Structural analysis of a plant glucosyl ceramidase (GBA2)-like enzyme from arabidopsis thaliana by cryogenic electron microscopy (CRYO-EM)

Glucosylceramide (GlcCer) is a common glycosphingolipid, that accumulates in cells in response to Gaucher disease, diabetes, and skin disorders in human but it is not well understood in plants. In animals, GlcCer is catabolized by glucosylcerebrosidase 1 and 2 (GBA1 and GBA2). GBA1 is a lysosomal enzyme in glycoside hydrolase (GH) family GH30, while GBA2 is a membrane-associated cytoplasmic protein in family GH116. Currently, there are no experimental structures of eukaryotic GH116 homologues. Although the bacterial TxGH116 β-glucosidase structure was determined by x-ray crystallography, it does not hydrolyze glucosylceramides, unlike the animal and plant enzymes. Therefore, we have investigated the structure of plant GH116 (At4GH116) by Cryo-EM single particle analysis. The recombinant At4GH116 protein was produced in E. coli with a 6x His-tag and purified by immobilized metal affinity chromatography followed by S200 size exclusion chromatography. Cryo-EM conducted at Mahidol University, Thailand, enabled the capture of detailed images of At4GH116, indicating strong potential for high-resolution structure determination. Initial map generation indicated a unique hexameric structure, consistent with previous high resolution size exclusion chromatography analysis. Solving the first eukaryotic structure of a GH116 enzyme will generate a template for improved modeling of human GBA2, with implications for treating human diseases, such as Gaucher disease and hereditary spastic paraplegia.