High-throughput screening of cell lysates for ganglioside synthesis.
A high-throughput screen to detect the synthesis of natural and non-natural gangliosides by cell lysates has been developed and automated. Utilizing the binding specificity of cholera toxin B-subunit for the oligosaccharide moiety of the ganglioside G M1, the synthesis of sugar-sphingolipid glycosidic linkages was detected using a modified enzyme-linked immunosorbent assay (ELISA)/enzyme-linked lectin assay (ELLA). The screen was optimized and validated for high-throughput screening of cell lysates by evaluating different vectors, promoters, substrates and detection strategies. The extent of ganglioside synthesis was found to be proportional to enzyme concentration and length of incubation time. As a test of the finalized screen efficacy, individual colonies from a saturation mutagenesis library of nucleophile mutants of an endoglycoceramidase were screened to identify the most active enzyme for ganglioside synthesis. This screen should find general application in assaying both glycolipid biosynthesis and glycolipid hydrolysis, as it is highly sensitive and can be used with crude cell extracts.
Hancock
Susan M
SM
Department of Chemistry, University of British Columbia, Vancouver, BC, Canada.
Tarling
Chris A
CA
Withers
Stephen G
SG
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D002384:Catalysis; D002417:Cattle; D002457:Cell Extracts / Q000032:analysis / Q000737:chemistry*; D002772:Cholera Toxin / Q000378:metabolism; D005459:Fluorides / Q000737:chemistry / Q000378:metabolism; D005732:Gangliosides / Q000096:biosynthesis*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism; D051379:Mice; D013110:Sphingosine / Q000378:metabolism; D019809:Streptavidin / Q000378:metabolism
D000906:Antibodies; D002457:Cell Extracts; D005732:Gangliosides; D001710:Biotin; D002772:Cholera Toxin; D019809:Streptavidin; D006735:Horseradish Peroxidase; D006026:Glycoside Hydrolases; D013110:Sphingosine; D005459:Fluorides