Ritz Valentina, Alfalah Marwan, Zimmer Klaus-Peter, Schmitz Jacques, Jacob Ralf, Naim Hassan Y
Institute of Physiological Chemistry, School of Veterinary Medicine Hannover, Germany.
Gastroenterology. 2003 Dec;125(6):1678-85. doi: 10.1053/j.gastro.2003.09.022.
BACKGROUND & AIMS: Congenital sucrase-isomaltase deficiency (CSID) is an autosomal recessive human disorder characterized by reduced activities of the brush border enzyme sucrase-isomaltase (SI). Here, we elucidate the pathogenesis of a new variant of CSID at the cellular and molecular level.
Assessment of the CSID phenotype was achieved by enzymatic activity measurements, biosynthetic labeling of intestinal biopsy specimens, immunoprecipitation of SI, and immunoelectronmicroscopy. The putative mutation was identified by sequencing of the SI cDNA isolated by RT-PCR from intestinal biopsy samples. The function of the mutation was verified by immunoprecipitation and confocal microscopy of transiently transfected cells.
Biosynthetic labeling and immunoelectron microscopy reveal a predominant localization of SI in the endoplasmic reticulum (ER) similar to phenotype I of CSID. Unlike phenotype I, however, a partial conversion of SI to a complex glycosylated mature form takes place. The SI cDNA in this phenotype revealed 3 mutations, 2 of which, Val to Phe at residue 15 and Ala to Thr at residue 231, had no effect on the structure or function of SI. By contrast, the third mutation resulted in an exchange of leucine by proline at position 620 (L620P) and revealed in transfected COS cells structural features and subcellular localization similar to the phenotype identified in the patient's enterocytes.
This is the first identification at the molecular and subcellular levels of a novel variant of CSID in which SI accumulates predominantly in the ER, and a minor proportion is further processed and transported to the apical membrane of enterocytes.
先天性蔗糖酶 - 异麦芽糖酶缺乏症（CSID）是一种常染色体隐性人类疾病，其特征为刷状缘酶蔗糖酶 - 异麦芽糖酶（SI）的活性降低。在此，我们在细胞和分子水平阐明了一种新的CSID变体的发病机制。
通过酶活性测量、肠道活检标本的生物合成标记、SI的免疫沉淀以及免疫电子显微镜评估CSID表型。通过对从肠道活检样本中经逆转录 - 聚合酶链反应（RT - PCR）分离的SI cDNA进行测序来鉴定推定的突变。通过对瞬时转染细胞进行免疫沉淀和共聚焦显微镜检查来验证突变的功能。
生物合成标记和免疫电子显微镜显示SI主要定位于内质网（ER），类似于CSID的I型表型。然而，与I型表型不同的是，SI会部分转化为复杂糖基化的成熟形式。该表型的SI cDNA显示有3个突变，其中2个突变，即第15位残基的缬氨酸变为苯丙氨酸以及第231位残基的丙氨酸变为苏氨酸，对SI的结构或功能没有影响。相比之下，第三个突变导致第620位的亮氨酸被脯氨酸替换（L620P），并且在转染的COS细胞中显示出与在患者肠细胞中鉴定出的表型相似的结构特征和亚细胞定位。
这是首次在分子和亚细胞水平鉴定出一种新型的CSID变体，其中SI主要在内质网中积累，一小部分进一步加工并转运至肠细胞的顶端膜。
