Expression and regulation of phospholipase A2 in venom gland of the chinese honeybee,Apis cerana cerana

Abstract

Phospholipase A(2) (PLA(2)) is one of the components of bee venom with a wide range of pharmacological functions. It operates as a major allergen working with other venom components to defend the colony from intruder. In the present study, the cDNA sequence of the Ac-pla(2) gene from cDNA library of the venom gland of Apis cerana was compared with the amplified corresponding region of genomic DNA. The result showed that the Ac-pla(2) gene consisted of four exons and three introns. Southern blot showed that the Ac-pla(2) gene was a single copy per haploid genome. The most active transcription period was during the first 8 days of adults, which correspondingly was the period of sharp increase of PLA(2) protein. ELISA analysis revealed that the PLA(2) was undetectable in pupal stage and the newly eclosed adult, but increased sharply to a maximum of 10-12 mug per honeybee by 8-10 days of adult life, followed by a gradual decrease to 8 mug for the rest of adult life. Transcriptional or post transcriptional regulation is the key step for Ac-pla(2) expression. The early secreted Ac-PLA(2) showed a low degree of post-translational modification; with increasing age, glycosylation was detected by Western blot and glycoprotein staining analysis. Different post-translational modifications were found among different individuals in A. cerana when compared to A. mellifera.

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

“…Soldatova et al (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;Altmann et al, 1991;Soldatova et al, 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 results indicate that there was crossreaction 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 antiAmPLA 2 antibody, which was caused by the characters of BvPLA 2 glycosylation (Li et al, 2005). These results also indicate that the expressed AccPLA 2 protein can be glycosylated in Tn cells.…”

“…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 (Li et al, 2005). Altmann et al (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 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). 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.…”

Nonviral gene delivery to the liver

“…PLA2 is involved in several cellular signaling pathways, such as inflammation 346 and apoptosis 347 . PLA2 is also found in the venom of many venomous animals such as bees [348][349][350] , wasps 351 , spiders 352,353 , scorpions 354 , centipedes 355 , snakes 356 , gila monsters 357 , cuttlefish 358 , sea anemones 359 , cone snails 360 , and others, creating an interesting example of convergent evolution 361 . These PLA2s often have neurotoxic and hemorrhagic activity, although some venom PLA2s are toxic while having no catalytic activity, acting as ligands for receptors on neurons [362][363][364] .…”