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

我们之前对b4Gal-T1和突变体Met340His-b4Gal-T1与壳聚糖和各种三糖复合物的晶体学研究，以及酶动力学分析和MD模拟确定了b4Gal-T1的寡糖结合位点（见Project #s Z01 BC 009304）。为了更好地了解b4Gal-T1对n -聚糖中GlcNAc残基的分支特异性，我们将野生型人b4Gal-T1 （h-b4Gal-T1）和突变体met340hs -b4Gal-T1 （h-M340H-b4Gal-T1）与n -聚糖中含有五糖的GlcNAc和几种含三糖的GlcNAc配合物进行动力学和晶体学研究。结果表明，b4Gal-T1优先与1,2-1,6臂三糖相互作用，而不是与n -聚糖的1,2-1,3臂或1,2-1,3臂的双或三天线寡糖链相互作用（见2006项目# Z01 BC010041）。在目前的研究中，我们正在确定向糖蛋白e. IgG的双天线寡糖的转移。我们使用合成三糖作为受体模型系统，发现受体1,2,1 -3臂（GlcNAc-b1,2-Man-a1,3-Man）和1,4,4,3臂（GlcNAc-b1,4-Man-a1,3-Man）三糖对b4Gal-T1的Km比1,2,1,2,6臂三糖（GlcNAc-b1,2-Man-a1,6-Man）高10- 60倍。后者显示底物抑制浓度远低于其他受体底物。我们已经建立了发生糖蛋白（如IgG）的n -聚糖的一个或两个天线半乳糖化的条件。为了确定哪一个天线，1-3臂或1-6臂，或两者都是半乳糖化的，我们与Timothy Weybright博士（来自Timothy Veenstras博士小组，来自上油-弗雷德里克公司BPP质谱中心）合作，通过质谱分析分析了低聚糖产物，并确定在高浓度的IgG中只有1-3臂是半乳糖化的。我们跟踪了半乳糖向去唾液化、去半乳糖化单克隆抗体Asialo-agalacto-IgG的转移，该单克隆抗体在IgG的fc区每个重链上携带一个n -连接的低聚糖链。单克隆抗体经PNGaseF处理后的糖链质谱分析显示，含有n -连接Asn 297的复杂双天线链的糖链显示出不同的糖基化模式；G0糖型末端有两个GlcNAc残基，G1和G2糖型末端分别含有一个或两个半乳糖的低聚糖。通过质谱监测，建立了半乳糖基化完全（100%）生成G0糖型的条件。野生型&#61538；4Gal-T1将半乳糖转移到IgG的G0糖型（MW 1485） 6小时后，PNGaseF处理IgG后产物的质谱分析表明，半乳糖主要转移到一侧（G1糖型，MW 1647）。进一步的双天线聚糖链的MS/MS分析表明，野生型&#61538；4Gal-T1以更快的速度将半乳糖转移到附着在Man 1-3臂上的GlcNAc上。相比之下，突变体&#61538；1,4gal - t1 - y289l在孵育6小时后将GalNAc主要转移到双臂（G2糖型MW 1891）。PNGasesF处理单克隆抗体后G1糖型的MS/MS分析显示，突变体Y289L- b4Gal-T1以更快的速度将GalNAc转移到附着在Man 1-6臂上的GlcNAc上。加州大学圣迭戈分校Jeff Esko博士的实验室已经证明，一类合成的双糖具有抑制肿瘤转移的潜在作用。它们是b4GalT1的高亲和力底物，因此可以作为sialyl Lewis X （sLeX）合成的诱饵，sLeX是细胞粘附表位，在转移细胞中表达水平升高。迄今为止，他们发现的最有效的化合物是glcnac - b3gal -b- o -萘二烯乙醇。乙酰化的化合物被细胞吸收，o -去乙酰化，然后双糖在细胞表面糖缀合物上诱骗合成含slex的聚糖。在一些模型系统中，这导致肿瘤形成的抑制。正如埃斯科博士的实验室所观察到的，这种化合物的所有活性都取决于细胞中b4GalT(s)的作用，因为利用它的第一步涉及半乳糖基化。在他们用glcnac - b3gal -b- o -萘二烯乙醇作为b4Gal-T1的受体底物的体外研究中，他们显示了10vm的Km。这种对酶的高亲和力可能是它与b4Gal-T1结合的独特模式，类似于我们观察到的1,2,2,6臂三糖（见上文）。我们用Esko博士实验室提供的glcnac - b3gal -b- o -萘二烯乙醇对h-M340H-Gal-T1的晶体结构进行了分析，并确定了双糖与酶之间的相互作用模式。GnGl-NP与Met344His-Cys342Thr-Gal-T1分子的整体结合与之前观察到的三糖GlcNAcb1-2Mana1-6Mana的结合非常相似（见2006项目# Z01 BC 010041），表明其对酶的Km将与1-6臂三糖相似，Km为60 vM。这种三糖来源于双触角n-聚糖的1-6臂，从核心甘露糖到非还原末端的游离GlcNAc。在酶结合的1-6臂三糖中，中间甘露糖与蛋白质分子的相互作用最小，而末端甘露糖（即双触角n -聚糖的核心甘露糖残基）与Tyr282残基的芳香侧链进行广泛的堆叠相互作用。相比之下，双糖GnGl-NP中的β连接的Gal残基与Tyr282形成了广泛的堆叠相互作用，而末端的芳香萘残基在b4Gal-T1分子的低聚糖结合位点上产生了额外的相互作用，尽管很弱。因此，由于萘部分产生的额外相互作用，预计GnGl-NP双糖的Km值将低于1-6臂三糖，从而使其成为已知的对b4Gal-T1具有最高亲和力的受体底物。此外，b4Gal-T家族成员T5和T6具有与b4Gal-T1的Phe356残基对应的保守残基Tyr。这些家族成员中的Tyr残基有望通过Tyr残基的侧链羟基进行额外的氢键相互作用，这有望进一步降低b4Gal-T5和b4Gal-T6的GnGl-NP双糖的Km。通过晶体结构分析，我们能够理解双糖GnGl-NP对b4Gal-T1的高亲和力。此外，分析表明，未来的化学修饰，如在受体设计中加入结构水分子，在Gal的第2位上加入氨基或甲基化的氨基，甚至在萘环上适当取代极性基团，都可能提高受体底物的亲和力，从而更好地设计肿瘤转移的双糖抑制剂。

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.