Angata K, Yen T Y, El-Battari A, Macher B A, Fukuda M
Glycobiology Program, Cancer Research Center, The Burnham Institute, La Jolla, California 92037, USA.
J Biol Chem. 2001 May 4;276(18):15369-77. doi: 10.1074/jbc.M100576200. Epub 2001 Feb 7.
NCAM polysialylation plays a critical role in neuronal development and regeneration. Polysialylation of the neural cell adhesion molecule (NCAM) is catalyzed by two polysialyltransferases, ST8Sia II (STX) and ST8Sia IV (PST), which contain sialylmotifs L and S conserved in all members of the sialyltransferases. The members of the ST8Sia gene family, including ST8Sia II and ST8Sia IV are unique in having three cysteines in sialylmotif L, one cysteine in sialylmotif S, and one cysteine at the COOH terminus. However, structural information, including how disulfide bonds are formed, has not been determined for any of the sialyltransferases. To obtain insight into the structure/function of ST8Sia IV, we expressed human ST8Sia IV in insect cells, Trichoplusia ni, and found that the enzyme produced in the insect cells catalyzes NCAM polysialylation, although it cannot polysialylate itself ("autopolysialylation"). We also found that ST8Sia IV does not form a dimer through disulfide bonds. By using the same enzyme preparation and performing mass spectrometric analysis, we found that the first cysteine in sialylmotif L and the cysteine in sialylmotif S form a disulfide bridge, whereas the second cysteine in sialylmotif L and the cysteine at the COOH terminus form a second disulfide bridge. Site-directed mutagenesis demonstrated that mutation at cysteine residues involved in the disulfide bridges completely inactivated the enzyme. Moreover, changes in the position of the COOH-terminal cysteine abolished its activity. By contrast, the addition of green fluorescence protein at the COOH terminus of ST8Sia IV did not render the enzyme inactive. These results combined indicate that the sterical structure formed by intramolecular disulfide bonds, which bring the sialylmotifs and the COOH terminus within close proximity, is critical for the catalytic activity of ST8Sia IV.
NCAM多唾液酸化在神经元发育和再生中起关键作用。神经细胞黏附分子（NCAM）的多唾液酸化由两种多唾液酸转移酶催化，即ST8Sia II（STX）和ST8Sia IV（PST），它们含有在所有多唾液酸转移酶成员中保守的唾液酸基序L和S。ST8Sia基因家族的成员，包括ST8Sia II和ST8Sia IV，其独特之处在于唾液酸基序L中有三个半胱氨酸，唾液酸基序S中有一个半胱氨酸，并且在COOH末端有一个半胱氨酸。然而，尚未确定任何一种多唾液酸转移酶的结构信息，包括二硫键是如何形成的。为了深入了解ST8Sia IV的结构/功能，我们在昆虫细胞草地贪夜蛾中表达了人ST8Sia IV，发现昆虫细胞中产生的该酶催化NCAM多唾液酸化，尽管它不能自身多唾液酸化（“自多唾液酸化”）。我们还发现ST8Sia IV不会通过二硫键形成二聚体。通过使用相同的酶制剂并进行质谱分析，我们发现唾液酸基序L中的第一个半胱氨酸和唾液酸基序S中的半胱氨酸形成二硫桥，而唾液酸基序L中的第二个半胱氨酸和COOH末端的半胱氨酸形成第二个二硫桥。定点诱变表明，参与二硫桥的半胱氨酸残基发生突变会使该酶完全失活。此外，COOH末端半胱氨酸位置的改变消除了其活性。相比之下，在ST8Sia IV的COOH末端添加绿色荧光蛋白并没有使该酶失活。这些结果综合表明，分子内二硫键形成的空间结构，使唾液酸基序和COOH末端紧密靠近，对ST8Sia IV的催化活性至关重要。
