A prototype galectin-1 (also known as galecin-2) from large yellow croaker (Larimichthys crocea): Molecular and function study.

Galectin-1 (also known as galecin-2), one member of galectins family, has multiple functions as a pattern recognition receptor (PRR) in innate immune defense system. In the present study, LcGal-1, a prototype galectin, was identified and function investigated in large yellow croaker (Larimichthys crocea). LcGal-1 consists of one carbohydrate recognition domain (CRD), which contains two carbohydrate binding motifs HFNPR and WG-E-R. LcGal-1 had a ubiquitous tissues profile with the highest and lowest expression in spleen and muscle, respectively. Moreover, it was in cytoplasm and nucleus of head-kidney cells in large yellow croaker. RT-qRCR showed that P. plecoglossicida induced LcGal-1 up-regulated expression in liver and gills, and the results were validated by immunohistochemistry analysis. Additionally, the recombinant LcGal-1 (rLcGal-1) showed agglutinate activity on erythrocytes, and the histidine (His) in the HFNPR motif was a key locus to the activity. The agglutination effect of rLcGal-1 on erythrocytes could be inhibited by LPS, α-lactase and d-galactose. The rLcGal-1 was able to bind and agglutinate Gram+ and Gram-bacteria, and damage bacterial membrane as confirmed by PI staining and SEM observation. Transcriptome analysis showed that the overexpressed LcGal-1 in HEK 293T cells could induce 176 DGEs, including 172 boosting genes and 4 falling genes. Collectively, LcGal-1 was a key immune gene involved in the recognition, conjunction, and elimination of pathogens in L. crocea, as well as multiple physiological and pathological regulatory processes.

Centrosomal protein of 192 kDa (Cep192) fragment possesses bactericidal and parasiticidal activities in Larimichthys crocea.

A novel AMP Lc1773, derived from centrosomal protein of 192 kDa (Cep192), was isolated from Larimichthys crocea using a Bacillus subtilis system. After cDNA libraries construction, repeating selection of B. subtilis system, extraction of extracellular protein, and expression of recombinant protein, we found that B. subtilis 1773, extracellular protein, and rLc1773 had a strong potential to kill Vibrio. parahaemolyticus and V. vulnificus. Further analysis of the antibacterial mechanism revealed that rLc1773 not only disrupted the integrity of bacterial membrane (as confirmed by SEM, TEM, and confocal microscopy observation, and flow cytometry assays), resulting in bacterial cell membrane pore conformation, bacterial rupture, and leakage of cellular contents, but also targeted to block protein synthesis rather than damage nucleic acids (as confirmed by SDS-PAGE, enzyme expression, and gel retardation assays). In addition, rLc1773 had the ability to kill parasite Scuticociliatida in a high rate and low concentration. Critically, the antibacterial activity of rLc1773 had good thermal stability and UV radiation tolerance, but it was affected by pH 9-11 and diverse enzyme to some extent. Lc1773 had neither hemolysis on fish, shrimp, and rabbit erythrocytes，nor significant cytotoxicity. To our knowledge, Cep192 fragment was first demonstrated to possess bactericidal and parasiticidal activities.

Characteristics of structure, composition, mass spectra, and iron release from the ferritin of shark liver (Sphyrna zygaena).

The ferritin consists of a protein shell constructed of 24 subunits and an iron core. The liver ferritin of Sphyrna zygaena (SZLF) purified by column chromatography is a protein composed of eight ferritins containing varying iron numbers ranging from 400+/-20 Fe3+/SZLF to 1890+/-20 Fe3+/SZLF within the protein shell. Nature SZLF (SZLFN) consisting of holoSZLF and SZLF with unsaturated iron (SZLFUI) to have been purified with polyacrylamide gel electrophoresis (PAGE) exhibited five ferritin bands with different pI values ranging from 4.0 to 7.0 in the gel slab of isoelectric focusing (IEF). HoloSZLF purified by PAGE (SZLFE) not only had 1890+/-20 Fe3+/SZLFE but also showed an identical size of iron core observed by transmission electron microscopy (TEM). Molecular weight of approximately 21 kDa for SZLFE subunit was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Four peaks of molecular ions at mass/charge (m/z) ratios of 10611.07, 21066.52, 41993.16, and 63555.64 that come from the SZLFE were determined by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS), which were identified as molecular ions of the ferritin subunit (M+) and its polymers, namely, [M]2+, [M]+, [2M]+, and [3M]+, respectively. Both SZLFE and a crude extract from shark liver of S. zygaena showed similar kinetic characteristics of complete iron release with biphasic behavior. In addition, a combined technique of visible spectrometry and column chromatography was used for studying ratio of phosphate to Fe3+ within the SZLFE core. Interestingly, this ratio maintained invariable even after the iron release, which differed from that of other mammal ferritins.

Characteristics of trapping various organophosphorus pesticides with a ferritin reactor of shark liver (Sphyrna zygaena).

A reactor is composed of liver ferritin of Sphyrna zygaena (SZLF) and an oscillating bag. A reactive procedure for trapping various organphosphorus pesticides (OPs) with the SZLF reactor in the flowing water is described in detail, showing the maximal trapping numbers of 28 +/-1.0 dichlorovos/SZLF, 42 +/- 1.0 dimethoate/SZLF, and 55 +/- 1.0 methamido-phos/SZLF determined by a improved spectrophotometric method in 12 h. In addition, it is found that the OP numbers trapped by the reactor increase along with the incubation time and its concentration increment in the flowing water (or seawater), respectively. This trapping capacity is considered to depend on the composition of amino acids on the surface of the ferritin shell interior rather than the available volume within the shell. A novel pathway for trapping various OPs with the ferritin is suggested in reference to unstable characteristics of the protein subunits. We claim that the ferritin reactor will be employed to monitor the contamination level of various OPs in the flowing water continuously.