Molecular cloning and expression of a fifth type of alpha2,8-sialyltransferase (ST8Sia V). Its substrate specificity is similar to that of SAT-V/III, which synthesize GD1c, GT1a, GQ1b and GT3

Abstract

The cDNAs encoding a new alpha2,8-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 (GD3 synthase) and mouse ST8Sia III (Siaalpha2,3Galbeta1,4GlcNAcalpha2,8-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 alpha2, 8-sialyltransferase activity toward GM1b, GD1a, GT1b, and GD3, and synthesized GD1c, GT1a, GQ1b, and GT3, respectively. The apparent Km values for GM1b, GD1a, GT1b and GD3 were 1.1, 0.082, 0.070, and 0.28 mM, respectively. However, ST8Sia V did not exhibit activity toward GM3. Thus, the substrate specificity of ST8Sia V is different from those of ST8Sia I and III, both of which exhibit activity toward GM3. Transfection of the ST8Sia V gene into COS-7 cells, which express GD1a as a major glycolipid, led to the expression of determinants for monoclonal antibody 4F10, which recognizes GT1a and GQ1b, suggesting that ST8Sia V exhibits activity toward gangliosides GD1a and/or GT1b in vivo. The expression of the ST8Sia V gene was tissue- and developmental stage-specific, and was clearly different from those of other alpha2,8-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 alpha2,8-sialyltransferase involved in GD1c, GT1a, GQ1b, and GT3 synthesis.