Cloning, Expression And Functional Characterizition Of A Gene Family Of Astacin-like Metalloproteases From Jellyfish Cyanea Capillata

Jellyfish is an abundant marine zooplankton. Jellyfish tentacles are covered withnematocysts. When touching the human or animal body, nematocysts launch barbed wireto attack and release the venom. It was reported that jellyfish toxins mainly belong toproteins and peptides with strong biological activities and high toxicity, includinghemolytic activity, cardiovascular toxicity, enzyme activity, neurotoxicity and muscletoxicity. Therefore, the study on material basis of jellyfish toxins is very important toclarify the composition of the jellyfish toxins, to understand the mechanism of action, andto lay the foundation for making full use of the active ingredients.Because jellyfish toxin proteins have been proved to be thermally instable, proteinaggregated and complex, there are still many limitations for the separation andidentification of the jellyfish toxin proteins. The efficiency of traditional separation methodis not high in jellyfish toxin protein purification. At present, there are few of singlejellyfish toxin protein reported with definited sequence at home and abroad, and theunderstanding on its composition and structure is still limited. Therefore, for obtaining acomprehensive screening of functional genes closely related to jellyfish toxins biologicalactivity, we chose the jellyfish Cyanea capillata, a representative toxic jellyfish in thesoutheast coast of China, to build its tentacles tissue cDNA library by the SMARTtechnology. Through large-scale random sequencing and homology comparison, we foundthat there are three EST sequences encoding the amino acid sequences which are highlyhomologous to the astacin family of metalloendopeptidases, suggesting that the proteinsencoding by these three sequences belong to the family of astacin-like metalloproteases.Therefore, these three sequences were named CALP1(Cyanea capillata astacin-likemetalloproteases1), CALP2and CALP3, respectively.The metalloproteinases have been proven to exist in the venom of snakes and otherpoisonous animals and play an important role as toxin components. Hyunkyoung Leefound four types of jellyfish nematocyst venoms (Nemopilema nomurai, et al.) withgelatinolytic, caseinolytic, and fibrinolytic activities. Most of these enzymatic activitieswere disappeared by the treatment of1,10-phenanthroline, suggesting they might belong tometalloproteinases. It is the first time to demonstrate that there are a large number ofmetalloproteinases in the jellyfish toxins.With a strong proteolytic activity, astacin-like metalloproteases are typical zinc-dependent endopeptidases. It was reported that astacin-like metalloproteases candegrade extracellular matrix components and cause bleeding, which is consistent with apart of biology activity of jellyfish toxins. A gene family of astacin-like metalloproteaseswas found recently in the venom of the brown spider (genus Loxosceles), with thecapability of degrading extracellular matrix components, assisting in the diffusion of othertoxins. Based on these results, we presumed that the astacin-like metalloproteases familyexsit in the tentacles of the jellyfish Cyanea capillata and be likely to play an importantrole in the toxic effects of jellyfish toxins. In-depth study of this gene family would help toclarify the material basis and action mechanism of the biological activities of the jellyfishtoxins.In this study, we conducted a comprehensive analysis on the basic properties of thesethree sequences coding astacin-like metalloproteases with bioinformatics methods,constructed prokaryotic recombinant expression plasmids, explored the best induceconditions for expression, purified the target fusion protein, and further examined theiractivity and function.PartⅠ: Full-length sequence analysis of CALP gene familyBy NCBI online software, CALP gene sequences were recognized for complete openreading frame. Results showed that CALP1, CALP2and CALP3all had similar sequencefeatures to astacin-like metalloproteinases family, which contains a typical astacin-likedomain characterized by a sequence of two features: the Zn binding motifHEXXHXXGFXHEXXRXDRD and the methionine turn SXMHY, suggesting that theyare the new members of astacin-like metalloproteases family. CALP1, CALP2and CALP3contain304aa,313aa and333aa, respectively, with approximate molecular weights of35kDa,35kDa and38kDa. CALP2and CALP3contain signal peptides, and they are bothsecreted protein. CALP1does not contain a signal peptide and is located in the cytoplasm.These three molecules are all free from transmembrane domain, but there are manyphosphorylation, glycosylation sites and disulfide bonds. These three proteins are rich inhydrophilic regions, with less hydrophobic regions.In addition to astacin-like domain, CALP1and CALP3also contain ShK toxindomains at C terminus, with the characteristics of blocking K+channels and regulatingmembrane potential, The ShK domain has been proved to promote Ca2+influx by blockingthe potassium channel K+outflows, leading to an intracellular Ca2+overload and cells damage. Therefore, in addition to proteolysis, these two molecules are likely to play otherrole in jellyfish toxins.PartⅡ: Cloning of CALP gene family and a small amount of expression of thefusion proteinThe primers were designed according to the complete coding region (without signalpeptide) sequence of CALP1, CALP2and CALP3. NdeⅠ/NheⅠ and XcoⅠ wereselected respectively as double digestion sites. Prokaryotic expression plasmid pET24awas used to construct complete coding regions of CALP1, CALP2and CALP3recombinant expression vector with6*His tag at C-terminus, correctly identified byrestriction endonuclease and DNA sequencing. The constructed recombinant plasmidswere transformed into expression strain E.coli Rosetta (DE3), and a small amount ofexpression of the fusion proteins were induced in the condition of1mM IPTG,37℃,250rpm. With SDS-PAGE electrophoresis detection, there were CALP1, CALP2and CALP3target fusion protein with approximate molecular weights of33kDa,26kDa and32kDa,respectively. Moreover, the fusion proteins were also identified by6*His tag antibody.Western blot results showed that a single specific band with a molecular weight of33kDa,26kDa and32kDa, respectively, could be detected in bacterial lytic solutions, which areconsistent with the predicted weights of target proteins according to the sequences.Part Ⅲ: Large amounts of expression, purification and functionalcharacterization of CALP family1. Optimization of the inducing conditions for large expression of CALP fusionproteinIn order to obtain a large amounts of recombinant fusion proteins with biologicalactivities on the basis of a small amount of induce expression, we further explored andoptimized the inducing expression condition of CALP fusion proteins. We first analyzedthe best inducing conditions for soluble expression. The results showed that the Rosettastrain is more conducive to the expression of CALP fusion protein than BL21(DE3)strains. However, the expression levels of CALP1and CALP2were relatively lower, whileCALP3was not expressed solublely, which might be related to the many pairs of disulfide bonds and structural modification sites of CALP family, causing the difficulty in correctlyfolding their spatial structures in E. coli. Due to the low expression level of soluble fusionprotein, we further analyzed inducing conditions for inclusion bodies expression of CALPfusion protein. The results showed under the optimum expression conditions for eachtarget fusion protein, most of the proteins presented in the precipitation of bacteria lysatesin the form of inclusion bodies. The expression levels of CALP1and CALP2inclusionbodies were relatively higher, while CALP3was low. After washed, inclusion bodies weredissolved in the dissolving liquid with screened strongest dissolving power. Therecombinant proteins were purified with HisTrap HP gel affinity chromatography underdenaturing conditions. Western blot results showed that there were target protein bands ofCALP1, CALP2and CALP3with the approximate molecular weight of33kDa,26kDaand32kDa, respectively. The fusion proteins were then refolded by the chemical dilutionand physical dialysis methods.2. Proteolytic activity detection of C. capillata nematocyst venom (NV) and CALPfusion proteinGelatin, fibronectin and fibrinogen were chosen as substrates to detect the proteolyticactivity of NV, CALP1, CALP2, and CALP3fusion protein. Gelatin degradation resultsshowed that there was an apparent white band between95kD and72kD in NV treatedgroup, and the bands were significantly weakened with the presence of the inhibitor1,10-phenanthroline. The result suggested that NV had gelatinolytic activity. NV from C.capillata was also showed significant proteolytic activity on fibronectin and fibrinogen,which was demonstrated by the change of protein bands of fibronectin and fibrinogen, andthe proteolysis was also significantly inhibited by the inhibitor respectively. Tentacleextract (TE) also had obvious fibronectinolytic, fibrinogenolytic and gelatinolytic activities,but the activities were weaker than that of NV. We further detected the effect of tentacleextract from C. capillata on the morphology of adherent cells. Compared with the normalcontrol group, TE-treated renal tubular epithelial cells became round, the cell gap wassignificantly larger, and the cells detached from the bottom of the culture plate after1h. Itwas showed that the tentacle extract can degrade extracellular matrix components, whichwas consistent with the effect of other jellyfish toxins reported.We further made purified CALP fusion proteins renature by five renaturation methodsand detected their proteolytic activity. The results showed that the proteolytic activity of the fusion protein was relatively weak. This might be due to poor renaturation conditionsof inclusion bodies or the lack of modification of the proteins. Therefore, we also intend toexpress and purify CALP fusion proteins with Pichia pastoris eukaryotic expressionsystem in the following study.In summary, we found a CALP gene family in the cDNA library of tentacle from C.capillata through large-scale sequencing and homology analysis. The three members of thefamily were highly homologous to astacin-like metalloproteinases, containing conservativeastacin-like domain. We successfully constructed recombinant expression vector ofpET24a-CALP1, pET24a-CALP2and pET24a-CALP3, and the fusion proteins wereexpressed in E. coli Rosetta (DE3). We analyzed the optimal conditions for CALP solubleexpression and inclusion body expression. After purification, we used a variety of methodsto dissolve and renaturation of inclusion bodies. Proteolytic test results showed that the NVand TE from C. capillata had obvious fibronectinolytic, fibrinogenolytic and gelatinolyticactivities, but the proteolytic activity of refolded fusion proteins were relatively low, whichneed to further study with eukaryotic expression system.