The role of strand 1 of the C β-sheet in the structure and function of α1-antitrypsin

Serpins inhibit cognate serine proteases involved in a number of important processes including blood coagulation and inflammation. Consequently, loss of serpin function or stability results in a number of disease states. Many of the naturally occurring mutations leading to disease are located within strand 1 of the C beta-sheet of the serpin. To ascertain the structural and functional importance of each residue in this strand, which constitutes the so-called distal hinge of the reactive center loop of the serpin, an alanine scanning study was carried out on recombinant alpha(1)-antitrypsin Pittsburgh mutant (P1 = Arg). Mutation of the P10' position had no effect on its inhibitory properties towards thrombin. Mutations to residues P7' and P9' caused these serpins to have an increased tendency to act as substrates rather than inhibitors, while mutations at P6' and P8' positions caused the serpin to behave almost entirely as a substrate. Mutations at the P6' and P8' residues of the C beta-sheet, which are buried in the hydrophobic core in the native structure, caused the serpin to become highly unstable and polymerize much more readily. Thus, P6' and P8' mutants of alpha(1)-antitrypsin had melting temperatures 14 degrees lower than wild-type alpha(1)-antitrypsin. These results indicate the importance of maintaining the anchoring of the distal hinge to both the inhibitory mechanism and stability of serpins, the inhibitory mechanism being particularly sensitive to any perturbations in this region. The results of this study allow more informed analysis of the effects of mutations found at these positions in disease-associated serpin variants.     

Cavities of α1-antitrypsin that play structural and functional roles

The native form of inhibitory serine protease inhibitors (serpins) is strained, which is critical for their inhibitory activity. Previous studies on stabilizing mutations of alpha(1)-antitrypsin, a prototype of serpins, indicated that cavities provide a structural basis for the native strain of the molecule. We have systematically mapped the cavities of alpha(1)-antitrypsin that play such structural and functional roles by designing cavity-filling mutations at residues that line the walls of the cavities. Results show that energetically unfavorable cavities are distributed throughout the alpha(1)-antitrypsin molecule, and the cavity-filling mutations stabilized the native conformation at 8 out of 10 target sites. The stabilization effect of the individual cavity-filling mutations of alpha(1)-antitrypsin varied (0.2-1.9 kcal/mol for each additional methylene group) and appeared to depend largely on the structural flexibility of the cavity environment. Cavity-filling mutations that decreased inhibitory activity of alpha(1)-antitrypsin were localized in the loop regions that interact with beta-sheet A distal from the reactive center loop. The results are consistent with the notion that beta-sheet A and the structure around it mobilize when alpha(1)-antitrypsin forms a complex with a target protease.     

Conformational substates in enzyme mechanism: the 120 K structure of alpha-lytic protease at 1.5 A resolution.

Insight into the dynamic properties of alpha-lytic protease (alpha LP) has been obtained through the use of low-temperature X-ray crystallography and multiple-conformation refinement. Previous studies of alpha LP have shown that the residues around the active site are able to move significantly to accommodate substrates of different sizes. Here we show a link between the ability to accommodate ligands and the dynamics of the binding pocket. Although the structure of alpha LP at 120 K has B-factors with a uniformly low value of 4.8 A2 for the main chain, four regions stand out as having significantly higher B-factors. Because thermal motion should be suppressed at cryogenic temperatures, the high B-factors are interpreted as the result of trapped conformational substates. The active site residues that are perturbed during accommodation of different substrates are precisely those showing conformational substates, implying that substrate binding selects a subset of conformations from the ensemble of accessible states. To better characterize the precise nature of these substates, a protein model consisting of 16 structures has been refined and evaluated. The model reveals a number of features that could not be well-described by conventional B-factors: for example, 40% of the main-chain residue conformations are distributed asymmetrically or in discrete clusters. Furthermore, these data demonstrate an unexpected correlation between motions on either side of the binding pocket that we suggest is a consequence of "dynamic close packing." These results provide strong evidence for the role of protein dynamics in substrate binding and are consistent with the results of dynamic studies of ligand binding in myoglobin and ribonuclease A.     

青弋江鱼类分类群和功能群的α和β多样性纵向梯度格局

确定溪流鱼类多样性的时空分布格局可为鱼类多样性保护与管理提供科学基础。尽管溪流鱼类分类群多样性的纵向梯度格局已有大量报道, 但以鱼类生物学特征为基础的功能多样性研究较少。本文基于2009-2010年4个季度对青弋江1-5级溪流共15个样点的调查数据, 利用形态特征数据和食性构建了鱼类复合功能群, 研究了不同级别溪流间鱼类分类群和功能群组成及多样性的异同, 着重探讨了鱼类分类群和功能群的α和β多样性沿溪流纵向梯度的变化规律。采集到的56种鱼类可分为4个营养功能群和5个运动功能群, 共计14个“营养-运动”复合功能群。双因素交互相似性分析结果显示, 鱼类分类群和功能群组成都随河流级别显著变化, 但季节动态不显著; 双因素方差分析后发现, 鱼类分类群和功能群α、β多样性都随河流级别显著变化, 但受季节影响不显著。经回归分析, 分类群和功能群α多样性与河流级别大小呈显著的线性正相关, 但最大分类群α多样性出现于4级河流, 最大功能群α多样性在4级和5级河流间一致; 分类群和功能群β多样性与河流级别大小呈显著的二项式关系, 呈U型分布。分类群β多样性的空间变化主要取决于物种周转, 而功能群β多样性主要由嵌套所驱动。本研究表明, 沿着“上游-下游”的纵向梯度, 河流鱼类的α和β多样性的空间变化规律不同, 分类群和功能群α多样性的空间格局基本一致, 但分类群(主要是物种周转)和功能群β多样性(主要是功能嵌套)的空间变化过程的驱动机制不同。     

鳞杯伞α-半乳糖苷酶的提取纯化及酶学性质研究

采用离子交换层析和凝胶过滤层析对鳞杯伞子实体中的α-半乳糖苷酶进行纯化,得到了一种分子量为50 kDa的α-半乳糖苷酶,命名为CSG。纯化后的CSG纯化倍数为891.46倍,比活力为54.78 U/mg,得率为0.71%。通过BLAST比对液相色谱-串联质谱(LC-MS/MS)获得其肽段,发现其为GH27家族的α-半乳糖苷酶。CSG的最适pH为3.0,最适温度为50 ℃。在酸性范围pH 2.2-7.0和温度范围4-30 ℃有较好的稳定性。Mn²⁺、Cd²⁺、Cu²⁺对CSG有较强的抑制作用。半乳糖和蜜二糖对CSG的抑制类型为混合型抑制。化学修饰剂N-溴代琥珀酰亚胺显著降低CSG的活力,碳二亚胺对CSG具有显著的激活作用。该酶具有良好的蛋白酶抗性,且对棉子糖家族寡糖(RFOs)、瓜尔豆胶和赤槐豆胶均表现出良好的水解作用。     

浙江省主要亚热带森林群落类型物种和谱系水平的α和β多样性比较

了解不同森林群落类型的物种和谱系水平的α和β多样性, 有助于指导森林经营和生物多样性保护。本研究比较了浙江省内不同地点主要森林类型(包括常绿阔叶林、常绿落叶阔叶混交林、落叶阔叶林和针阔叶混交林)的物种α多样性和谱系α多样性, 以及物种β多样性和谱系β多样性。研究表明, 该地区主要森林类型的物种和谱系α多样性均存在较大差异, 但控制了空间和地形因子的作用后, 差异几乎全部消失; 森林类型内部及相互间的物种和谱系β多样性均存在显著差异, 同种森林类型内部的物种和谱系β多样性分别小于不同森林类型之间的物种和谱系β多样性, 且在控制了空间和地形因子的作用后, 以上差异仍然显著。本研究表明影响亚热带主要森林群落类型物种和谱系水平的α和β多样性的因素存在差异: α多样性可能主要受到空间和地形因子等的影响, 而β多样性则可能受到森林类型的重要影响。     

Synthesis and bioactivities evaluation of oleanolic acid oxime ester derivatives as α-glucosidase and α-amylase inhibitors

Different oleanolic acid (OA) oxime ester derivatives (3a-3t) were designed and synthesised to develop inhibitors against α-glucosidase and α-amylase. All the synthesised OA derivatives were evaluated against α-glucosidase and α-amylase in vitro. Among them, compound 3a showed the highest α-glucosidase inhibition with an IC₅₀ of 0.35 µM, which was ∼1900 times stronger than that of acarbose, meanwhile compound 3f exhibited the highest α-amylase inhibitory with an IC₅₀ of 3.80 µM that was ∼26 times higher than that of acarbose. The inhibition kinetic studies showed that the inhibitory mechanism of compounds 3a and 3f were reversible and mixed types towards α-glucosidase and α-amylase, respectively. Molecular docking studies analysed the interaction between compound and two enzymes, respectively. Furthermore, cytotoxicity evaluation assay demonstrated a high level of safety profile of compounds 3a and 3f against 3T3-L1 and HepG2 cells.

Highlights

1. Oleanolic acid oxime ester derivatives (3a–3t) were synthesised and screened against α-glucosidase and α-amylase.
2. Compound 3a showed the highest α-glucosidase inhibitory with IC50 of 0.35 µM.
3. Compound 3f presented the highest α-amylase inhibitory with IC50 of 3.80 µM.
4. Kinetic studies and in silico studies analysed the binding between compounds and α-glucosidase or α-amylase.

     

儿童孤独症相关基因NRXN1β启动子功能分析

Neurexins是神经特异性突触蛋白,Neurexin1β结构的异常与孤独症密切相关。为分析孤独症相关基因NRXN1β最小启动子和调节基因转录的功能元件,本文构建了含NRXN1β基因上游调控区不同区域的荧光素酶报告基因质粒,转染HEK293细胞后,利用检测双荧光素酶报告基因的转录活性以确定NRXN1β基因最小启动子区,进而筛选出相应的显著增强或抑制报告基因活性的功能区;同时,为鉴定顺式作用元件,利用基因定点突变技术对基因功能区内和临近DNA序列进行连续的碱基突变;最后,采用转录因子预测工具对启动子功能区内的转录调控元件进行分析。结果首次发现NRXN1β最小启动子区位于-88~+156 bp,-88~-73 bp和+156~+149 bp可增强启动子活性,+229~+419 bp可抑制启动子活性,且-84~-63 bp为能够显著性增强启动子活性的顺式作用元件,该区域可能存在DBP(Albumin D-site-binding protein,DBP)和ABF1(Autonomously replicating sequence-binding factor 1,ABF1)两个转录因子结合位点。     

In vitro and in silico studies of bis (indol-3-yl) methane derivatives as potential α-glucosidase and α-amylase inhibitors

In this paper, bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated for their inhibitory activity against α-glucosidase and α-amylase. All synthesised compounds showed potential α-glucosidase and α-amylase inhibitory activities. Compounds 5 g (IC₅₀: 7.54 ± 1.10 μM), 5e (IC₅₀: 9.00 ± 0.97 μM), and 5 h (IC₅₀: 9.57 ± 0.62 μM) presented strongest inhibitory activities against α-glucosidase, that were ∼ 30 times stronger than acarbose. Compounds 5 g (IC₅₀: 32.18 ± 1.66 µM), 5 h (IC₅₀: 31.47 ± 1.42 µM), and 5 s (IC₅₀: 30.91 ± 0.86 µM) showed strongest inhibitory activities towards α-amylase, ∼ 2.5 times stronger than acarbose. The mechanisms and docking simulation of the compounds were also studied. Compounds 5 g and 5 h exhibited bifunctional inhibitory activity against these two enzymes. Furthermore, compounds showed no toxicity against 3T3-L1 cells and HepG2 cells.

Highlights

1. A series of bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated inhibitory activities against α-glucosidase and α-amylase.
2. Compound 5g exhibited promising activity (IC₅₀ = 7.54 ± 1.10 μM) against α-glucosidase.
3. Compound 5s exhibited promising activity (IC₅₀ = 30.91 ± 0.86 μM) against α-amylase.
4. In silico studies were performed to confirm the binding interactions of synthetic compounds with the enzyme active site.

     

Phosphorylation of human CEACAM1-LF by PKA and GSK3β promotes its interaction with β-catenin

CEACAM1-LF, a homotypic cell adhesion adhesion molecule, transduces intracellular signals via a 72 amino acid cytoplasmic domain that contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and a binding site for β-catenin. Phosphorylation of Ser503 by PKC in rodent CEACAM1 was shown to affect bile acid transport or hepatosteatosis via the level of ITIM phosphorylation, but the phosphorylation of the equivalent residue in human CEACAM1 (Ser508) was unclear. Here we studied this analogous phosphorylation by NMR analysis of the ¹⁵N labeled cytoplasmic domain peptide. Incubation with a variety of Ser/Thr kinases revealed phosphorylation of Ser508 by GSK3bβ but not by PKC. The lack of phosphorylation by PKC is likely due to evolutionary sequence changes between the rodent and human genes. Phosphorylation site assignment by mass spectrometry and NMR revealed phosphorylation of Ser472, Ser461 and Ser512 by PKA, of which Ser512 is part of a conserved consensus site for GSK3β binding. We showed here that only after phosphorylation of Ser512 by PKA was GSK3β able to phosphorylate Ser508. Phosphorylation of Ser512 by PKA promoted a tight association with the armadillo repeat domain of β-catenin at an extended region spanning the ITIMs of CEACAM1. The kinetics of phosphorylation of the ITIMs by Src, as well dephosphorylation by SHP2, were affected by the presence of Ser508/512 phosphorylation, suggesting that PKA and GSK3β may regulate the signal transduction activity of human CEACAM1-LF. The interaction of CEACAM1-LF with β-catenin promoted by PKA is suggestive of a tight association between the two ITIMs of CEACAM1-LF.     

Evidence that Distinct States of the Integrin α6β1 Interact with Laminin and an ADAM

Integrins can exist in different functional states with low or high binding capacity for particular ligands. We previously provided evidence that the integrin α6β1, on mouse eggs and on α6-transfected cells, interacted with the disintegrin domain of the sperm surface protein ADAM 2 (fertilin β). In the present study we tested the hypothesis that different states of α6β1 interact with fertilin and laminin, an extracellular matrix ligand for α6β1. Using α6-transfected cells we found that treatments (e.g., with phorbol myristate acetate or MnCl₂) that increased adhesion to laminin inhibited sperm binding. Conversely, treatments that inhibited laminin adhesion increased sperm binding. Next, we compared the ability of fluorescent beads coated with either fertilin β or with the laminin E8 fragment to bind to eggs. In Ca²⁺-containing media, fertilin β beads bound to eggs via an interaction mediated by the disintegrin loop of fertilin β and by the α6 integrin subunit. In Ca²⁺-containing media, laminin E8 beads did not bind to eggs. Treatment of eggs with phorbol myristate acetate or with the actin disrupting agent, latrunculin A, inhibited fertilin bead binding, but did not induce laminin E8 bead binding. Treatment of eggs with Mn²⁺ dramatically increased laminin E8 bead binding, and inhibited fertilin bead binding. Our results provide the first evidence that different states of an integrin (α6β1) can interact with an extracellular matrix ligand (laminin) or a membrane-anchored cell surface ligand (ADAM 2).     

Elongation of the BH8 β-hairpin peptide: Electrostatic interactions in β-hairpin formation and stability

An elongated version of the de novo designed beta-hairpin peptide, BH8, has allowed us to gain insight into the role of electrostatic interactions in beta-hairpin stability. A Lys-Glu electrostatic pair has been introduced by adding a residue at the beginning and at the end of the N-terminal and C-terminal strands, respectively, of the beta-hairpin structure, in both orientations. The two resulting peptides and controls having Ala residues at these positions and different combinations of Ala with Lys, or Glu residues, have been analyzed by nuclear magnetic resonance (NMR), under different pH and ionic strength conditions. All of the NMR parameters, in particular the conformational shift analysis of Calpha protons and the coupling constants, (3)J(HNalpha), correlate well and the population estimates are in reasonable agreement among the different methods used. In the most structured peptides, we find an extension of the beta-hairpin structure comprising the two extra residues. Analysis of the pH and salt dependence shows that ionic pairs contribute to beta-hairpin stability. The interaction is electrostatic in nature and can be screened by salt. There is also an important salt-independent contribution of negatively charged groups to the stability of this family of beta-hairpin peptides.     

Development of selective protease inhibitors via engineering of the bait region of human α2-macroglobulin

Human α₂-macroglobulin (A2M) is an abundant protease inhibitor in plasma, which regulates many proteolytic processes and is involved in innate immunity. A2M’s unique protease-trapping mechanism of inhibition is initiated when a protease cleaves within the exposed and highly susceptible “bait region.” As the wild-type bait region is permissive to cleavage by most human proteases, A2M is accordingly a broad-spectrum protease inhibitor. In this study, we extensively modified the bait region in order to identify any potential functionally important elements in the bait region sequence and to engineer A2M proteins with restrictive bait regions, which more selectively inhibit a target protease. A2M in which the bait region was entirely replaced by glycine-serine repeats remained fully functional and was not cleaved by any tested protease. Therefore, this bait region was designated as the “tabula rasa” bait region and used as the starting point for further bait region engineering. Cleavage of the tabula rasa bait region by specific proteases was conveyed by the insertion of appropriate substrate sequences, e.g., basic residues for trypsin. Screening and optimization of tabula rasa bait regions incorporating matrix metalloprotease 2 (MMP2) substrate sequences produced an A2M that was specifically cleaved by MMPs and inhibited MMP2 cleavage activity as efficiently as wild-type A2M. We propose that this approach can be used to develop A2M-based protease inhibitors, which selectively inhibit target proteases, which might be applied toward the clinical inhibition of dysregulated proteolysis as occurs in arthritis and many types of cancer.     

Molecular docking analysis of stachydrine and sakuranetin with IL-6 and TNF-α in the context of inflammation

Inflammation is a process triggered by pro-inflammatory cytokines and anti-inflammatory molecules. Therefore, it is of interest to document the anti-inflammatory activity of Stachydrine and Sakuranetin against the inflammatory target proteins IL-6 and TNF-α by using molecular docking analysis. Both compounds showed good binding features with the selected target proteins. Compared to Sakuranetin, the Stachydrine have low binding energy and good hydrogen bond interactions. Hence, data show that Stachydrine possessed high and specific inhibitory activity on tumor necrosis factor-α and interleukin-6.     

Induction of beige‐like adipocyte markers and functions in 3T3‐L1 cells by Clk1 and PKCβII inhibitory molecules

Excessive dietary intake of fat results in its storage in white adipose tissue (WAT). Energy expenditure through lipid oxidation occurs in brown adipose tissue (BAT). Certain WAT depots can undergo a change termed beiging where markers that BAT express are induced. Little is known about signalling pathways inducing beiging. Here, inhibition of a signalling pathway regulating alternative pre‐mRNA splicing is involved in adipocyte beiging. Clk1/2/4 kinases regulate splicing by phosphorylating factors that process pre‐mRNA. Clk1 inhibition by TG003 results in beige‐like adipocytes highly expressing PGC1α and UCP1. SiRNA for Clk1, 2 and 4, demonstrated that Clk1 depletion increased UCP1 and PGC1α expression, whereas Clk2/4 siRNA did not. TG003‐treated adipocytes contained fewer lipid droplets, are smaller, and contain more mitochondria, resulting in proton leak increases. Additionally, inhibition of PKCβII activity, a splice variant regulated by Clk1, increased beiging. PGC1α is a substrate for both Clk1 and PKCβII kinases, and we surmised that inhibition of PGC1α phosphorylation resulted in beiging of adipocytes. We show that TG003 binds Clk1 more than Clk2/4 through direct binding, and PGC1α binds to Clk1 at a site close to TG003. Furthermore, we show that TG003 is highly specific for Clk1 across hundreds of kinases in our activity screen. Hence, Clk1 inhibition becomes a target for induction of beige adipocytes.     

NEDD4L-induced β-catenin ubiquitination suppresses the formation and progression of interstitial pulmonary fibrosis via inhibiting the CTHRC1/HIF-1α axis

Interstitial pulmonary fibrosis (IPF) is a severe progressive lung disease with limited therapeutic options and poor prognosis. Initially, we found the downregulated level of neural precursor cell expressed developmentally down-regulated 4-like protein (NEDD4L) in IPF-related expression microarray dataset, and this study was thus performed to explore the molecular mechanism of NEDD4L in IPF. The expression of NEDD4L was subsequently validated in lung tissues of IPF patients and mouse models. Then, mouse primary lung fibroblasts (LFs) were collected for in vitro functional experiments, with CCK-8, Transwell, and immunofluorescence assays used to examine the viability, migration, and differentiation of LFs. The in vitro findings were further assessed using in vivo mouse models. The expression of NEDD4L was down-regulated in lung tissues of IPF patients and mouse models. Overexpression of NEDD4L restricted the formation and progression of IPF in mice and attenuated the proliferative, invasive and differentiative abilities of LFs. Further, NEDD4L halted LFs activity by enhancing β-catenin ubiquitination and down-regulating the CTHRC1/HIF-1α axis. Also, in vivo experiments then validated that NEDD4L silencing repressed β-catenin ubiquitination and activated the CTHRC1/HIF-1α axis, thereby aggravating IPF in mice. NEDD4L may suppress the formation and progression of IPF through augmenting β-catenin ubiquitination and inhibiting the CTHRC1/HIF-1α axis.     

Unusual β1-4-galactosidase activity of an α1-6-mannosidase from Xanthomonas manihotis in the processing of branched hybrid and complex glycans

Mannosidases are a diverse group of glycoside hydrolases that play crucial roles in mannose trimming of oligomannose glycans, glycoconjugates, and glycoproteins involved in numerous cellular processes, such as glycan biosynthesis and metabolism, structure regulation, cellular recognition, and cell–pathogen interactions. Exomannosidases and endomannosidases cleave specific glycosidic bonds of mannoside linkages in glycans and can be used in enzyme-based methods for sequencing of isomeric glycan structures. α1-6-mannosidase from Xanthomonas manihotis is known as a highly specific exoglycosidase that removes unbranched α1-6 linked mannose residues from oligosaccharides. However, we discovered that this α1-6-mannosidase also possesses an unexpected β1-4-galactosidase activity in the processing of branched hybrid and complex glycans through our use of enzymatic reactions, high performance anion-exchange chromatography, and liquid chromatography mass spectrometric sequencing. Our docking simulation of the α1-6-mannosidase with glycan substrates reveals potential interacting residues in a relatively shallow pocket slightly differing from its homologous enzymes in the glycoside hydrolase 125 family, which may be responsible for the observed higher promiscuity in substrate binding and subsequent terminal glycan hydrolysis. This observation of novel β1-4-galactosidase activity of the α1-6-mannosidase provides unique insights into its bifunctional activity on the substrate structure-dependent processing of terminal α1-6-mannose of unbranched glycans and terminal β1-4-galactose of hybrid and complex glycans. The finding thus suggests the dual glycosidase specificity of this α1-6-mannosidase and the need for careful consideration when used for the structural elucidation of glycan isomers.     

人β-半乳糖苷酶R299L突变体的获得与活性研究*

目的: GM1神经节苷脂贮积症是一种由半乳糖苷酶beta 1(galactosidase beta 1, GLB1)基因突变引起的β-半乳糖苷酶(β-galactosidase,β-gal)活性降低导致的严重的溶酶体贮积病。该病以进行性、致命性神经退行性病变为特征,目前尚无有效的治疗手段,AAV载体介导的基因治疗被认为是最有希望的治疗方法。通过基因定点突变获得具有较高β-gal活性的GLB1突变体,以期用于后续AAV介导的基因治疗。方法: 对人类和其他6种脊椎动物GLB1基因进行多序列比对分析,筛选出部分氨基酸位点进行定点突变,采用携带突变位点的重组质粒和AAV9载体转染或感染HEK-293细胞,比较突变体与未突变体的活性差异。对GM1模型鼠注射携带coGLB1-R299L的rAAV9病毒,探究该突变体的体内活性表达。结果: 从15个突变体中筛选出coGLB1-R299L突变体,经质粒转染导入细胞后,其β-gal活性比具有野生型氨基酸序列的coGLB1增加了30%~40%。AAV体外感染实验中,rAAV9-coGLB1-R299L组的β-gal活性较未感染的细胞对照组提升了约2.2倍。体内结果显示,rAAV9-coGLB1-R299L在模型鼠体内广泛表达,心脏、肝脏、脾脏、肺、脑组织中β-gal活性显著提升。结论: 获得了具有更高β-gal活性的突变体coGLB1-R299L,初步探究了rAAV9-coGLB1-R299L的体外表达效果和模型鼠体内β-半乳糖苷酶的表达与分布,为该突变体应用于AAV介导的GM1神经节苷脂病治疗奠定基础。