Processing of lysozyme at distinct loops by pepsin: a novel action for generating multiple antimicrobial peptide motifs in the newborn stomach

C-type lysozyme (cLZ) is an antimicrobial enzyme that plays a major defense role in many human secretions. Recently, we have identified a helix-loop-helix antimicrobial peptide fragment of cLZ. This finding suggests that processing by coexisting proteases might be a relevant physiological process for generating peptides that contribute to the in vivo mucosal defense role of cLZ. In this study, we found that pepsin, under condition relevant to the newborn stomach (pH 4.0), generated various peptides from cLZ with potent bactericidal activity against several strains of Gram-negative and Gram-positive bacteria. Microsequencing and mass spectral analysis revealed that pepsin cleavage occurred at conserved loops within the alpha-domain of cLZ. We found that the bactericidal domain, which was isolated by gel filtration and reversed-phase HPLC, contains two cationic alpha-helical peptides generated from a helix-loop-helix domain (residues 1-38 of cLZ) by nicking at leucine17. A third peptide consisting of an alpha-helix (residues 18-38) and a two-stranded beta-sheet (residues 39-56) structure was also identified. These peptides share structural motifs commonly found in different innate immune defenses. Functional cellular studies with outer membrane-, cytoplasmic membrane vitality- and redox-specific fluorescence dyes revealed that the lethal effect of the isolated antimicrobial peptides is due to membrane permeabilization and inhibition of redox-driven bacterial respiration. The results provide the first demonstration that pepsin can fine-tune the antimicrobial potency of cLZ by generating multiple antimicrobial peptide motifs, delineating a new molecular switch of cLZ in the mucosal defense systems. Finally, this finding offers a new strategy for the design of antibiotic peptide drugs with potential use in the treatment of infectious diseases.     

Inhibition of DNA polymerization and DNA transcription to RNA by seminal plasma peptides

An oligopeptide fraction purified from the extracellular compartment of bull semen and strongly interacting with DNA was shown to hinder mononucleotide polymerizations to DNA and RNA in vitro. The fraction, collectively called seminal plasma inhibitor, was active in the endogenous DNA and RNA polymerase reactions of the nuclei from rat hepatocytes and in the analogous nucleotide polymerizations catalyzed by purified enzymes of bacterial origin. The type of the induced inhibition was studied using the RNA polymerase from Escherichia coli as a representative nucleotidyl transferase. In the enzymatic polycondensation of mononucleotides, the seminal plasma inhibitor appeared to exert its effect mainly by a competitive inhibition for the utilization of DNA templates without specificity with respect to the source and the base sequence of DNA. Concavities of the plots of V0/Vi versus the amounts of inhibitor in the nucleotide polymerizing reactions and of the Dixon plots in the assays of RNA polymerase from E. coli suggested that the isolated oligopeptide fraction contained more than one active molecular species with differential effects at low and high doses. Preliminary results on the microheterogeneity of the seminal plasma inhibitor supported this contention.     

Characterization of a monoclonal antibody to a conserved epitope on human seminal vesicle-specific peptides: a novel probe/marker system for semen identification

A novel sperm-coating antigen from the human seminal vesicles was discovered. We identified a monoclonal antibody MHS-5, recognizing an epitope with characteristics of a forensic semen marker: conservation in all vasectomized or normal semen samples tested (421); absence in all human tissues or biological fluids other than semen; and immunolocalization on the surface of ejaculated sperm. Western blots of ejaculates allowed to liquefy for 5 min demonstrated the MHS-5 epitope to be located on peptides of a wide range of molecular masses from 69 to 8 kDa. After 15 h of semen liquefaction, immunoreaction peptides of higher molecular mass were undetectable in semen, while peptides of lower molecular mass from 8 to 21 kDa retained antigenicity. Three peptides of 10, 11.9, and 13.7 kDa were the most immunoreactive species in semen liquified for 15 h. Using the MHS-5 monoclonal, an enzyme-linked immunosorbent assay (ELISA) was developed sensitive to 1 ng of seminal protein. This assay showed that the MHS-5 antigen was undetectable in semen of common domestic animals and monkeys but was present in chimpanzee, gorilla, and orangutan semen. ELISA of homogenates from human organs and reproductive tissues demonstrated the antigen only in samples of seminal vesicles. Epididymal sperm obtained at vasovasostomy lacked the MHS-5 epitope, a fact that, together with immunolocalization on ejaculated sperm, demonstrated that the MHS-5 antigen functions as a "sperm-coating antigen." The MHS-5 monoclonal detected semen in sexual-assault evidence obtained six months previously and in mixtures of semen with vaginal or cervical fluid. Assay systems employing the MHS-5 monoclonal may be useful for identification of semen in sexual-assault casework. The MHS-5 epitope resides on novel seminal vesicle-specific peptides whose functions, aside from sperm coating, are uncharacterized.     

Induction of non-lamellar lipid phases by antimicrobial peptides: a potential link to mode of action

Antimicrobial peptides are naturally produced by numerous organisms including insects, plants and mammals. Their non-specific mode of action is thought to involve the transient perturbation of bacterial membranes but the molecular mechanism underlying the rearrangement of the lipid molecules to explain the formation of pores and micelles is still poorly understood. Biological membranes mostly adopt planar lipid bilayers; however, antimicrobial peptides have been shown to induce non-lamellar lipid phases which may be intimately linked to their proposed mechanisms of action. This paper reviews antimicrobial peptides that alter lipid phase behavior in three ways: peptides that induce positive membrane curvature, peptides that induce negative membrane curvature and peptides that induce cubic lipid phases. Such structures can coexist with the bilayer structure, thus giving rise to lipid polymorphism induced upon addition of antimicrobial peptides. The discussion addresses the implications of induced lipid phases for the mode of action of various antimicrobial peptides.     

Analysis of cytokines in seminal plasma and blood sera of patients with chronic prostatitis during immunotherapy with natural complex of cytokines and antimicrobial peptides

Levels of pro- and anti-inflammatory cytokines (TNFalpha, IL-6, IL-8, IL-10) were studied in blood sera and seminal plasma of healthy volunteers and patients with chronic bacterial prostatitis (CBP) or chronic abacterial prostatitis (CABP). Assessment of effect of immunotropic drug Superlymph with direct antimicrobial action for treatment of mentioned groups of patients was performed. It was shown that seminal plasma of patients with CBP and CABP contained higher levels of IL-6 compared with healthy subjects. IL-8 level was increased in small part of patients with CBP and CABP. Changes in cytokine status of patients with CBP and CABP that occurred during treatment and manifested in decrease of proinflammatory cytokines levels (IL-6 and IL-8) and increase of antiinflammatory cytokine level (IL-10) point to reduction of inflammatory process in prostate. Clinical effect of complex treatment in patients with CBP considering eradication of pathogen and shortening of duration of antibacterial treatment amounted 95%. Monotherapy of patients with CABP with Superlymph was effective in 72%.     

Phosphorylation of p38 by GRK2 at the docking groove unveils a novel mechanism for inactivating p38MAPK

p38 Mitogen-activated protein kinases (MAPK) are a family of Ser/Thr kinases that regulate important cellular processes such as stress responses, differentiation, and cell-cycle control . Activation of MAPK is achieved through a linear signaling cascade in which upstream kinases (MAPKKs) dually phosphorylate MAPKs at a conserved 3-amino-acid motif (Thr-X-Tyr) . G-protein-coupled receptor kinases (GRKs) are known to selectively phosphorylate G-protein-coupled receptors (GPCRs) and thus trigger desensitization . We report that GRK2 is a novel inactivating kinase of p38MAPK. p38 associates with GRK2 endogenously and is phosphorylated by GRK2 at Thr-123, a residue located at its docking groove. Mimicking phosphorylation at this site impairs the binding and activation of p38 by MKK6 and diminishes the capacity of p38 to bind and phosphorylate its substrates. Accordingly, p38 activation is decreased or increased when cellular GRK2 levels are enhanced or reduced, respectively. Changes in GRK2 levels and activity can modify p38-dependent processes such as differentiation of preadipocytic cells and LPS-induced cytokine release, enhanced in macrophages from GRK2(+/-) mice. Phosphorylation of p38 at a region key for its interaction with different partners uncovers a new mechanism for the regulation of this important family of kinases.     

Induction of autolysis in starved Escherichia coli cells by seminalplasmin, an antimicrobial protein from bovine seminal plasma

Abstract A pair of relA ⁺ and relA E. coli strains, otherwise isogenic, were studied with regard to the susceptibility of starved cells to lysis induced by the natural peptide seminalplasmin. Starved relA cells were more sensitive to seminalplasmin-induced lysis when compared to starved relA ⁺ cells. Nevertheless, pronounced lysis of starved relA ⁺ cells was observed with increase in the concentration of seminalplasmin. In conctrast, ampicillin could not lyse starved relA ⁺ cells even at very high concentrations. Further, seminalplasmin could cause loss of viability and degradation of peptidoglycan in starved relA ⁺ cells. These observations suggest that, unlike many other antibiotics, seminalplasmin can induce autolysis under the conditions of a stringent response.     

Identification of novel GAPDH-derived antimicrobial peptides secreted by Saccharomyces cerevisiae and involved in wine microbial interactions

Saccharomyces cerevisiae plays a primordial role in alcoholic fermentation and has a vast worldwide application in the production of fuel-ethanol, food and beverages. The dominance of S. cerevisiae over other microbial species during alcoholic fermentations has been traditionally ascribed to its higher ethanol tolerance. However, recent studies suggested that other phenomena, such as microbial interactions mediated by killer-like toxins, might play an important role. Here we show that S. cerevisiae secretes antimicrobial peptides (AMPs) during alcoholic fermentation that are active against a wide variety of wine-related yeasts (e.g. Dekkera bruxellensis) and bacteria (e.g. Oenococcus oeni). Mass spectrometry analyses revealed that these AMPs correspond to fragments of the S. cerevisiae glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein. The involvement of GAPDH-derived peptides in wine microbial interactions was further sustained by results obtained in mixed cultures performed with S. cerevisiae single mutants deleted in each of the GAPDH codifying genes (TDH1-3) and also with a S. cerevisiae mutant deleted in the YCA1 gene, which codifies the apoptosis-involved enzyme metacaspase. These findings are discussed in the context of wine microbial interactions, biopreservation potential and the role of GAPDH in the defence system of S. cerevisiae.     

Hainanenins: a novel family of antimicrobial peptides with strong activity from Hainan cascade-frog, Amolops hainanensis

Antimicrobial peptides (AMPs) secreted by amphibian skin represent an important innate immune defense strategy. There are more than 340 species in the family of Ranidae worldwidely, and from which nearly 100 families of AMPs comprising between 8 and 48 amino acid (aa) residues have been characterized. In current work, two novel AMPs were purified from the skin secretion of Hainan cascade-frog, Amolops hainanensis, and 31 cDNA sequences encoding 10 novel AMPs belonging to 4 families were cloned from the constructed skin cDNA library of A. hainanensis. Among these 10 AMPs, 5 peptides represent the prototypes of a novel amphibian AMP family. According to the generic name of the species of origin, they were designated as hainanenin-1-5. Each of them consists of 21 aa residues with a C-terminal disulphide loop of 7 residues between Cys(15) and Cys(21). Two of them (hainanenin-1 and 5) were then synthesized and their in vitro activities were screened, including antimicrobial, hemolytic and antioxidant activities. The results showed that hainanenin-1 and 5 possessed strong and broad-spectrum antimicrobial activities against Gram-positive, Gram-negative bacteria and fungi, including a large number of clinically isolated drug-resistant pathogenic microorganisms, and slight antioxidant activity. Undesirably, hainanenin-1 and 5 exhibited strong hemolytic activity on human erythrocytes. The discovery of hainanenins and their great antimicrobial potency provides new templates for anti-infective agent design.     

Interaction of seminal plasma proteins with cell surface antigens: Presence of a CD4-binding glycoprotein in human seminal plasma

We report in this paper the presence in the human seminal plasma of a glycoprotein capable of binding to CD4, a surface antigen expressed on the surface of T-cells, macrophages, and sperm cells, which acts as a coreceptor in antigen-mediated T-cell activation and as a receptor for the AIDS virus, HIV-1. This protein, namely gp17 (apparent MW = 17,500 Da), was purified by affinity chromatography and characterized by SDS/PAGE analysis. Its binding to CD4 was inhibited by anti-CD4 mAbs directed against V1, a region of CD4 implicated in the binding to MHC class II antigens and to the HIV-1 envelope protein gp120, but not by mAbs directed against other CD4 determinants. The presence of a CD4-masking factor in human seminal plasma may be relevant to the modulation of maternal immunity at insemination and to the control of sexual transmission of HIV-1.     

Recombinant production of antimicrobial peptides in Escherichia coli: a review

Antimicrobial peptides are of great interest due to their potential application as novel antibiotics. Large quantities of highly purified peptides are required to meet the needs of basic research and clinical trials. Compared with isolation from natural sources and chemical synthesis, recombinant approach offers the most cost-effective means for large-scale peptide manufacture. Among the systems available for heterologous protein production, Escherichia coli has been the most widely used host. Antimicrobial peptides produced in E. coli are often expressed as fusion proteins, a strategy necessary to mask these peptides' lethal effect towards the host and protect them from proteolytic degradation. The present article reviews commonly used fusion partners (e.g., solubility-enhancing, aggregation-promoting and self-cleavable carriers, etc.), cleavage methods and optimization options for antimicrobial peptides production in E. coli. In addition, the various approaches developed to generate recombinant human antimicrobial peptide LL-37, which offer excellent examples demonstrating effective production strategies, were briefly discussed.     

Activation of the mitogen-activated protein kinase p38 by human cytomegalovirus infection through two distinct pathways: a novel mechanism for activation of p38

Recent evidence indicates activated mitogen-activated protein kinase (MAPK) p38 has a critical function in human cytomegalovirus (HCMV) viral DNA replication in infected human fibroblasts. To elucidate the mechanism of HCMV-mediated p38 activation, we have performed a detailed analysis of p38 activation and the kinases associated with this activation at different times postinfection. We demonstrate that p38 kinase activity is strongly increased following viral infection. Inhibition of this activity significantly inhibited HCMV-induced hyperphosphorylation of pRb and phosphorylation of heat shock protein 27, suggesting that p38 activation is involved in virus-mediated changes in host cell metabolism throughout the course of infection. We then provide evidence that p38 activation is mediated by different mechanisms at early times versus later times of infection. At early times of infection (8 to 14 h postinfection [hpi]), when p38 activation is first observed, no significant activation of the three kinases which can directly phosphorylate p38 (namely, MKK3, MKK6, and MKK4) is detected. Using vectors which express dominant negative proteins, we demonstrate that basal MKK6 kinase activity is necessary for HCMV-mediated p38 activation at these early times of infection (12 hpi). Then, we use ATP depletion to show that at 12 hpi, HCMV inhibits dephosphorylation of activated p38. These two experiments suggest that HCMV activates p38 by inhibition of dephosphorylation of p38. In contrast to early times of infection, at later times of infection (48 to 72 hpi), increased MKK3/6, but not MKK4, activity is observed. These results indicate that at early times of HCMV infection, increased steady-state levels of activated p38 is mediated at least in part by inhibition of dephosphorylation of p38, while at later times of infection p38 activation is due to increased activity of the upstream kinases MKK3 and MKK6. These findings indicate that HCMV has developed multiple mechanisms to ensure activation of the MAPK p38, a kinase critical to viral infection.     

Oriented samples: a tool for determining the membrane topology and the mechanism of action of cationic antimicrobial peptides by solid-state NMR

Biophysical Reviews - Overuse and misuse of antibiotics have led bacteria to acquire several mechanisms of resistance. In response to this, researchers have identified natural antimicrobial...     

Overall assessment of antimicrobial peptides in piglets: a set of meta-analyses

Developing alternatives to antibiotics is an urgent need in livestock production. Antimicrobial peptides (AMPs) are regarded as powerful antibiotic substitutes (ASs) because AMPs have broad-spectrum antimicrobial activities and growth-promoting ability. Here, we aimed to comprehensively assess the effects of AMPs on the growth performance, diarrhea rate, intestinal morphology and immunity of healthy or challenged piglets, compared with an antibiotics group or negative control group. We performed a set of meta-analyses of feeding trials from database inception to 27 May 2019. Among the 1379 identified studies, 20 were included in our meta-analyses (56 arms and 4067 piglets). The meta-analyses revealed that (1) compared with the negative control group, AMPs significantly improved the healthy piglets’ average daily gain (ADG), average daily feed intake (ADFI), gain : feed ratio (G/F), levels of immune globulin (Ig) IgM and IgG, and intestinal villus height : crypt depth ratio (V/C) (P < 0.05). Meanwhile, AMPs significantly increased the challenged piglets’ ADG, ADFI, G/F and V/C of the jejunum and ileum, and notably deceased the diarrhea rate (P < 0.05); (2) compared with antibiotics group, the effects of AMPs were slightly weaker than those of antibiotics in the healthy piglets, but AMPs have similar effects to those of antibiotics in challenged piglets. In a higher purity, the optimal dose of AMPs may be approximately 0.01%. Our findings indicate that AMPs can improve piglet growth performance, enhance immunity, benefit intestinal morphology and decrease the diarrheal rate. AMPs could be great ASs especially under infection conditions.     

Permeabilization of raft-containing lipid vesicles by delta-lysin: a mechanism for cell sensitivity to cytotoxic peptides

Delta-lysin is a linear, 26-residue peptide that adopts an alpha-helical, amphipathic structure upon binding to membranes. Delta-lysin preferentially binds to mammalian cell membranes, the outer leaflets of which are enriched in sphingomyelin, cholesterol, and unsaturated phosphatidylcholine. Mixtures including these lipids have been shown to exhibit separation between liquid-disordered (l(d)) and liquid-ordered (l(o)) domains. When rich in sphingomyelin and cholesterol, these ordered domains have been called lipid "rafts". We found that delta-lysin binds poorly to the l(o) (raft) domains; therefore, in mixed-phase lipid vesicles, delta-lysin preferentially binds to the l(d) domains. This leads to the concentration of delta-lysin in l(d) domains, enhancing peptide aggregation and, consequently, the rate of peptide-induced dye efflux from lipid vesicles. The efficient lysis of eukaryotic cells by delta-lysin can thus be attributed not to specific delta-lysin-cholesterol or delta-lysin-sphingomyelin interactions but, rather, to the exclusion of delta-lysin from ordered rafts. The degree to which the kinetics of dye efflux are enhanced in mixed-phase vesicles over those observed in pure, unsaturated phosphatidylcholine vesicles directly reflects the amount of l(d) phase present in mixed-phase systems. This effect of lipid domains has broader consequences, beyond the hemolytic efficiency of delta-lysin. We discuss the hypothesis that bacterial sensitivity to antimicrobial peptides may be determined by a similar mechanism.     

NMEGylation: a novel modification to enhance the bioavailability of therapeutic peptides

We have evaluated "NMEGylation"--the covalent attachment of an oligo-N-methoxyethylglycine (NMEG) chain--as a new form of peptide/protein modification to enhance the bioavailability of short peptides. OligoNMEGs are hydrophilic polyethylene glycol-like molecules made by solid-phase synthesis, typically up to 40 monomers in length. They have been studied as nonfouling surface coatings and as monodisperse mobility modifiers for free-solution conjugate capillary electrophoresis. However, polyNMEGs have not been demonstrated before this work as modifiers of therapeutic proteins. In prior published work, we identified a short peptide, "C20," as a potential extracellular inhibitor of the fusion of human respiratory syncytial virus with mammalian cells. The present study was aimed at improving the C20 peptide's stability and solubility. To this end, we synthesized and studied a series of NMEGylated C20 peptide-peptoid bioconjugates comprising different numbers of NMEGs at either the N- or C-terminus of C20. NMEGylation was found to greatly improve this peptide's solubility and serum stability; however, longer polyNMEGs (n > 3) deleteriously affected peptide binding to the target protein. By incorporating just one NMEG monomer, along with a glycine monomer as a flexible spacer, at C20's N-terminus (NMEG-Gly-C20), we increased both solubility and serum stability greatly, while recovering a binding affinity comparable to that of unmodified C20 peptide. Our results suggest that NMEGylation with an optimized number of NMEG monomers and a proper linker could be useful, more broadly, as a novel modification to enhance bioavailability and efficacy of therapeutic peptides.     

Novel purification method for mammalian seminal plasma phospholipid-binding proteins reveals the presence of a novel member of this family of protein in stallion seminal fluid

A family of bull seminal plasma (BSP) phospholipid-binding proteins (BSP proteins), potentiate heparin- and HDL-induced capacitation. The homologous proteins have been purified from stallion and boar seminal plasma, and detected in low concentrations in other mammalian seminal plasma. In this study, we developed a new isolation method for mammalian seminal plasma choline phospholipid-binding proteins wherein they are present in low concentrations. The method is based on the interaction of this family of proteins with egg yolk low-density lipoprotein fraction (LDF). In order to demonstrate the feasibility of the method, we incubated LDF with alcohol precipitates of bull, boar, and stallion seminal plasma. LDF were re-isolated by ultracentrifugation along with bound proteins. LDF with associated proteins were dialyzed, lyophilized, and delipidated. BSP homologous proteins were finally purified by p-aminophenyl phosphorylcholine (PPC)-agarose and/or gelatin-agarose chromatographies, and analyzed by SDS-PAGE. With this new protocol, phospholipid-binding proteins of bull, boar, and stallion seminal plasma were recovered almost 100%. A new 12 kDa stallion seminal plasma protein of the same family was also isolated and partially sequenced. The radio-immunoassay (RIA) data showed that 10 mg of LDF can bind all BSP proteins present in 120 mg of alcohol precipitated BSP proteins. These results confirm the efficiency of the method and that the LDF step could be used for the isolation of all BSP proteins homologs from different mammalian species.     

Characterization by cDNA cloning of the mRNA of a new growth factor from bovine seminal plasma: acidic seminal fluid protein.

A cDNA expression library in lambda gt11 prepared from cDNA derived of seminal vesicle tissue was screened by means of monospecific rabbit anti-aSFP IgG. The sequence of clone pTF21, containing an insert of 668 bp comprised an open reading frame from position 7 to 411 terminated by two stop codons. From this sequence a protein of 134 amino acid residues can be deduced. The mature aSFP was preceded by a signal peptide of 20 amino acids length. The protein sequence contains no signal for N-glycosylation. The molecular weight calculated from the amino acid sequence is 12922 Da. The start codon ATG is part of the sequence AAGATGA which fulfills the criteria of an initiation consensus sequence. The coding region was followed by 257bp of the complete 3'-untranslated region (3'UTR). A putative polyadenylation signal AATAAT, although not of the standard type, is observed at position 650. According to Northern analysis, aSFP mRNA is expressed in seminal vesicle tissue, ampulla and weakly in tissue of epididymis, but not in testis or other bovine tissue. aSFP is specified by a single copy gene. Attempts to detect homologies to known protein sequences were not successful.