Naim H Y, Sterchi E E, Lentze M J
Department of Gastroenterology, Children's Hospital of the University of Berne, Switzerland.
J Biol Chem. 1988 Dec 25;263(36):19709-17.
Maltase-glucoamylase (MGA) was immunoprecipitated from detergent extracts of brush border membranes of the human small intestinal mucosa. Electrophoretic analysis of the precipitates under denaturing conditions revealed a single polypeptide of Mr = 335,000 in the presence or absence of reducing agents. Cross-linking of brush border membranes with the homobifunctional reagent dithiobis(succinimidylpropionate) did not result in considerable changes in the electrophoretic pattern of MGA. In contrast, aminopeptidase N, used in these studies as a control glycoprotein of the brush border membrane revealed dimeric structures of its single subunit in the presence of dithiobis(succinimidylpropionate). These data suggest that MGA is expressed in the human small intestinal brush border as a monomeric polypeptide. The biosynthesis of MGA was studied by pulse-labeling of human intestinal biopsy specimens or mucosal explants in organ culture. Continuous labeling with [35S]methionine for 30 min revealed a single polypeptide high mannose precursor of Mr = 285,000 (MGAh) which matures after 4 h of labeling to the Mr = 335,000 as judged by the susceptibility of these two forms to endo-beta-N-acetylglucosaminidase H. Owing to the absence of pancreatic secretions in the culture medium and the isolation of an identical species from nonlabeled mucosa, this result indicates that the Mr = 335,000 does not undergo an in situ extracellular cleavage by intraluminal proteases. Further, biosynthetically labeled, intracellularly cleaved polypeptides corresponding to the high mannose precursor or mature forms of MGA were not detected. The mature form of MGA (MGAm) bears in addition to N-linked glycans also O-glycosidically linked oligosaccharides. In fact, endo-beta-N-acetylglucosaminidase F/glycopeptidase F treatment of MGAm followed by chemical deglycosylation with trifluoromethanesulfonic acid revealed approximately 35,000 daltons of O-linked sugars. Furthermore, MGAm as well as its N-linked sugars-depleted form bound to Helix pomatia lectin which has specificity toward Gal-GalNAc structures. In addition, the data were suggestive of a post-translational O-glycosylation of the molecule since (i) the high mannose precursor of MGA did not bind to H. pomatia lectin and (ii) its endo-beta-N-acetylglucosaminidase H or endo-beta-N-acetylglucosaminidase F/glycopeptidase F form displayed an apparent molecular weight similar to that obtained upon endo-beta-N-acetylglucosaminidase F/glycopeptidase F/trifluoromethanesulfonic acid deglycosylation. Finally, pulse-chase experiments revealed a relatively slow rate of post-translational processing of MGA in comparison to aminopeptidase N.(ABSTRACT TRUNCATED AT 400 WORDS)
麦芽糖酶 - 葡糖淀粉酶（MGA）是从人小肠黏膜刷状缘膜的去污剂提取物中免疫沉淀得到的。在变性条件下对沉淀物进行电泳分析，发现在有或没有还原剂的情况下，均存在一条分子量为335,000的单一多肽。用同双功能试剂二硫代双（琥珀酰亚胺丙酸酯）对刷状缘膜进行交联，并未导致MGA电泳图谱发生显著变化。相比之下，在这些研究中用作刷状缘膜对照糖蛋白的氨肽酶N，在二硫代双（琥珀酰亚胺丙酸酯）存在的情况下显示出其单个亚基的二聚体结构。这些数据表明，MGA在人小肠刷状缘中以单体多肽形式表达。通过对人肠道活检标本或器官培养中的黏膜外植体进行脉冲标记来研究MGA的生物合成。用[35S]甲硫氨酸连续标记30分钟，揭示了一条分子量为285,000的单一多肽高甘露糖前体（MGAh），根据这两种形式对内切β - N - 乙酰葡糖胺酶H的敏感性判断，标记4小时后其成熟为分子量335,000。由于培养基中没有胰腺分泌物，并且从未标记的黏膜中分离出相同的物种，该结果表明分子量335,000的形式不会被腔内蛋白酶原位细胞外切割。此外，未检测到与MGA的高甘露糖前体或成熟形式相对应的生物合成标记的细胞内切割多肽。MGA的成熟形式（MGAm）除了N - 连接聚糖外还带有O - 糖苷连接的寡糖。事实上，用内切β - N - 乙酰葡糖胺酶F/糖肽酶F处理MGAm，然后用三氟甲磺酸进行化学去糖基化，揭示了约35,000道尔顿的O - 连接糖。此外，MGAm及其N - 连接糖耗尽形式与对Gal - GalNAc结构具有特异性的苹果螺凝集素结合。此外，这些数据表明该分子存在翻译后O - 糖基化，因为（i）MGA的高甘露糖前体不与苹果螺凝集素结合，并且（ii）其内切β - N - 乙酰葡糖胺酶H或内切β - N - 乙酰葡糖胺酶F/糖肽酶F形式显示出的表观分子量与内切β - N - 乙酰葡糖胺酶F/糖肽酶F/三氟甲磺酸去糖基化后得到的相似。最后，脉冲追踪实验表明，与氨肽酶N相比，MGA的翻译后加工速率相对较慢。（摘要截断于400字）
