D'Agrosa R M, Hubbes M, Zhang S, Shankaran R, Callahan J W
Research Institute, Hospital for Sick Children, Ontario, Canada.
Biochem J. 1992 Aug 1;285 ( Pt 3)(Pt 3):833-8. doi: 10.1042/bj2850833.
Lysosomal beta-galactosidase (beta-Gal) occurs either alone in monomeric and dimeric forms, or in a high-M(r) complex with at least two additional proteins. One is neuraminidase and the second is the protective protein, which has also been shown to possess carboxypeptidase activity. beta-Gal activity is deficient in GM1-gangliosidosis as a primary defect, and is secondarily affected in galactosialidosis (GS), where the primary defect is the absence of protective protein activity. Fibroblasts from three patients with GM1-gangliosidosis, type 1, showed markedly reduced amounts of beta-Gal cross-reacting material (CRM), and a fourth appeared to have normal levels. A patient with type 2 GM1-gangliosidosis was also found to be CRM-normal. These findings demonstrate that patients with GM1-gangliosidosis type 1 are heterogeneous with respect to the level of residual beta-Gal protein. Fibroblasts from four patients with GS were strongly CRM-positive with an anti-beta-Gal antibody, as was a sample of brain from one of these patients, suggesting that the loss of beta-Gal activity is linked to a subtler change in the primary structure of the enzyme than has been previously thought. While three GS cell lines displayed reduced carboxypeptidase activity (to 32-42% of the control), one cell line was completely devoid of activity, demonstrating that while carboxypeptidase activity is a property of the protective protein this action is distinct and separate from its protective role. On direct immunoprecipitation with anti-beta-Gal antibody, a portion of the total carboxypeptidase activity co-precipitated with beta-Gal from extracts of normal and GM1-gangliosidosis cells, consistent with the presence of the complex in these cells. However, no carboxypeptidase activity was precipitable with this antibody from GS fibroblasts, suggesting the absence of complex from these cells. To examine this further, the various forms of beta-Gal were resolved by h.p.l.c. molecular-sieve chromatography. Three forms of beta-Gal activity were resolved in normal cells: a complex, a dimer and a monomer. Residual beta-Gal activity of GS cells resolved into two of these forms, the complex and the monomer. In normal and GM1-gangliosidosis cells a portion of the total carboxypeptidase activity co-chromatographed with the complex while the bulk of the activity occurred in a single 36,000-M(r) peak. Only the low-M(r) carboxypeptidase activity was detected in GS cells. This confirms our results on immunoprecipitation indicating that portions of the beta-Gal and the carboxypeptidase activities exist outside the complex in normal, GM1-gangliosidosis and GS cells. In summary, the loss of protective protein function from GS cells results in disproportionate loss of the dimeric and monomeric forms of beta-Gal activity, but does not result in the complete degradation of the protein.
溶酶体β-半乳糖苷酶（β-Gal）以单体和二聚体形式单独存在，或与至少两种其他蛋白质形成高分子量复合物。一种是神经氨酸酶，另一种是保护蛋白，该蛋白也已被证明具有羧肽酶活性。β-Gal活性在GM1神经节苷脂病中作为主要缺陷而缺乏，在半乳糖唾液酸贮积症（GS）中则受到继发性影响，后者的主要缺陷是缺乏保护蛋白活性。三名1型GM1神经节苷脂病患者的成纤维细胞显示β-Gal交叉反应物质（CRM）的量明显减少，第四名患者的CRM水平似乎正常。一名2型GM1神经节苷脂病患者也被发现CRM正常。这些发现表明，1型GM1神经节苷脂病患者在残余β-Gal蛋白水平上存在异质性。四名GS患者的成纤维细胞与抗β-Gal抗体呈强CRM阳性，其中一名患者的脑样本也是如此，这表明β-Gal活性的丧失与该酶一级结构中比先前认为的更细微的变化有关。虽然三个GS细胞系的羧肽酶活性降低（至对照的32 - 42%），但一个细胞系完全没有活性，这表明虽然羧肽酶活性是保护蛋白的特性，但这种作用与其保护作用是不同且分开的。用抗β-Gal抗体直接免疫沉淀时，正常和GM1神经节苷脂病细胞提取物中的一部分总羧肽酶活性与β-Gal共沉淀，这与这些细胞中存在复合物一致。然而，用该抗体从GS成纤维细胞中无法沉淀出羧肽酶活性，表明这些细胞中不存在复合物。为了进一步研究这一点，通过高效液相色谱分子筛色谱法分离了β-Gal的各种形式。在正常细胞中分离出三种β-Gal活性形式：一种复合物、一种二聚体和一种单体。GS细胞的残余β-Gal活性解析为其中两种形式，即复合物和单体。在正常和GM1神经节苷脂病细胞中，一部分总羧肽酶活性与复合物共色谱，而大部分活性出现在一个单一的36,000分子量峰中。在GS细胞中仅检测到低分子量的羧肽酶活性。这证实了我们免疫沉淀的结果，表明在正常、GM1神经节苷脂病和GS细胞中，β-Gal和羧肽酶活性的一部分存在于复合物之外。总之，GS细胞中保护蛋白功能的丧失导致β-Gal活性的二聚体和单体形式不成比例地丧失，但不会导致蛋白质完全降解。
