Pohl S, Hoffmann A, Rüdiger A, Nimtz M, Jaeken J, Conradt H S
Gesellschaft für Biotechnologische Forschung, Department of Protein Glycosylation, Braunschweig, Germany.
Glycobiology. 1997 Dec;7(8):1077-84. doi: 10.1093/glycob/7.8.1077.
Human beta-trace protein is a major intrathecally synthesized polypeptide constituent of human cerebrospinal fluid. We have previously shown that this protein is almost quantitatively modified with biantennary complex-type N-linked oligosaccharides which show "brain-type" glycosylation characteristics (Hoffmann,A. et al., J. Neurochem., 63, pp. 2185-2191, 1994). In the present study human beta-trace protein from the cerebrospinal fluid (CSF) of patients with carbohydrate-deficient glycoprotein syndrome (CDGS) due to phosphomannomutase (PMM) deficiency and N-acetyl-glucosaminyltransferase II (GlcNAc-T II) deficiency as well as from control individuals was studied by Western blot analysis. The protein from pooled CSFs was purified by immunoaffinity chromatography. The protein from the five patients with CDGS PMM deficiency showed three protein bands upon SDS-PAGE analysis corresponding to the di-, mono-, and unglycosylated polypeptide forms. Carbohydrate structural analysis of the enzymatically liberated N-glycans was performed applying mapping by HPAEC-PAD, methylation analysis as well as MALD/TOF-MS. Essentially identical oligosaccharide structures were detected in beta-TP from type I patients and control adult pooled CSF. The beta-trace protein from two patients with GlcNAc-T II deficiency showed a single di-N-glycosylated protein band with a significantly lower molecular weight than the di-glycosylated polypeptide from control patients and the beta-trace protein from pooled adult CSF. Beta-TP from GlcNAc-T II deficiency patients shared only three oligosaccharides out of the 13 observed in beta-TP from controls or patients with PMM deficiency. The major oligosaccharide structures of the glycoprotein from patients with GlcNAc-T II deficiency were found to be monoantennary asialo- or monosialylated lactosamine-type chains with proximal fucose and bisecting GlcNAc.
人β-微球蛋白是人类脑脊液中主要的鞘内合成多肽成分。我们之前已经表明，该蛋白几乎被具有“脑型”糖基化特征的双天线复合型N-连接寡糖定量修饰（霍夫曼，A.等人，《神经化学杂志》，63卷，第2185 - 2191页，1994年）。在本研究中，通过蛋白质印迹分析对来自因磷酸甘露糖变位酶（PMM）缺乏和N-乙酰葡糖胺基转移酶II（GlcNAc-T II）缺乏导致的碳水化合物缺乏糖蛋白综合征（CDGS）患者以及对照个体的脑脊液（CSF）中的人β-微球蛋白进行了研究。通过免疫亲和色谱法纯化来自合并脑脊液的蛋白质。对来自五名PMM缺乏型CDGS患者的蛋白质进行SDS-PAGE分析时显示出三条蛋白带，分别对应双糖基化、单糖基化和未糖基化的多肽形式。对酶解释放的N-聚糖进行碳水化合物结构分析，采用高效阴离子交换色谱-脉冲安培检测法（HPAEC-PAD）进行图谱分析、甲基化分析以及基质辅助激光解吸/电离飞行时间质谱（MALD/TOF-MS）。在I型患者的β-TP和对照成人合并脑脊液中检测到基本相同的寡糖结构。两名GlcNAc-T II缺乏患者的β-微球蛋白显示出一条单一的双N-糖基化蛋白带，其分子量明显低于对照患者的双糖基化多肽以及成人合并脑脊液中的β-微球蛋白。与对照或PMM缺乏患者的β-TP中观察到的13种寡糖相比，GlcNAc-T II缺乏患者的β-TP仅共有三种寡糖。发现GlcNAc-T II缺乏患者糖蛋白的主要寡糖结构为单天线去唾液酸或单唾液酸化乳糖胺型链，带有近端岩藻糖和平分型GlcNAc。
