level. Decreased coagulant/antigen ratios indicate abnormal species secretion in heterozygous subjects which varies between individuals. However, in contrast to overexpression in experimental models, disruption of the AαCys36-BβCys65 disulfide bond did not result in the appearance of Aα-Bβ-γ moieties in vivo. A 188 kDa molecule reacting only with anti Aα and anti Bβ chains was found in the plasma of the AαCys45Tyr variant. Heterozygous carriers of Aα chain mutations usually have normal fibrinogen levels, in contrast to the AαCys36Gly, AαCys36Arg and AαCys45Tyr variants that are shown here to cause hypofibrinogenemia.