Characterization and antimicrobial evaluation of SpPR-AMP1, a proline-rich antimicrobial peptide from the mud crab Scylla paramamosain

Antimicrobial peptide (AMP) is an important molecule in the innate immune system. Here, we report the cloning and functional studies of proline-rich AMPs (PR-AMPs) from the three species of mud crab: Scylla paramamosain, S. serrata, and the swimming crab Portunus pelagicus. The deduced peptides revealed that they contain the putative signal peptides and encode for mature peptides, which contain sequence architecture similar to a 6.5-kDa proline-rich AMP of the shore crab, Carcinus maenas which showed similarity with the bactenecin7. Tissue distribution analysis indicated that the SpPR-AMP1 was expressed in a wide range of adult tissues, with the highest expression levels in the crab hemocyte. Challenge experiments showed that the levels of SpPR-AMP1 mRNA expression were up-regulated in the hemocyte after peptidoglycan stimulation. To evaluate the biological properties of mature SpPR-AMP1, peptides were chemically synthesized and recombinantly expressed. SpPR-AMP1 showed strong antibacterial activity against both Gram-positive bacteria Micrococcus luteus and Gram-negative bacteria Vibrio harveyi. The results indicate that the SpPR-AMP1 plays a role in crab immunity.     

Canine cathelicidin (K9CATH): gene cloning, expression, and biochemical activity of a novel pro-myeloid antimicrobial peptide

Cathelicidins, a group of cationic peptides found in leukocytes and epithelial cells, play a central role in the early innate immune defense against infection. Although these host defense peptides have been reported in several mammalian species, including primates, no cathelicidins have been identified in carnivores. Here we report the cloning, tissue expression and biological activity of a novel canine cathelicidin (K9CATH). The full-length cDNA sequence of K9CATH encodes a predicted 172 amino acid pre-propeptide that is 60–70% similar to other mammalian cathelicidins. Mass spectrometry analysis confirmed that the 38 aa mature K9CATH peptide was present in neutrophil granule contents. Synthetic K9CATH displayed broad antimicrobial activity against Gram-positive bacteria (Listeria monocytogenes, and Staphylococcus aureus; MICs (minimal inhibitory concentrations) 0.5 and 50 μM, respectively), Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Salmonella serotype Typhimurium, Pseudomonas aeruginosa, Proteus mirabilis; MICs 1.25 μM, Salmonella serotype Enteritidis; MIC 0.5 μM, and Neisseria gonorrhoeae; MIC 0.06 μM), and yeast (Candida albicans; MIC 12.5–50 μM). K9CATH demonstrated high antimicrobial activity against Ureaplasma canigenitalium, and lower activity against Ureaplasma urealyticum (MIC 0.06 and 50 μM, respectively). Similar to its ovine congener SMAP-29, K9CATH possesses salt-independent antimicrobial activity and LPS binding capacity. K9CATH displayed minimal hemolytic activity against human, dog and chicken erythrocytes. The potency and broad antimicrobial activity of K9CATH suggest that this peptide may act as a fundamental contributor to the innate immune responses in this carnivore species     

A highly divergent gene cluster in honey bees encodes a novel silk family

The pupal cocoon of the domesticated silk moth Bombyx mori is the best known and most extensively studied insect silk. It is not widely known that Apis mellifera larvae also produce silk. We have used a combination of genomic and proteomic techniques to identify four honey bee fiber genes (AmelFibroin1-4) and two silk-associated genes (AmelSA1 and 2). The four fiber genes are small, comprise a single exon each, and are clustered on a short genomic region where the open reading frames are GC-rich amid low GC intergenic regions. The genes encode similar proteins that are highly helical and predicted to form unusually tight coiled coils. Despite the similarity in size, structure, and composition of the encoded proteins, the genes have low primary sequence identity. We propose that the four fiber genes have arisen from gene duplication events but have subsequently diverged significantly. The silk-associated genes encode proteins likely to act as a glue (AmelSA1) and involved in silk processing (AmelSA2). Although the silks of honey bees and silkmoths both originate in larval labial glands, the silk proteins are completely different in their primary, secondary, and tertiary structures as well as the genomic arrangement of the genes encoding them. This implies independent evolutionary origins for these functionally related proteins.     

Differential antimicrobial peptide gene expression patterns during early chicken embryological development

The adaptive immune system is not completely developed when chickens hatch, so the innate immune system has evolved a range of mechanisms to deal with early pathogenic assault. Avian β-defensins (AvBDs) and cathelicidins (CTHLs) are two major sub-classes of antimicrobial peptides (AMPs) with a fundamental role in both innate and adaptive immune responses. In this study, we demonstrate distinct expression patterns of innate immune genes including – Toll-like receptors (TLRs) (TLR2, TLR15 and TLR21, but not TLR4), the complete repertoire of AvBDs, CTHLs and both pro- and anti-inflammatory cytokines (IL1B, IL8, IFNG and IL10) during early chicken embryonic development. AvBD9 was significantly increased by over 150 fold at day 9; and AvBD10 was increased by over 100 fold at day 12 in the abdomen of the embryo, relative to day 3 expression levels (P < 0.01). In contrast, AvBD14 was preferentially expressed in the head of the embryo. This is the first study to demonstrate differential patterns of AMP gene expression in the sterile environment of the developing embryo. Our results propose novel roles for AMPs during development and reveal the innate preparedness of developing embryos for pathogenic assault in ovo, or post-hatching.     

Organization and expression analysis of the zebrafish hepcidin gene, an antimicrobial peptide gene conserved among vertebrates

Hepcidin is an antimicrobial peptide and iron-regulatory molecule that is conserved among vertebrates. Mutations or over-expression of the human hepcidin gene have been found in patients with hemochromatosis and refractory anemia. To further understand the function and regulation of hepcidin, animal models are needed. We sequenced cDNA, genes and upstream regions of zebrafish hepcidin and analyzed gene expression by kinetic PCR. Zebrafish hepcidin genes consist of two introns and three exons that encode a prepropeptide (91 amino acids). The amino acid sequences and gene organization were remarkably conserved between zebrafish and other species. Elevated gene expression was observed in abdominal organs, skin, and heart in fish that developed signs of infection following bacterial injection. Zebrafish may be a suitable model organism for further study of hepcidin gene regulation.     

A novel pseudoautosomal human gene encodes a putative protein similar to Ac-like transposases

We report the cloning of a novel gene, called Tramp, in the Xp/Yp PAR region that has a functional homologue on the Y chromosome and escapes X-inactivation. This gene encodes, within a single exon, a putative protein that has amino acid similarity with transposases of the Ac family. Flanking this gene we have identified putative terminal inverted repeats (TIRs) and a duplicate target site, suggesting that it may be an ancient transposable element. The nucleotide differences in these sites and the TIR-binding inactivity of the putative Tramp protein suggest that this element is not an autonomous transposon. In the human genome, the Tramp protein may be involved in the transposition of other transposable elements, like medium reiterated frequency repeats, or it could be specialized in the acquisition of a new cellular function.      

A novel antimicrobial peptide from the sea hare Dolabella auricularia

The sea hare Dolabella auricularia is a marine shell-less gastropod. Four cytotoxic glycoproteins named dolabellanin A, C, E and P were found in the animal previously. An antimicrobial factor was newly isolated from the sea hare's body-wall including skin and mucus. This factor is a novel peptide which consists of 33 amino acid residues, and is called dolabellanin B2. Dolabellanin B2 was cytotoxically effective against some pathogenic microorganisms at a concentration of 2.5-100 microg/ml.     

Cloning and sequencing of a novel human gene which encodes a putative hydroxylase

Using a conventional two-hybrid technique with MAWD as bait protein, a novel full-length cDNA was isolated and sequenced from a human liver cDNA library. This cDNA consists of 2575 base pairs and has a predicted open reading frame encoding 255 amino acids. Overall, it is similar to the catalytic enzyme PHZF, catalyzing the hydroxylation of phenazine-1-carboxylic acid to 2-hydroxy-phenazine-1-carboxylic acid. Polymerase chain reaction-based mapping with both a monochromosomal hybrid panel and radiation hybrid cell panels placed the gene to human chromosome 10q21.1 near the marker D10S210. Received: December 15, 2000 / Accepted: January 22, 2001     

Pleurocidin,a novel antimicrobial peptide,induces human mast cell activation through the FPRL1 receptor

Pleurocidins are a novel family of α-helical cationic antimicrobial peptides (CAPs) that are structurally and functionally similar to cathelicidins, one of the major CAP families. As cathelicidins stimulate mast cell chemotaxis and mediator release, we postulated that pleurocidins similarly activate mast cells. A screen of 20 pleurocidin peptides revealed that some were capable of degranulating the human mast cell line LAD2 (Laboratory of Allergic Diseases 2). Pleurocidin NRC-04 caused LAD2 to adhere, migrate, degranulate, and release cysteinyl leukotrienes and prostaglandin D₂. Moreover, pleurocidin increased intracellular Ca²⁺ mobilization in mast cells and induced the production of proinflammatory chemokines such as monocyte chemotactic protein-1/C-C motif chemokine ligand 2 (CCL2) and macrophage inflammatory protein-1β/CCL4. Our evaluation of possible cellular mechanisms suggested that G proteins, phosphoinositol-3 kinase (PI3K), phospholipase C (PLC), and phosphokinase C (PKC) were involved in pleurocidin-induced mast cell activation as evidenced by the inhibitory effects of pertussis toxin (G protein inhibitor), wortmanin (PI3K inhibitor), U-73122 (PLC inhibitor), and Ro-31-8220 (PKC inhibitor), respectively. We also found that human mast cells expressed the N-formyl-peptide receptor 1 (FPRL1) receptor and FPRL1-specific inhibitor affected pleurocidin-mediated activation of mast cell. Our finding that the novel CAP pleurocidin activated human mast cell through G protein–coupled receptor signaling suggests that this peptide might have immunomodulatory functions.     

A novel Toll like receptor with two TIR domains (HcToll-2) is involved in regulation of antimicrobial peptide gene expression of Hyriopsis cumingii

Animal Toll-like receptors (TLRs) are involved in innate immunity. Toll proteins are generally transmembrane proteins. In this study, an atypical Toll-like receptor (HcToll-2) was identified from the triangle-shell pearl mussel Hyriopsis cumingii, which belongs to phylum Mollusca. Unlike the typical Toll like receptors with extracellular leucine-rich repeats (LRRs), transmembrane, and intracellular Toll/interleukin-1 receptor (TIR) domains, HcToll-2 has two homologous TIR domains located at the C-terminal (designated as HcTIR1 and HcTIR2) and lacks a transmembrane domain. Phylogenetic analysis showed that HcTIR1 was clustered with TIR of sea anemone Toll, and HcTIR2 was clustered with TIR of Drosophila Toll. HcToll-2 mRNA could be detected in the hepatopancreas and was upregulated after challenge with Escherichia coli and Staphylococcus aureus. Recombinant HcLRR protein with GST tag could bind to bacteria and also to LPS and PGN. Over-expression of both HcTIR1 and HcTIR2 induced drosomycin genes in Drosophila S2 cells. RNAi analysis showed that HcToll-2 was required for the expression of theromacin, which is a cysteine-rich antimicrobial peptide (AMP) gene. This research is the first report of an atypical Toll-like receptor HcToll-2 involved in antibacterial immunity through induction of AMP expression.     

A mouse Y chromosome gene encoded by a region essential for spermatogenesis and expression of male-specific minor histocompatibility antigens

A new mouse Y chromosome gene, Smcy, has been isolated fromthe region encoding Spy, a spermato-genesis gene and Hya andSdma, the genes that, respectively, control the expression ofthe male specific minor histocompatibility antigen H-Y, as measuredby specific T-cell assays and the serologically detected maleantigen SDMA. Smcy is well conserved on the Y in mouse, manand even marsupials. It is expressed in all adult male tissuestested and can also be detected during mouse development fromas early as two cells. In addition, its human Y homologue, SMCY,is expressed in multiple tissues and maps to the same Yq deletioninterval as the human H-Y antigen controlling locus, HY.