The human beta-defensin-3, an antibacterial peptide with multiple biological functions

A group of interesting molecules called defensins exhibit multiple functions but have been primarily recognized to possess a broad spectrum of antimicrobial activities. Studies have reported two different types of defensins (α and β) from human and animals, a cyclic θ defensin from rhesus, and several defensin-like peptides from plants. There is no amino acid sequence homology between these peptides, but they all contain three Cys-Cys disulfide linkages while the connectivities are different. Human β-defensin-3 (HβD-3) is the most recently discovered member of the host-defense peptide family that has attracted much attention. This molecule is expressed either constitutively or induced upon a challenge, and a growing evidence indicates the involvement of such molecules in adaptive immunity as well. It has been shown to exhibit antibacterial activities towards Gram-negative and Gram-positive bacteria as well as an ability to act as a chemo-attractant. Analysis of NMR structural data suggested a symmetrical dimeric form of this peptide in solution, which consists of three β strands and a short helix in the N-terminal region. While the disulfide linkages are known to provide the structural stability and stability against proteases, the biological relevance of this dimeric form was contradicted by another biological study. Since there is considerable current interest in developing HβD-3 for possible pharmaceutical applications, studies to further our understanding on the determinants of antibacterial activities and immunomodulatory function of HβD-3 are considered to be highly significant. The knowledge of its biosynthetic regulation will also help in understanding the role of HβD-3 in immunity. This article presents an overview of the expression and regulation of HβD-3 in humans, and the structure-function correlations among HβD-3 and its modified peptides are discussed emphasizing the functional importance. The future scope for studies on HβD-3 and design of short potent antimicrobial peptides, based on the native HβD-3 molecule, that do not interfere in the immunomodulatory function is also outlined.     

Human β-Defensin 4 with Non-Native Disulfide Bridges Exhibit Antimicrobial Activity

Human defensins play multiple roles in innate immunity including direct antimicrobial killing and immunomodulatory activity. They have three disulfide bridges which contribute to the stability of three anti-parallel β-strands. The exact role of disulfide bridges and canonical β-structure in the antimicrobial action is not yet fully understood. In this study, we have explored the antimicrobial activity of human β-defensin 4 (HBD4) analogs that differ in the number and connectivity of disulfide bridges. The cysteine framework was similar to the disulfide bridges present in μ-conotoxins, an unrelated class of peptide toxins. All the analogs possessed enhanced antimicrobial potency as compared to native HBD4. Among the analogs, the single disulfide bridged peptide showed maximum potency. However, there were no marked differences in the secondary structure of the analogs. Subtle variations were observed in the localization and membrane interaction of the analogs with bacteria and Candida albicans, suggesting a role for disulfide bridges in modulating their antimicrobial action. All analogs accumulated in the cytosol where they can bind to anionic molecules such as nucleic acids which would affect several cellular processes leading to cell death. Our study strongly suggests that native disulfide bridges or the canonical β-strands in defensins have not evolved for maximal activity but they play important roles in determining their antimicrobial potency.     

Analysis and separation of residues important for the chemoattractant and antimicrobial activities of beta-defensin 3

beta-Defensins are important in mammalian immunity displaying both antimicrobial and chemoattractant activities. Three canonical disulfide intramolecular bonds are believed to be dispensable for antimicrobial activity but essential for chemoattractant ability. However, here we show that HBD3 (human beta-defensin 3) alkylated with iodoactemide and devoid of any disulfide bonds is still a potent chemoattractant. Furthermore, when the canonical six cysteine residues are replaced with alanine, the peptide is no longer active as a chemoattractant. These findings are replicated by the murine ortholog Defb14. We restore the chemoattractant activity of Defb14 and HBD3 by introduction of a single cysteine in the fifth position (Cys V) of the beta-defensin six cysteine motif. In contrast, a peptide with a single cysteine at the first position (Cys I) is inactive. Moreover, a range of overlapping linear fragments of Defb14 do not act as chemoattractants, suggesting that the chemotactic activity of this peptide is not dependent solely on an epitope surrounding Cys V. Full-length peptides either with alkylated cysteine residues or with cysteine residues replaced with alanine are still strongly antimicrobial. Defb14 peptide fragments were also tested for antimicrobial activity, and peptides derived from the N-terminal region display potent antimicrobial activity. Thus, the chemoattractant and antimicrobial activities of beta-defensins can be separated, and both of these functions are independent of intramolecular disulfide bonds. These findings are important for further understanding of the mechanism of action of defensins and for therapeutic design.     

Development of disulfide peptide mapping and determination of disulfide structure of recombinant human osteoprotegerin chimera produced in Escherichia coli

Recombinant human osteoprotegerin chimera is a 90-kDa protein containing a human IgG Fc domain fused to human osteoprotegerin. The molecule is a dimer linked by two intermolecular disulfide bonds and contains eleven intramolecular disulfide bonds per monomer. A cysteine-rich region in osteoprotegerin contains nine disulfide bridges homologous to the cysteine-rich signature structure of the tumor necrosis factor receptor/nerve growth factor receptor superfamily. In this report, we have developed peptide mapping procedures suitable to generate disulfide-containing peptides for disulfide structure assignment of the fusion molecule. The methods employed included proteolytic digestion using endoproteinases Glu-C and Lys-C in combination followed by LC-MS analyses. Disulfide linkages of peptide fragments containing a single disulfide bond were assigned by sequence analysis via detection of (phenylthiohydantoinyl) cystine and/or by MS analysis. Disulfide bonds of a large, core fragment containing three peptide sequences linked by four disulfides were assigned after generation of smaller disulfide-linked peptides by a secondary thermolysin digestion. Disulfide structures of peptide fragments containing two disulfide bonds were assigned using matrix-assisted laser desorption ionization mass spectrometry with postsource decay. Both the inter- and intramolecular disulfide linkages of the chimeric dimer were confirmed.     

Conformational landscape and pathway of disulfide bond reduction of human alpha defensin

Human alpha defensins are a class of antimicrobial peptides with additional antiviral activity. Such antimicrobial peptides constitute a major part of mammalian innate immunity. Alpha defensins contain six cysteines, which form three well defined disulfide bridges under oxidizing conditions. Residues C3-C31, C5-C20, and C10-C30 form disulfide pairs in the native structure of the peptide. The major tissue in which HD5 is expressed is the crypt of the small intestine, an anaerobic niche that should allow for substantial pools of both oxidized and (partly) reduced HD5. We used ion mobility coupled to mass spectrometry to track the structural changes in HD5 upon disulfide bond reduction. We found evidence of stepwise unfolding of HD5 with sequential reduction of the three disulfide bonds. Alkylation of free cysteines followed by tandem mass spectrometry of the corresponding partially reduced states revealed a dominant pathway of reductive unfolding. The majority of HD5 unfolds by initial reduction of C5-C20, followed by C10-C30 and C3-C31. We find additional evidence for a minor pathway that starts with reduction of C3-C31, followed by C5-C20 and C10-C30. Our results provide insight into the pathway and conformational landscape of disulfide bond reduction in HD5.     

Three-dimensional structure of RK-1: a novel alpha-defensin peptide

NMR spectroscopy and simulated annealing calculations have been used to determine the three-dimensional structure of RK-1, an antimicrobial peptide from rabbit kidney recently discovered from homology screening based on the distinctive physicochemical properties of the corticostatins/defensins. RK-1 consists of 32 residues, including six cysteines arranged into three disulfide bonds. It exhibits antimicrobial activity against Escherichia coli and activates Ca(2+) channels in vitro. Through its physicochemical similarity, identical cysteine spacing, and linkage to the corticostatins/defensins, it was presumed to be a member of this family. However, RK-1 lacks both a large number of arginines in the primary sequence and a high overall positive charge, which are characteristic of this family of peptides. The three-dimensional solution structure, determined by NMR, consists of a triple-stranded antiparallel beta-sheet and a series of turns and is similar to the known structures of other alpha-defensins. This has enabled the definitive classification of RK-1 as a member of this family of antimicrobial peptides. Ultracentrifuge measurements confirmed that like rabbit neutrophil defensins, RK-1 is monomeric in solution, in contrast to human neutrophil defensins, which are dimeric.     

Three-dimensional structure of RTD-1, a cyclic antimicrobial defensin from Rhesus macaque leukocytes

Most mammalian defensins are cationic peptides of 29-42 amino acids long, stabilized by three disulfide bonds. However, recently Tang et al. (1999, Science 286, 498-502) reported the isolation of a new defensin type found in the leukocytes of rhesus macaques. In contrast to all the other defensins found so far, rhesus theta defensin-1 (RTD-1) is composed of just 18 amino acids with the backbone cyclized through peptide bonds. Antibacterial activities of both the native cyclic peptide and a linear form were examined, showing that the cyclic form was 3-fold more active than the open chain analogue [Tang et al. (1999) Science 286, 498-502]. To elucidate the three-dimensional structure of RTD-1 and its open chain analogue, both peptides were synthesized using solid-phase peptide synthesis and tert-butyloxycarbonyl chemistry. The structures of both peptides in aqueous solution were determined from two-dimensional (1)H NMR data recorded at 500 and 750 MHz. Structural constraints consisting of interproton distances and dihedral angles were used as input for simulated-annealing calculations and water refinement with the program CNS. RTD-1 and its open chain analogue oRTD-1 adopt very similar structures in water. Both comprise an extended beta-hairpin structure with turns at one or both ends. The turns are well defined within themselves and seem to be flexible with respect to the extended regions of the molecules. Although the two strands of the beta-sheet are connected by three disulfide bonds, this region displays a degree of flexibility. The structural similarity of RTD-1 and its open chain analogue oRTD-1, as well as their comparable degree of flexibility, support the theory that the additional charges at the termini of the open chain analogue rather than overall differences in structure or flexibility are the cause for oRTD-1's lower antimicrobial activity. In contrast to numerous other antimicrobial peptides, RTD-1 does not display any amphiphilic character, even though surface models of RTD-1 exhibit a certain clustering of positive charges. Some amide protons of RTD-1 that should be solvent-exposed in monomeric beta-sheet structures show low-temperature coefficients, suggesting the possible presence of weak intermolecular hydrogen bonds.     

Chemical synthesis of beta-defensins and LEAP-1/hepcidin.

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 beta-defensins 1 and 2 (hBD-1, hBD-2) as well as of the novel murine beta-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.     

Human Antimicrobial Peptides: Defensins, Cathelicidins and Histatins

Antimicrobial peptides, which have been isolated from many bacteria, fungi, plants, invertebrates and vertebrates, are an important component of the natural defenses of most living organisms. The isolated peptides are very heterogeneous in length, sequence and structure, but most of them are small, cationic and amphipathic. These peptides exhibit broad-spectrum activity against Gram-positive and Gram-negative bacteria, yeasts, fungi and enveloped viruses. A wide variety of human proteins and peptides also have antimicrobial activity and play important roles in innate immunity. In this review we discuss three important groups of human antimicrobial peptides. The defensins are cationic non-glycosylated peptides containing six cysteine residues that form three intramolecular disulfide bridges, resulting in a triple-stranded β-sheet structure. In humans, two classes of defensins can be found: α-defensins and β-defensins. The defensin-related HE2 isoforms will also be discussed. The second group is the family of histatins, which are small, cationic, histidine-rich peptides present in human saliva. Histatins adopt a random coil conformation in aqueous solvents and form α-helices in non-aqueous solvents. The third group comprises only one antimicrobial peptide, the cathelicidin LL−37. This peptide is derived proteolytically from the C-terminal end of the human CAP18 protein. Just like the histatins, it adopts a largely random coil conformation in a hydrophilic environment, and forms an α-helical structure in a hydrophobic environment.     

Dimeric analogs of immunosuppressive decapeptide fragment of ubiquitin

Our previous studies revealed that ubiquitin and its decapeptide fragment with the LEDGRTLSDY sequence, located on the exposed molecule loop, strongly suppressed the immune response. This suggested that the loop may serve as a functional epitope of ubiquitin molecule and that a possible mechanism of biological action of the synthesized peptides is associated with interfering in interactions of ubiquitin with other molecules. Ubiquitin is known to exist in oligomeric forms, which can interact with various oligomeric receptors. We designed and synthesized new dimeric analogs of the ubiquitin fragment, to probe whether dimeric peptides may have higher affinity towards the ubiquitin receptors responsible for immunosuppression, which are believed to form oligomeric structures. Three dimerization strategies, N‐terminus to N‐terminus, C‐terminus to C‐terminus, and N‐terminus to C‐terminus (head‐to‐tail) via PEG derivatives were used to synthesize the dimeric peptides on solid support. In the course of our research, we developed a new and straightforward procedure of dimerization where α‐amino groups of the C‐terminal lysine residues of two peptide fragments were linked by PEG spacer directly on solid support. The effect of dimeric analogs on the immunological response was tested in the AFC in vitro experiment. The immunological tests showed that the head‐to‐tail dimerization caused a more profound increase in the biological activity than other tested dimerization methods. Our results suggest that such orientation of peptide components may correspond to orientation of the hypothetic ubiquitin receptors responsible for the immunomodulatory activity. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

β-Defensin 1 gene variability among non-human primates

Defensins are a recently described family of peptides that play an important role in innate immunity. Recent studies have shown that defensins exhibit a broad spectrum of antimicrobial activities against bacteria and fungi. Three families have been identified so far in mammals, alpha-defensins, beta-defensins and theta-defensins, presumably derived from a common ancestral defensin. A long-term study on the evolution of these multigene families among primates has been undertaken to investigate: (1) the degree of interspecific differentiation; (2) the genetic mechanisms responsible for the variability of these molecules; and (3) the possible role of different environmental factors in their evolution. Nucleotide sequences have been obtained from great and lesser apes, several African and Asian catarrhine monkeys and one New World monkey. A comparison of rates of synonymous and nonsynonymous (amino-acid changing) nucleotide substitution indicates that the primate beta-defensin 1 gene evolved under a pattern of random nucleotide substitution as predicted by the neutral theory of molecular evolution. These results are not consistent with the hypothesis that the primate beta-defensin 1 gene has diversified in response to changes in the microbial species to which a given host is exposed. Analyses of interpecific variability have yielded some insights about the pattern of molecular evolution of the gene among primates. Humans and great apes present high levels of sequence similarity, differing in only one amino acid residue in the mature peptide. Compared with these taxa, hylobatids and cercopithecids exhibit 3-4 amino acid substitutions, some of which increase the net charge of the active molecule.     

Amino-terminal dimerization of peptides on the solid support. Synthesis and biological activity of the immunosuppressive HLA-DR fragments linked by poly(ethylene glycol)s

The nonapeptide fragment of the HLA-DR molecule, located in the exposed loop of the beta chain (164-172) and having the sequence VPRSGEVYT, suppresses the immune response. On the basis of the three-dimensional structure of the HLA-DR superdimer, we designed new dimeric analogs in which the VPRSGEVYT peptides are linked through their N-termini by poly(ethylene glycol) linkers of different lengths and are able to mimic the dimeric nature of the immunosuppressive fragments of HLA class II molecules. The analogs were synthesized using standard solid-phase peptide synthesis protocols. The dimerization was achieved by cross-linking the N-terminal positions of the peptides, attached to an MBHA resin, with alpha,omega-bis(acetic acid) poly(ethylene glycol), activated by esterification with pentafluorophenol. Our results demonstrate that the amino-terminal dimerization of the peptide results in enhanced immunosuppressive activity and that the potency of the conjugates depends on the length of the poly(ethylene glycol) linker. MS/MS analysis of the obtained dimeric peptides is also presented.     

Insect immunity. Isolation from the lepidopteran Heliothis virescens of a novel insect defensin with potent antifungal activity

Lepidoptera have been reported to produce several antibacterial peptides in response to septic injury. However, in marked contrast to other insect groups, no inducible antifungal molecules had been described so far in this insect order. Surprisingly, also cysteine-rich antimicrobial peptides, which predominate in the antimicrobial defense of other insects, had not been discovered in Lepidoptera. Here we report the isolation from the hemolymph of immune induced larvae of the lepidopteran Heliothis virescens of a cysteine-rich molecule with exclusive antifungal activity. We have fully characterized this antifungal molecule, which has significant homology with the insect defensins, a large family of antibacterial peptides directed against Gram-positive strains. Interestingly, the novel peptide shows also similarities with the antifungal peptide drosomycin from Drosophila. Thus, Lepidoptera appear to have built their humoral immune response against bacteria on cecropins and attacins. In addition, we report that Lepidoptera have conferred antifungal properties to the well conserved structure of antibacterial insect defensins through amino acid replacements.     

The Structure and Stability of the Monomorphic HLA-G Are Influenced by the Nature of the Bound Peptide

The highly polymorphic major histocompatibility complex class Ia (MHC-Ia) molecules present a broad array of peptides to the clonotypically diverse αβ T-cell receptors. In contrast, MHC-Ib molecules exhibit limited polymorphism and bind a more restricted peptide repertoire, in keeping with their major role in innate immunity. Nevertheless, some MHC-Ib molecules do play a role in adaptive immunity. While human leukocyte antigen E (HLA-E), the MHC-Ib molecule, binds a very restricted repertoire of peptides, the peptide binding preferences of HLA-G, the class Ib molecule, are less stringent, although the basis by which HLA-G can bind various peptides is unclear. To investigate how HLA-G can accommodate different peptides, we compared the structure of HLA-G bound to three naturally abundant self-peptides (RIIPRHLQL, KGPPAALTL and KLPQAFYIL) and their thermal stabilities. The conformation of HLA-G^KGPPAALTL was very similar to that of the HLA-G^RIIPRHLQL structure. However, the structure of HLA-G^KLPQAFYIL not only differed in the conformation of the bound peptide but also caused a small shift in the α2 helix of HLA-G. Furthermore, the relative stability of HLA-G was observed to be dependent on the nature of the bound peptide. These peptide-dependent effects on the substructure of the monomorphic HLA-G are likely to impact on its recognition by receptors of both innate and adaptive immune systems.     

The N-hydroxysuccinimide ester of Boc-[S-(3-nitro-2-pyridinesulfenyl)]-cysteine: a heterobifunctional cross-linking agent

Synthetic cysteine-containing peptides were unidirectionally conjugated to albumin via disulfide bonds using the S-(3-nitro-2-pyridinesulfenyl) derivative of cysteine. This method employs the N-hydroxysuccinimide ester of Boc-[S-(3-nitro-2-pyridinesulfenyl)]-cysteine, a protected amino acid derivative used in peptide synthesis, as a heterobifunctional cross-linking agent. The disulfide bonds in the conjugates are formed by the reaction of free thiols with S-(3-nitro-2-pyridinesulfenyl) groups. Bovine albumin was conjugated in this manner to several synthetic peptides derived from human fibrin. Amino acid analysis of these conjugates demonstrated incorporations of from 6 to 11 peptide molecules per molecule of protein.     

The beta 2-microglobulin dissociation rate is an accurate measure of the stability of MHC class I heterotrimers and depends on which peptide is bound.

Stable, recombinant, water-soluble complexes of HLA-A2 and HLA-B27 were reconstituted from 125I-labeled beta 2-microglobulin (beta 2m), a synthetic peptide, and HLA H chain fragments expressed as inclusion bodies in the Escherichia coli cytoplasm. Using this system, we were able to show: 1) the t1/2 of beta 2m dissociation from HLA complexes at 37 degrees C varied from approximately 40 h to less than 1 h, depending on the peptide employed for reconstitution. Peptide length and composition were found to be critical factors in determining the beta 2m dissociation rate. Endogenous peptides form complexes that are about as stable as those formed with typical antigenic peptides. 2) Peptide exchange reactions, in which an exogenous peptide replaces the peptide that is already bound by the class I molecule, proceed readily for complexes that have rapid beta 2m dissociation rates. Thus, difficulties in demonstrating peptide binding to complexes that contain endogenous peptides can be attributed to the stability of the endogenous peptide/class I molecule complex. 3) The peptide exchange reaction does not require concomitant beta 2m dissociation. 4) Distal parts of the class I molecule, which are not directly involved in peptide binding or beta 2m binding, have a major impact on the stability of class I molecules. Thus, these studies show that the dissociation rate of beta 2m is an excellent measure of how tightly a given peptide binds to class I MHC molecules, that the ability to bind peptide is tightly coupled to the binding of beta 2m and vice versa, and that regions of the molecule distal from the binding site influence the stability of peptide binding.     

Assembly of HLA DR1 molecules translated in vitro: binding of peptide in the endoplasmic reticulum precludes association with invariant chain.

MHC class II molecules usually bind peptides in the endocytic pathway, but can also present endogenous peptides from newly synthesized proteins in a chloroquine-insensitive manner, suggesting that peptide binding might occur in the endoplasmic reticulum (ER). We used in vitro translation of HLA-DR1 class II molecules in the presence of microsomes to study peptide binding in the ER. Formation of functional class II molecules in vitro depends on formation of disulfide bridges in alpha and beta chains. The class II alpha beta heterodimers made by in vitro translation resemble class II molecules synthesized in cells in (i) their reactivity with conformation-specific antibodies, (ii) their assembly with Ii chain homotrimers, (iii) the generation of SDS-stable dimers upon peptide binding and (iv) their specificity of peptide binding. The assembly of class II molecules occurs via an alpha beta intermediate and can occur post-translationally, but only in intact microsomes. Class II alpha beta heterodimers are able to bind peptides in ER-derived microsomes, a process that precludes subsequent association of class II molecules with Ii chain. This mechanism might explain presentation of endogenous peptides by class II molecules.     

Three new antimicrobial peptides from the scorpion Pandinus imperator

Three novel cysteine-free venom peptides, which were referred to as Pantinin-1, Pantinin-2 and Pantinin-3, respectively, have been identified from the scorpion Pandinus imperator by cDNA cloning strategy. The precursor of each peptide consists of a signal peptide, a mature peptide with no disulfide bridges, and an acidic propeptide with a typical processing signal. Each of the three peptides is an α-helical, cationic and amphipathic molecule with 13 or 14 amino acid residues. Their amino acid sequences are homologous to those of some 13-mer antimicrobial peptides isolated from scorpions. Antimicrobial assay showed that all the three peptides possess relatively strong activities against Gram-positive bacteria and a fungus, but have very weak antimicrobial activities against Gram-negative bacteria. Toxicity assay showed that the three peptides exhibit very low or mild hemolytic activities against human red blood cells. It is interesting to see that Pantinin-3 is able to potently inhibit the growth of vancomycin-resistant Enterococcus (VRE) S13, a pathogen that can cause a number of human infections; this suggests that Pantinin-3 has great potential to be applied in the treatment of VRE infections. Our findings gain new insights into the structure/function relationships of the small linear cationic antimicrobial peptides from scorpions, and provide new templates for designing of antimicrobial agents targeting antibiotic-resistant pathogenic bacteria.     

Determination of the disulfide structure of sillucin, a highly knotted, cysteine-rich peptide, by cyanylation/cleavage mass mapping

The disulfide structure of sillucin, a highly knotted, cysteine-rich, antimicrobial peptide, isolated from Rhizomucor pusillus, has been determined to be Cys2--Cys7, Cys12--Cys24, Cys13--Cys30, and Cys14--Cys21 by disulfide mass mapping based on partial reduction and CN-induced cleavage enabled by cyanylation. The denatured 30-residue peptide was subjected to partial reduction by tris(2-carboxyethyl)phosphine hydrochloride at pH 3 to produce a mixture of partially reduced sillucin species; the nascent sulfhydryl groups were immediately cyanylated by 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate. The cyanylated species, separated and collected during reversed phase high-performance liquid chromatography, were treated with aqueous ammonia, which cleaved the peptide chain on the N-terminal side of cyanylated cysteine residues. The CN-induced cleavage mixture was analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry before and after complete reduction of residual disulfide bonds in partially reduced and cyanylated species to mass map the truncated peptides to the sequence. Because the masses of the CN-induced cleavage fragments of both singly and doubly reduced and cyanylated sillucin are related to the linkages of the disulfide bonds in the original molecule, the presence of certain truncated peptide(s) can be used to positively identify the linkage of a specific disulfide bond or exclude the presence of other possible linkages.     

Dimeric half-molecules of human fibrinogen are joined through disulfide bonds in an antiparallel orientation

Human fibrinogen is a dimer composed of two identical halves. Each dimeric half contains three peptide chains (alpha, beta, and gamma) linked by disulfide bonds. The two half-molecules are joined by three disulfide bonds, one between the two alpha-chains (residue alpha-28) and two between the two gamma-chains (residues gamma-8 and gamma-9). In the absence of any difinitive experimental evidence, it has been presumed that the joined halves were aligned in a parallel orientation similar to the situation found in immunoglobulins. We have now determined that the two gamma-chains--hence, the dimeric halves--are connected in an antiparallel manner. A tryptic peptide containing gamma-chain residues 6-14 was isolated as a disulfide-linked dimer from CNBr-treated fragment E. Synthetic peptides corresponding to this sequence were prepared, from which parallel and antiparallel dimers were constructed. During the syntheses, cysteine thiol groups were protected as p-methoxybenzyl and acetamidomethyl sulfides; the peptides were dimerized by selective deprotection and disulfide bond formation. First, the p-methoxybenzyl groups were removed by liquid hydrogen fluoride and the newly exposed thiols oxidized in the presence of potassium ferricyanide. Then the monocystine compound was converted to the double-cystine product by iodolytic cleavage of the acetamidomethyl group with concomitant disulfide bond formation. This selectivity was used to prepare peptide dimers which modeled both parallel and antiparallel arrangements. The antiparallel-oriented synthetic peptide was indistinguishable from the native tryptic peptide as judged by elution from reverse-phase high-performance liquid chromatography and circular dichroism spectroscopy. The parallel-oriented synthetic peptide differed from the native material by both criteria.