Naim H Y, Lentze M J
Institute of Microbiology, Heinrich-Heine University of Düsseldorf, Germany.
J Biol Chem. 1992 Dec 15;267(35):25494-504.
Lactase-phlorizin hydrolase (LPH) is an integral intestinal brush border membrane glycoprotein responsible for the hydrolysis of lactose, the primary carbohydrate in mammalian milk. To assess the role of N- and O-glycosylation on the function of LPH, lectin-binding experiments combined with enzymatic and chemical deglycosylation of purified LPH molecules were performed. These investigations provided evidence for the existence of two forms of brush border LPH, an N-glycosylated molecule (LPHN) and an N- and O-glycosylated molecule (LPHN/O). These two forms could be discriminated on the basis of (i) their binding capacity to Helix pomatia lectin, which has high specificity toward O-linked oligosaccharides, and (ii) their deglycosylation patterns with endo-beta-N-acetylglucosaminidase F/GF, O-glycanase, and trifluoromethanesulfonic acid. Interestingly, both forms have identical Km values (approximately 14 mM) when assayed with lactose, but hydrolyze this substrate at different rates. Thus, the N- and O-glycosylated form exhibits almost a 4-fold higher Vmax than that of the N-glycosylated enzyme (3.28 nM/min versus 0.90 nM/min) and is therefore enzymatically more active than the latter. Sequential affinity chromatography of glycopeptides derived from [3H]mannose-labeled LPHN and LPHN/O on lectin columns revealed similar patterns of N-linked glycosylation of both forms indicating that the presence of O-linked oligosaccharides did not affect or alter the processing of N-linked oligosaccharides. O-Linked glycosylation of LPH appears to occur in the Golgi apparatus, since the earliest detectable forms of LPH, the mannose-rich precursor (pro-LPH) is not O-glycosylated. In view of the fact that differentiation of intestinal crypt cells to mature epithelial cells is accompanied by significant phenotypical, morphological, and structural alterations, including changes in the levels of several Golgi glycosyl, -sialyl, galactosyl-, and N-acetylgalactosaminyltransferases, and since O-glycosylation is a Golgi event, we suggest that the generation of LPHN and LPHN/O is strongly linked to differentiation of intestinal cells. Finally, the variations in the enzymatic activity of the two forms propose a role for O-glycosylation in posttranslational regulation of LPH activity.
乳糖酶 - 根皮苷水解酶（LPH）是一种整合于肠刷状缘膜的糖蛋白，负责水解乳糖，乳糖是哺乳动物乳汁中的主要碳水化合物。为了评估N - 糖基化和O - 糖基化对LPH功能的作用，我们进行了凝集素结合实验，并结合了对纯化的LPH分子进行酶促和化学去糖基化处理。这些研究为刷状缘LPH存在两种形式提供了证据，一种是N - 糖基化分子（LPHN），另一种是N - 和O - 糖基化分子（LPHN/O）。这两种形式可以根据以下两点进行区分：（i）它们与对O - 连接寡糖具有高特异性的苹果螺凝集素的结合能力；（ii）它们用内切 - β - N - 乙酰葡糖胺酶F/GF、O - 聚糖酶和三氟甲磺酸进行去糖基化的模式。有趣的是，当用乳糖进行测定时，这两种形式具有相同的Km值（约14 mM），但水解该底物的速率不同。因此，N - 和O - 糖基化形式的Vmax比N - 糖基化酶的Vmax高近4倍（3.28 nM/分钟对0.90 nM/分钟），因此其酶活性比后者更高。对源自[3H]甘露糖标记的LPHN和LPHN/O的糖肽在凝集素柱上进行连续亲和层析，结果显示两种形式的N - 连接糖基化模式相似，这表明O - 连接寡糖的存在不会影响或改变N - 连接寡糖的加工过程。LPH的O - 连接糖基化似乎发生在高尔基体中，因为最早可检测到的LPH形式，即富含甘露糖的前体（前体LPH）没有O - 糖基化。鉴于肠隐窝细胞分化为成熟上皮细胞伴随着显著的表型、形态和结构改变，包括几种高尔基体糖基转移酶、唾液酸转移酶、半乳糖基转移酶和N - 乙酰半乳糖胺基转移酶水平的变化，并且由于O - 糖基化是高尔基体的事件，我们认为LPHN和LPHN/O的产生与肠细胞的分化密切相关。最后，这两种形式的酶活性变化表明O - 糖基化在LPH活性的翻译后调节中起作用。
