A novel antimicrobial peptide from salivary glands of the hard tick, Ixodes sinensis

A novel antimicrobial peptide named as ixosin was isolated from the salivary glands of the hard tick, Ixodes sinensis, by gel filtration, ion exchange chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Its amino acid sequence was determined as GLHKVMREVLGYERNSYKKFFLR by Edman degradation and its molecular weight was 2870.5 analyzed by fast atom bombardment (FAB) mass spectrometry. This is the first antimicrobial peptide from ticks that lacks cysteine in its primary structure. The cDNA encoding ixosin was cloned by cDNA library screening. The predicted protein from the cDNA sequence composed of 79 amino acids including mature ixosin. Purified ixosin exerted its antimicrobial activities against bacteria and fungi. No similarity was found by BLAST search to any database entries and, thus, our findings describe a novel antimicrobial peptide.     

A novel defensin‐like peptide from salivary glands of the hard tick,Haemaphysalis longicornis

A novel defensin‐like antimicrobial peptide named longicornsin was isolated from the salivary glands of the hard tick, Haemaphysalis longicornis, using a 10‐kDa cut‐off Centriprep filter and reversed‐phase high‐performance liquid chromatography (RP‐HPLC). Its amino acid sequence was determined as DFGCGQGMIFMCQRRCMRLYPGSTGFCRGFRCMCDTHIPLRPPFMVG by Edman degradation. The cDNA encoding longicornsin was cloned by cDNA library screening. The predicted protein from the cDNA sequence was composed of 78 amino acids including a mature longicornsin. It showed similarity with defensin‐like peptides from other ticks by BLAST search. Different from most other tick defensin‐like peptides, longicornsin had a C‐terminal extension. Purified longicornsin exerted potent antimicrobial activities against bacteria and fungi. Interestingly, it even showed strong antimicrobial ability against drug‐resistant microorganisms and Helicobacter pylori. The results of this study indicated that longicornsin is a potential candidate for novel antimicrobial drug design.     

A novel antimicrobial peptide from skin secretions of the earthworm, Pheretima guillelmi (Michaelsen)

A novel lumbricin-like antimicrobial peptide named lumbricin-PG was isolated from skin secretions of the earthworm, Pheretima guillelmi (Michaelsen), using a procedure of one step Sephadex G-50 gel filtration and one step C₈ reverse-phase high-performance liquid chromatography (RP-HPLC). Its amino acid sequence was determined as FSRYARMRDSRPWSDRKNNYSGPQFTYPPEKAPPEKLIKWNN EGSPIFEMPAEGGHIEP by Edman degradation combined with cDNA cloning and mass spectrometry analysis. The cDNA encoding lumbricin-PG was cloned by cDNA library screening. The predicted protein from the cDNA sequence was composed of 73 amino acid residues including a mature lumbricin-PG and predicted signal peptide. It showed similarity with lumbricin antimicrobial peptide from the earthworm, Lumbricus rubellus by BLAST search. Purified lumbricin-PG exerted potential antimicrobial activities against bacteria and fungi; it showed weak hemolysis activity against human and rabbit red cells.     

Recombinant expression of human cathelicidin (hCAP18/LL-37) in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The constitutive expression of human cathelicidin LL-37 antimicrobial peptide was achieved using the methylotrophic yeast, Pichia pastoris. An LL-37 cDNA clone was amplified by PCR using human fetal cDNA library as template. The 111 bp fragment encoding mature LL-37 gene was subcloned into pGAPZ-E, an episomal form of the pGAPZB vector incorporating PARS1. It was then transformed into the P. pastoris X-33 strain for intracellular expression. A small peptide with a molecular mass of about 5 kDa was detected by 17% peptide-PAGE analysis. The recombinant LL-37 peptide was purified from the gel and its amino acid sequence was determined by LC-ESI-MS/MS analysis. The initiating amino acid, methionine, was still attached to the N-terminal region of recombinant LL-37. LL-37 crude extract from P. pastoris showed an antimicrobial activity against Micrococcus luteus as the test strain. The successful expression of human LL-37 indicates that the system may be applicable to the expression of other human defensins without resorting to fusion protein constructions.     

Identification and structure-activity relationship of an antimicrobial peptide of the palustrin-2 family isolated from the skin of the endangered frog Odorrana ishikawae

Recently, we identified nine novel antimicrobial peptides from the skin of the endangered anuran species, Odorrana ishikawae, to assess its innate immune system. In this study an additional antimicrobial peptide was initially isolated based on antimicrobial activity against Escherichia coli. The new antimicrobial peptide belonging to the palustrin-2 family was named palustrin-2ISb. It consists of 36 amino acid residues including 7 amino acids C-terminal to the cyclic heptapeptide Rana box domain. The peptide's primary structure suggests a close relationship with the Chinese odorous frog, Odorrana grahami. The cloned cDNA encoding the precursor protein contained a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg processing site and the C-terminal precursor antimicrobial peptide. It also contained 3 amino acid residues at the C-terminus not found in the mature peptide. Finally, the antimicrobial activities against four microorganisms (E. coli, Staphylococcus aureus, methicillin-resistant S. aureus and Candida albicans) were investigated using several synthetic peptides. A 29 amino acid truncated form of the peptide, lacking the 7 amino acids C-terminal to the Rana box, possessed greater antimicrobial activities than the native structure.     

Identification and bioactivity evaluation of a novel bradykinin inhibitory peptide from the skin secretion of Chinese large odorous frog,Odorrana livida

A novel peptide was isolated from the skin secretion of Chinese large odorous frog, Odorrana livida, and was named as Rana‐BI. The cDNA sequencing was obtained by ‘shotgun’ cloning. The amino acid sequence of the mature peptide was identified as Gly‐Leu‐Leu‐Ser‐Gly‐Lys‐Ser‐Val‐Lys‐Gly‐Ser‐Ile‐OH by automated Edman degradation, and the molecular weight of the peptide was confirmed to be 1144.68 Da by MALDI‐TOF and liquid chromatography/MS. Subsequently, the bioactivity of synthetic peptide was evaluated by smooth muscle assay using isolated rat bladder preparation. It was demonstrated that Rana‐BI inhibited the contraction of rat bladder induced by bradykinin. Comparing with other peptides by searching from database, the primary structure of Rana‐BI showed high similarity with that of an antimicrobial peptide of Rana family (12/12 residues). These data revealed a novel biological function of this peptide. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Characterization of antimicrobial peptides isolated from the skin of the Chinese frog, Rana dybowskii

The skins of amphibians secrete small antimicrobial peptides that fight infection and are being explored as potential alternatives to conventional antibiotics. In this study we combined mass spectrometry with cDNA sequencing to examine antimicrobial peptides in skin secretions from the Chinese frog Rana dybowskii. Thirteen peptides having precursor sequences that resemble known antimicrobial peptides from this genus were identified, ten of which were members of previously described peptide families based on their primary structures; i.e., brevinin-1, Japonicin-1, brevinin-2 and temporin. The other three peptides from R. dybowskii, which were named dybowskin-1CDYa, dybowskin-2 CDYa and dybowskin-2CDYb, had different amino acid compositions and little sequence similarity to known antimicrobial peptides. The carboxyl terminus of dybowskin-1CDY lacked amidation and is therefore clearly distinct from temporin peptides, whereas dybowskin-2CDYa and dybowskin-2CDYb consisted of 18 amino acids and were rich in Arg residues. Chemically synthesized peptides corresponding to mature dybowskin-1CDYa and dybowskin-2CDYa had strong antimicrobial activity and caused little hemolysis of human erythrocytes, suggesting they may serve as interesting templates for the development of novel antibiotics.     

Molecular Cloning and Expression of Yak (Bos grunniens) Lactoferrin cDNA in Pichia pastoris

cDNA encoding lactoferrin from yak was isolated by RT-PCR and then sequenced. The cloned cDNA (2127 bp) encodes a 709 amino acid precursor molecule of yak lactoferrin with a signal peptide of 19 amino acids. The yak lactoferrin cDNA was expressed in Pichia pastoris. The recombinant protein, purified by Ni-NTA affinity column, had a molecular weight of 76 kDa and reacted with an antibody raised against native bovine lactoferrin. The iron-binding behavior and antimicrobial activity of the purified protein indicated that it was correctly folded and functional.     

Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A

We recently reported the primary structures, antimicrobial activities and cDNA precursors of nine novel antimicrobial peptides from the skin of the endangered anuran species, Odorranaishikawae. Their cDNA clones revealed a highly conserved approximately 60 bp region upstream of the start codon. This conserved region was used in the “shotgun” cDNA cloning method to reveal additional cDNAs encoding novel antimicrobial peptides of O.ishikawae. After sequencing 344 clones, we identified novel 13 cDNAs encoding dermal peptides in addition to the previously identified nine antimicrobial peptides. These 13 unique cDNAs encoded precursor proteins each containing a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg/Lys processing site and a dermal peptide at the C-terminus. The dermal peptides were members of the palustrin-2 (two peptides; termed palustrin-2ISc and palustrin-2ISd), nigrocin-2 (one peptide; nigrocin-2ISc), brevinin-1 (one peptide; brevinin-1ISa), odorranain-M (one peptide; odorranain-MISa) and entirely novel peptides (eight peptides; ishikawain-1-8). Although palustrin-2ISd and odorranain-MISa had few antimicrobial activities, palustrin-2ISc and nigrocin-2ISc possessed a broad-spectrum of growth inhibition against bacteria. Brevinin-1ISa had the most potent antimicrobial activities against the Gram-positive bacteria and the fungus but not the Gram-negative bacterium, Escherichiacoli. However, eight novel peptides showed no growth inhibition against these microorganisms.     

A novel myotropic peptide from the skin secretions of the tree frog, Polypedates pingbianensis

A novel myotropic peptide, polypedatein, was purified and characterized from the skin secretions of the tree frog, Polypedates pingbianensis. Its primary structure, TLLCKYFAIC, was determined by Edman degradation and mass spectrometry. Polypedatein was subjected to bioassays including myotropic, antimicrobial, and serine protease inhibitory activities, which are related with many amphibian skin bioactive peptides. It was found to elicit concentration-dependent contractile effects on isolated rat ileum. cDNA clones encoding the precursor of polypedatein were isolated by screening a skin cDNA library of P. pingbianensis and then sequenced. The amino acid sequence deduced from the cDNA sequences matches well with the result from Edman degradation. BLAST search revealed that the sequence of polypedatein did not show similarity to known protein or peptide sequences. Especially, polypedatein does not contain conserved structural motifs of other amphibian myotropic peptides, such as bradykinins, bombesins, cholecystokinin (CCK), and tachykinins, indicating that polypedatein belongs to a novel amphibian myotropic peptide family. The signal peptide of the precursor encoding polypedatein shows significant sequence identity to that of other amphibian skin defensive peptides, such as antimicrobial peptides, bradykinins, lectins, and serine protease inhibitors, suggesting that polypedatein belongs to a novel amphibian myotropic peptide family. Polypedatein is also the first bioactive peptide from the genus of the frog, Polypedates.     

An antimicrobial peptide of the earthworm Pheretima tschiliensis: cDNA cloning, expression and immunolocalization

A cDNA encoding a putative antimicrobial peptide (named PP-1) was obtained using a rapid amplification of cDNA ends from the Asian earthworm, Pheretima tschiliensis. PP-1 showed 77.6% homology with the antimicrobial peptide lumbricin I isolated from the earthworm Lumbricus rubellus. PP-1 lacked an obvious signal peptide sequence. RT-PCR analysis demonstrated that this gene was expressed mainly in the body wall. PP-1 was expressed in Escherichia coli as a fusion protein with a maltoze-binding protein. A polyclonal antiserum was raised in mice using this recombinant fusion protein as antigen. Immunohistochemical studies showed that PP-1 was only in the mucus of the epidermis.     

Novel short antimicrobial peptide isolated from Xenopus laevis skin

A rich source of bioactive peptides, including a large number of antimicrobial peptides, has been found in amphibian skin. In this study, a novel short antimicrobial peptide was purified from Xenopus laevis skin and characterised through reversed‐phase high‐performance liquid chromatography, Edman degradation and matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry. The peptide was composed of six amino acids with a sequence of DEDLDE and thus named X. laevis antibacterial peptide‐P2 (XLAsp‐P2). Transmission electron microscopy revealed that this peptide showed potential antimicrobial abilities against bacteria by damaging the bacterial cell membrane. XLAsp‐P2 maybe inhibit bacterial growth by binding to the microbial genomic DNA. The peptide also exhibited a weak haemolytic activity against rabbit red blood cells. Therefore, XLAsp‐P2 is a novel short anionic antibacterial peptide with broad activities. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

A novel antimicrobial peptide from amphibian skin secretions of Odorrana grahami

A novel antimicrobial peptide named odorranain-NR was identified from skin secretions of the diskless odorous frog, Odorrana grahami. It is composed of 23 amino acids with an amino acid sequence of GLLSGILGAGKHIVCGLTGCAKA. Odorranain-NR was classified into a novel family of antimicrobial peptide although it shared similarity with amphibian antimicrobial peptide family of nigrocin. Odorranain-NR has an unusual intramolecular disulfide-bridged hexapeptide segment that is different from the intramolecular disulfide-bridged heptapeptide segment at the C-terminal end of nigrocins. Furthermore, the -AKA fragment at the C-terminal of odorranain-NR is also different from nigrocins. Three different cDNAs encoding two odorranain-NR precursors and only one mature odorranain-NR was cloned from the cDNA library of the skin of O. grahami. This peptide showed antimicrobial activities against tested microorganisms except Escherichia coli (ATCC25922). Its antimicrobial mechanisms were investigated by transmission electron microcopy. Odorranain-NR exerted its antimicrobial functions by various means depending on different microorganisms.     

Purification and characterization of a D‐mannose specific lectin from the green marine alga,Bryopsis plumosa

A d ‐mannose specific lectin was purified from the green marine alga, Bryopsis plumosa (Huds.) Ag. The lectin agglutinated horse and sheep erythrocytes. Matrix assisted laser desorption/ionization time of flight mass spectrometry, size exclusion chromatography, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and two dimensional gel electrophoresis (2DE) results showed that the lectin was a monomer with molecular weight of 17 kDa and pI 7.3. The agglutinating activity was inhibited by d ‐mannose (1 mM), α‐methyl‐D‐mannose (4 mM) and l ‐fucose (8 mM). d ‐glucose (125 mM) showed weak inhibition. The lectin did not need divalent cations for agglutinating activity. N‐terminal amino acid sequence of the lectin was analyzed. As the lectin was novel, we named it BPL‐2 (Bryopsis plumosa lectin 2). Full cDNA sequence of BPL‐2 was obtained using cDNA library. It was comprised of 624 bp of open reading frame and 167 bp/57 bp of 3′/5′ untranslated regions as well as N‐terminal signal peptide. No antimicrobial activity of BPL‐2 was observed in four bacteria strains tested.     

Purification of a novel antibacterial short peptide in earthworm Eisenia foetida

Earthworms live in an environment with abundantpathogens. These pathogens are, firstly, bacteria living inwater or soil that are ingested during feeding or introducedinto the body following injury. Parasites, particularlylarval forms, which represent the dissemination phase,are another important group of potentially pathogenicagents. During the course of evolution, earthworms havedeveloped defense strategies against these living patho-gens [1,2]. Earthworms lack true antibodies and hence anada…     

Nucleotide sequence and characterization of a cDNA encoding the acetohydroxy acid isomeroreductase from Arabidopsis thaliana

The primary structure of acetohydroxy acid isomeroreductase from Arabidopsis thaliana was deduced from two overlapping cDNA. The full-length cDNA sequence predicts an amino acid sequence for the protein precursor of 591 residues including a putative transit peptide of 67 amino acids. Comparison of the A. thaliana and spinach acetohydroxy acid isomeroreductases reveals that the sequences are conserved in the mature protein regions, but divergent in the transit peptides and around their putative processing site.