Jiehui Chen

Identification and agglutination properties of hemocyanin from the mud crab (Scylla serrata)

Infectious diseases have significantly delayed the growth of crab aquaculture. Identification of the immune molecules and characterization of the defense mechanisms will be pivotal to the reduction of these diseases. Hemocyanin is an important non-specific immune protein present in the hemolymph of both mollusks and arthropods. However, little is known about the hemocyanin from the mud crab Scylla serrata. In this study, we identified the S. serrata hemocyanin using affinity proteomics and investigated its agglutinative properties. The results showed that S. serrata hemocyanin consists of five subunits with molecular weights of 70, 72, 75, 76 and 80 kDa, respectively. It demonstrated agglutination activities against seven bacterial species at concentrations ranging from 7.5 to 30 μg/ml. Agglutination was inhibited by 50-200 mM of N-acetylneuraminic acid, α-d-glucose, d-galactose and d-xylose. The 76 kDa subunit was identified as the protein that primarily binds bacterial cells and we speculate that it functions as the agglutinating subunit. We showed that outer membrane proteins (Omp) of bacteria could completely inhibit agglutination and that the agglutination activities of hemocyanin against Escherichia coli ▵OmpA and ▵OmpX mutants were significantly decreased, suggesting that these two Omps may be important ligands of hemocyanin. Together, the data collectively suggests that the 76 kDa subunit of S. serrata hemocyanin mediates agglutination through recognition of OmpA and OmpX proteins in bacteria.

SNPs of hemocyanin C-terminal fragment in shrimp Litopenaeus vannamei.

In this study, we identified a variable region in the C‐terminus of hemocyanin from the shrimp Litopenaeus vannamei (2288–2503 bp, HcSC) by sequence alignments. A total of 13 SNPs were identified by PCR‐SSCP and HcSC clone sequencing. The SSCP patterns of HcSC could be modulated in Vibro parahaemolyticus‐treated shrimps. A novel SSCP band with four SNP sites was identified in V. parahaemolyticus‐resistant shrimps. More importantly, three of these four SNPs introduced variations in amino acid sequence and possibly secondary structure of the HcSC polypeptide and resulted in a higher agglutinative activity against seven pathogenic bacteria. These results suggest that the C‐terminus of shrimp L. vannamei hemocyanin possesses SNPs, which may be related to shrimp resistance to different pathogens.

Identification and characterization of the related immune-enhancing proteins in crab Scylla paramamosain stimulated with rhubarb polysaccharides.

Recently, considerable interest has been focused on immunostimulants to reduce diseases in crab aquaculture. However, information regarding to the related immune-enhancing proteins in crabs is not available yet. In this study, rhubarb polysaccharides were tested for enhancement of the immune activity in crab Scylla paramamosain. Compared with those in the control group, values of, phenoloxidase (PO), alkaline phosphatase (AKP) and alkaline phosphatasein (ACP) activity in the, experimental group were improved significantly 4 d after the treatment. Furthermore, 15 and 17 altered proteins from haemocytes and hepatopancreas, respectively, were found in rhubarb polysaccharide-treated crabs using 2-DE approach. Of these, hemocyanin, chymotrypsin, cryptocyanin, C-type lectin receptor, and ferritin protein were identified by mass spectrometry. In addition, RT-PCR, analysis showed that the mRNA levels of hemocyanin and chymotrypsin increased about 2.4- and 1.4-fold in the experiment group. Moreover, the hemocyanin gene in S. paramamosain (SpHMC) was, cloned and characterized. SpHMC contains one open reading frame of 2022 bp and encodes a polypeptide of 673 amino acids. It is clustered into one branch along with crab hemocyanin in a phylogenetic tree. The mRNA transcripts of SpHMC were detected mainly in the tissues of, hepatopancreas, hemocyte and intestines, and its levels were up-regulated significantly in hemocytes, of S. paramamosain treated with Vibrio parahemolyticus, Beta streptococcus or poly I:C for 6-48 h. Taken together, these studies found 5 related immune-enhancing proteins and a novel heomcyanin homologue with potential pathogen-resistant activities in crab.

Evidences of abundant hemocyanin variants in shrimp Litopenaeus vannamei

Hemocyanin (HMC) is a multifunctional immune molecule present in mollusks and arthropods and functions as an important antigen non-specific immune protein. Our previous evidences demonstrated that Litopenaeus vannamei HMC might display extensive molecular diversities. In this study, bioinformatics analysis showed dozens of variant sequences of the HMC subunit with higher molecular weight from L. vannamei (LvHMC). Three variant fragments, named as LvHMCV1-3, which shared 85-99% nucleotide identity with that of the classical form of LvHMC (AJ250830.1), were cloned and characterized. Spatial expression profiles showed that LvHMCV1-3 had different tissue-specific distribution, which were affected by stimulation with six pathogenic bacteria, including Escherichia coli K12, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio fluvialis, Streptococcus pyogenes and Staphylococcus aureus, with each variant fragment showing a specific stress pattern to different bacterial pathogens. Full length cDNA of LvHMCV3 was further cloned and characterized. The deduced amino acid sequence shared 92% identity with that of LvHMC, possessed a conserved structure characteristic of the HMC family and could be clustered into one branch along with other arthropod HMC in a phylogenetic tree. In addition, the recombinant protein of LvHMCV3 (rLvHMCV3) showed obvious agglutination activities against three aquaculture pathogenic bacteria including E. coli K12, V. parahaemolyticus and S.aureus at concentrations ranging from 31.25-62.5g/mL. It also showed obvious antibacterial activity against V. parahaemolyticus at concentrations 0.02-0.5mg/mL, and possessed the best inhibitive effects compared with those of rLvHMCV4 and rLvHMC. Co-injection of V. parahaemolyticus and rLvHMCV3 in L. vannamei showed significant decrease of the mortality rate at 24-72h after injection. Therefore, these studies suggested that L. vannamei had abundant HMC variants, which possessed obvious resistance to pathogenic infection and might specifically target on different pathogens in shrimp.

Hemolytic Properties of Hemocyanin from Mud Crab Scylla serrata

ABSTRACT Recently, infectious diseases have seriously inhibited the aquaculture of mud crab Scylla serrata in southeastern China. A better understanding of the immune molecules and defense mechanisms may be beneficial in reducing the harmful nature of these diseases. Available data show that hemocyanin (HMC) is a copper-containing respiratory protein present in the hemolymph of both mollusc and arthropod, and that it plays multiple roles in immune defense. In the current study, HMC from S. serrata (HMC-C) was isolated, and its hemolytic properties were investigated. The HMC-C shows hemolytic activities against vertebrate erythrocytes. The hemolysis displays dependency on pH, temperature, divalent cation, and HMC-C concentration. Complete hemolysis occurred at a concentration of 0.1 mg/mL, pH 5.0, and temperature of 37°C in the presence of calcium. Furthermore, all 5 subunits of HMC-C were detected in the solubilized incubation products of erythrocytes with HMC-C, and the hemolysis could be inhibited to different degrees by osmoprotectants of various molecular masses. Together, our data suggest that HMC-C mediates hemolysis by inserting all 5 subunits into the erythrocyte membrane, causing cell rupture through a colloidosmotic mechanism.

Litopenaeus vannamei hemocyanin exhibits antitumor activity in S180 mouse model in vivo

Hemocyanin is a multifunctional glycoprotein, which also plays multiple roles in immune defense. While it has been demonstrated that hemocyanin from some mollusks can induce potent immune response and is therefore undergoing clinical trials to be used in anti-tumor immunotherapy, little is currently known about how hemocyanin from arthropods affect tumors. In this study we investigated the anti-tumor activity of hemocyanin from Litopenaeus vannamei on Sarcoma-180 (S180) tumor-bearing mice model. Eight days treatment with 4mg/kg bodyweight of hemocyanin significantly inhibited the growth of S180 up to 49% as compared to untreated. Similarly, histopathology analysis showed a significant decrease in tumor cell number and density in the tissues of treated mice. Moreover, there was a significant increase in immune organs index, lymphocyte proliferation, NK cell cytotoxic activity and serum TNF-α level, suggesting that hemocyanin could improve the immunity of the S180 tumor-bearing mice. Additionally, there was a significant increase in superoxide dismutase (SOD) activity and a decrease in the level of malondialdehyde (MDA) in serum and liver, which further suggest that hemocyanin improved the anti-oxidant ability of the S180 tumor-bearing mice. Collectively, our data demonstrated that L. vannamei hemocyanin had a significant antitumor activity in mice.

Characterization of a Novel Hemolytic Activity of Human IgG Fractions Arising from Diversity in Protein and Oligosaccharide Components

Human IgG is a well-established multifunctional antigen specific immunoglobulin molecule of the adaptive immune system. However, an antigen nonspecific immunological function of human IgG has never been reported. In this study, human IgG was isolated using ammonium sulfate fractional precipitation and diethylaminoethanol (DEAE) cellulose 52 ion exchange chromatography, from which h-IgG and hs-IgG fractions were purified on the basis of their differential binding to rabbit anti-shrimp hemocyanin antibody (h) and rabbit anti-shrimp hemocyanins small subunit antibody (hs), respectively. We found that h-IgG had a higher hemolytic activity than hs-IgG against erythrocytes from humans, rabbits, mice and chickens, whereas the control IgG showed negligible activity. h-IgG could interact directly with erythrocyte membranes, and this interaction was suppressed by high molecular weight osmoprotectants, showing that it may follow a colloid-osmotic mechanism. In comparative proteomics and glycomics studies, h-IgG and hs-IgG yielded 20 and 5 significantly altered protein spots, respectively, on a 2-D gel. The mean carbohydrate content of h-IgG and hs-IgG was approximately 3.6- and 2-fold higher than that of IgG, respectively, and the α-d-mannose/α-d-glucose content was in the order of h-IgG>hs-IgG>IgG. In this study, a novel antigen nonspecific immune property of human IgG was investigated, and the diversity in the protein constituents and glycosylation levels may have functional signficance.

Litopenaeus vannamei attenuates white spot syndrome virus replication by specific antiviral peptides generated from hemocyanin

ABSTRACT Recent studies have shown that hemocyanin plays immune‐related functions apart from its canonical respiratory function. While shrimp hemocyanin is found to generate antimicrobial peptides, antiviral related peptides have not been reported. In the present study, the serum of white spot syndrome virus (WSSV) infected Litopenaeus vannamei analyzed by two‐dimensional gel electrophoresis, revealed 45 consistently down‐regulated protein spots and 10 up‐regulated protein spots. Five of the significantly up‐regulated spots were identified as hemocyanin derived peptides. One of the five peptides, designated LvHcL48, was further characterized by analyzing its primary sequence via Edman N‐terminal sequencing, C‐terminal sequencing and amino acid sequence alignment. LvHcL48 was found to be a 79 amino acid fragment (aa584‐662) from the C‐terminal domain of L. vannamei hemocyanin protein (ADZ15149). Both in vivo and in vitro functional studies revealed that LvHcL48 has immunological activities, as recombinant LvHcL48 protein (rLvHcL48) significantly inhibited the transcription of the WSSV genes wsv069 and wsv421 coupled with a significant reduction in WSSV copy numbers. Further analysis showed that LvHcL48 could interact with the WSSV envelope protein 28 (VP28). Our present data therefore reveals the generation of an antiviral hemocyanin derived peptide LvHcL48 from WSSV infected shrimp, which binds to the envelope protein VP28 of WSSV. HighlightsWe found five hemocyanin‐derived peptides ˜10 kDa in size from WSSV‐challenged shrimp.a novel peptide LvHcL48 with 8.9 kDa could inhibit WSSV transcription and proliferation.LvHcL48 might bind to the viral envelope protein VP28 of WSSV.

Functional domains of Litopenaeus vannamei transglutaminase and their involvement in immunoregulation in shrimp

&NA; Shrimps, which mainly rely on their innate immune system to response to infectious pathogens, have clottable proteins as an important component of this system. While transglutaminases (TGase) are found in Litopenaeus vannamei and constitute part of the coagulation system, the specific immune‐related roles played by its functional domains in the immunoregulation of shrimp has not been well understood. In the present study, we report that the Ig‐like domain of L. vannamei transglutaminase (TGase‐C) is the main immune‐related domain among the three functional domains, as it had higher bacterial agglutinative activity against Vibrio parahaemolyticus and Streptococcus iniae. Using Co‐immunoprecipitation and LC‐MS/MS analysis, TGase‐C was shown to interact with 474 proteins, of which 52 proteins were annotated to L. vannamei. More than half of the L. vannamei annotated proteins have immune‐related functions, including apoptosis. Further analysis using pull‐down assay revealed that TGase‐C interacted with CAP‐3 (a homologue of caspase 3). In addition, siRNA‐mediated knockdown of LvTGase significantly (p < 0.01) increased the expression level of LvCAP‐3 coupled with a significant (p < 0.01) increase in caspase 3/7 activity, suggesting that probably LvTGase participates in shrimp immune response by modulating the activity of LvCAP‐3. These findings thus suggest the Ig‐like functional domain of L. vannameis transglutaminase is the domain that is involved in immunoregulation in shrimp. HighlightsThe Ig‐like region of Litopenaeus vannamei TGase (TGase‐C) is the main immune‐related functional domain.TGase‐C has bacteria agglutination activity and can interact with immune‐related proteins.TGase‐C modulates LvCAP‐3 expression to regulate apoptosis.