RESUMO
Poria cocos (P. cocos) has long been used as traditional Chinese medicine and triterpenoids are the most important pharmacologically active constituents of this fungus. Farnesyl pyrophosphate synthase (FPS) is a key enzyme of triterpenoids biosynthesis. The gene encoding FPS was cloned from P. cocos by degenerate PCR, inverse PCR and cassette PCR. The open reading frame of the gene is 1086 bp in length, corresponding to a predicted polypeptide of 361 amino acid residues with a molecular weight of 41.2 kDa. Comparison of the P. cocos FPS deduced amino acid sequence with other species showed the highest identity with Ganoderma lucidum (74%). The predicted P. cocos FPS shares at least four conserved regions involved in the enzymatic activity with the FPSs of varied species. The recombinant protein was expressed in Pichia pastoris and purified. Gas chromatography analysis showed that the recombinant FPS could catalyze the formation of farnesyl diphosphate (FPP) from geranyl diphosphate (GPP) and isopentenyl diphosphate (IPP). Furthermore, the expression profile of the FPS gene and content of total triterpenoids under different stages of development and methyl jasmonate treatments were determined. The results indicated that there is a positive correlation between the activity of FPS and the amount of total triterpenoids produced in P. cocos.
Assuntos
Genes Fúngicos , Geraniltranstransferase/genética , Poria/enzimologia , Poria/genética , Triterpenos/metabolismo , Acetatos/farmacologia , Sequência de Aminoácidos , Sequência de Bases , Biocatálise/efeitos dos fármacos , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Cromatografia Gasosa , Clonagem Molecular , Ciclopentanos/farmacologia , Eletroforese em Gel de Poliacrilamida , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Geraniltranstransferase/isolamento & purificação , Dados de Sequência Molecular , Oxilipinas/farmacologia , Filogenia , Pichia/efeitos dos fármacos , Pichia/metabolismo , Poria/efeitos dos fármacos , Poria/crescimento & desenvolvimento , Regiões Promotoras Genéticas/genética , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Edible fungus Poria cocos (Schw.) Wolf is a cooking material that has myriad health benefits. However, its active constituents have not been well-defined. We previously purified an immunomodulatory protein, PCP, from P. cocos and described its biochemical features and its ability to activate primary macrophage via TLR4. In this study, we cloned the gene of PCP and demonstrated its ability to activate Th1 response in cell cultures and in mice. The complete cDNA sequence of PCP consisted of 807 bp, which included a 579 bp coding sequence that encoded 194 amino acids. With the addition of co-stimulatory CD3/CD28 signals, PCP significantly increased the surface expression of CD44 and CD69 on effector T cells. PCP could also up-regulate T-bet and STAT4 expressions and IFN-γ and IL-2 secretions. Oral administration of PCP suppressed the production of both total and OVA-specific IgG1 in serum and enhanced the amounts of serum and OVA-specific IgG2a and Th1-related cytokine production in BALB/c splenocytes. In addition, oral administration of PCP significantly reduced IL-4 and IgE expressions in a murine model of atopic dermatitis. In conclusion, these results provide evidence that PCP could regulate mammalian immune cells and reveal their pharmaceutical potential in developing therapeutic strategies against Th2-mediated immune disorders.
Assuntos
Clonagem Molecular , Dermatite Atópica/tratamento farmacológico , Proteínas Fúngicas/administração & dosagem , Proteínas Fúngicas/genética , Imunoglobulina E/imunologia , Poria/química , Células Th1/imunologia , Células Th2/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Dermatite Atópica/genética , Dermatite Atópica/imunologia , Feminino , Proteínas Fúngicas/imunologia , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Interleucina-4/genética , Interleucina-4/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Poria/genética , Poria/imunologiaRESUMO
Squalene synthase (SQS) catalyzes the condensation of two molecules of farnesyl diphosphate to give presqualene diphosphate and the subsequent rearrangement to form squalene. The gene encoding squalene synthase was cloned from Poria cocos by degenerate PCR and inverse PCR. The open reading frame of the gene is 1,497 bp, which encodes 499 amino acid residues. A phylogenetic analysis revealed that P. cocos SQS belonged to the fungus group, and was more closely related to the SQS of Ganoderma lucidum than other fungi. The treatment of P. cocos with methyl jasmonate (MeJA) significantly enhanced the transcriptional level of P. cocos sqs gene and the content of squalene in P. cocos. The transcriptional level of sqs gene was approximately fourfold higher than the control sample and the squalene content reached 128.62 µg/g, when the concentration of MeJA was 300 µM after 72 h induction.