Heterologous expression and functional characterization of the ligand-binding domain of oxysterol-binding protein from Aspergillus oryzae
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Oxysterol-binding proteins (OSBPs) comprise a family of sterol-binding proteins. In this study, we focused on AoOSBP1, one of the five OSBP proteins identified from the industrial fungus Aspergillus oryzae. The temporal expression pattern analysis showed that the expression of AoOSBP1, in both gene and protein levels, was stably expressed throughout the developmental stages, while was upregulated during the accelerated growth stage. The immunofluorescence observation revealed that AoOSBP1 protein was mainly distributed in the conidiophore, indicating its underlying role in spore formation. The ligand-binding domain of AoOSBP1, namely OSBP-related domain (ORD), was heterologously expressed in Escherichia coli and purified. The binding assay carried out using microscale thermophoresis showed that the recombinant AoORD protein exhibited binding affinity for ergosterol, and exhibited much higher affinity to oxysterols (25-hydroxycholesterol and 7-ketocholesterol) and phytosterols (β-sitosterol and stigmasterol). By contrast, MBP tag as the negative control showed no binding affinity for sterols. The present work demonstrates that AoORD domain in AoOSBP1 is capable of binding sterols, plays an underlying role in sterols transportation, and may participate in spore formation.
KeywordsAspergillus oryzae Oxysterol-binding protein Ergosterol Microscale thermophoresis binding assay
This work was supported by National Natural Science Foundation of China (NSFC) (Grant Nos. 31460447 and 31700068), Natural Science Foundation of Jiangxi Province (20171BAB214004 and 20181BAB214001), and Science and Technology Research Project of Jiangxi Provincial Department of Education (Grant Nos. GJJ170660, GJJ180600, and GJJGJJ170692).
- 5.Machida M, Asai K, Sano M, Tanaka T, Kumagai T, Terai G, Kusumoto KI, Arima T, Akita O, Kashiwagi Y, Abe K, Gomi K, Horiuchi H, Kitamoto K, Kobayashi T, Takeuchi M, Denning DW, Galagan JE, Nierman WC, Yu J, Archer DB, Bennett JW, Bhatnagar D, Cleveland TE, Fedorova ND, Gotoh O, Horikawa H, Hosoyama A, Ichinomiya M, Igarashi R, Iwashita K, Juvvadi PR, Kato M, Kato Y, Kin T, Kokubun A, Maeda H, Maeyama N, Maruyama JI, Nagasaki H, Nakajima T, Oda K, Okada K, Paulsen I, Sakamoto K, Sawano T, Takahashi M, Takase K, Terabayashi Y, Wortman JR, Yamada O, Yamagata Y, Anazawa H, Hata Y, Koide Y, Komori T, Koyama Y, Minetoki T, Suharnan S, Tanaka A, Isono K, Kuhara S, Ogasawara N, Kikuchi H (2005) Genome sequencing and analysis of Aspergillus oryzae. Nature. 438:1157–1161CrossRefGoogle Scholar
- 8.Pietrangelo A, Pietrangelo ND (2018) Golgi-localization of oxysterol binding protein-related protein 4L (ORP4L) is regulated by ligand binding. J Cell Sci 19:131Google Scholar
- 26.Beh CT, Cool L, Phillips J et al (2001) Overlapping functions of the yeast oxysterol-binding protein homologues. Genet. 157:1117–1140Google Scholar