Kawano T, Koyama S, Takematsu H, Kozutsumi Y, Kawasaki H, Kawashima S, Kawasaki T, Suzuki A
Department of Membrane Biochemistry, Tokyo Metropolitan Institute of Medical Science.
J Biol Chem. 1995 Jul 7;270(27):16458-63. doi: 10.1074/jbc.270.27.16458.
Cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) hydroxylase, which is the key enzyme for the synthesis of N-glycolylneuraminic acid (NeuGc), has been purified from the cytosolic fraction of mouse liver, as described in our previous paper. The amino acid sequences of the purified CMP-NeuAc hydroxylase, and peptides obtained by lysylendopeptidase digestion, were used to synthesize specific oligonucleotide primers. A mouse cDNA clone of the enzyme was obtained by a combination of the polymerase chain reaction and rapid amplification of cDNA ends. The sequence of the clone contained an open reading frame coding for a protein of 577 amino acids with a predicted molecular mass of 66 kDa. The deduced sequence included the amino acid sequences obtained for the purified enzyme and peptides, and a complete match was obtained for 159 residues. The enzyme has neither a signal peptide sequence nor a membrane spanning domain, which is consistent with localization of the enzyme in the cytosol. Transfection of a cDNA construct to COS-1 cells increased the enzyme activity and the amount of NeuGc. Comparison of the sequence with GenBank data indicated that no similar sequence has been reported so far. Northern blot analysis of various mouse tissues with the enzyme cDNA as a probe indicated that expression of NeuGc is related to the level of CMP-NeuAc hydroxylase mRNA. On Southern blot analysis with the same probe, cross-hybridizing bands were detected in the human and fish genomes.
如我们之前的论文所述，胞苷单磷酸 - N - 乙酰神经氨酸（CMP - NeuAc）羟化酶是合成N - 羟乙酰神经氨酸（NeuGc）的关键酶，已从小鼠肝脏的胞质部分中纯化出来。利用纯化的CMP - NeuAc羟化酶以及经赖氨酰内肽酶消化得到的肽段的氨基酸序列，合成了特异性寡核苷酸引物。通过聚合酶链反应和cDNA末端快速扩增相结合的方法，获得了该酶的小鼠cDNA克隆。该克隆的序列包含一个编码577个氨基酸的开放阅读框，预测分子量为66 kDa。推导的序列包括纯化酶和肽段的氨基酸序列，并且有159个残基完全匹配。该酶既没有信号肽序列也没有跨膜结构域，这与该酶定位于胞质溶胶中是一致的。将cDNA构建体转染到COS - 1细胞中可提高酶活性和NeuGc的量。将该序列与GenBank数据进行比较表明，目前尚未报道过类似序列。用该酶的cDNA作为探针，对各种小鼠组织进行Northern印迹分析表明，NeuGc的表达与CMP - NeuAc羟化酶mRNA的水平相关。用相同探针进行Southern印迹分析时，在人类和鱼类基因组中检测到了交叉杂交带。
