Beaulieu J F, Nichols B, Quaroni A
Division of Biological Sciences, Cornell University, Ithaca, New York 14853.
J Biol Chem. 1989 Nov 25;264(33):20000-11.
Expression and synthesis of sucrase-isomaltase (SI) were studied in human jejunum and in the colon tumor cell lines Caco-2 and HT-29. Twelve monoclonal antibodies produced against the adult human intestinal enzyme were shown to recognize specifically SI by immunoprecipitation of 14C-labeled membrane proteins, analysis of enzyme activities in the immunoprecipitates, and immunoblotting. These antibodies produced markedly different patterns of immunofluorescent staining of the intestinal mucosa. Three of them were specific for the absorptive villus cells, while the other nine also stained the luminal membrane of the proliferative crypt cells, with different intensities which paralleled their ability to recognize SI in immunoblots. Sequential immunoprecipitation of SI solubilized from purified brush borders or entire jejunum with four selected antibodies demonstrated the presence of different forms of the enzyme, expressed by either villus or crypt cells. Two immunologically distinct forms of high mannose precursor (hmP1 and hmP2) were also identified in both jejunal mucosa and colon tumor cells. They were present as monomers and their immunological differences were preserved under various ionic and pH conditions. Pulse-chase studies indicated that, in Caco-2 cells, hmP1 is converted into hmP2 within 30 min of chase, and hmP2 is then processed into the complex-glycosylated precursor destined for the brush border membrane. hmP1 was immunologically related to the mature SI present in crypt cells and lacked the epitopes specific for mature SI expressed by villus cells. These results demonstrated that sucrase-isomaltase is synthesized by both crypt and villus cells, but processing of the cotranslationally glycosylated high mannose precursor is dependent on the state of differentiation of the enterocytes. This may represent a general mechanism for the regulation of expression of differentiated cell products at the post-translational level.
研究了蔗糖酶 - 异麦芽糖酶（SI）在人空肠以及结肠肿瘤细胞系Caco - 2和HT - 29中的表达与合成情况。通过对14C标记的膜蛋白进行免疫沉淀、分析免疫沉淀物中的酶活性以及进行免疫印迹，结果显示，针对成人肠道酶产生的12种单克隆抗体能够特异性识别SI。这些抗体在肠黏膜上产生了明显不同的免疫荧光染色模式。其中三种抗体对吸收性绒毛细胞具有特异性，而另外九种抗体也能对增殖性隐窝细胞的腔面膜进行染色，染色强度不同，这与它们在免疫印迹中识别SI的能力相关。用四种选定的抗体对从纯化的刷状缘或整个空肠中溶解的SI进行连续免疫沉淀，结果表明存在由绒毛或隐窝细胞表达的不同形式的该酶。在空肠黏膜和结肠肿瘤细胞中还鉴定出了两种免疫上不同的高甘露糖前体（hmP1和hmP2）。它们以单体形式存在，并且在各种离子和pH条件下都保留了免疫差异。脉冲追踪研究表明，在Caco - 2细胞中，hmP1在追踪30分钟内转化为hmP2，然后hmP2被加工成运往刷状缘膜的复杂糖基化前体。hmP1在免疫上与隐窝细胞中存在的成熟SI相关，并且缺乏绒毛细胞表达的成熟SI的特异性表位。这些结果表明，蔗糖酶 - 异麦芽糖酶由隐窝细胞和绒毛细胞共同合成，但共翻译糖基化的高甘露糖前体的加工取决于肠上皮细胞的分化状态。这可能代表了在翻译后水平调节分化细胞产物表达的一种普遍机制。
