RESUMEN
Onoceroids are a rare family of triterpenes. One representative onoceroid is ambrein, which is the main component of ambergris used as a traditional medicine. We have previously identified the onoceroid synthase, BmeTC, in Bacillus megaterium and succeeded in creating ambrein synthase by introducing mutations into BmeTC. Owing to the structural similarity of ambrein to vitamin D, a molecule with diverse biological activities, we hypothesized that some of the activities of ambergris may be induced by the binding of ambrein to the vitamin D receptor (VDR). We demonstrated the VDR binding ability of ambrein. By comparing the structure-activity relationships of triterpenes with both the VDR affinity and osteoclastic differentiation-promoting activity, we observed that the activity of ambrein was not induced via the VDR. Therefore, some of the activities of ambergris, but not all, can be attributed to its VDR interaction. Additionally, six unnatural onoceroids were synthesized using the BmeTC reactions, and these compounds exhibited higher VDR affinity than that of ambrein. Enzymatic syntheses of onoceroid libraries will be valuable in creating a variety of bioactive compounds beyond ambergris.
Asunto(s)
Ámbar Gris , Triterpenos , Ámbar Gris/química , Receptores de Calcitriol , Triterpenos/farmacología , Naftoles/química , Vitamina DRESUMEN
Pichia pastoris is a popular eukaryotic system employed for the fast, simple and inexpensive production of recombinant protein including biotherapeutics such as human albumin. The CH2 domain of human Immunoglobulin G (IgG) is a promising scaffold for developing novel therapeutics. To accelerate the research of CH2 domain, we have established a procedure to highly express human CH2 domain (â¼150 mg/l) as well as human Fc (â¼30 mg/l) in yeast P. pastoris. The procedure yields, simultaneously, a major glycosylated (â¼70%) and non-glycosylated (â¼30%) fractions. They can be easily separated with high purity. Although both forms of CH2 domain have essentially the same secondary structure, the presence of the glycan increased the thermal stability of the CH2 domain by about 5°C as determined from calorimetry. The purified glycosylated CH2 domain elicited polyclonal antibodies in mouse, recognizing not only the CH2 domain, but also recombinant human Fc and the commercial IgG1 antibody Rituxan. Protein A and Protein G binding to the kink region between CH2 domain and CH3 domain of human Fc are used to purify therapeutic proteins. Therefore, these antibodies are candidates to develop a novel affinity material to purify human antibodies using their CH2 domain.
Asunto(s)
Anticuerpos/metabolismo , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Saccharomycetales/metabolismo , Animales , Formación de Anticuerpos , Electroforesis en Gel de Poliacrilamida/métodos , Femenino , Glicosilación , Humanos , Fragmentos Fc de Inmunoglobulinas/biosíntesis , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB C , Pichia/metabolismo , Polisacáridos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismoRESUMEN
Ambergris, a sperm whale metabolite, has long been used as a fragrance and traditional medication, but it is now rarely available. The odor components of ambergris result from the photooxidative degradation of the major component, ambrein. The pharmacological activities of ambergris have also been attributed to ambrein. However, efficient production of ambrein and odor compounds has not been achieved. Here, we constructed a system for the synthesis of ambrein and odor components. First, we created a new triterpene synthase, "ambrein synthase," for mass production of ambrein by redesigning a bacterial enzyme. The ambrein yields were approximately 20 times greater than those reported previously. Next, an efficient photooxidative conversion system from ambrein to a range of volatiles of ambergris was established. The yield of volatiles was 8-15%. Finally, two biological activities, promotion of osteoclast differentiation and prevention of amyloid ß-induced apoptosis, were discovered using the synthesized ambrein.