Bouhours J F, Bouhours D
Institut National de la Santé et de la Recherche Médicale, Unité 76, Paris, France.
J Biol Chem. 1989 Oct 15;264(29):16992-9.
An enzymatic activity responsible for the hydroxylation of CMP-NeuAc into CMP-N-glycolylneuraminic acid (CMP-NeuGc) was found in the cytosolic fraction after cellular fractionation of the mucosa of rat small intestine. It was maximum in the presence of NADPH or NADH, but it was reduced by 50% by addition of 1 mM EDTA. The Km value for CMP-NeuAc was 0.6 microM. The CMP-NeuAc hydroxylase activity paralleled the expression of the GM3 (NeuGc) phenotype in the epithelium of the small intestine and was not measurable in the mutant rats BN and SHR that only expressed GM3 (NeuAc). Furthermore, the only form of CMP-sialic acid present in the intestinal mucosa of the mutants was CMP-NeuAc, whereas in the other strains CMP-NeuGc accounted for 70-85% of the native CMP-sialic acids. Wild-type and CMP-NeuAc hydroxylase-deficient inbred rats were mated. Individuals of F1 and backcross generations were typed for the phenotypes GM3(NeuGc)/GM3(NeuAc) and the activity of CMP-NeuAc hydroxylase in the small intestine. It was found that the expression of NeuGc in GM3 depends on a single autosomal dominant gene and correlates with the activity of CMP-NeuAc hydroxylase. Two tissues other than small intestine, kidney and spleen, which expressed GM3(NeuGc) in BN and SHR, also expressed the CMP-NeuAc hydroxylase activity, as in the other strains. It was concluded that the key enzyme responsible for the presence of NeuGc in GM3 is a CMP-NeuAc hydroxylase and that mutant rats carry a defect that is specific to intestine. The comparative analysis of the respective contribution of NeuGc and NeuAc to the glycoprotein sialic acids of the small intestine showed that CMP-NeuAc hydroxylase is also responsible for part of the NeuGc present in the glycoproteins. However, the occurrence of 20-30% of NeuGc in the intestinal glycoproteins of the CMP-NeuAc hydroxylase-deficient rats indicated that there is another enzyme providing intestinal glycoproteins with NeuGc and operating under a different genetic control.
在对大鼠小肠黏膜进行细胞分级分离后，在胞质部分发现了一种负责将CMP - NeuAc羟基化为CMP - N - 羟乙酰神经氨酸（CMP - NeuGc）的酶活性。在存在NADPH或NADH的情况下该活性最高，但加入1 mM EDTA后活性降低50%。CMP - NeuAc的Km值为0.6 microM。CMP - NeuAc羟化酶活性与小肠上皮中GM3（NeuGc）表型的表达平行，在仅表达GM3（NeuAc）的突变大鼠BN和SHR中无法检测到该活性。此外，突变体小肠黏膜中存在的唯一形式的CMP - 唾液酸是CMP - NeuAc，而在其他品系中，CMP - NeuGc占天然CMP - 唾液酸的70 - 85%。将野生型和CMP - NeuAc羟化酶缺陷的近交系大鼠进行交配。对F1代和回交后代个体进行GM3（NeuGc）/GM3（NeuAc）表型分型以及小肠中CMP - NeuAc羟化酶活性检测。结果发现，GM3中NeuGc的表达取决于一个常染色体显性基因，并且与CMP - NeuAc羟化酶的活性相关。除小肠外，在BN和SHR中表达GM3（NeuGc）的另外两个组织，肾脏和脾脏，也表达CMP - NeuAc羟化酶活性，与其他品系情况相同。得出的结论是，负责GM3中存在NeuGc的关键酶是CMP - NeuAc羟化酶，并且突变大鼠存在特定于肠道的缺陷。对NeuGc和NeuAc对小肠糖蛋白唾液酸各自贡献的比较分析表明，CMP - NeuAc羟化酶也负责糖蛋白中部分NeuGc的存在。然而，在CMP - NeuAc羟化酶缺陷大鼠的肠道糖蛋白中出现20 - 30%的NeuGc表明，存在另一种为肠道糖蛋白提供NeuGc且受不同遗传控制的酶。
