A novel strategy for the synthesis of the cysteine-rich protective antigen of the malaria merozoite surface protein (MSP-1)

The most promising antigen for a protective malaria vaccine is a cysteine-rich domain at the carboxyl terminus of the merozoite surface protein (MSP-1). Passive transfer of anti-MSP-1 antibody or immunization of MSP-1 against infection challenge confers protection in primate and rodent models. The antigen belongs to the three-disulfide epidermal growth factor (EGF) family based on the alignment of the six cysteines. In the K1 strain there are, however, only four cysteines corresponding to the four carboxyl cysteines of EGF. Furthermore, disulfide pairing would produce a non-EGF pattern. Because this cysteine-rich antigen is conformation-dependent, and reduction of the disulfide bonds abolishes antigenicity, we used a synthetic analog to investigate the probable disulfide pairing of this antigen. This paper describes the synthesis, folding and disulfide pairings of two 50-residue cysteine-rich peptides. One contains two disulfides (VK-50) derived from the native sequence of MSP-1 of the Thailand K1 strain (aa 1629–1679). The other contains an EGF-like, three-disulfide [Cys-9,14]VK-50 peptide. Both peptides were synthesized by a solid-phase method using Fmoc-chemistry. The crude peptide of VK-50 was folded, and the disulfide was oxidized by the DMSO method to obtain a structure with an expected disulfide pairing of 3–4, and 5–6. The specific pairing pattern of 1–3, 2–4 and 5–6 in [Cys 9,14]VK-50 corresponding to EGF in [Cys 9,14]VK-50 was obtained using a ‘knowledge-based’ (KB) strategy for their formation. Purified VK-50 and [Cys-9,14]VK-50 had the correct molecular weight, as shown by Cf-252 fission ionization mass-spectrometry. The disulfide pairings were confirmed by enzymatic digestion. These two peptides can be conjugated to the multiple peptide antigen core for immunization. The immunological results will allow us to conclude the correct disulfide pairing and the conformational importance of this antigen.     

Chemical synthesis of β‐defensins and LEAP‐1/hepcidin

Abstract: A large and steadily growing subfamily of antimicrobially active peptides of animals and plants is formed by the defensins, which are highly disulfide‐bonded, cationic peptides with a molecular mass of about 4 kDa. The synthesis of the human β‐defensins 1 and 2 (hBD‐1, hBD‐2) as well as of the novel murine β‐defensins 7 and 8 (mBD‐7 and mBD‐8) is reported. The peptides were synthesized by solid‐phase peptide synthesis using fluorenylmethoxycarbonyl chemistry. The linear products were oxidized in the presence of the cysteine/cystine redox system to the biologically active molecules. The correct disulfide connectivity of the resulting cyclic products was partly verified by mass spectrometry and sequence analysis of the fragments obtained after tryptic cleavage. In addition, the recently discovered antimicrobially active human peptide LEAP‐1/hepcidin, which contains four disulfide bonds, was successfully synthesized and subsequently oxidized. For Liver‐expressed anti microbial peptide (LEAP)‐1/hepcidin and hBD‐1, the identity of native and synthetic peptides was demonstrated by high‐pressure liquid chromatography and capillary electrophoretic analysis. The general synthetic procedure is suitable to rapidly perform the total chemical synthesis of novel fully bioactive defensins, which are expected to be identified soon, as well as of structurally modified analogs.     

二硫化碳对大鼠F_2代后遗影响的研究

本文报道大鼠妊娠7～14d以Cs_2染毒对F_2代的后遗影响。结果表明,CS_2对F_2代生长发育指标及F_1代脏器系数无明显影响,但对胎鼠有致畸作用,并可延续到第二代,以骨骼畸形为主,主要为胸骨缺失,其次为枕骨骨化延缓、肋骨延长和囱门增大等,且发生率几乎同胎鼠的F_1代。由此认为CS_2对后代的影响,主要是对F_2代的致畸作用较明显。     

Reinvestigation of the disulfide bridge arrangement in human pro-opiomelanocortin N-terminal segment (hNT 1–76)

The cystine bridge structure of the amino-terminal fragment of human proopiomelanocortin has been reinvestigated. Highly purified amino-terminal fragment 1–76 was rapidly isolated from human pituitaries using only reverse-phase liquid chromatography (RP-HPLC). This peptide was then subjected to trypsin and V8-protease digestion and the products separated by RP-HPLC> and subjected to amino acid and microsequence analysis. The results show that disulfide bridges link Cys-2 to Cys-24 and Cys-8 to Cys-20. Amino acid analysis and amino sugar determination confirm (i) the previously proposed sequence and (ii) the suggestion of the presence of two glycosylation sites in this molecule. These are most probably located at Thr-45 (O-glycosylation) and at Asn-65 (N-glycosylation).     

Injectable in situ cross-linking hyaluronic acid/carboxymethyl cellulose based hydrogels for drug release

A series of injectable in situ cross-linking hyaluronic acid/carboxymethyl cellulose based hydrogels (HA/CMC) was prepared via disulfide bonds by the oxidation of dissolved oxygen. The results showed that HA/CMC hydrogels exhibited tunable gelling time, appropriate rheology properties, high swelling ratio, good stability, and sustained drug release ability. The gelling time of HA/CMC hydrogels ranged from 1.4 to 7.0 min, and the values of the storage modulus, complex shear modulus, dynamic viscosity, and yield stress of HA3/CMC3 hydrogel were about 5869 Pa, 5870 Pa, 587 Pa·s, and 1969 Pa, respectively. The degradation percentage of HA1/CMC1, HA2/CMC2, and HA3/CMC3 hydrogels were about 60, 49, and 41% after incubating 42 days, and the in vitro cumulative release percentage of BSA from HA1/CMC1, HA2/CMC2, and HA3/CMC3 drug-loaded hydrogels were about 99, 91, and 82% after 30 days. The series of injectable in situ cross-linking HA/CMC hydrogels exhibited good comprehensive performance, signifying that these hydrogels could be potentially used in the fields of short- and medium-term controlled drug release, cell encapsulation, regenerative medicine, and tissue engineering.     

A folding-dependent mechanism of antimicrobial peptide resistance to degradation unveiled by solution structure of distinctin

Many bioactive peptides, presenting an unstructured conformation in aqueous solution, are made resistant to degradation by posttranslational modifications. Here, we describe how molecular oligomerization in aqueous solution can generate a still unknown transport form for amphipathic peptides, which is more compact and resistant to proteases than forms related to any possible monomer. This phenomenon emerged from 3D structure, function, and degradation properties of distinctin, a heterodimeric antimicrobial compound consisting of two peptide chains linked by a disulfide bond. After homodimerization in water, this peptide exhibited a fold consisting of a symmetrical full-parallel four-helix bundle, with a well secluded hydrophobic core and exposed basic residues. This fold significantly stabilizes distinctin against proteases compared with other linear amphipathic peptides, without affecting its antimicrobial, hemolytic, and ion-channel formation properties after membrane interaction. This full-parallel helical orientation represents a perfect compromise between formation of a stable structure in water and requirement of a drastic structural rearrangement in membranes to elicit antimicrobial potential. Thus, distinctin can be claimed as a prototype of a previously unrecognized class of antimicrobial derivatives. These results suggest a critical revision of the role of peptide oligomerization whenever solubility or resistance to proteases is known to affect biological properties.     

Locations of the beta1 transmembrane helices in the BK potassium channel

BK channels are composed of α-subunits, which form a voltage- and Ca²⁺-gated potassium channel, and of modulatory β-subunits. The β1-subunit is expressed in smooth muscle, where it renders the BK channel sensitive to [Ca²⁺]_i in a voltage range near the smooth-muscle resting potential and slows activation and deactivation. BK channel acts thereby as a damped feedback regulator of voltage-dependent Ca²⁺ channels and of smooth muscle tone. We explored the contacts between α and β1 by determining the extent of endogenous disulfide bond formation between cysteines substituted just extracellular to the two β1 transmembrane (TM) helices, TM1 and TM2, and to the seven α TM helices, consisting of S1–S6, conserved in all voltage-dependent potassium channels, and the unique S0 helix, which we previously concluded was partly surrounded by S1–S4. We now find that the extracellular ends of β1 TM2 and α S0 are in contact and that β1 TM1 is close to both S1 and S2. The extracellular ends of TM1 and TM2 are not close to S3–S6. In almost all cases, cross-linking of TM2 to S0 or of TM1 to S1 or S2 shifted the conductance–voltage curves toward more positive potentials, slowed activation, and speeded deactivation, and in general favored the closed state. TM1 and TM2 are in position to contribute, in concert with the extracellular loop and the intracellular N- and C-terminal tails of β1, to the modulation of BK channel function.     

Physicochemically stable cholera toxin B subunit pentamer created by peripheral molecular constraints imposed by de novo-introduced intersubunit disulfide crosslinks

We attempted to generate a physicochemically stable cholera toxin B subunit (CTB) by de novo-introduction of intersubunit disulfide bonds between adjacent subunits. Genes encoding double mutant CTB (dmCTB) encompassing a pair of amino acids to be replaced with cysteine residues either at the N-terminal (T1C/T92C, Q3C/T47C), C-terminal (F25C/N103C, Y76C/N103C), or at the internal α-helix region (L77C/T78C), were engineered. One mutant with the N-terminal constraint [dmCTB(T1C/T92C)], expressed as pentamer retained monosialoganglioside G(M1) (GM1) binding affinity, and exhibited robust thermostability. However, when the mutant CTB was heat-treated in the presence of a reducing agent, the thermostable phenotype was abolished, indicating the observed phenotype is due to the introduction of intersubunit disulfide bonds. The mutant CTB also exhibited a strong acid stability at a pH as low as 1.2, as well as stability against incubation with sodium dodecyl sulfate at concentrations as high as 10%. Furthermore, intranasal administration of the mutant CTB to mice induced CTB-specific serum IgG even after heat treatment, while the wildtype CTB failed to show such heat-resistant mucosal immunogenicity. This study demonstrated that an enterotoxin B subunit could be transformed into a physicochemically stable pentamer by the de novo-introduction of peripherally arranged intersubunit disulfide crosslinks, which may prove to be a useful strategy for the development of molecularly stable enterotoxin B subunit-based vaccines and delivery molecules.     

Enhancing protein stability with extended disulfide bonds

Disulfide bonds play an important role in protein folding and stability. However, the cross-linking of sites within proteins by cysteine disulfides has significant distance and dihedral angle constraints. Here we report the genetic encoding of noncanonical amino acids containing long side-chain thiols that are readily incorporated into both bacterial and mammalian proteins in good yields and with excellent fidelity. These amino acids can pair with cysteines to afford extended disulfide bonds and allow cross-linking of more distant sites and distinct domains of proteins. To demonstrate this notion, we preformed growth-based selection experiments at nonpermissive temperatures using a library of random β-lactamase mutants containing these noncanonical amino acids. A mutant enzyme that is cross-linked by one such extended disulfide bond and is stabilized by ∼9 °C was identified. This result indicates that an expanded set of building blocks beyond the canonical 20 amino acids can lead to proteins with improved properties by unique mechanisms, distinct from those possible through conventional mutagenesis schemes.We are developing strategies that begin to address the question of whether an expanded genetic code provides an evolutionary advantage to an organism. For example, it has been recently shown that a unique noncanonical amino acid mutation in TEM-1 β-lactamase significantly increases the enzyme’s catalytic activity for the substrate cephalexin, a result that cannot be recapitulated by substitution of canonical amino acids at this site (1). This same enzyme has been reengineered to be dependent on a noncanonical active site residue for activity, a dependency that was maintained for hundreds of generations without escape (2). Furthermore, addition of noncanonical amino acid building blocks to an unbiased library of ribosomally synthesized cyclic peptides provided a selective advantage in the evolution of inhibitors of cytotoxic intracellular proteases (3). Here we begin to explore whether noncanonical amino acids can provide Escherichia coli a selective growth advantage by increasing the thermal stability of essential proteins.Cysteine is unique among the 20 canonical amino acids in that it can form reversible covalent cross-links in proteins. Disulfide bonds can stabilize monomeric and multisubunit proteins (4), play a role in catalysis (5, 6), and regulate protein activity (7); because of these unique properties, disulfide bonds are highly conserved in protein evolution (8, 9). There has been considerable success in the use of structure-based design to engineer disulfide bonds into proteins for both biopharmaceutical and industrial applications (10, 11). However, the sites in proteins that can be cross-linked by a cystine disulfide are typically constrained to a distance between the two β-carbons of ∼5.5 Å (10) and a near 90° dihedral angle for the disulfide bond (12). Thus, the relatively long distances that might be required to bridge distinct protein domains or subunits, or steric constraints at specific sites may preclude the introduction of a natural disulfide bond. These challenges led us to explore whether we could overcome the geometrical constraints of the cysteine disulfide by genetically encoding noncanonical amino acids (NCAAs) with longer thiol-containing side chains.To this end, we designed a series of tyrosine derivatives (SetY, SprY, and SbuY) with para-substituted aliphatic thiols of various lengths (Fig. 1A). The calculated length between the two β-carbons of the SbuY-Cys disulfide bond is 14 Å when fully extended, which is significantly longer than a natural cystine cross-link. We genetically encoded these NCAAs in bacterial and mammalian cells by suppressing the nonsense codon TAG with an orthogonal, amber suppressor aminoacyl-tRNA synthetase (RS)/tRNA pair (13). Because statistical analyses of proteins have suggested that stabilizing disulfide mutations are most often found in regions of higher mobility near the protein surface and associated with longer loop lengths (>25 residues) (10, 14), we reasoned that these more flexible, extended disulfides might facilitate the introduction of stabilizing disulfide bonds into proteins. Using a N-terminal truncated β-lactamase as a model system (15), we carried out a growth-based selection under nonpermissive temperatures with a library of mutants in which the thiol-containing NCAAs were randomly incorporated, and identified a mutant enzyme cross-linked by an extended disulfide bond that is stabilized by ∼9 °C.Open in a separate windowFig. 1.Genetic incorporation of NCAAs containing long-side-chain thiols. (A) Structure of O-(2-mercaptoethyl)-l-tyrosine (SetY), O-(3-mercaptopropyl)-l-tyrosine (SprY), and O-(4-mercaptobutyl)-l-tyrosine (SbuY). (B) SDS/PAGE analysis of purified GFP (134TAG) expressed in E. coli DH10B using the MbXYRS/tRNA^pyl pair in the presence or absence of 1 mM NCAAs. GFP mutants were expressed in LB medium and purified by standard Ni-NTA affinity chromatography. (C) Mass spectral analysis of GFP mutants containing the corresponding NCAAs. The calculated masses for the mutant GFPs containing SetY, SprY, and SbuY are 28,005 Da, 28,019 Da, and 28,033 Da, respectively. (D) Fluorescence microscopy (10×) of 293T cells expressing an EGFP mutant (Tyr39TAG) in the presence or absence of NCAAs. 293T cells were transiently cotransfected with pCMV-XYRS and pEGFP-Tyr39TAG and were grown in DMEM supplemented with 10 FBS in the presence or absence of 250 μM NCAAs.