Identification and functional characterization of intracellular sialidase NeuA3 from Streptomyces avermitilis

Sialidases (EC 3.2.1.18), glycosidases that cleave the linkages whereby sialic acids are attached to glycoconjugates, are found in most bacterial species. Because sialidases can convert polysialogangliosides to monosialoganglioside GM1, they have potential clinical application for treatment of human neurological and other disorders including Alzheimer's disease, Parkinson's disease, and spinal cord injury. Sialidases with high substrate specificity are desirable for more efficient GM1 production. In this study, the sialidase neuA3 gene from the non-pathogenic bacterium Streptomyces avermitills, which is commonly used for industrial applications, was analyzed, cloned, and expressed in E. coli BL21 (DE3). Purified NeuA3 enzyme was characterized using 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (4-MUN) as a synthetic substrate. NeuA3 has a low molecular weight (similar to 38 kDa), showed strong stability in the presence of various divalent metal ions and temperature and pH values, preferentially cleaved alpha 2,3- and alpha 2,6-linked sialic acids from gangliosides, and efficiently converted crude porcine brain gangliosides to GM1. NeuA3 treatment of malignant human bladder cancer cells YTS-1 presented enhanced cell surface expression of GM1. The novel sialidase NeuA3 will be useful for functional studies of sialylated oligosaccharides and other sialoglycoconjugates, especially for studying the functions of GM1 in cancer research. (C) 2015 Elsevier Ltd. All rights reserved.