Datta A K, Chammas R, Paulson J C
Department of Molecular Biology and Molecular and Experimental Medicine, Scripps Research Institute, San Diego, California 92037, USA.
J Biol Chem. 2001 May 4;276(18):15200-7. doi: 10.1074/jbc.M010542200. Epub 2001 Jan 29.
The sialyltransferase gene family is comprised of 16 cloned enzymes. All members contain two conserved protein domains, termed the S- and L-sialylmotifs, that participate in substrate binding. Of only six invariant amino acids, two are cysteines, with one found in each sialylmotif. Although the recombinant soluble form of ST6Gal I has six cysteines, quantitative analysis indicated the presence of only one disulfide linkage, and thiol reducing agents dithiothreitol and beta-mercaptoethanol inactivated the enzyme. Analysis of site-directed mutants showed that alanine or serine mutants of invariant Cys(181) or Cys(332) exhibit no detectable activity, either by direct assay or by staining of the transfected cells with Sambucus nigra agglutinin, which recognizes the product NeuAcalpha2,6Galbeta1,4GlcNAc on glycoproteins. In contrast, alanine mutations of charged residues adjacent to either cysteine showed little or no effect on enzyme activity. Immunofluorescence microscopy showed that although the wild type sialyltransferase is properly localized in the Golgi apparatus, the inactive cysteine mutants are retained in the endoplasmic reticulum. The results suggest that the invariant cysteine residues in the L- and S-sialylmotifs participate in the formation of an intradisulfide linkage that is essential for proper conformation and activity of ST6Gal I.
唾液酸转移酶基因家族由16种已克隆的酶组成。所有成员都包含两个保守的蛋白质结构域，即S-和L-唾液酸基序，它们参与底物结合。在仅有的六个不变氨基酸中，有两个是半胱氨酸，每个唾液酸基序中各有一个。尽管重组可溶性形式的ST6Gal I有六个半胱氨酸，但定量分析表明仅存在一个二硫键，并且硫醇还原剂二硫苏糖醇和β-巯基乙醇会使该酶失活。对定点突变体的分析表明，不变半胱氨酸（181）或半胱氨酸（332）的丙氨酸或丝氨酸突变体，无论是通过直接测定还是用黑接骨木凝集素对转染细胞进行染色（该凝集素识别糖蛋白上的产物NeuAcalpha2,6Galbeta1,4GlcNAc），均未显示出可检测到的活性。相比之下，与任一胱氨酸相邻的带电荷残基的丙氨酸突变对酶活性几乎没有影响。免疫荧光显微镜检查表明，尽管野生型唾液酸转移酶正确定位于高尔基体中，但无活性的半胱氨酸突变体保留在内质网中。结果表明，L-和S-唾液酸基序中的不变半胱氨酸残基参与了二硫键内连接的形成，这对于ST6Gal I的正确构象和活性至关重要。
