Single Disulphide Bond Research Articles

Redox homeostasis in Mycobacterium tuberculosis is modulated by a novel actinomycete‐specific transcription factor

Mycobacterium tuberculosis (Mtb) has evolved diverse cellular processes in response to the multiple stresses it encounters within the infected host. We explored available TnSeq datasets to identify transcription factors (TFs) that are essential for Mtb survival inside the host. The analysis identified a single TF, Rv1332 (AosR), conserved across actinomycetes with a so‐far uncharacterized function. AosR mitigates phagocyte‐derived oxidative and nitrosative stress, thus promoting mycobacterial growth in the murine lungs and spleen. Oxidative stress induces formation of a single intrasubunit disulphide bond in AosR, which in turn facilitates AosR interaction with an extracytoplasmic‐function sigma factor, SigH. This leads to the specific upregulation of the CysM‐dependent non‐canonical cysteine biosynthesis pathway through an auxiliary intragenic stress‐responsive promoter, an axis critical in detoxifying host‐derived oxidative and nitrosative radicals. Failure to upregulate AosR‐dependent cysteine biosynthesis during the redox stress causes differential expression of 6% of Mtb genes. Our study shows that the AosR‐SigH pathway is critical for detoxifying host‐derived oxidative and nitrosative radicals to enhance Mtb survival in the hostile intracellular environment.

Escherichia coli Lipopolysaccharide Modulates Biological Activities of Human-β-Defensin Analogues but Not Non-Ribosomally Synthesized Peptides.

Human-β-defensins (HBD1-3) are antibacterial peptides containing three disulphide bonds. In the present study, the effect of Escherichia coli lipopolysaccharide (LPS) on the antibacterial activities of HBD2-3, C-terminal analogues having a single disulphide bond, Phd1-3, and their corresponding myristoylated analogues MPhd1-3 were investigated. The effect of LPS on the activities of linear amphipathic peptides melittin, LL37 and non-ribosomally synthesized peptides, polymyxin B, alamethicin, gramicidin A, and gramicidin S was also examined. The antibacterial activity of HBD 2-3, Phd1-3, and MPhd1-3 in the presence of LPS against E. coli and Staphylococcus aureus was inhibited. While LPS inhibited the antibacterial activity of LL37, the inhibition of melittin activity was partial. The hemolytic activity exhibited by MPhd1, MPhd3, melittin, and LL37 was inhibited in the presence of LPS. HBD2-3, Phd1-3, and MPhd1-3 also showed endotoxin neutralizing activity. The antibacterial and hemolytic activities of polymyxin B, alamethicin, gramicidin A, and gramicidin S were not inhibited in the presence of LPS. Fluorescence assays employing dansyl cadaverine showed that HBD2-3 and defensin analogues bind to LPS more strongly as compared to alamethicin, gramicidin A, and gramicidin S. Electron microscopy images indicated that peptides disintegrate the structure of LPS. The inhibition of the antibacterial activity of native defensins and analogues in the presence of LPS indicates that the initial interaction with the bacterial surface is similar. The native defensin sequence or structure is also not essential, although cationic charges are necessary for binding to LPS. Hydrophobic interaction is the main driving force for association of non-ribosomally synthesized polymyxin B, alamethicin, gramicidin A, and gramicidin S with LPS. It is likely that these peptides rapidly insert into membranes and do not interact with the bacterial cell surface, whereas cationic peptides such as β-defensin and their analogues, melittin and LL37, first interact with the bacterial cell surface and then the membrane. Our results suggest that evaluating interaction of antibacterial and hemolytic peptides with LPS is a compelling way of elucidating the mechanism of bacterial killing or hemolysis.

One step Purification and Characterisation of Abrin Toxin from Abrus Precatorius Seeds

Abrin is a plant toxin obtained from Abrus precatorius seeds. It belongs to the type II ribosomal inactivating proteins (RIPs) consisting of two chains namely, catalytically active A chain and sugar binding B chain linked by a single disulphide bond. Due to high toxicity of abrin, its exposure or consumption can lead to serious public health problems. In the present work, we have extracted and purified the abrin toxin from Abrus precatorius seeds. The toxin was purified using a single step anion exchange chromatography. The purified protein was characterized by SDS-PAGE and MALDI- TOF to confirm its purity. The toxicity of purified abrin toxin was also confirmed by injecting the toxin in mice. The purified protein was further used to raise antibodies in mice and characterized by indirect Enzyme Linked Immunosorbent Assay. The results of present study established the use of single step ion exchange chromatography to purify abrin toxin for further development of its detection system.

Substitution of cysteines in the yeast viral killer toxin K1 precursor reveals novel insights in heterodimer formation and immunity

The killer toxin K1 is a virally encoded fungal A/B toxin acting by disrupting plasma membrane integrity. The connection of α and β constitutes a critical feature for toxin biology and for decades the formation of three disulphide bonds linking the major toxin subunits was accepted as status quo. Due to the absence of experimental evidence, the involvement of each cysteine in heterodimer formation, K1 lethality and immunity was systematically analysed. Substitution of any cysteine in α led to a complete loss of toxin dimer secretion and toxicity, whereas K1 toxin derivatives carrying mutations of C248, C312 or the double mutation C248-312 were active against spheroplasted cells. Importantly, substitution of the C95 and C107 in the toxin precursor completely abolished the mediation of functional immunity. In contrast, K1 toxicity, i.e. its ionophoric effect, does not depend on the cysteine residues at all. In contrast to the literature, our data imply the formation of a single disulphide bond involving C92 in α and C239 in β. This finding not only refines the current model stated for decades but also provides new opportunities to elucidate the mechanisms underlying K1 toxicity and immunity at the molecular level.

Subcellular Trafficking and Functional Relationship of the HSV-1 Glycoproteins N and M.

The herpes simplex virus type 1 (HSV-1) glycoprotein N (gN/UL49.5) is a type I transmembrane protein conserved throughout the herpesvirus family. gN is a resident of the endoplasmic reticulum that in the presence of gM is translocated to the trans Golgi network. gM and gN are covalently linked by a single disulphide bond formed between cysteine 46 of gN and cysteine 59 of gM. Exit of gN from the endoplasmic reticulum requires the N-terminal core of gM composed of eight transmembrane domains but is independent of the C-terminal extension of gM. Co-transport of gN and gM to the trans Golgi network also occurs upon replacement of conserved cysteines in gM and gN, suggesting that their physical interaction is mediated by covalent and non-covalent forces. Deletion of gN/UL49.5 using bacterial artificial chromosome (BAC) mutagenesis generated mutant viruses with wild-type growth behaviour, while full deletion of gM/UL10 resulted in an attenuated phenotype. Deletion of gN/UL49.5 in conjunction with various gM/UL10 mutants reduced average plaque sizes to the same extent as either single gM/UL10 mutant, indicating that gN is nonessential for the function performed by gM. We propose that gN functions in gM-dependent as well as gM-independent processes during which it is complemented by other viral factors.

A capillary electrophoretic–mass spectrometric method for the assessment of octreotide stability under stress conditions

A capillary zone electrophoretic-electrospray ion trap mass spectrometric method has been developed to assess the stability and pathways of degradation of the cancer therapeutic octapeptide, octreotide. As a somatostatin analogue, octreotide contains a single disulphide bond linking Cys2–Cys7 with the structure of ▪. Resolution of octreotide from its degradation products was achieved using a capillary zone electrophoretic method with bare fused silica capillaries, a 10mM ammonium formate buffer, pH 3.20, at 25°C and an applied voltage of 25kV. An ion trap low energy collision induced dissociation procedure was applied for the characterization of the chemical structures of the degradation products derived from an acidic, alkaline, neutral and thermal solution treatment of octreotide. The results so obtained indicated that linear octreotide degradation products were formed under acidic and alkaline conditions, due to the hydrolysis of a ring amide bond and a hitherto unknown desulfurization of the Cys–Cys disulfide bond, respectively. Degradation under neutral conditions occurred via cleavage of the exocyclic N-((2R,3R)-1,3-dihydroxybutan-2-yl) amide bond which also preceded the ring amide hydrolysis under acidic conditions. The developed method was further successfully applied to assess the kinetics of these octreotide degradations. Overall, this method is suitable for the rapid and precise assessment of the stability and quality control of octreotide as a synthetic peptide-based pharmaceutical product, and has led to the discovery of a new Cys–Cys disulfide degradation pathway.

Reanalysis of parabiosis of obesity mutants in the age of leptin

In this study we set out to explain the differing effects of parabiosis with genetically diabetic (db) mice versus administration of recombinant leptin. Parabiosis of db mutant, which overexpress leptin, to wildtype (WT) or genetically obese (ob) mice has been reported to cause death by starvation, whereas leptin infusions do not produce lethality at any dose or mode of delivery tested. Leptin is not posttranslationally modified other than a single disulphide bond, raising the possibility that it might require additional factor(s) to exert the maximal appetite-suppressing effect. We reconfirmed the lethal effect of parabiosis of db mutant on WT mice and further showed that this lethality could not be rescued by administration of ghrelin or growth hormone. We then initiated a biochemical fractionation of a high-molecular-weight leptin complex from human plasma and identified clusterin as a major component of this leptin-containing complex. However, in contrast to previous reports, we failed to observe a leptin-potentiating effect of either exogenous or endogenous clusterin, and parabiosis of db clusterin(-/-) double-mutant to WT mice still caused lethality. Intriguingly, in parabiotic pairs of two WT mice, leptin infusion into one of the mice led to an enhanced starvation response during calorie restriction as evidenced by increased plasma ghrelin and growth-hormone levels. Moreover, leptin treatment resulted in death of the parabiotic pairs. These data suggest that the appetite suppression in WT mice after parabiosis to db mutants is the result of induced hyperleptinemia combined with the stress or other aspect(s) of the parabiosis procedure.

RTA-specific monoclonal antibodies neutralize ricin toxin intracellularly (53.13)

Abstract According to the current paradigm, antibodies neutralize protein toxins by either preventing toxin binding to cell surfaces or promoting toxin clearance via Fc-receptor mediated uptake. In the case of the biothreat agent ricin, although this toxin uses its B-subunit (RTB) to gain cell entry the majority of antibodies are produced against its enzymatic A-subunit (RTA). We have previously demonstrated using a collection of monoclonal antibodies (mAbs) against RTA that neutralizing activity and protection in a mouse model is associated with epitope specificity, and not mAb affinity or Ig subclass. We now put forth evidence to indicate that at least two RTA-specific mAbs, R70 and IB2, neutralize ricin intracellulary. Neither R70 nor IB2 prevented ricin binding to cell surfaces, as measured by flow cytometry. Moreover, R70 (and likely IB2) retained toxin neutralizing activity in a Vero cell assay when added after ricin had attached to cell surfaces. Using in vitro assays, we also found that R70 and IB2 affected the ability of protein disulfide isomerase to reduce the single disulphide bond linking RTA to RTB and interfered with RTA’s capacity to arrest protein synthesis. We are currently using a combination of techniques to define more precisely the mechanism by which these and other RTA-specific mAbs neutralize ricin.

Binding of peptides corresponding to the carboxy-terminal region of human-β-defensins-1–3 with model membranes investigated by isothermal titration calorimetry

Human-β-defensins HBD-1–3 are important components of the innate immune system. Synthetic peptides Phd-1–3 with a single disulphide bond, spanning the cationic C-terminal region of HBD-1–3, have antimicrobial activity. The interaction of Phd-1–3 with model membranes was investigated using isothermal titration calorimetry (ITC) and steady-state fluorescence polarization to understand the biophysical basis for the mechanism of antimicrobial action. Calorimetric titration of POPE:POPG (7:3) vesicles with peptides at 25°C and 37°C showed complex profiles with two distinct regions of heat changes. The data indicate binding of Phd-1–3 at 37°C to both negative and zwitterionic lipid vesicles is exothermic with low enthalpy values (ΔH~−1.3 to −2.8kcal/mol) as compared to amphipathic helical antibacterial peptides. The adsorption of peptides to negatively charged lipid membranes is modulated by electrostatic interactions that are described by surface partition equilibrium model using Gouy–Chapman theory. However, this model could not explain the isotherms of peptide binding to zwitterionic lipid vesicles. Fluorescence polarization of TMA-DPH (1-[4-(trimethylammonio) phenyl]-6-phenyl-1,3,5-hexatriene) and DPH (1,6-diphenyl-1,3,5-hexatriene) located in the head group and acyl chain region respectively, indicates that the peptides interact with interfacial region of negatively charged membranes. Based on the results obtained, we conclude that adsorption of cationic peptides Phd-1–3 on lipid surface do not result in conformational change or pore formation. It is proposed that interaction of Phd-1–3 with the negatively charged lipid head group causes membrane destabilization, which in turn affects the efficient functioning of cytoplasmic membrane proteins in bacteria, resulting in cell death.

Cupryphans, metal‐binding, redox‐active, redesigned conopeptides

Contryphans are bioactive peptides, isolated from the venom of marine snails of the genus Conus, which are characterized by the short length of the polypeptide chain and the high degree of unusual post-translational modifications. The cyclization of the polypeptide chain through a single disulphide bond, the presence of two conserved Pro residues, and the epimerization of a Trp/Leu residue confer to Contryphans a stable and well-defined structure in solution, conserved in all members of the family, and tolerant to multiple substitutions. The potential of Contryphans as scaffolds for the design of redox-active (macro)molecules was tested by engineering a copper-binding site on two different variants of the natural peptide Contryphan-Vn. The binding site was designed by computational modeling, and the redesigned peptides were synthesized and characterized by optical, fluorescence, electron spin resonance, and nuclear magnetic resonance spectroscopy. The novel peptides, named Cupryphan and Arg-Cupryphan, bind Cu(2+) ions with a 1:1 stoichiometry and a K(d) in the 100 nM range. Other divalent metals (e.g., Zn(2+) and Mg(2+)) are bound with much lower affinity. In addition, Cupryphans catalyze the dismutation of superoxide anions with an activity comparable to other nonpeptidic superoxide dismutase mimics. We conclude that the Contryphan motif represents a natural robust scaffold which can be engineered to perform different functions, providing additional means for the design of catalytically active mini metalloproteins.

Interplays Between Covalent Modifications in the Endoplasmic Reticulum Increase Conformational Diversity in Nascent Prion Protein

Prion protein (PrP), the causative agent of Transmissible Spongiform Encephalopathies, is synthesized in the endoplasmic reticulum (ER) where it undergoes numerous covalent modifications. Here we investigate the interdependence and regulation of PrP oxidative folding, N-glycosylation, and GPI addition in diverse ER conditions. Our results show that formation of the single disulphide bond is a pivotal event, essential for PrP transport, and can occur post-translationally. Retarding its formation enhances N-glycosylation and GPI-anchoring. In contrast, lowering ER Ca2+ concentration inhibits N-glycosylation and GPI-anchoring. These data reveal tight interplays between the different ER covalent modifications, which collectively increase of PrP conformational diversity and may be important for its propagation.

Characterisation of the thiol–disulphide chemistry of desmopressin by LC, μ-LC, LC-ESI-MS and Maldi-Tof

To date, the majority of therapeutic peptides and proteins have to be administered via parenteral routes, which are painful and inconvenient. In order to gain sufficient high blood concentrations after oral application, various barriers in the gastrointestinal tract have to be overcome. Apart from a poor membrane uptake and intense enzymatic degradation, this study will demonstrate that thiol-disulphide reactions are an underestimated essential part of the presystemic metabolism. Glutathione, integrative part of the antioxidant defence system in the gastrointestinal tract, may play an important role in the inactivation of orally given peptides and proteins. In order to verify this hypothesis, desmopressin which bears a single disulphide bond was used as model peptide drug. Desmopressin was incubated with GSH in various concentrations, and the extent of thiol/disulphide exchange reactions between the peptide drug and GSH was investigated in dependence on pH and ratio of reactants determined as a function of time via HPLC, LC-MS and Maldi-Tof-MS analyses. Results showed that desmopressin is degraded by 1% reduced glutathione at pH 4 and pH 5.5. In presence of 0.01%, 0.1% and 1% of reduced glutathione 6.1%, 19.4% and 52.1% of desmopressin, respectively, were degraded. The masses of the conjugates after deconvolution measured by liquid chromatography and electrospray ionisation mass spectrometric detection were m/z 1069.67, m/z 1376.50, m/z 1683.40 and m/z 2138. These molecular masses, confirmed by Maldi-Tof-MS analysis, correspond with the masses of conjugates expected in theory. Under defined conditions, these results reveal that thiol-disulphide exchange reactions have a considerable impact on the alteration of peptide drugs and proteins.

Dynamics in the Unfolded State of β 2-microglobulin Studied by NMR

Many proteins form amyloid-like fibrils in vitro under conditions that favour the population of partially folded conformations or denatured state ensembles. Characterising the structural and dynamic properties of these states is crucial towards understanding the mechanisms of self-assembly in amyloidosis. The aggregation of beta2-microglobulin (beta2m) into amyloid fibrils in vivo occurs in the condition known as dialysis-related amyloidosis (DRA) and the protein has been shown to form amyloid-like fibrils under acidic conditions in vitro. We have used a number of 1H-15N nuclear magnetic resonance (NMR) experiments in conjunction with site-directed mutagenesis to study the acid-unfolded state of beta2m. 15N NMR transverse relaxation experiments reveal that the acid-denatured ensemble, although predominantly unfolded at the N and C termini, contains substantial non-native structure in the central region of the polypeptide chain, stabilised by long-range interactions between aromatic residues and by the single disulphide bond. Relaxation dispersion studies indicate that the acid-unfolded ensemble involves two or more distinct species in conformational equilibrium on the micro- to millisecond time-scale. One of these species appears to be hydrophobically collapsed, as mutations in an aromatic-rich region of the protein, including residues that are solvent-exposed in the native protein, disrupt this structure and cause a consequent decrease in the population of this conformer. Thus, acid-unfolded beta2m consists of a heterogeneous ensemble of rapidly fluctuating species, some of which contain stable, non-native hydrophobic clusters. Given that amyloid assembly of beta2m proceeds with lag kinetics under the conditions of this study, a rarely populated species such as a conformer with non-native aromatic clustering could be key to the initiation of amyloidosis.

Critical cysteine residues for regulation of integrin alphaIIbbeta3 are clustered in the epidermal growth factor domains of the beta3 subunit.

Chemical or enzymic reduction/oxidation of integrin cysteine residues (e.g. by reducing agents and protein disulphide isomerase) may be a mechanism for regulating integrin function. It has also been proposed that unique cysteine residues in the integrin beta3 subunit are involved in the regulation of alphaIIbbeta3. In the present study, we studied systematically the role of disulphide bonds in beta3 on the ligand-binding function of alphaIIbbeta3 by mutating individual cysteine residues of beta3 to serine. We found that the disulphide bonds that are critical for alphaIIbbeta3 regulation are clustered within the EGF (epidermal growth factor) domains. Interestingly, disrupting only a single disulphide bond in the EGF domains was enough to activate alphaIIbbeta3 fully. In contrast, only two (of 13) disulphide bonds tested outside the EGF domains activated alphaIIbbeta3. These results suggest that the disulphide bonds in the EGF domains should be intact to keep alphaIIbbeta3 in an inactive state, and that there is no unique cysteine residue in the EGF domain critical for regulating the receptor. The cysteine residues in the EGF domains are potential targets for chemical or enzymic reduction.

Solution Structure of the Third TB Domain from LTBP1 Provides Insight into Assembly of the Large Latent Complex that Sequesters Latent TGF-β

Almost all TGF-β is secreted as part of a large latent complex. This complex is formed from three molecules, a latent transforming growth factor-β binding protein (LTBP), which plays roles in targeting and activation, a latency associated peptide (LAP), which regulates latency, and the TGF-β cytokine. LAP is the TGF-β pro-peptide that is cleaved intracellularly prior to secretion, and TGF-β binds non-covalently to LAP. Formation of the large latent complex is important for the efficient secretion of TGF-β. Previous studies have revealed that the LTBP–LAP interaction is mediated by intracellular exchange of a single disulphide bond within the third, and only the third, TB domain (TB3) with LAP. We have previously reported the structure of a homologous TB domain from fibrillin-1. However, TB3 contains a two amino acid insertion, not found in fibrillin-1 TB domains, which is not amenable to molecular modelling. In order to clarify the basis of TB domain function, we have determined the solution NMR structure of TB3LTBP1. Comparison with the fibrillin-1 TB domain reveals that the two-residue insertion is associated with a significant increase in solvent accessibility of one of the disulphide bonds (linking the second and sixth cysteine residues). Site-directed mutagenesis and NMR studies indicate that this is the only disulphide bond that can be removed without perturbing the TB domain fold. Furthermore, a ring of negatively charged residues has been identified that surrounds this disulphide bond. Homology modelling suggests that the surface properties of TB3 domains from different LTBP isoforms correlate with binding activities. This research provides testable hypotheses regarding the molecular basis of complex formation between LTBPs and LAPs.

PrP knock-out and PrP transgenic mice in prion research.

Spongiform encephalopathies such as scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle or Creutzfeldt-Jacob disease (CJD) and Gerstmann-Sträussler-Scheinker syndrome (GSS) in humans is caused by a transmissible agent designated prion. The 'protein only' hypothesis proposes that the prion consists partly or entirely of a conformational isoform of the normal host protein PrP(C), designated PrP(*)(1) and that the abnormal conformer, when introduced into the organism, causes the conversion of PrP(C) into a likeness of itself. PrP(*) may be congruent with PrP(Sc), a protease-resistant, aggregated conformer of PrP that accumulates mainly in brain of almost all prion-infected organisms. PrP(C) consists of a flexible N-terminal half, comprising Cu(2+)-binding octapeptide repeats, and a globular domain consisting of three alpha-helices, one short antiparallel beta-sheet and a single disulphide bond. It is anchored at the outer cell-surface by a glycosyl phosphatidylinositol (GPI) tail and is present in almost all tissues, however, mainly in brain. Compelling linkage between the prion and PrP was established by biochemical and genetic data and led to the prediction that animals devoid of PrP should be resistant to experimental scrapie and fail to propagate infectivity. This prediction was indeed borne out, adding substantial support to the 'protein only' hypothesis. In addition, the availability of PrP knock-out mice provided an approach to carry out reverse genetics on PrP, both in regard to prion disease and to its physiological role.

Molecular cloning and biochemical characterization of rabbit factor XI

Human factor XI, a plasma glycoprotein required for normal haemostasis, is a homodimer (160 kDa) formed by a single interchain disulphide bond linking the Cys-321 of each Apple 4 domain. Bovine, porcine and murine factor XI are also disulphide-linked homodimers. Rabbit factor XI, however, is an 80 kDa polypeptide on non-reducing SDS/PAGE, suggesting that rabbit factor XI exists and functions physiologically either as a monomer, as does prekallikrein, a structural homologue to factor XI, or as a non-covalent homodimer. We have investigated the structure and function of rabbit factor XI to gain insight into the relation between homodimeric structure and factor XI function. Characterization of the cDNA sequence of rabbit factor XI and its amino acid translation revealed that in the rabbit protein a His residue replaces the Cys-321 that forms the interchain disulphide linkage in human factor XI, explaining why rabbit factor XI is a monomer in non-reducing SDS/PAGE. On size-exclusion chromatography, however, purified plasma rabbit factor XI, like the human protein and unlike prekallikrein, eluted as a dimer, demonstrating that rabbit factor XI circulates as a non-covalent dimer. In functional assays rabbit factor XI and human factor XI behaved similarly. Both monomeric and dimeric factor XI were detected in extracts of cells expressing rabbit factor XI. We conclude that the failure of rabbit factor XI to form a covalent homodimer due to the replacement of Cys-321 with His does not impair its functional activity because it exists in plasma as a non-covalent homodimer and homodimerization is an intracellular process.

Role of the single disulphide bond of beta(2)-microglobulin in amyloidosis in vitro.

The aggregation of beta(2)-microglobulin (beta(2)m) into amyloid fibrils occurs in the condition known as dialysis-related amyloidosis (DRA). The protein has a beta-sandwich fold typical of the immunoglobulin family, which is stabilized by a highly conserved disulphide bond linking Cys25 and Cys80. Oxidized beta(2)m forms amyloid fibrils rapidly in vitro at acidic pH and high ionic strength. Here we investigate the role of the single disulphide bond of beta(2)m in amyloidosis in vitro. We show that reduction of the disulphide bond destabilizes the native protein such that non-native molecules are populated at neutral pH. These species are prone to oligomerization but do not form amyloid fibrils when incubated for up to 8 mo at pH 7.0 in 0.4 M NaCl. Over the pH range 4.0-1.5 in the presence of 0.4 M NaCl, however, amyloid fibrils of reduced beta(2)m are formed. These fibrils are approximately 10 nm wide, but are shorter and assemble more rapidly than those produced from the oxidized protein. These data show that population of non-native conformers of beta(2)m at neutral pH by reduction of its single disulphide bond is not sufficient for amyloid formation. Instead, association of one or more specific partially unfolded molecules formed at acid pH are necessary for the formation of beta(2)m amyloid in vitro. Further experiments will now be needed to determine the role of different oligomeric species of beta(2)m in the toxicity of the protein in vivo.

Kell, Kx and the McLeod syndrome

The antigens of the Kell blood group system are carried on a 93 kDa type II glycoprotein encoded by a single gene on chromosome 7 at 7q33. XK is a 50·9 kDa protein that traverses the membrane ten times and derives from a single gene on the X chromosome at Xp21. A single disulphide bond, Kell Cys 72-XK Cys 347, links Kell to XK. The Kell component of the Kell/XK complex is important in transfusion medicine since it is a highly polymorphic protein, carrying over 23 different antigens, that can cause severe reactions if mismatched blood is transfused and in pregnant mothers antibodies to Kell may elicit serious fetal and neonatal anaemia. The different Kell phenotypes are all caused by base mutations leading to single amino acid substitutions. By contrast the XK component carries a single blood group antigen, termed Kx. The physiological functions of Kell and XK have not been fully elucidated but Kell is a zinc endopeptidase with endothelin-3-converting enzyme activity and XK has the structural characteristics of a membrane transporter. Lack of Kx, the McLeod phenotype, is associated with red cell acanthocytosis, elevated levels of serum creatine phosphokinase and late onset forms of muscular and neurological defects.

High-resolution structure of a potent, cyclic proteinase inhibitor from sunflower seeds

Proteinaceous serine proteinase inhibitors are widespread throughout the plant kingdom where they play an important role in protection against pests and pathogens. Here, we describe the isolation and characterisation of a novel 14 amino acid residue cyclic peptide from sunflower seeds, which is a potent inhibitor of trypsin ( K i = 100 pM). The crystal structure of this peptide in complex with bovine β-trypsin shows both sequence and conformational similarity with the trypsin-reactive loop of the Bowman-Birk family of serine proteinase inhibitors. This inhibitor, however, is unique in being monofunctional, cyclic and far shorter (14 amino acid residues) than inhibitors belonging to this family (typically 60–70 amino acid residues). The high potency of this peptide is likely to arise from the considerable structural rigidity achieved through its cyclic nature which is further stabilised by a single internal disulphide bond. This study helps delineate the minimal unit required for effective peptide inhibitors of serine proteinases, and will assist in the further design of inhibitors to this widespread class of enzymes.