Expression of a bee venom phospholipase A₂ from Apis cerana cerana in the baculovirus-insect cell

Expression of a bee venom phospholipase A 2 from Apis cerana cerana in the baculovirus-insect cell 1 first-author †E-mail:shenlirong@zju.edu.cn †E-mail:shenlirong@zju.edu.cn shenlirong@zju.edu.cn 1 1 1 2 1 Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310029, China Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310029, China Department of Agriculture and Natural Resources, Delaware State University, Dover, DE 19901, USA Department of Agriculture and Natural Resources, Delaware State University, Dover, DE 19901, USA Bee venom phospholipase A 2 (BvPLA 2 ) is a lipolytic enzyme that catalyzes the hydrolysis of the sn-2 acyl bond of glycerophospholipids to liberate free fatty acids and lysophospholipids. In this work, a new BvPLA 2 (AccPLA 2 ) gene from the Chinese honeybee ( Apis cerana cerana ) venom glands was inserted into bacmid to construct a recombinant transfer vector. Tn-5B-4 (Tn) cells were transfected with the recombinant bacmid DNA for expression. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed a double band with molecular weights of 16 and 18 kDa. Products of hexahistidine AccPLA 2 fusion protein accumulated up to 5.32% of the total cellular proteins. The AccPLA 2 fusion protein was cross reactive with the anti-AmPLA 2 (BvPLA 2 of the European honeybee, Apis mellifera ) polyclonal serum. The reaction resulted in a double glycosylation band, which agrees with the band generated by the native AmPLA 2 in Western blot analysis. The PLA 2 activity of the total extracted cellular protein in the hydrolyzing egg yolk is about 3.16 μmol/(min·mg). In summary, the recombinant AccPLA 2 protein, a native BvPLA 2 -like structure with corresponding biological activities, can be glycosylated in Tn cells. These findings provided fundamental knowledge for potential genetic engineering to produce AccPLA 2 in the pharmaceutical industry. Apis cerana cerana Bee venom phospholipase A 2 (BvPLA 2 ) Insect cell Expression Introduction 1 Phospholipases A 2 (PLA 2 s; EC 3.1.1.4) are a large super-family of lipolytic enzymes that catalyze the hydrolysis of the sn-2 acyl bond of glycerophospholipids to liberate free fatty acids and lysophospholipids (Mingarro et al., 1995 1995 ). PLA 2 s have important functions in the cellular processes that modulate the release of arachidonic acid and the precursor of eicosanoids of potent inflammatory mediators (Rodriguez de Turco et al., 2002 2002 ). Additionally, PLA 2 s play a critical role in host defense, atherosclerosis, signal transduction processes, membrane remodeling (Murakami and Kudo, 2002 2002 ; Dennis, 1994 1994 ; 1997 1997 ), and delaying oxidant-induced cell death (Zhao et al., 2001 2001 ). PLA 2 s are also associated with numerous human disorders such as rheumatoid arthritis, autoimmune uveitis, respiratory distress syndrome, myocardial infarction, and septic and endotoxic shock (Mukherjee et al., 1994 1994 ). PLA 2 s enzymes arise from a variety of sources including the mammalian pancreas, reptile venom and insect venom, and synovial fluids (Scott et al., 1990b 1990b ). These enzymes have been systematically classified into 15 closely structure-related groups on the basis of the level of homology among their nucleotide and amino acid sequences. Based on their properties, PLA 2 s can be historically classified into secretory (sPLA 2 ), cytosolic Ca 2+ -dependent (cPLA 2 ), and cytosolic Ca 2+ -independent (iPLA 2 ). Group I gobra/krait venom and mammalian pancreas, Group II crotalid and viper venom, and Group III bee/lizard/scorpion venom are all secretory, low-molecular mass (13–18 kDa) and Ca 2+ -dependent PLA 2 s (Balsinde et al., 2002 2002 ). Bee venom PLA 2 (BvPLA 2 ) is a typical Group III sPLA 2 member and makes up approximately 12% of the dry weight of venom in the European honeybee, Apis mellifera (Habermann, 1972 1972 ; Dennis, 1997 1997 ). BvPLA 2 is the most lethal honeybee venom peptide. It acts as an allergen cooperating with other components to defend the colony against predators and intruders (Schmidt, 1995 1995 ; King and Spangfort, 2000 2000 ). The activity of BvPLA 2 can be enhanced by melittin, the most abundant constituent of honey bee venom (Mingarro et al., 1995 1995 ). BvPLA 2 has a wide variety of pharmacological properties including anti-human immunodeficiency virus (HIV) activity, neurotoxicity, myo-toxicity, and neurite outgrowth induction (Fenard et al., 1999 1999 ; 2001 2001 ; Nakashima et al., 2004 2004 ). A nucleotide sequence of BvPLA 2 (AmPLA 2 ) from the European honeybee has been determined. The deduced amino acid sequence of AmPLA 2 consists of a signal peptide of 18 amino acid residues (preregion of PLA 2 ), a proregion of 15 residues, and a mature peptide of 134 amino acid residues. The mature peptide contains 10 cystine residues which can form 5 disulfide bonds (Kuchler et al., 1989 1989 ; Shipolini et al., 1974a 1974a ; 1974b 1974b ). The crystal structure and catalyzing activity of PLA 2 were also well documented (Annand et al., 1996 1996 ; Scott et al., 1990a 1990a ; 1990b 1990b ). A synthetic gene encoding the mature peptide of AmPLA 2 was expressed in Escherichia coli , but the biological activities of the expressed protein were low because it can form an inclusion body with incorrect folding in the prokaryotic cell (Dudler et al., 1992 1992 ). The similar phenomenon was found for another recombinant bee venom allergen, hyaluronidase (BvHya) from the European honeybee. The biological activity of the BvHya expressed in E. coli was only 20%–30% of that of the BvHya expressed in the baculovirus-infected insect cells (Soldatova et al., 1998 1998 ). The Asiatic honeybee, Apis cerana , is another commonly-domesticated species distributed in southern and southeastern Asia. It has been appreciated that the European honeybee derived from its closed relative Asiatic honeybee in western or central Asia and subsequently spread into Europe and Africa (Oldroyd and Wongsiri, 2006 2006 ; Whitfield et al., 2006 2006 ). The Chinese honeybee, Apis cerana cerana , one subspecies of Asiatic honeybee, has more than 3000 years of history. The total number of the Chinese honeybee colonies maintained by Chinese farmers is 1.2 million, while European honeybee has as much as 7.8 million colonies in recent years. This species was the only breeding honeybee in China before the European honeybee was introduced at the beginning of the last century. The Chinese honeybee has unique biological characters and behaviors that are significantly different from the European honeybee, and has played important roles in the formation of the unique botanical layer in China by pollination (Zhang et al., 2008 2008 ; Xu et al., 2009 2009 ). We previously amplified the BvPLA 2 (AccPLA 2 ) gene that encodes a mature peptide of the Chinese honeybee (Shen et al., 2002 2002 ). The expression of AccPLA 2 appears from 5 to 42 d of the worker bee of the Chinese honeybee. PLA 2 modification between the Chinese honeybee and the European honeybee was compared (Li et al., 2005 2005 ). The aim of the present study was to investigate the expression, glycosylation, immunologic properties, and the enzymatic activity of the recombinant AccPLA 2 in insect cells. Materials and methods 1 Materials 2 The plasmid, pGEM®-AcPLA 2 , was constructed as previously described (Shen et al., 2002 2002 ). Trichoplusia ni cell line (Tn-5B-4 (Tn) cell) was maintained in our laboratory. New Zealand white rabbits were purchased from the animal center of Chinese Traditional Medical Institute of Zhejiang. The restriction enzymes Eco RI and Hin dIII, DL2000 and DL15 000 markers were purchased from TaKaRa Company (Dalian, China). The commercially-purified native AmPLA 2 was purchased from Sigma Company (St. Louis, MO, USA). The T4 DNA ligase, X-gal, isopropyl beta-D-thiogalactopyranoside (IPTG), Lipofectin, TNM-FH insect cell medium and bovine calf serum, the transfer vector pFastBacHTa, and E. coli strains DH10B were purchased from Invitrogen Corporation (Carlsbad, USA). The Taq DNA polymerase, protein molecular marker, nitrocellulose filter (NC filter), and the goat anti-rabbit IgG (Fc) conjugate were purchased from Sino-Promega Company (Shanghai, China). Plasmid DNA extraction kit was purchased from Omega Bio-Tek Corporation (Norcross, USA). The calcium chloride, sodium deoxycholate, and bovine serum albumin (BSA) were purchased from Sangon Company (Shanghai, China). All the biochemical reagents were of highest commercially available purity. Construction of baculovirus transfer vector and recombinant bacmid 2 The pGEM®-AccPLA 2 was amplified in E. coli TG1 cells and extracted with a plasmid DNA extraction kit. The Eco RI/ Hin dIII-digested cDNA fragment of AccPLA 2 from pGEM®-AccPLA 2 was directionally inserted into the transfer vector pFastBacHTa. The recombinant transfer vectors were identified by using digestion and polymerase chain reaction (PCR) with the primers AccF1 5′-A GAATTC ATGATAATATATCCAGGAACG-3′ ( Eco RI site was added and underlined) and AccR1 5′-G AAGCTT AATACTTGCGAAGATCG-3′ ( Hin dIII site was added and underlined) of AccPLA 2 , following the described procedures (Sambrook and Russell, 2002 2002 ). Then it was sequenced with a DNA sequencer (performed by Sangon Company of Shanghai). After transformed into competent E. coli DH10B cells, the AccPLA 2 fragment in recombinant transfer vector was transposed to the recombinant baculovirus shuttle vector, bacmid, by Tn7 transposition. We extracted DNA from the positive clones that had been verified by PCR using primers AccF1 and AccR1 of AccPLA 2 , M13F1 (5′-GTAAAACGACGGCCAGT) and M13R1 (5′-AACAGCTATGACCATG) of PUC/M13, following the described procedures (Luckow et al., 1993 1993 ; Shang et al., 2007 2007 ). Culture of insect cells and Lipofectin-mediated transfection 2 Tn cells were used for the routine transfection and propagation of recombinant bacmid. TNM-FH medium supplemented with 10% (v/v) fetal bovine calf serum (complete medium) was used for propagation of the insect cells. The recombinant bacmid DNA was introduced into Tn cells mediated using a Lipofectin kit according to the supplier’s instruction. Then the cells were incubated with serum-free medium. After the cells were exposed to DNA-lipid-medium for 24 h, the medium was replaced by the complete medium. The 2-d-old conditioned medium was collected. After transfection, Tn cells were incubated for 24 h and the medium was replaced with serum-free medium. The 2-d-old conditioned medium was collected. After centrifugation at 1500× g at 4 °C for 3 min, the pellet of infected cells was stored at −80 °C, and the supernatant containing the virus particles was used to propagate in Tn cells. After propagation for 4 generations, the genomic viral DNA isolated from the infected cells was confirmed by PCR with primers AccF1 and AccR1. Preparation of anti-AmPLA 2 polyclonal serum 2 New Zealand white rabbits were immunized with the mixture of 300 mg commercially-purified native AmPLA 2 and 1 ml of phosphate buffered saline (PBS)/complete Freund’s adjuvant emulsion. The rabbits were bled three weeks after primary immunization with 200 mg of the enzyme emulsified in incomplete Freund’s adjuvant (1 ml), followed by injection (200 mg of enzyme in 1 ml of saline) for three weeks. The effect value of the serum extracted from the blood sample after one week of the second enhanced immunization was detected with native AmPLA 2 by the gel diffused protocol as described (Sambrook and Russell, 2002 2002 ). The serum was stored at −80 °C. Recombinant protein analysis 2 The intracellular proteins of the infected cells, a negative control (normal insect cells), and positive control (native AmPLA 2 ) were analyzed using 10% (v/v) sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described (Sambrook and Russell, 2002 2002 ). Gels were stained with Coomassie brilliant blue R250. The concentration of expressed fusion protein was measured by thin layer scanning of the SDS-PAGE gel. For Western blots, the separated unstained proteins were transferred onto unmodified NC membrane in a Bio-Rad transblot apparatus. The fusion protein was analyzed using rabbit antiserum against native AmPLA 2 prepared as above. The proteins on the blot were detected using goat anti-rabbit IgG (Fc) conjugate as described (Sambrook and Russell, 2002 2002 ). Assay of PLA 2 activity 2 Titrimetric assay of PLA 2 was based on a modified method used by Owen et al. ( 1990 1990 ). The fresh egg yolk substrate blended in 200 ml of distilled and deionized water was prepared on daily basis. Five milliliters of egg yolk substrate was mixed with 1 ml of 3×10 −2 mol/L calcium chloride, 1 ml of 1.3×10 −2 mol/L sodium deoxycholate, 1 ml of 5% (v/v) BSA, and 2 ml of water. This mixture was heated to 37 °C and NaOH was added to bring the pH value of mixture to 8.0 in a thermostatted microreaction vessel. A pH meter system was used to perform pH-stat titration with the pH value held at 8.0 by addition of 0.005 mol/L of NaOH. The concentration of the solubilized proteins of the extracts of the infected cells was measured and then the extracts were added to the substrate mixture. The rate of release of fatty acids was determined by the amount of added NaOH to maintain the pH at 8.0. One unit of PLA 2 activity is defined as the amount of enzyme required to hydrolyze 1 μmol of substrate per minute at pH 8.0 and 37 °C. The purified native AmPLA 2 and native bee venom powder diluted in distilled and deionized water, respectively, were used as positive control. The extracts of the normal Tn cells were used as negative control. Results 1 Construction of baculovirus transfer vector 2 The Eco RI/ Hin dIII-digested cDNA fragment of AccPLA 2 from pGEM®-AccPLA 2 was inserted into the transfer vector pFastBacHTa (Fig. 1 1 ). The products of restricted digestion and PCR of the recombinant plasmid had an approximate 400-bp band, which is identical to the AccPLA 2 fragment (GenBank accession No. AF321087). Nucleotide sequencing confirmed that the inserted fragment is a correct open-reading frame (ORF) for protein expression (Fig. 2 2 ). In accordance with the schematic representation of the recombinant transfer vector pFastBac-AccPLA 2 , the inserted gene was under the control of the Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin promoter. The expressed gene was designed to generate a fusion protein composed of hexahistidine resides (6×His) at amino-terminus, which allows a single-step purification using immobilized Ni 2+ ion affinity chromatography. The deduced amino acid sequence (matured peptide) is given below the nucleotide sequence. Forward primer and reversed primer for PCR are indicated with arrows, respectively Construction of recombinant of bacmid and transfection of insect cells 2 After the recombinant transfer vector pFastBac-AccPLA 2 was transformed into E. coli DH10B competent cells that contain baculovirus shuttle vector bacmid, the recombinant colonies were further cultured. Then the PCR identification was conducted with the bacmid DNA isolated from the positive colonies as a template using primers AccPLA 2 and PUC/M13, respectively. Two fragments, about 400 bp (Fig. 3a 3a ) and 2700 bp (Fig. 3b 3b ), were generated. These fragments are identical to AccPLA 2 fragment within the multiple cloning sites (MCSs) and the bacmid fragment of 2300 bp within the left and right ends of bacterial transposon Tn7 plus AccPLA 2 fragment, which confirms that the recombinant bacmid, rBacmid-AccPLA 2 , had been correctly constructed. (a) AccPLA 2 primer. Lane 1: rBacmid-AccPLA 2 PCR; Lane 2: pGEM-AccPLA 2 PCR product; Lane M: DNA molecular weight marker (DL2000); (b) PUC/M13 primer: Lane 1: rBacmid-AccPLA 2 PCR product; Lane M: DNA molecular weight marker (DL15 000) With mediation of the Lipofectin, the rBacmid-AccPLA 2 DNA was introduced into Tn cells in medium with free bovine calf serum. AccPLA 2 appeared in 3 d after infection of rBacmid-AccPLA 2 DNA. After centrifugation for harvesting cells, the amplified rBacmid in Tn cells, rTnV-Bac-AccPLA 2 , in supernatants was used to propagate. PCR identification of rTnV-Bac-AccPLA 2 2 After propagation for four generations, PCR identification was performed using the rTnV-Bac-AccPLA 2 genomic DNA isolated from the infected cells as a template. The agarose electrophoretic analysis profile showed that the PCR products were of approximate 400 bp, identical with AccPLA 2 gene, confirming that rTnV-Bac-AccPLA 2 had been propagated in Tn cells. SDS-PAGE and Western blot analysis 2 The SDS-PAGE analysis showed that the expressed product has a double band of 16 to 18 kDa (Fig. 4a 4a ). The 18-kDa band agrees with the expected molecular weight for the expressed fusion protein of AccPLA 2 (134 amino acids, 10.45 kDa) and 25 amino acids of N -terminal 6×His tag protein (2.8 kDa), a spacer region and rTEV protease cleavage site (3 kDa). (a) SDS-PAGE analysis: Lane M: protein molecular weight marker proteins; Lane 1: native AmPLA 2 ; Lane 2: extracts from Tn cells infected with rBacmid-AccPLA 2 (the expressed bands were indicated with arrows); Lane 3: extracts from normal Tn cells; (b) Western blot: Lane 4: native AmPLA 2 ; Lane 5: extracts from Tn cells infected by rBacmid-AccPLA 2 ; Lane 6: extracts from normal Tn cells The gel diffusion detection showed that there was precipitation between the anti-AmPLA 2 polyclonal serum (1:8, 1:16, 1:32, 1:64, and 1:128) and antigen (1:1000). It has been confirmed that the prepared rabbit antibody could cross-react with native AmPLA 2 . Following resolution on SDS-PAGE, the proteins of the infected cells were transferred electrophoretically into unmodified NC membrane probed with the anti-AmPLA 2 polyclonal serum (1:3000). The expressed fusion protein was recognized by the AmPLA 2 -antibody, which confirms the expression of AccPLA 2 proteins, and shows that the Tn cells infected with the recombinant virus exhibited a double-immunoreaction band with molecular weights of 16 to 18 kDa (Fig. 4b 4b ). Meanwhile, the blots of native AmPLA 2 and native bee venom (positive control) yielded a similar double-band. By contrast, no immunoreactivity was detected in negative control. The results indicate that there was cross-reaction between the expressed fusion protein in Tn cells and anti-AmPLA 2 polyclonal serum. One mixed venom sample of Chinese honeybee was reported to have three bands detected for the natural AccPLA 2 with molecular weight 15 kDa using the anti-AmPLA 2 antibody, which was caused by the characters of BvPLA 2 glycosylation (Li et al., 2005 2005 ). These results also indicate that the expressed AccPLA 2 protein can be glycosylated in Tn cells. Enzymatic activity assay of the expressed fusion protein 2 The hydrolyzation analysis of the total infected cellular protein on egg yolk substrate revealed that the PLA 2 enzymatic activity is about 3.16 μmol/(min∙mg) (Table 1 1 ). The result demonstrates that the crude fusion protein extracts of AccPLA 2 expressed in Tn cells could directly yield enzymatic activity of hydrolyzation on egg yolk substrate. The crude enzymatic activity of the pure fusion protein of AccPLA 2 was estimated at 63.3 μmol/(min∙mg), which is about 20 times that of the total protein of the cells. However, the enzymatic activity of the purified AccPLA 2 from Tn cells cannot be detected because of the low expression of AccPLA 2 . Samples Enzymatic activity (μmol/(min·mg)) Native AmPLA 2 193.75±8.84 Native bee venom 15.84±1.18 Extracts from Tn cells infected with rBacmid-AccPLA 2 3.16±1.13 Extracts from normal Tn cells Not detected Discussion 1 Previous studies have concluded that the amino acid sequences deduced from the cDNAs of AccPLA 2 and AmPLA 2 are composed of 134 amino acids with 5 disulfide bridges, 2 catalytic domains, and 3 calcium binding sites. The sequences of AccPLA 2 and AmPLA 2 share 95% homologies (Shen et al., 2002 2002 ). This study confirmed that the activity and immunological properties of AccPLA 2 fusion protein expressed in Tn cells are in agreement with those of the native AmPLA 2 isolated from the worker bee venom glands of the European honeybee. Based on the molecular and biochemical homologies of AccPLA 2 and AmPLA 2 , it could be speculated that the recombinant protein of AccPLA 2 might have a native-like conformation in Tn cells. Baculovirus-mediated expression in insect cells is well established for the production of recombinant glycoproteins. With regard to protein folding and post-translational processing, insect cells are second only to mammalian cell lines. Evidence indicated that many processing events known in mammalian systems also occurred in insects (Luckow et al., 1993 1993 ; Altmann et al., 1999 1999 ). Numerous experiments were conducted with insect cells. For instance, human β-interferon and O -glycan core 2β-1,6- N -acetylglucosaminyltransferase (C2GnT) with high levels of activity have been successfully expressed in Sf-9 cells (Smith et al., 1983 1983 ; Toki et al., 1997 1997 ), and Tn cells showed high glycosylation potential for the recombinant insect glycoprotein, BvHya from the European honeybee (Soldatova et al., 1998 1998 ). However, its capability to produce human-like complex type N -glycans has been a matter of controversy for many years. The lack of complex glycosylation has limited the use of the insect cell baculovirus expression vector system. Tn cells have been found to produce a small amount of galactose-terminated N -glycans, which prevent glycoproteins from being used as therapeutics proteins (Altmann et al., 1999 1999 ). Therefore, two novel cell lines, A7S from Pseudaletia unipuncta and DpN1 from Danaus plexippus that possess the capability of complex glycosylation, can overcome such a problem (Palomares et al., 2003 2003 ). However, the similar problems should not be taken account of for the naturally occurring glycoproteins such as BvPLA 2 and BvHya from insects to be expressed in Tn cell system. The Bac-to-Bac system and Tn cells may serve as a general probe for elucidation of some of the regulatory factors influencing glycosylation of insect protein. BvPLA 2 is the most important bee venom glycoprotein with a single oligosaccharide attached to asparagin-13, which is responsible for its enzymatic activity and allergenicity (Scott et al., 1990a 1990a ; 1990b 1990b ; Kubelka et al., 1993 1993 ). Prior to this study, BvPLA 2 has not been expressed in insect cells. Here, the AccPLA 2 gene was expressed in Tn cells. The expressed fusion protein was active, indicating that Tn cells could express properly modified AccPLA 2 . The appearance of recombinant protein in SDS-PAGE and Western blot is a double band and the glycosylation capacity of Bac-to-Bac system and Tn cells for AccPLA 2 was verified, which provides further insights into the advantages of baculovirus and its active foreign eukaryotic insect protein. Our previous work on the difference of BvPLA 2 glycosylation among individuals of Chinese honeybee showed that AccPLA 2 was glycosylated during 8–12 d old at adult stage, and the AccPLA 2 in venom was a mixture of three different glycosides. The sequencing for all three bands of proteins excised from the NC membrane of Western blot showed that they had the same N -terminal sequence, IIYPGTLW, which is the N -terminal sequence of the mature AccPLA 2 . The difference in the molecular weights of the three bands was caused by their different glycosylations (Li et al., 2005 2005 ). Altmann et al. ( 1991 1991 ) found that AmPLA 2 from the European honeybee consisted of three isoforms with approximate molecular masses of 16, 18, and 20 kDa, respectively. The sequencing data on N -terminal amino acid sequence showed that the PLA 2 -18 and PLA 2 -20 carried oligosaccharide residues, and PLA 2 -16 escaped glycosylation during biosynthesis. The electrophoretic separation of the three isoforms was based on structural features of glycosylation of AmPLA 2 . Soldatova et al. ( 1998 1998 ) also found that recombinant BvHya expressed in Tn cells appeared as a double band of 43 to 44 kDa with different glycosylations. Our results are consistent with these previous reports (Li et al., 2005 2005 ; Altmann et al., 1991 1991 ; Soldatova et al., 1998 1998 ). Since the glycosylation of recombinant protein is an important factor that affects protein function, the present study combined with our previous findings may lay a new scientific basis for the molecular biological utilization of AccPLA 2 in the future. The present study showed a low level of expression of AccPLA 2 protein in Tn cells, which might be caused by the allergen and toxic characters of BvPLA 2 and may have harmed insect cells. It also showed that the splicing time of the insect cells infected by the bacmid containing AccPLA 2 protein is much less than that of the cells infected by the bacmids containing other foreign genes. In conclusion, we constructed Tn cells by transfecting the recombinant bacmid DNA that was inserted in a new BvPLA 2 (AccPLA 2 ) gene from the Chinese honeybee ( Apis cerana cerana ) venom glands to express the recombinant AccPLA 2 protein, a native BvPLA 2 -like structure with corresponding biological activities that can be glycosylated in Tn cells. Our work may provide fundamental knowledge for potential genetic engineering to produce AccPLA 2 in the pharmaceutical industry. References F Altmann , V Kubelka , E Staudacher , K Uhl , L Marz . Characterization of the isoforms of phospholipase A 2 from honeybee venom . Insect Biochem , 1991 . 21 ( 5 ): 467 - 472 . DOI: 10.1016/0020-1790(91)90099-Z http://doi.org/10.1016/0020-1790(91)90099-Z . Characterization of the isoforms of phospholipase A2 from honeybee venom F Altmann , E Staudacher , IBH Wilson , L Marz . Insect cells as hosts for the expression of recombinant glycoproteins . Glycoconjugate J , 1999 . 16 ( 2 ): 109 - 123 . DOI: 10.1023/A:1026488408951 http://doi.org/10.1023/A:1026488408951 . Insect cells as hosts for the expression of recombinant glycoproteins RR Annand , M Kontoyianni , JE Penzotti , T Dudler , TP Lybrand , MH Gelb . Active site of bee venom phospholipase A 2 : the role of histidine-34, As-partate-64 and Tyrosine-87 . Biochemistry , 1996 . 35 ( 14 ): 4591 - 4601 . 8605210 DOI: 10.1021/bi9528412 http://doi.org/10.1021/bi9528412 . Active site of bee venom phospholipase A2: the role of histidine-34, As-partate-64 and Tyrosine-87 J Balsinde , MV Winstead , EA Dennis . Phospholipase A 2 regulation of arachidonic acid mobilization . FEBS Lett , 2002 . 531 ( 1 ): 2 - 6 . 12401193 DOI: 10.1016/S0014-5793(02)03413-0 http://doi.org/10.1016/S0014-5793(02)03413-0 . Phospholipase A2 regulation of arachidonic acid mobilization EA Dennis . Diversity of group types, regulation and function of phospholipase A 2 . J Biochem , 1994 . 269 ( 18 ): 13057 - 13060 . . Diversity of group types, regulation and function of phospholipase A2 EA Dennis . A growing phospholipase A 2 superfamily of signal transduction enzymes . Trends Biochem Sci , 1997 . 22 ( 1 ): 1 - 2 . 9020581 DOI: 10.1016/S0968-0004(96)20031-3 http://doi.org/10.1016/S0968-0004(96)20031-3 . A growing phospholipase A2 superfamily of signal transduction enzymes T Dudler , WQ Chen , SS Wang , T Schneider , RR Annand , RO Dempcy , R Crameri , M Gmachl , M Suter , MH Gelb . High-level expression in Escherichia coli and rapid purification of enzymatically active honey bee venom phospholipase A 2 . Biochim Biophys Acta , 1992 . 1165 ( 2 ): 201 - 210 . 1450215 . High-level expression in Escherichia coli and rapid purification of enzymatically active honey bee venom phospholipase A2 D Fenard , G Lambeau , E Valentin , JC Lefebvre , M Lazdunski , A Doglio . Secreted phospholipases A 2 , a new class of HIV inhibitors that block virus entry into host cells . J Clin Invest , 1999 . 104 ( 5 ): 611 - 618 . 10487775 DOI: 10.1172/JCI6915 http://doi.org/10.1172/JCI6915 . Secreted phospholipases A2, a new class of HIV inhibitors that block virus entry into host cells D Fenard , G Lambeau , T Maurin , JC Lefebvre , A Doglio . A peptide derived from bee venom-secreted phospholipase A 2 inhibits replication of T-cell tropic HIV-1 strains via interaction with the CXCR4 chemokine receptor . Mol Pharmacol , 2001 . 60 ( 2 ): 341 - 347 . 11455021 . A peptide derived from bee venom-secreted phospholipase A2 inhibits replication of T-cell tropic HIV-1 strains via interaction with the CXCR4 chemokine receptor E Habermann . Bee and wasp venoms . Science , 1972 . 177 ( 4046 ): 314 - 322 . 4113805 DOI: 10.1126/science.177.4046.314 http://doi.org/10.1126/science.177.4046.314 . Bee and wasp venoms TP King , MD Spangfort . Structure and biology of stinging insect venom allergens . Int Arch Allergy Imm , 2000 . 123 ( 2 ): 99 - 106 . DOI: 10.1159/000024440 http://doi.org/10.1159/000024440 . Structure and biology of stinging insect venom allergens V Kubelka , F Almann , E Staudacher , V Tretter , L Marz , K Hard , JP Kamerling , JF Vliegenthart . Primary structures of the N -linked carbohydrate chains from honeybee venom phospholipase A 2 . Eur J Biochem , 1993 . 213 ( 3 ): 1193 - 1204 . 8504812 DOI: 10.1111/j.1432-1033.1993.tb17870.x http://doi.org/10.1111/j.1432-1033.1993.tb17870.x . Primary structures of the N-linked carbohydrate chains from honeybee venom phospholipase A2 K Kuchler , M Gmachl , MJ Sippl , G Krell . Analysis of the cDNA for phospholipase A 2 from honeybee venom glands-the deduced amino acid sequence reveals homology to the corresponding vertebrate enzymes . Eur J Biochem , 1989 . 184 ( 1 ): 249 - 254 . 2776767 DOI: 10.1111/j.1432-1033.1989.tb15014.x http://doi.org/10.1111/j.1432-1033.1989.tb15014.x . Analysis of the cDNA for phospholipase A2 from honeybee venom glands-the deduced amino acid sequence reveals homology to the corresponding vertebrate enzymes JH Li , CX Zhang , LR Shen , ZH Tang , JA Cheng . Expression and regulation of phospholipase A 2 in venom gland of the Chinese honeybee, Apis cerana cerana . Arch Insect Biochem Physiol , 2005 . 60 ( 1 ): 1 - 12 . 16116618 DOI: 10.1002/arch.20075 http://doi.org/10.1002/arch.20075 . Expression and regulation of phospholipase A2 in venom gland of the Chinese honeybee, Apis cerana cerana VA Luckow , SC Lee , GF Barry , PO Olins . Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli . J Virol , 1993 . 67 ( 8 ): 4566 - 4579 . 8392598 . Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli I Mingarro , E Prez-Paya , C Pinilla , JR Appel , RA Houghten , SE Blondelle . Activation of bee venom phospholipase A 2 through a peptide-enzyme complex . FEBS Lett , 1995 . 372 ( 1 ): 131 - 134 . 7556634 DOI: 10.1016/0014-5793(95)00964-B http://doi.org/10.1016/0014-5793(95)00964-B . Activation of bee venom phospholipase A2 through a peptide-enzyme complex AB Mukherjee , L Miele , N Pattabiraman . Phospholipases A 2 enzymes: regulation and hysiological role . Biochem Pharmacol , 1994 . 48 ( 1 ): 1 - 10 . 8043009 DOI: 10.1016/0006-2952(94)90216-X http://doi.org/10.1016/0006-2952(94)90216-X . Phospholipases A2 enzymes: regulation and hysiological role M Murakami , I Kudo . Phospholipase A 2 . J Biochem , 2002 . 131 ( 2 ): 285 - 292 . 11872155 . Phospholipase A2 S Nakashima , K Kitamoto , M Arioka . The catalytic activity, but not receptor binding, of PLA 2 s plays a critical role for neurite outgrowth induction in PC12 cells . Brain Res , 2004 . 1015 ( 1/2 ): 207 - 211 . 15223389 DOI: 10.1016/j.brainres.2004.04.069 http://doi.org/10.1016/j.brainres.2004.04.069 . The catalytic activity, but not receptor binding, of PLA2s plays a critical role for neurite outgrowth induction in PC12 cells BP Oldroyd , S Wongsiri . Asian Honeybees. Biology, Conservation, and Human Interactions , 2006 . : Cambridge, Massachusetts, USA Harvard University Press , 36 - 64 . . Asian Honeybees. Biology, Conservation, and Human Interactions MD Owen , LA Pfaff , RE Reisman , J Wypych . Phospholipase A 2 in venom extracts from honey bees ( Apis mellifera L.) of different ages . Toxicon , 1990 . 28 ( 7 ): 813 - 820 . 2219143 DOI: 10.1016/S0041-0101(09)80004-4 http://doi.org/10.1016/S0041-0101(09)80004-4 . Phospholipase A2 in venom extracts from honey bees (Apis mellifera L.) of different ages LA Palomares , CE Joosten , PR Hughes , RR Granados , ML Shuler . Novel insect cell line capable of complex N -glycosylation and sialylation of recombinant proteins . Biotechnol Prog , 2003 . 19 ( 1 ): 185 - 192 . 12573024 DOI: 10.1021/bp025598o http://doi.org/10.1021/bp025598o . Novel insect cell line capable of complex N-glycosylation and sialylation of recombinant proteins EB Rodriguez de Turco , FR Jackson , MA DeCoster , M Kolko , NG Bazan . Glutamate signalling and secretory phospholipase A 2 modulate the release of arachidonic acid from neuronal membranes . J Neurosci Res , 2002 . 68 ( 5 ): 558 - 567 . 12111845 DOI: 10.1002/jnr.10239 http://doi.org/10.1002/jnr.10239 . Glutamate signalling and secretory phospholipase A2 modulate the release of arachidonic acid from neuronal membranes J Sambrook , DW Russell . Molecular Cloning: A Laboratory Manual , 2002 . : New York, USA Cold Spring Harbor Laboratory Press , . Molecular Cloning: A Laboratory Manual JO Schmidt . Toxinology of venoms from the honeybee genus Apis . Toxicon , 1995 . 33 ( 7 ): 917 - 927 . 8588216 DOI: 10.1016/0041-0101(95)00011-A http://doi.org/10.1016/0041-0101(95)00011-A . Toxinology of venoms from the honeybee genus Apis DL Scott , SP White , Z Otwinowski , W Yuan , MH Gelb , PB Sigler . Interfacial catalysis: the mechanism of phospholipase . Science , 1990 . 250 ( 4987 ): 1541 - 1546 . 2274785 DOI: 10.1126/science.2274785 http://doi.org/10.1126/science.2274785 . Interfacial catalysis: the mechanism of phospholipase DL Scott , Z Otwinowski , MH Gelb , PB Sigler . Crystal structure of bee-venom phospholipase A 2 in a complex with a transition-state analogue . Science , 1990 . 250 ( 4987 ): 1563 - 1566 . 2274788 DOI: 10.1126/science.2274788 http://doi.org/10.1126/science.2274788 . Crystal structure of bee-venom phospholipase A2 in a complex with a transition-state analogue JY Shang , YM Shao , GJ Lang , G Yuan , ZH Tang , CX Zhang . Expression of two types of acetylcholinesterase gene from the silkworm, Bombyx mori , in insect cells . Insect Sci , 2007 . 14 ( 6 ): 443 - 449 . . Expression of two types of acetylcholinesterase gene from the silkworm, Bombyx mori, in insect cells LS Shen , CZ Zhang , JA Cheng . Cloning and sequencing of genes encoding phospholipase A 2 from the venom of Apis cerana cerana and A. mellifera . J Agric Biotechnol , 2002 . 10 ( 1 ): 29 - 32 . (in Chinese) . Cloning and sequencing of genes encoding phospholipase A2 from the venom of Apis cerana cerana and A. mellifera RA Shipolini , GL Callewaert , RC Cottrell , CA Vernon . The amino-acid sequence and carbohydrate content of phospholipase A 2 from bee venom . Eur J Biochem , 1974 . 48 ( 2 ): 465 - 476 . 4448181 DOI: 10.1111/j.1432-1033.1974.tb03787.x http://doi.org/10.1111/j.1432-1033.1974.tb03787.x . The amino-acid sequence and carbohydrate content of phospholipase A2 from bee venom RA Shipolini , S Doonan , CA Vernon . The disulphide bridges of phospholipase A 2 from bee venom . Eur J Biochem , 1974 . 48 ( 2 ): 477 - 483 . 4614976 DOI: 10.1111/j.1432-1033.1974.tb03788.x http://doi.org/10.1111/j.1432-1033.1974.tb03788.x . The disulphide bridges of phospholipase A2 from bee venom GE Smith , MD Summers , M Fraser . Production of human belta interferon in insect cell with baculovirus expression vectors . J Mol Cell Biol , 1983 . 3 ( 12 ): 2156 - 2165 . . Production of human belta interferon in insect cell with baculovirus expression vectors LN Soldatova , R Crameri , M Gmachl , DM Kemeny , M Schmidt , M Weber , UR Mueller . Superior biologic activity of the recombinant bee venom allergen hyaluronidase expressed in baculovirus infected insect cells as compared with Escherichia coli . J Allergy Clin Immunol , 1998 . 101 ( 5 ): 691 - 698 . 9600508 DOI: 10.1016/S0091-6749(98)70179-4 http://doi.org/10.1016/S0091-6749(98)70179-4 . Superior biologic activity of the recombinant bee venom allergen hyaluronidase expressed in baculovirus infected insect cells as compared with Escherichia coli D Toki , M Sarkar , B Yip , F Reck , D Joziasse , M Fukuda , H Schachter , I Brockhausen . Expression of stable human O -glycan core 2β-1,6- N -acetylgluco-saminyltransferase in Sf9 insect cells . Biochem J , 1997 . 325 ( 1 ): 63 - 69 . 9224630 . Expression of stable human O-glycan core 2β-1,6-N-acetylgluco-saminyltransferase in Sf9 insect cells CW Whitfield , SK Behura , SH Berlocher , AGJ Clark , S Johnston , WS Sheppard , DR Smith , AV Suarez , D Weaver , ND Tsutsui . Thrice out of Africa: ancient and recent expansions of the honey bee, Apis mellifera . Science , 2006 . 314 ( 5799 ): 642 - 645 . 17068261 DOI: 10.1126/science.1132772 http://doi.org/10.1126/science.1132772 . Thrice out of Africa: ancient and recent expansions of the honey bee, Apis mellifera P Xu , M Shi , XX Chen . Antimicrobial peptide evolution in the Asiatic honey bee Apis cerana . PLoS ONE , 2009 . 4 ( 1 ): e4239 19156201 DOI: 10.1371/journal.pone.0004239 http://doi.org/10.1371/journal.pone.0004239 . Antimicrobial peptide evolution in the Asiatic honey bee Apis cerana CX Zhang , XD Tang , JA Cheng . The utilization and industrialization of insect resources in China . Entomol Res , 2008 . 38 ( S1 ): S38 - S47 . DOI: 10.1111/j.1748-5967.2008.00173.x http://doi.org/10.1111/j.1748-5967.2008.00173.x . The utilization and industrialization of insect resources in China M Zhao , UT Brunk , JW Eaton . Delayed oxidant-induced cell death involves activation of phospholipase A 2 . FEBS Lett , 2001 . 509 ( 3 ): 399 - 404 . 11749963 DOI: 10.1016/S0014-5793(01)03184-2 http://doi.org/10.1016/S0014-5793(01)03184-2 . Delayed oxidant-induced cell death involves activation of phospholipase A2 We are grateful to Prof. Chuan-xi ZHANG and Prof. Jia-an CHENG in the Institute of Insect Science, Zhejiang University for their kind instruction in experiment. We are also grateful to Dr. Jian-xiang WU in Zhejiang University for his assistance in preparing the antibody against AmPLA 2 . We would like to acknowledge Dr. Jia-cai WU in Zhejiang University for his technical assistance, and Dr. Zhen-hua LIU in Tufts University and Dr. Song-bai LIU in Harvard University for editing the manuscript. 10.1631/jzus.B0900254 Project (No. 2007AA10Z324) supported by the National High-Tech Research and Development Program (863) of China 2009-08-08 2010-03-17 2010-01-28 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2010 2010 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 05 11