Molecular cloning and expression of a fifth type of alpha,8-sialyltransferase (ST8Sia V) - Its substrate specificity is similar to that of SAT-V/III, which synthesize G(D1c), G(T1a), G(Q1b) and G(T3)

The cDNAs encoding a new alpha 2,8-sialyltransferase (ST8Sia V) were cloned from a mouse brain cDNA library by means of a polymerase chain reaction-based method using the nucleotide sequence information on mouse ST8Sia I (G(D3) synthase) and mouse ST8Sia III (Sia alpha 2,3Gal beta 1,4GlcNAc alpha 2,8-sialyltransferase) both of which exhibit activity toward glycolipids. The predicted amino acid sequence of ST8Sia V shows 36.1% and 15.0% identity to those of mouse ST8Sia I and III, respectively. The recombinant protein A-fused ST8Sia V expressed in COS-7 cells exhibited an alpha 2,8-sialyltransferase activity toward G(M1b), G(D1a), G(T1b), and G(D3), and synthesized G(D1c), G(T1a), G(Q1b), and G(T3), respectively. The apparent K-m values for G(M1b), G(D1a), G(T1b) and G(D3) were 1.1, 0.082, 0.070, and 0.28 mM, respectively. However, ST8Sia V did not exhibit activity toward G(M3). Thus, the substrate specificity of ST8Sia V is different from those of ST8Sia I and III, both of which exhibit activity toward G(M3). Transfection of the ST8Sia V gene into COS-7 cells, which express G(D1a) as a major glycolipid, led to the expression of determinants for monoclonal antibody 4F10, which recognizes G(T1a) and G(Q1b), suggesting that ST8Sia V exhibits activity toward gangliosides G(D1a) and/or G(T1b) in vivo. The expression of the ST8Sia V gene was tissue- and developmental stage-specific, and was clearly different from those of other alpha 2,8-sialyltransferase genes. The ST8Sia V gene was strongly expressed in the brain and weakly in other tissues such as the liver. In addition, its expression was greater in the adult than fetal brain. These results strongly indicate that ST8Sia V is a candidate for SAT-V, the alpha 2,8-sialyltransferase involved in G(D1c), G(T1a), G(Q1b), and G(T3) synthesis.