Büller H A, Rings E H, Pajkrt D, Montgomery R K, Grand R J
Department of Pediatrics, Floating Hospital, New England Medical Center, Boston, Massachusetts.
Gastroenterology. 1990 Mar;98(3):667-75. doi: 10.1016/0016-5085(90)90287-b.
Age-specific changes in glycosylation of rat intestinal lactase-phlorizin hydrolase were analyzed using enzyme immunoprecipitated from microvillus membranes of suckling, weaning, and adult rats, and carbohydrate moieties were examined by lectin affinity binding, metabolic labeling, and neuraminidase treatment. Lectin binding indicated the presence of N-linked and O-linked oligosaccharide chains containing mannose and galactose throughout development. An age-dependent shift in sialic acid and fucose was seen during the period of weaning; no fucose was detectable in lactase-phlorizin hydrolase until after the rats were 20 days of age, whereas sialic acid was reduced in adult lactase-phlorizin hydrolase. The presence of sialic acid in suckling intestines and fucose in adult was confirmed by metabolic labeling with appropriate radioactive precursors. Sodium dodecyl phosphate-polyacrylamide gel electrophoresis analysis of immunoprecipitated lactase-phlorizin hydrolase from the proximal and mid small intestine showed two bands of approximately 220 and 130 kilodaltons in all age groups. In the distal part of the adult small intestine, lactase-phlorizin hydrolase appeared as two bands of similar size to those found in the proximal and mid portions. In contrast, during the suckling and weaning periods, these distal bands were approximately 225 and 135 kilodaltons. [35S]-methionine labeling and fluorography of neonatal intestines confirmed these observations. The size difference between proximal and distal small intestines was virtually eliminated by neuraminidase treatment. These data indicate that the core structure of microvillus membrane lactase-phlorizin hydrolase, consisting of both N-linked and O-linked oligosaccharides, remains constant during development, although terminal sugars shift from predominantly sialic acid during the suckling period to fucose in adulthood. This alteration in glycosylation of the protein occurs in a different pattern from the postweaning decline in lactase specific activity. Consequently, age-dependent changes in glycosylation cannot account for the decrease in lactase-phlorizin hydrolase-specific activity observed during development.
利用从乳鼠、断奶大鼠和成鼠的微绒毛膜中免疫沉淀的酶，分析了大鼠肠道乳糖酶 - 根皮苷水解酶糖基化的年龄特异性变化，并通过凝集素亲和结合、代谢标记和神经氨酸酶处理对碳水化合物部分进行了检测。凝集素结合表明在整个发育过程中存在含有甘露糖和半乳糖的N - 连接和O - 连接寡糖链。在断奶期间观察到唾液酸和岩藻糖的年龄依赖性变化；在大鼠20日龄之前，乳糖酶 - 根皮苷水解酶中未检测到岩藻糖，而成人乳糖酶 - 根皮苷水解酶中的唾液酸减少。用适当的放射性前体进行代谢标记证实了乳鼠肠道中唾液酸的存在和成鼠中岩藻糖的存在。对来自近端和中小肠的免疫沉淀乳糖酶 - 根皮苷水解酶进行十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分析，结果显示在所有年龄组中均有两条带，分子量约为220和130千道尔顿。在成年小肠的远端部分，乳糖酶 - 根皮苷水解酶呈现出两条与近端和中部发现的带大小相似的带。相比之下，在哺乳和断奶期间，这些远端带的分子量约为225和135千道尔顿。新生小肠的[35S] - 甲硫氨酸标记和荧光显影证实了这些观察结果。神经氨酸酶处理几乎消除了近端和远端小肠之间的大小差异。这些数据表明，微绒毛膜乳糖酶 - 根皮苷水解酶的核心结构由N - 连接和O - 连接寡糖组成，在发育过程中保持不变，尽管末端糖从哺乳期间的主要唾液酸转变为成年期的岩藻糖。蛋白质糖基化的这种改变与断奶后乳糖酶比活性的下降模式不同。因此，糖基化的年龄依赖性变化不能解释发育过程中观察到的乳糖酶 - 根皮苷水解酶特异性活性的下降。
