Analysis of the Biogenesis of Heparan Sulfate Acetyl-CoA:α-Glucosaminide N-Acetyltransferase Provides Insights into the Mechanism Underlying Its Complete Deficiency in Mucopolysaccharidosis IIIC
Analysis of the Biogenesis of Heparan Sulfate Acetyl-CoA:α-Glucosaminide N-Acetyltransferase Provides Insights into the Mechanism Underlying Its Complete Deficiency in Mucopolysaccharidosis IIIC
复制标题
DOI：
10.1074/jbc.m110.141150
复制
发表时间：
2010-10-08
影响因子：
4.8
通讯作者：
Pshezhetsky, Alexey V.
中科院分区：
文献类型：
--
作者：
Durand, Stephanie;Feldhammer, Matthew;Pshezhetsky, Alexey V.
硫酸乙酰肝素乙酰辅酶A：α-氨基葡萄糖苷N-乙酰转移酶（HGSNAT）催化溶酶体中硫酸乙酰肝素的跨膜乙酰化，这是其进一步催化所需的。人类HGSNAT的遗传性缺乏导致硫酸乙酰肝素的溶酶体储存，并导致严重的神经退行性疾病粘多糖样变性IIIC（MPS IIIC）。以前我们克隆了HGSNAT基因，鉴定了MPS IIIC患者的分子缺陷，发现所有错义突变都阻止了酶的正常折叠和运输。在目前的研究中，我们发现HGSNAT是作为一种无催化活性的77-kDa前体合成的，它通过一种接头蛋白介导的途径转运到溶酶体，该途径涉及其C-端保守的基于酪氨酸和二亮氨酸的溶酶体靶向信号。在N-末端胞质环中，基于二亮氨酸的信号起作用。在溶酶体中，前体被切割成29-kDa N-末端α链和48-kDa C-末端β链，并组装成类似于440-kDa寡聚体的活性。亚基通过酶的溶酶体腔环中至少两个半胱氨酸残基（Cys（123）和Cys（434））之间的二硫键保持在一起。我们推测，蛋白水解切割允许活性位点中的亲核残基His（269）接近细胞质中的底物乙酰辅酶A，以进一步将乙酰基转移到硫酸乙酰肝素上的末端葡糖胺。总之，我们的研究结果确定了溶酶体内寡聚化和蛋白水解裂解作为HGSNAT功能激活的两个关键步骤。
Heparan sulfate acetyl-CoA:alpha-glucosaminide N-acetyltransferase (HGSNAT) catalyzes the transmembrane acetylation of heparan sulfate in lysosomes required for its further catabolism. Inherited deficiency of HGSNAT in humans results in lysosomal storage of heparan sulfate and causes the severe neurodegenerative disease, mucopolysaccharidosis IIIC (MPS IIIC). Previously we have cloned the HGSNAT gene, identified molecular defects in MPS IIIC patients, and found that all missense mutations prevented normal folding and trafficking of the enzyme. Therefore characterization of HGSNAT biogenesis and intracellular trafficking became of central importance for understanding the molecular mechanism underlying the disease and developing future therapies.In the current study we show that HGSNAT is synthesized as a catalytically inactive 77-kDa precursor that is transported to the lysosomes via an adaptor protein-mediated pathway that involves conserved tyrosine-and dileucine-based lysosomal targeting signals in its C-terminal cytoplasmic domain with a contribution from a dileucine-based signal in the N-terminal cytoplasmic loop. In the lysosome, the precursor is cleaved into a 29-kDa N-terminal alpha-chain and a 48-kDa C-terminal beta-chain, and assembled into active similar to 440-kDa oligomers. The subunits are held together by disulfide bonds between at least two cysteine residues (Cys(123) and Cys(434)) in the lysosomal luminal loops of the enzyme. We speculate that proteolytic cleavage allows the nucleophile residue, His(269), in the active site to access the substrate acetyl-CoA in the cytoplasm, for further transfer of the acetyl group to the terminal glucosamine on heparan sulfate. Altogether our results identify intralysosomal oligomerization and proteolytic cleavage as two steps crucial for functional activation of HGSNAT.