Characterization of two crustin antimicrobial peptides from the freshwater crayfish Pacifastacus leniusculus

The two bacteria-induced crustin genes, Plcrustin1 and Plcrustin2, previously found in the hemocyte cDNA library of Pacifastacus leniusculus, contain the open reading frames of 357 bp encoding a putative protein of 118 amino acid residues and 330 bp encoding a putative protein of 109 amino acid residues, respectively. The carboxyl-terminal part of the two crustins possesses, respectively, 7 and 8 conserved cysteine residues representation of a WAP domain that is found in carcinins and crustins in other several crustaceans. The amino acid sequences of Plcrustin1 and Plcrustin2 show that they belong to type I crustins. In order to characterize their properties and biological activities, the two recombinant crustin proteins were produced in the Escherichia coli expression system. Antimicrobial assays showed that the growth of only one Gram-positive bacterium, Micrococcus luteus M1 11, was inhibited by the recombinant Plcrustin1 and Plcrustin2 with MIC of about 0.07-0.27 μM and 3.5-8 μM, respectively. In addition, the study of inhibition mechanism revealed that the antimicrobial activity of the two recombinant crustin proteins was a result of bactericidal effect. However, the two crustins did not exhibit the inhibitory activities against trypsin, chymotrypsin, elastase and subtilisin A.     

Molecular cloning of crustins from the hemocytes of Brazilian penaeid shrimps

Crustins are antimicrobial peptides initially identified in the hemocytes of the crab Carcinus maenas (11.5-kDa peptide or carcinin) and recently also recognized in penaeid shrimps and other crustacean species. The aim of this study was to identify sequences encoding for crustins from the hemocytes of four Brazilian penaeid species: Farfantepenaeus paulensis, Farfantepenaeus subtilis, Farfantepenaeus brasiliensis and Litopenaeus schmitti. Using primers based on consensus nucleotide alignment of crustins from different crustaceans, cDNA sequences coding for crustins in all indigenous penaeid species were amplified. The obtained four crustin sequences encoded for peptides containing a hydrophobic N-terminal region rich in glycine repeats and a C-terminal part with 12 cysteine residues and a conserved whey acidic protein domain. All obtained crustin sequences showed high amino acidic similarity among each other and with crustins from litopenaeid shrimps (76-98%). This is the first report of crustins in native Brazilian penaeid shrimps.     

A double WAP domain (DWD)-containing protein with proteinase inhibitory activity in Chinese white shrimp, Fenneropenaeus chinensis

The whey major component, whey acidic protein (WAP), has one or more WAP domains characterized by a four-disulfide core (4-DSC) structure. These kinds of proteins are involved in multiple functions, including proteinase inhibitor activity, antimicrobial activity, ATPase inhibitor activity, and regulatory function in cell proliferation. Recent research indicates that WAP domain-containing proteins play an important role in the innate immunity of crustaceans. In this study, a novel double WAP domain (DWD)-containing protein named Fc-DWD was found for the first time in Chinese white shrimp, Fenneropenaeus chinensis. The open reading frame of Fc-DWD encodes a protein of 117 amino acids, including a signal peptide of 16 amino acids and two WAP domains. The predicted molecular mass of the mature protein is 12.78 kDa with an estimated pI of 8.49. The first WAP domain, named WAP 1, composed of 49 amino acids locates in the amino-terminal of Fc-DWD, and the second WAP domain, named WAP 2, composed of 45 amino acids locates in the carboxy-terminal. Fc-DWD mRNA was upregulated in hemocytes, hepatopancreas, gills, and stomach of bacteria- and virus-challenged shrimp. Results of the binding assay showed that rFc-DWD could bind to both Gram-negative bacteria and Gram-positive bacteria. rWAP 1 could only bind to Gram-positive bacteria, but rWAP 2 could bind to both Gram-negative and positive bacteria. Moreover, rFc-DWD exhibited proteinase inhibitory activity against the secretory proteinase(s) from Bacillus subtilis and Pseudomonas aeruginosa. All of these findings suggest that Fc-DWD may play an important role in enabling the host defense to execute its proteinase inhibitory activity against pathogens.     

Multiple isoforms of immune-related genes from hemocytes and eyestalk cDNA libraries of swimming crab Portunus trituberculatus

Expressed sequence tags (ESTs) analysis has been shown to be an efficient approach not only for gene discovery, but also for gene expression profiles performance. Two full-length enriched cDNA libraries were constructed from hemocytes and eyestalk of Portunus trituberculatus, respectively, and randomly sequenced to collect genomic information and identify genes involved in immune defense response. A total of 99 unigenes including 64 unigenes (6.00% of 1066 unigenes) in hemocytes library and 35 unigens (6.86% of 510 unigenes) in eyestalk library are identified to be immune genes. These genes are categorized into six classes, viz. antimicrobial peptides, redox proteins, melanization related proteins, chaperone proteins, clottable proteins and other immune factors. The content and category of immune genes in eyestalk library indicate eyestalk might have unrecognized role in crab immunity. Five immune genes containing multiple protein isoforms are identified and characterized, including anti-lipopolysaccharide factor (PtALF1-7), crustin (PtCrustin1-3), thioredoxin (PtTrx1-2), clip domain serine proteinase (PtcSP1-5) and kazal-type proteinase inhibitor (PtKPI1-4). Sequence alignment and phylogenetic analysis reveal PtALF1-7 contain two conserved cysteine residues and might be encoded by multiple genomic loci. PtCrustin1-3 share the consensus cysteine motif and are considered as Type I crustins. PtTrx1 possesses the critical structural cysteine residue C?3 of Trx-1, while PtTrx2 has the N-terminal mitochondrial translocation signal of Trx-2. Sequence analysis shows PtcSP1-5 contain one clip domain and one partial SP catalytic triad domain. PtKPI1-4 present one typical Kazal domain consisting of six conserved cysteine residues. Some protein isoforms are tissue-specific, which might suggest they have different origins and perform diverse functions. Except PtALF1-3 and PtCrustin1, the other isoformes in this study are firstly identified from P. trituberculatus. Especially, PtTrx2 are firstly identified from crustaceans. Our research will provide useful genomic information of P. trituberculatus and be helpful in understanding the molecular mechanisms of crab immunity.     

Adaptive evolution of crustin antimicrobial peptides in decapods

Antimicrobial peptides (AMPs) are present in a wide range of taxonomic groups and played a crucial role in host adaptation to a diverse array of ever-changing pathogens. Crustin, a cysteine-rich cationic AMP, is known to exhibit antimicrobial activity against Gram-positive and Gram-negative bacteria in decapods. Given their important role in host-immune defense, a large proportion of amino acid substitutions in crustin AMPs are expected to be fixed by natural selection. Utilizing the complete coding nucleotide sequence data of crustin, the present study revealed the pervasive role of positive Darwinian selection in the evolution and divergence of crustin AMPs in decapods. Approximately, 20–35?% of codons in two phylogenetically distinct groups of closely related crustins in Penaeid shrimps are shown to have evolved under positive selection. Interestingly, several of these positively selected sites are located at the carboxyl-terminal region, the region that directly interacts with the invading pathogens. Pathogen-mediated selection pressure could be the likely cause for such an accelerated rate of amino acid substitutions and could have contributed to the structural and functional diversification of crustin AMPs in several taxa.     

Molecular cloning and characterization of crustin from mud crab<Emphasis Type="Italic"> Scylla paramamosain</Emphasis>

Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. In the present study, we report the identification and characterization of a crustin (CrusSp) from the hemocyte of mud crab, Scylla paramamosain using an expressed sequence tag (EST) and rapid amplification cDNA end (RACE) approaches. Analysis of the nucleotide sequence revealed seven different variances of the CrusSp cDNA in mud crab. The open reading frame encodes a protein of 111 amino acids with 21 residues signal sequence. The predicted molecular mass of the mature protein (90 amino acids) is 10.27 kDa with an estimated pI of 8.54. Analysis of the protein domain features indicated typical conserved cysteine residues containing a single whey acidic protein (WAP) domain at the C-terminus. A neighbour-joining tree showed that S. paramamosain crustin is closely related to other crustin homologues, and displays the highest similarity to crustin antimicrobial peptide in shore crab Carcinus maenas. Four exons and three introns were identified within the 999 bp genomic DNA sequence of CrusSp. Tissue distribution analysis showed that CrusSp was highly expressed in hemocytes, gills, intestines and muscle but it was not expressed in hepatopancreas and eyestalks. To gain insight into the in vitro antimicrobial activities of CrusSp, the mature peptide coding region was cloned into E. coli for heterologous expression. The recombinant CrusSp could inhibit the growth of gram-positive bacteria but had no inhibition activity against gram-negative bacteria. These results indicated the involvement of CrusSp in the innate immunity of S. paramamosain.     

Molecular evolution of monotreme and marsupial whey acidic protein genes

Whey acidic protein (WAP), a major whey protein present in milk of a number of mammalian species has characteristic cysteine-rich domains known as four-disulfide cores (4-DSC). Eutherian WAP, expressed in the mammary gland throughout lactation, has two 4-DSC domains, (DI-DII) whereas marsupial WAP, expressed only during mid-late lactation, contains an additional 4-DSC (DIII), and has a DIII-D1-DII configuration. We report the expression and evolution of echidna (Tachyglossus aculeatus) and platypus (Onithorhynchus anatinus) WAP cDNAs. Predicted translation of monotreme cDNAs showed echidna WAP contains two 4-DSC domains corresponding to DIII-DII, whereas platypus WAP contains an additional domain at the C-terminus with homology to DII and has the configuration DIII-DII-DII. Both monotreme WAPs represent new WAP protein configurations. We propose models for evolution of the WAP gene in the mammalian lineage either through exon loss from an ancient ancestor or by rapid evolution via the process of exon shuffling. This evolutionary outcome may reflect differences in lactation strategy between marsupials, monotremes, and eutherians, and give insight to biological function of the gene products. WAP four-disulfide core domain 2 (WFDC2) proteins were also identified in echidna, platypus and tammar wallaby (Macropus eugenii) lactating mammary cells. WFDC2 proteins are secreted proteins not previously associated with lactation. Mammary gland expression of tammar WFDC2 during the course of lactation showed WFDC2 was elevated during pregnancy, reduced in early lactation and absent in mid-late lactation.     

Structural and functional differences of Litopenaeus vannamei crustins

Penaeid crustins were described in Litopenaeus vannamei and L. setiferus as proteins belonging to an antibacterial peptide family with similar sequences but different sizes. Six crustin-coding clones were isolated from a cDNA library from L. vannamei hemocytes, sequenced and compared. Two different isoforms (named I and P) were found, based on two nucleotide differences that produce one change in amino acid sequence (Ile/Pro). Other single differences in nucleotide sequences were also noted, but they did not change the translated product. The mRNA steady state levels of crustin I, but not of crustin P, were down regulated by Vibrio alginolyticus inoculation. Thus, the differences among penaeid crustins seem to be associated with one amino acid substitution, which affects their expression after bacterial inoculation. By structural similarity, shrimp crustins seem to belong to an antibacterial WAP-domain containing protein family.     

Molecular cloning and characterization of three crustins from the Chinese white shrimp,Fenneropenaeus chinensis

Antibacterial peptides crustins are the effector molecules of innate immunity in decapods. In this study, three crustin cDNA sequences (Fc-crus 1, Fc-crus 2, and Fc-crus 3) were cloned from the Chinese white shrimp Fenneropenaeus chinensis. The full-length cDNAs of Fc-crus 2 and 3 are 473 bp and 574 bp, respectively. The deduced peptides of Fc-crus 2 and 3 contain a signal peptide and a crustin domain at the C-terminal formed by twelve conserved cysteine residues. The partial sequence of Fc-cru 1 is 575 bp long and the deduced amino acids also contain a crustin domain. The expression profiles of these three crustins were studied with RT-PCR. Fc-crus 1 and Fc-crus 2 constitutively expressed in hemocytes with high levels, and the expression level is increased in the heart, stomach, intestine and ovaries when shrimp was challenged with Vibrio anguillarum, The expression of Fc-crus 1 and Fc-crus 2 was detected in each developmental stage. Fc-crus 3 was constitutively expressed in the ovaries and induced as an expression in the stomach. Unlike Fc-crus 1 and Fc-crus 2, the mRNA of Fc-crus 3 was not detected in the developmental stages extending from nauplii and mysis to post-larvae. The recombinant proteins containing mature Fc-crus 2 and Fc-crus 3 were recombinantly expressed in Escherichia coli and respectively purified. The antibacterial assays revealed that the recombinant mFc-crus could inhibit the growth of Gram-positive bacteria in vitro.     

Crustin,a WAP domain containing antimicrobial peptide from freshwater prawn Macrobrachium rosenbergii: Immune characterization

Crustin (MrCrs) was sequenced from a freshwater prawn Macrobrachium rosenbergii. The MrCrs protein contains a signal peptide region at N-terminus between 1 and 22 and a long whey acidic protein domain (WAP domain) at C-terminus between 57 and 110 along with a WAP-type ‘four-disulfide core’ motif. Phylogenetic results show that MrCrs is clustered together with other crustacean crustin groups. MrCrs showed high sequence similarity (77%) with crustin from Pacific white shrimp Litopenaeus vannamei and Japanese spiny lobster Panulirus japonicas. I-TASSER uses the best structure templates to predict the possible structures of MrCrs along with PDB IDs such as 2RELA and 1FLEI. The gene expressions of MrCrs in both healthy M. rosenbergii and those infected with virus including infectious hypodermal and hematopoietic necrosis virus (IHHNV) and white spot syndrome virus (WSSV) and bacteria Aeromonas hydrophila (Gram-negative) and Enterococcus faecium (Gram-positive) were examined using quantitative real time PCR. To understand its biological activity, the recombinant MrCrs gene was constructed and expressed in Escherichia coli BL21 (DE3). The recombinant MrCrs protein agglutinated with the bacteria considered for analysis at a concentration of 25 μg/ml, except Lactococcus lactis. The bactericidal results showed that the recombinant MrCrs protein destroyed all the bacteria after incubation, even less than 6 h. These results suggest that MrCrs is a potential antimicrobial peptide, which is involved in the defense system of M. rosenbergii against viral and bacterial infections.     

Amphioxus connectin exhibits merged structure as invertebrate connectin in I-band region and vertebrate connectin in A-band region

Connectin is an elastic protein found in vertebrate striated muscle and in some invertebrates as connectin-like proteins. In this study, we determined the structure of the amphioxus connectin gene and analyzed its sequence based on its genomic information. Amphioxus is not a vertebrate but, phylogenetically, the lowest chordate. Analysis of gene structure revealed that the amphioxus gene is approximately 430 kb in length and consists of regions with exons of repeatedly aligned immunoglobulin (Ig) domains and regions with exons of fibronectin type 3 and Ig domain repeats. With regard to this sequence, although the region corresponding to the I-band is homologous to that of invertebrate connectin-like proteins and has an Ig-PEVK region similar to that of the Neanthes sp. 4000K protein, the region corresponding to the A-band has a super-repeat structure of Ig and fibronectin type 3 domains and a kinase domain near the C-terminus, which is similar to the structure of vertebrate connectin. These findings revealed that amphioxus connectin has the domain structure of invertebrate connectin-like proteins at its N-terminus and that of vertebrate connectin at its C-terminus. Thus, amphioxus connectin has a novel structure among known connectin-like proteins. This finding suggests that the formation and maintenance of the sarcomeric structure of amphioxus striated muscle are similar to those of vertebrates; however, its elasticity is different from that of vertebrates, being more similar to that of invertebrates.     

Invertebrate trypsins: a review

Food protein hydrolysis, a crucial step in digestion, is catalyzed by trypsin enzymes from the digestive apparatus of invertebrates. Trypsin appeared early in evolution and occurs in all phyla and, in the digestive systems of invertebrates, it became the most abundant proteinase. As in vertebrates, invertebrate trypsin is also present in several forms (isoenzymes). Its physiological importance in food protein digestion in several invertebrate species has emerged with compelling evidence; and several other physiological functions, such as regulation of digestive functions, are now settled. Recent advances in the knowledge of invertebrate trypsin synthesis, regulation, genetics, catalytic characteristics; structure, evolution, as well as inhibition, especially in non-Drosophilidae insects and in some crustaceans are reviewed. Most of the existing information is largely based on the use of several tools, including molecular techniques, to answer many still open questions and solve medical, agricultural, and food quality problems.