Irie S, Minguell J J, Tavassoli M
University of Mississippi School of Medicine, Veterans Administration Medical Center, Jackson 39216.
Biochem J. 1989 Apr 15;259(2):427-31. doi: 10.1042/bj2590427.
We have previously shown that the liver endothelium can desialylate the glycoprotein transferrin (Tf). In the present work we provide evidence that asialotransferrin obtained by this means behaves differently on Ricinus communis agglutinin (RCA120) lectin affinity chromatography from asialotransferrin obtained by either neuraminidase treatment or acid hydrolysis. Purified rat transferrin was radiolabelled either with 125I (protein moiety) or with 3H (sialyl residues), and subsequently saturated with iron. It was then passed through an RCA120-agarose column to isolate the fully sialylated component. Sialylated Tf was then desialylated either by incubation with purified rat liver endothelium or, in vitro, by neuraminidase treatment or by acid hydrolysis. The protein was again subjected to RCA120 column chromatography. Although both neuraminidase treatment and acid hydrolysis almost completely desialylated the glycoprotein (as evidenced by near absence of 3H label), the glycoprotein was not retained by the RCA120-agarose column. By contrast, liver endothelium partially desialylated the glycoprotein, but this desialylated fraction was retained by the RCA120-agarose column. These results suggest that desialylation with neuraminidase or acid hydrolysis may be inadequate for functional studies of asialotransferrin.
我们之前已经表明，肝脏内皮细胞能够使糖蛋白转铁蛋白（Tf）去唾液酸化。在本研究中，我们提供的证据表明，通过这种方式获得的去唾液酸转铁蛋白在蓖麻凝集素（RCA120）凝集素亲和色谱上的行为与通过神经氨酸酶处理或酸水解获得的去唾液酸转铁蛋白不同。纯化的大鼠转铁蛋白用125I（蛋白质部分）或3H（唾液酸残基）进行放射性标记，随后用铁饱和。然后将其通过RCA120-琼脂糖柱以分离完全唾液酸化的组分。然后通过与纯化的大鼠肝脏内皮细胞孵育，或在体外通过神经氨酸酶处理或酸水解使唾液酸化的Tf去唾液酸化。该蛋白质再次进行RCA120柱色谱分析。尽管神经氨酸酶处理和酸水解几乎完全使糖蛋白去唾液酸化（3H标记几乎不存在证明了这一点），但该糖蛋白未被RCA120-琼脂糖柱保留。相比之下，肝脏内皮细胞使糖蛋白部分去唾液酸化，但这种去唾液酸化的部分被RCA120-琼脂糖柱保留。这些结果表明，用神经氨酸酶或酸水解进行去唾液酸化可能不足以用于去唾液酸转铁蛋白的功能研究。
