Vine D T, McGovern M M, Schuchman E H, Haskins M E, Desnick R J
J Clin Invest. 1982 Feb;69(2):294-302. doi: 10.1172/jci110452.
The molecular pathology of the deficient arylsulfatase B activity in feline mucopolysaccharidosis (MPS) VI was investigated. Compared with the highly purified normal feline hepatic enzyme, the purified MPS VI residual activity had a 100-fold higher Michaelis constant (K(m)), an altered electrophoretic mobility, half the apparent native molecular weight, and markedly decreased thermo-, cryo-, and pH stabilities. Molecular weight and alkylation studies were consistent with the normal enzyme being a homodimer and the residual MPS VI enzyme a monomer. When incubated with various sulfhydryl reagents, the residual specific activity was enhanced several-fold, whereas the activity of the purified normal enzyme was un-affected or slightly inhibited. In the presence of dithiothreitol (DTT) and cysteamine, a lysosomotropic aminothiol, the residual activity had an electrophoretic mobility and native molecular weight similar to those of the normal feline enzyme. These findings suggested that the monomeric residual enzyme was dimerized in the presence of thiol-reducing agents. To determine if thiol-induced subunit association could therapeutically increase the residual activity and degrade the accumulated dermatan sulfate, in vitro and in vivo experiments were undertaken. When 2 mM DTT or cysteamine was incubated with heparinized whole blood from an MPS VI cat, the leukocyte residual arylsulfatase B activity increased 11- and 20-fold, respectively, and the accumulated dermatan sulfate was degraded in the presence of both thiol reagents. Intravenous administration of DTT (50 mg/kg) effected an immediate, but transient, increase in leukocyte residual activity; however, the substrate levels were not significantly decreased. In contrast, intravenous administration of cysteamine (15 mg/kg) increased leukocyte residual activity more than sixfold 30 min postinfusion; concomitantly, the leukocyte substrate was decreased to 35% of the initial level immediately after infusion and to about 45% of preinfusion values during the 120-min period studied. These results suggest that the defective residual activity in feline MPS VI can be therapeutically manipulated by thiol-induced subunit association. Furthermore, this animal analog provides a prototype for the investigation of human inborn errors of metabolism resulting from enzymatic defects that might be amenable to enzyme manipulation therapy.
对猫黏多糖贮积症（MPS）VI中芳基硫酸酯酶B活性缺乏的分子病理学进行了研究。与高度纯化的正常猫肝酶相比，纯化的MPS VI残余活性的米氏常数（K(m)）高100倍，电泳迁移率改变，表观天然分子量减半，热稳定性、冷冻稳定性和pH稳定性显著降低。分子量和烷基化研究表明，正常酶为同二聚体，而残余的MPS VI酶为单体。当与各种巯基试剂一起孵育时，残余比活性提高了几倍，而纯化的正常酶的活性未受影响或略有抑制。在二硫苏糖醇（DTT）和溶酶体促渗氨基硫醇半胱胺存在的情况下，残余活性的电泳迁移率和天然分子量与正常猫酶相似。这些发现表明，单体残余酶在巯基还原剂存在的情况下会二聚化。为了确定巯基诱导的亚基缔合是否能通过治疗增加残余活性并降解积累的硫酸皮肤素，进行了体外和体内实验。当将2 mM DTT或半胱胺与一只MPS VI猫的肝素化全血一起孵育时，白细胞中芳基硫酸酯酶B的残余活性分别增加了11倍和20倍，并且在两种巯基试剂存在的情况下，积累的硫酸皮肤素都被降解。静脉注射DTT（50 mg/kg）使白细胞残余活性立即但短暂增加；然而，底物水平没有显著降低。相比之下，静脉注射半胱胺（15 mg/kg）在输注后30分钟使白细胞残余活性增加了六倍多；同时，白细胞底物在输注后立即降至初始水平的35%，在研究的120分钟内降至输注前值的约45%。这些结果表明，猫MPS VI中缺陷的残余活性可以通过巯基诱导的亚基缔合进行治疗性调控。此外，这种动物模型为研究可能适用于酶调控治疗的酶缺陷导致人类先天性代谢错误提供了一个原型。
