PATHOPHYSIOLOGY OF CARBOHYDRATE-DEFICIET GLYCOPROTEIN SYNDROME

碳水化合物缺乏糖蛋白综合征（CDGS）是一种常染色体隐性遗传病，其特征是血清中出现有糖链缺陷的天冬酰胺- n -糖蛋白。CDGS I型的基本缺陷在1997年发现于磷酸腺苷转氨酶2基因。CDGS异常糖蛋白仅在血清糖蛋白中发现。为了鉴定细胞内糖基化异常，我们研究了来自3名日本CDGS i型患者的培养成纤维细胞。在这个项目中，我们研究了在培养成纤维细胞中合成的溶酶体酶的糖链。我们未能在培养基和细胞中鉴定出几种溶酶体酶中有糖链缺陷的异常部分。然后我们研究了β-己糖氨酸酶的细胞内加工，发现55-kDa成熟形式的α链的蛋白质和mRNA水平。提示CDG成纤维细胞内糖蛋白（包括b-己糖氨基酶）的加工可能发生改变。在培养的成纤维细胞中，我们研究了使用标记甘露糖形成全尺寸脂联寡糖（LLO）的途径，发现患者成纤维细胞需要6倍浓度的甘露糖来形成暗尺寸的LLO。筛选了多种恢复CDGS I型成纤维细胞LLO合成的试剂，发现添加醇对恢复全尺寸LLO的形成最有效。这些现象可能是由于添加多醇激活了多醇磷酸甘糖合成酶，因为如我们之前的论文所述，多醇和LLO水平在CDGS I型成纤维细胞中降低（J Biol Chem 272, 6868-6875, 1997）。

Carbohydrate-deficient glycoprotein syndrome (CDGS) is an autosomal recessive disorders characterized by appearance of serum asparagine-N-linked glycoprotein with defective sugar chain. The basic defect of CDGS type I has been identified in the phosphomannomutase 2 gene in 1997. Abnormal glycoproteins in CDGS have been identified only in serum glycoproteins. To identify intracellular glycosylation abnormalities, we have been studied in cultured fibroblasts from three Japanese patients with CDGS type I. In this project we have studied sugar chains of lysosomal enzymes synthesized in cultured fibroblasts. We have failed to identify the abnormal fractions with defective sugar chains in several lysosomal enzymes in media and in cells. Then we have studied intrecellular processing of β-hexosaminidase and found that protein and mRNA levels of a 55-kDa mature form of the α chain. It was suggested that intracellular processing of glycoproteins including b-hexosaminidase in CDG fibroblasts might be altered. In cultured fibroblasts we have studied a pathway to form full-sized lipid linked oligosaccharide (LLO) using labeled mannose and found patient fibroblasts required six times higher concentrations of mannose to form dull-sized LLO. Various reagents restoring the LLO syn thesis in CDGS type I fibroblasts were screened and the addition of dolichol was found to be most effective to restore the formation of the full-sized LLO. These phenomena may be due to activation of dolichol phosphomannose synthase by addition of dolichol, since the level of dolichol and LLO are decreased in CDGS type I fibroblasts as described in our previous paper (J Biol Chem 272, 6868-6875, 1997).