龐貝氏症患者及帶原者酵素活性差異性之原因

Abstract

龐貝氏症(Pompe’s disease)是一種肝醣儲積症(Glycogen storage disease, GSD)，又稱為肝醣儲積症第二型(GSD II)。是由於先天缺乏溶小體酵素-酸性麥芽糖酶(Acid Maltase，又稱acid α-glucosidase, GAA)，使得進入溶小體的肝醣無法被分解而持續堆積，進而影響到細胞的功能。臨床上依發病年齡分為嬰兒型、青少年型及成人型。龐貝氏症確定診斷的方法為測定皮膚纖維芽細胞或血液中GAA酵素活性。通常患者的酵素活性很低(<5%)，而帶原者的活性會介於正常人和患者之間。臨床上發現，部分帶原者的酵素活性偏低，約只有正常人5 ~ 10%，可能會增加檢測困難度。因此，我們認為可能存在某些因數會影響酸性麥芽糖酶的表現，如基因多型性或其他機轉，影響到酸性麥芽糖酶的蛋白質穩定性。在本實驗室近年來的研究，發現熱休克蛋白Hsp27對於突變蛋白GTP cyclohydrolase I的dominant-negative effect會有影響。Hsp27是一種small heat shock蛋白，和蛋白質摺疊、細胞骨架之穩定性，以及細胞凋亡有關。突變酸性麥芽糖酶可能在某些情況下，如Hsp27存在下，其蛋白質穩定性會有所改變，也可能會受到其他因數的影響，導致細胞酵素活性降到如此的低。 本研究進行的方法(1)分析所有個案的GAA酵素活性、mRNA與蛋白質表現量。(2)藉著基因序列分析找尋是否有基因多型性的存在，與突變蛋白GAA酵素活性具有關聯性。(3)以表現Hsp27的質體轉染龐貝氏症患者或是帶原者的皮膚纖維芽細胞，瞭解Hsp27對於突變蛋白GAA酵素活性是否具有調控的能力。 本實驗結果證實GAA酵素活性表現與蛋白質表現量有正相關性。也證實GAA突變蛋白的存在。RNA實驗結果顯示，帶原者的GAA蛋白表現量和其mRNA間並無絕對相關性存在。以蛋白結構推測，GAA突變蛋白與正常蛋白具有差異性。雖然基因多型性可能會影響GAA突變蛋白的穩定性，在基因序列分析中，也發現許多序列多型性，並形成三個區塊，但並無特殊的基因多型性與酵素活性間有關聯性。Hsp27 -S3D實驗中發現Hsp27-S3D會降低患者與帶原者的酵素活性。 影響GAA酵素活性的因數，還是有其他基因多型性，或其他分子伴隨者調控的可能。本研究主要目的希望能應用於未來的臨床診斷及治療，在臨床上提供更精確診斷，治療上能有其他新藥物。我們的研究指出蛋白質的穩定性是影響酵素活性的重要因數。可能在基因的其他位置，例如是intron仍存在著有影響的序列多型性。或是細胞內的其他熱休克蛋白會影響GAA蛋白的穩定性，這些都還待進一步之研究。

Pompe’s disease is a lysosomal storage disease involving the storage of glycogen. It is used to be called type II glycogenosis. The etiology of this disease is the deficiency of acid maltase (or acid alpha-glucosidase, GAA). The deficiency of GAA leads to a progressive storage of glycogen in the lysosome, which affects the function of the cells. According to the onset clinically, there are three subtypes – infantile, juvenile, and adult type. The diagnosis of Pompe’s disease depends on the measurement of GAA activity in either skin fibroblast or in peripheral blood mononuclear cells. Theoretically, GAA activities in obligatory carriers should be 50% of normal. However, we frequently met carriers with GAA activities as low as 5~10% of normal. The current hypothesis is the presence of some important factors, ex. polymorphism or some other mechanisms that will alter the stability of GAA. In our previous studies, Hsp27 can alter the stability of the GTP-cyclohydrolase I protein. Hsp27 is a member of the small HSP that is involved in protein folding, stability of the cytoskeleton, and cell apoptosis. It is possible that the stability of GAA will be changed under certain circumferences, and then causes the excessive low GAA activity in some individuals. In this study, we are going to perform the assays: (1) the GAA activity, mRNA, and protein expression; (2) the gene polymorphism analysis; (3) to overexpress Hsp27 in skin fibroblasts from either Pompe’s patients or the carriers. We will observe the changes of GAA protein by the western blot analysis, and also GAA activities by enzyme assays, to see if these parameters will be changed by the expression of Hsp27. Special interests will be on Pompe’s carriers with excessive low GAA activities. In the study, there is a positive correlation between the GAA activity and the protein expression. From the RNA study, there is no correlation between the expression of the GAA protein and of the mRNA. From the structure analysis, the polymorphism (V816I) may influence the stability of mutant GAA protein. We also find some polymorphisms and form three blocks in the gene polymorphism study. But, no relation between the polymorphisms and the GAA activity. The data of the Hsp27 study shows Hsp-S3D will decrease the GAA activity, either in the patients or carriers. There still exists the possibility of some other factors that will influence the GAA activity, ex. Polymorphisms, or heat shock proteins. This study will contribute significantly the knowledge of the pathogenesis of disease, the mechanism of changes in protein stability, the diagnostic technology, and also deeply to the future treatment of the diseases, including those with similar molecular mechanisms. Our data showed the stability of GAA protein is one important factor to the GAA activity. Maybe some polymorphisms exists in some position of the GAA gene, ex. Intron, that will play some roles. Or, the other Hsp in the cells may affect the stability of the GAA protein. Those all will be needed further study.