Oligosaccharide Substrate and Inhibitor Interactions with beta-1,4-Gal-T1

寡糖底物和抑制剂与 β-1,4-Gal-T1 的相互作用

• 批准号: 7732974
• 负责人: Pradman K Qasba
• 金额: $ 6.55万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7732974
• 关键词: Affinity; Binding; Binding Sites; Biological Models; Biological Process; Carbohydrates; Cell Adhesion; Cell surface; Cells; Chemicals; Class; Collaborations; Complex; Condition; Dimerization; Disaccharides; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzyme Kinetics; Enzymes; Epitopes; Exhibits; Family member; Focal Adhesion Kinase 1; Future; Galactose; Galactosyltransferases; Glycoconjugates; Glycolipids; Glycoproteins; Homo; Hour; Human; Hydrogen Bonding; Immunoglobulin G; In Vitro; Integrins; Kinetics; Lead; Link; Mannose; Mass Spectrum Analysis; Modification; Monitor; Monoclonal Antibodies; Monosaccharides; Naphthalene; Naphthalenes; Neoplasm Metastasis; Oligosaccharides; Pattern; Play; Pliability; Polysaccharides; Positioning Attribute; Proteins; Rate; Role; Side; Specificity; Structure; Trisaccharides; Tyrosine Phosphorylation; Upper arm; Ursidae Family; Water; amino group; cell growth; chitobiose; crosslink; design; glycosyltransferase; human disease; hydroxyl group; improved; inhibitor/antagonist; lactosamine; link protein; man; mutant; preference; response; simulation; tumor

Our previous crystallographic studies on b4Gal-T1 and of the mutant Met340His-b4Gal-T1 in complex with chitobiose and various trisaccharides, together with the enzyme kinetic analysis and MD simulations defined the oligosaccharide binding site of b4Gal-T1 (see Project #s Z01 BC 009304). For a better understanding of the branch specificity of b4Gal-T1 towards the GlcNAc residues of N-glycans, the kinetic and crystallographic studies with the wild-type human b4Gal-T1 (h-b4Gal-T1) and the mutant Met340His-b4Gal-T1 (h-M340H-b4Gal-T1), in complex with a GlcNAc containing pentasaccharide and several GlcNAc containing trisaccharides present in N-glycans, showed that b4Gal-T1 preferentially interacts with the 1,2-1,6-arm trisaccharide rather than with the 1,2-1,3-arm or 1,4-1,3-arm of a bi- or tri-antennary oligosaccharide chain of N-glycan (see 2006 project # Z01 BC010041) In the present studies we are determining the transfer to a biantennary oligosaccharide of a glycoprotein e. IgG. Transfer preferences of b4Gal-T1 to the 1-3 or 1-6 arm of a biantennary glycan We showed, using synthetic trisaccharides as acceptor model systems, that the acceptor 1,2-1,3-arm (GlcNAc-b1,2-Man-a1,3-Man) and 1,4-1,3-arm (GlcNAc-b1,4-Man-a1,3-Man) trisaccharides have a 10- to 60-fold higher Km for b4Gal-T1 than the 1,2-1,6-arm trisaccharide (GlcNAc-b1,2-Man-a1,6-Man), latter shows substrate inhibition at concentrations that are much lower than for other acceptor substrates. We have established conditions under which the galactosylation of one or both antennas of an N-glycan of a glycoprotein, e.g., IgG, occur. To establish which antenna, 1-3-arm or 1-6-arm, or both, is galactosylated we have, in collaboration with Dr. Timothy Weybright (from Dr. Timothy Veenstras group, of the Mass Spectrometry Center, BPP, SAIC-Frederick, Inc.), analyzed the oligosaccharide products by MS/MS analysis and established that at high concentrations of IgG only 1-3-arm is galactosylated. We have followed the transfer of galactose to de-sialated, de-galactosylated monoclonal antibodies, Asialo-agalacto-IgG, which carry a single N-linked oligosaccharide chain on each heavy chain of the IgG at the Fc-region. MS analysis of the glycan chains after PNGaseF treatment of the monoclonal antibodies, bearing glycans of complex bi-antennary chains N-linked to Asn 297, show various glycosylated patterns; G0 glycoform having two terminal GlcNAc residues, and G1 and G2 glycoforms which bear oligosaccharides with one or two terminal galactose, respectively. As monitored by MS analysis, the conditions for the complete de-galactosylation (100 %) to G0 glycoform was established. After 6 hr transfer of galactose by the wild type 4Gal-T1 to G0 glycoform (MW 1485) of IgG, the MS analyses of the product after PNGaseF treatment of IgG, show that galactose is mainly transferred to one arm (G1 glycoform, MW 1647). Further MS/MS analysis of the bi-antennary glycan chain shows that the wild type 4Gal-T1 transfers galactose at a faster rate to the GlcNAc attached to the Man 1-3 arm. In contrast, the mutant 1,4Gal-T1-Y289L at 6 hours incubation transfers GalNAc mainly to both arms (G2 glycoform MW 1891). MS/MS analysis of the G1 glycoform after PNGasesF treatment of the monoclonal antibodies show that the mutant Y289L- b4Gal-T1 transfers GalNAc at a faster rate to the GlcNAc attached to the Man 1-6 arm. Crystal structure of the h-M340H-Gal-T1 in complex with the disaccharide GlcNAc-b3Gal-b-O-napthalenemethanol A class of synthetic disaccharides has been shown as potential inhibitors of tumor metastasis by Dr. Jeff Esko's lab in UC, San Diego. They are high affinity substrates for b4GalT1 and thus act as decoys for the synthesis of sialyl Lewis X (sLeX ), the cell adhesion epitope, which is expressed at elevated levels in metastatic cells. The most effective compound they have identified to date is GlcNAc-b3Gal-b-O-napthalenemethanol. The acetylated compound is taken up by the cells, O-deacetylated, and then the disaccharide decoys the synthesis of sLeX-containing glycans on cell surface glycoconjugates. In several model systems this results in an inhibition of tumor formation. As Dr. Esko's lab has observed, all the activity of the compound depends on the action of b4GalT(s) in the cell, since the first step in its utilization involves galactosylation. In their in vitro studies with GlcNAc-b3Gal-b-O-napthalenemethanol as an acceptor substrate for b4Gal-T1, they show a Km of 10 vM. This high affinity for the enzyme may be its unique mode of binding to b4Gal-T1 similar to the one we have observed with the 1,2-1,6-arm trisaccharide (see above). We have carried the crystal structure of h-M340H-Gal-T1 with GlcNAc-b3Gal-b-O-napthalenemethanol, provided by Dr. Esko's lab, and determined the mode of interaction between the disaccharide and the enzyme. The overall binding of GnGl-NP to the Met344His-Cys342Thr-Gal-T1 molecule is quite similar to the binding of the tri-saccharide GlcNAcb1-2Mana1-6Mana, observed earlier (see 2006 project # Z01 BC 010041), suggesting its Km for the enzyme will be similar to that of the 1-6 arm tri-saccharide, a Km of 60 vM. This tri-saccharide is derived from the 1-6 arm of the biantennary N-glycan, starting from the core mannose to the free GlcNAc at the non-reducing end. In the enzyme-bound 1-6 arm tri-saccharide, the middle mannose exhibits the least interactions with the protein molecule, while the terminal mannose (i.e., the core mannose residue of the biantennary N-glycans) makes extensive stacking interactions with the aromatic side chain of the Tyr282 residue.In contrast, the beta-linked Gal residue in the disaccharide GnGl-NP forms extensive stacking interactions with Tyr282, and the terminal aromatic naphthalene residue makes additional interactions, although weak, in the oligosaccharide binding site of the b4Gal-T1 molecule. Therefore, due to the additional interactions that arise from the naphthalene moiety, it is expected that GnGl-NP disaccharide will have a lower Km than that of the 1-6 arm tri-saccharide, thus making it the best known acceptor substrate with the highest affinity for b4Gal-T1. Furthermore, the b4Gal-T family members T5 and T6 have the conserved residue Tyr corresponding to the Phe356 residue of the b4Gal-T1. The Tyr residue in these family members is expected to make additional hydrogen bonding interactions via the side-chain hydroxyl group of the Tyr residue, which is expected to further lower the Km of the GnGl-NP disaccharide for b4Gal-T5 and b4Gal-T6. From the crystal structure analysis we are able to understand the high affinity of the disaccharide GnGl-NP for b4Gal-T1. Furthermore, the analysis suggests that future chemical modifications, such as incorporating the structural water molecule in the acceptor design, amino or methylated amino group at the second position of Gal or even appropriate substitutions of polar groups at the naphthalene ring, may improve the affinity of the acceptor substrate and lead to better design of the disaccharide inhibitors for the tumor metastasis.

我们先前对B4GAL-T1和突变体MET340HIS-B4GAL-T1进行的晶体学研究与壳聚糖和各种三糖，以及酶动力学分析和MD模拟定义了B4GAL-T1的寡糖结合位点（请参阅Project Z01 BC 009304）。为了更好地理解B4GAL-T1对N-Glycans的GlcNAC残基的分支特异性，使用野生型人B4GAL-T1（H-B4GAL-T1）的动力学和晶体学研究N-glycans中存在的三糖表明B4GAL-T1优先与1,2-1,6臂三糖相互作用，而不是与1,2-1,3臂或1,2-1,3臂或1,4-1,3臂两臂两臂两臂两臂两臂二糖，两年一的两年代或三年期寡糖链链链N-糖的n-Glycan链链的N-聚糖链的n-Glycan链条（请参阅2006 project with 2006 project with 2006 project with 2006 bc z01 bc01 bc01 bc01 bc01 bc01 bc0110041）糖蛋白E的寡糖。 IgG。 我们使用合成三糖作为受体模型系统表明，B4GAL-T1对双聚糖的1-3或1-6臂的转移偏好表明受体1,2-1,3-ARM（GLCNAC-B1,2-MAN-A1-A1,3-MAN）和1,4-1,4-1,3-ARM（GLCNAC-B1,4-MAN-MAN-MAN-MAN-MAN-MAN-MAN-MAN-MAN） B4GAL-T1比1,2-1,6臂三糖（GlcNAC-B1,2-MAN-A1,6-MAN）高60倍，后者在浓度下显示底物抑制均低于其他受体底物。我们已经确定了将糖蛋白N-聚糖的一个或两个天线的半乳糖基化（例如IgG）发生的。 为了确定哪个天线，1-3臂或1-6臂或两者是半乳糖基化的，我们与Timothy Weybright博士（来自BPP，SAIC-Frederick，Inc。的Timothy Weybright博士（来自Timothy veenstras Group，Timothy veenstras Group博士），仅通过MS/MS分析，并通过MS/MS分析了IS-MS Ansporty-IS-IS-IS-IS-IS-IS-Ans-ig in-MS Ansigations IS-IS-Ans Agn Agn Agn Ansered Ansered Ansered Ansered Ansered Ansered Ansered Ansered An半乳糖基化。我们遵循了半乳糖向去除，去半乳糖基化的单克隆抗体Asialo-Agalacto-IgG的转移，后者在FC-Regregion的IgG的每个重链上携带单个N连接的寡糖链。 PNGASEF处理单克隆抗体后的聚糖链的MS分析，其具有复杂的双疗链N-链接为ASN 297的复杂双链链，显示了各种糖基化模式；具有两个末端GLCNAC残基的G0糖型，以及分别带有一个或两个末端半乳糖的G1和G2糖型。如MS分析所监测的，建立了完全去乳糖基化（100％）对G0糖基型的条件。通过野生型4GAL-T1转移半乳糖为IgG的G0糖衣（MW 1485）后，pngasef处理IgG后对产物的MS分析表明，半乳糖主要转移到一个ARM上（G1 Glycoform，MW 1647）。对双抗聚糖链的进一步MS/MS分析表明，野生型4GAL-T1以更快的速度传输半乳糖，向连接到男子1-3 ARM的GlcNAC。相比之下，在6小时孵育时，突变体1,4Gal-T1-Y289L主要将Galnac转移到两个臂上（G2 Glycoform MW 1891）。 PNGASEF治疗单克隆抗体后G1糖基型的MS/MS分析表明，突变体Y289L-B4GAL-T1以更快的速率转移GalNAC，以与GlcNAC相连的GlcNAC速度更快地连接到MAN 1-6 ARM的GLCNAC。 与二糖GlcNAC-B3GAL-B-O-NAPTHANENEMETHANOL A-M340H-GAL-T1在复合物中的晶体结构已显示出Jeff Esko博士在San Diego的UC的Jeff Esko博士在UC的LAB。 它们是B4GALT1的高亲和力底物，因此充当诱饵的诱饵，用于合成siAllyl Lewis X（SLEX），细胞粘附表位，在转移性细胞中升高的水平表达。他们迄今为止确定的最有效的化合物是GlcNAC-B3GAL-B-O-裸甲醇。乙酰化化合物被细胞占用，O-二乙酰化，然后二糖诱饵将含SLEX的聚糖的合成在细胞表面糖缀合物上。在几种模型系统中，这会导致抑制肿瘤形成。正如Esko博士的实验室所观察到的那样，该化合物的所有活性都取决于B4GALT在细胞中的作用，因为其利用的第一步涉及半乳糖基化。在他们对GlcNAC-B3GAL-B-O-氯甲醇作为B4GAL-T1的受体底物的体外研究中，它们的公里为10 vm。这种对酶的高亲和力可能是其与B4GAL-T1结合的独特模式，类似于我们使用1,2-1,6臂三糖观察到的酶（见上文）。我们携带了由Esko博士实验室提供的H-M340H-GAL-T1与GlcNAC-B3GAL-B-O-氯甲醇的晶体结构，并确定了二糖和酶之间的相互作用方式。 GNGL-NP与MET344HIS342THR-GAL-T1分子的总体结合与前面观察到的三糖GlcNACB1-2MANA1-6MANA的结合非常相似（请参见2006 Project＃Z01 BC 010041），建议与60的KM相似，与1-SAC相似。 VM。该三糖来自双胞质N-聚糖的1-6臂，从核心甘露糖开始到非还原端的自由GlcNAC。 In the enzyme-bound 1-6 arm tri-saccharide, the middle mannose exhibits the least interactions with the protein molecule, while the terminal mannose (i.e., the core mannose residue of the biantennary N-glycans) makes extensive stacking interactions with the aromatic side chain of the Tyr282 residue.In contrast, the beta-linked Gal residue in the disaccharide GnGl-NP形成与Tyr282的广泛堆叠相互作用，而末端芳香族萘残基在B4GAL-T1分子的寡糖结合位点中产生了额外的相互作用，尽管较弱，但虽然较弱。 因此，由于萘部分引起的其他相互作用，预计GNGL-NP二糖的Km将比1-6 ARM三糖的Km低，从而使其成为最知名的受体底物，对B4GAL-T1的亲和力最高。此外，B4GAL-T家族成员T5和T6具有与B4GAL-T1的PHE356残基相对应的保守残基Tyr。预计这些家族成员中的Tyr残基将通过Tyr残基的侧链羟基进行额外的氢键相互作用，预计该基团将进一步降低B4GAL-T5和B4GAL-T6的GNGL-NP二糖的Km。从晶体结构分析中，我们能够理解二糖GNGL-NP对B4GAL-T1的高亲和力。此外，分析表明，未来的化学修饰，例如将结构水分子纳入受体设计，氨基或甲基化的氨基群，在GAL的第二个位置，甚至在萘环处的极性组的适当取代，可能会改善受体底物的亲和力，并为Tumor Metastasis提供更好的disaccharide抑制剂设计。