Utilizing Glycosyltransferases for Bioconjugation

利用糖基转移酶进行生物共轭

• 批准号: 8552799
• 负责人: Pradman K Qasba
• 金额: $ 20.9万
• 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/8552799
• 关键词: Affect; Amino Acids; Antibodies; Antigens; Avastin; Azides; Bacterial Toxins; Binding; Biological; Biological Models; Biotin; Blood Coagulation Factor VII; C-terminal; Carbohydrates; Catalytic Domain; Cattle; Chemicals; Complex; Contrast Media; Coupled; Coupling; Detection; Drug Delivery Systems; Engineering; Enzyme-Linked Immunosorbent Assay; Enzymes; Epidermal Growth Factor; Escherichia coli; Exhibits; Extracellular Domain; Fab domain; Fluorescence-Activated Cell Sorting; Galactose; Galactosyltransferases; Glutathione S-Transferase; Glycoconjugates; Glycolipids; Glycoproteins; Growth Factor; Human; Immunoglobulin G; Immunotherapeutic agent; In Vitro; Inclusion Bodies; Ketones; Laboratories; Lectin; Legal patent; Ligands; Link; Liposomes; Magnetic Resonance Imaging; Measures; Methodology; Methods; Modification; Monitor; Monoclonal Antibodies; Mutate; Mutation; N-Acetylgalactosaminyltransferases; N-Acetylglucosaminyltransferases; Nucleotides; Ovalbumin; Peptide N-glycohydrolase F; Peptides; Polypeptide N-acetylgalactosaminyltransferase; Polysaccharides; Positioning Attribute; Post-Translational Protein Processing; Preparation; Proteins; Reaction; Roche brand of rituximab; Roche brand of trastuzumab; Site; Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Streptavidin; Structure; System; Techniques; Testing; Therapeutic; Therapeutic Monoclonal Antibodies; Time; Transferase; UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase; Uridine Diphosphate Sugars; analog; asialofetuin; base; design; functional group; glycosyltransferase; infliximab; lactosamine; milligram; mutant; notch protein; novel; polypeptide; protein aminoacid sequence; sugar; sugar nucleotide; tool

Utilization of wild-type and mutant glycosyltransferase for linking glycoconjugates via glycan moieties: The mutant enzymes that have been generated in our laboratory can transfer a sugar residue with a chemically reactive unique functional group to a sugar moiety of a glycoprotein or glycolipids (glycoconjugates). Also wild-type polypeptide-alpha-N-acetylgalactosaminyltransferase (ppGalNAc-T2) that has been expressed in E. coli and in vitro folded from inclusion bodies transfers GalNAc moiety with a chemically reactive unique functional group to a polypeptide chain.The b4Gal-T1 enzyme transfers Gal from UDP-Gal to GlcNAc present at the non-reducing end of an acceptor substrate, and its double mutant R228K-Y289L-b4Gal-T1 exhibits better GlcNAc-transferase activity where it transfers GlcNAc from UDP-GlcNAc to the same acceptor substrate. In the present study we find that this double-mutant enzyme can transfer C2-keto-Glc from UDP-C2-keto-Glc; however, only to GlcNAc, not to its analogue, C2-keto-Glc. Furthermore, we also show that the two wild-type N-acetylglucosaminyltransferases, human b3GN-T2 that synthesizes the poly-N-acetyl-lactosamine, and the human MFng that is involved in the synthesis of glycan of epidermal growth factor (EGF) repeats of the extracellular domains of the Notch receptor, which accommodate the N-acetyl group of the donor sugar GlcNAc, can also transfer the C2-modified Glc, C2-keto-Glc, to their corresponding acceptors, LacNAc on the N-glycans of Asialofetuin (ASF) and O-fucosylated EGF repeat from Factor VII, respectively. Thus our results suggest that the N-acetyl groups of the donor sugars GlcNAc and GalNAc of the N-acetylglucosaminyl- and N-acetylgalactosaminyl-transferases are generally embedded in a cavity or a hydrophobic pocket which can also accommodate a ketone group or an azido group in the N-acetyl-binding pocket. The transfer of a modified sugar residue that has a chemical handle by the mutant or wild type glycosyltransferases to a specific sugar residue on a glycoconjugate or to a specific site in a polypeptide engineered in the non-glycoprotein makes it possible to link bioactive molecules via the modified sugar residue. We have tested this strategy, using a few model systems described here and demonstrate the feasibility of this approach.The presence of a unique modified sugar moiety with a chemical handle at a specific site on a glycoconjugate or a non-glycoprotein makes it possible to transfer galactose derivative to GlcNAc residues on the glycan chains of Ovalbumin and IgG with the mutant enzyme Tyr289Leu and coupling of the aminooxy-biotin or aminooxy- fluoroprobes to the modified galactose residue: We showed that the mutant Tyr289Leu-Gal-T1 enzyme can transfer the C2-ketone derivative of galactose from its UDP derivative to the GlcNAc residue on the N-linked glycan chain on Ovalbumin or to an IgG molecule which does not have a fully matured N-glycan chain. The transfer is followed by coupling to the ketone group at the C2 position of galactose with the biotinylated aminooxy ligand, which was then detected by chemiluminescence after treating with the streptavidin-HRP system. The wild-type enzyme can not utilize the ketone derivative of galactose. That the biotinylated aminooxy ligand is linked only to the N-glycan chain of Ovalbumin has been confirmed by treatment of the proteins after the transfer of ketone derivatives with PNGase F, which removes N-glycan chains from the protein. We have followed the transfer of the modified sugars, like 2-keto-galactose or 2-azido-galactose, by MS analysis of the sugar chain, before and after the transfer reaction with IgGs, e.g Avastin, Remicade, Rituxan and Herceptin, and established the conditions where the transfer of modified sugar is nearly 100%. MALDI-TOF methodology was used to monitor the conditions for the complete de-galactosylation of the IgGs (100%) to G0 glycoform and the re-galactosylation to G2 glycoform. Using mutant enzyme b4Gal-T1-Y289L, modified sugars were transferred from the respective UDP-derivatives to the de-galactosylated MAb. The MAb carrying modified sugar could be completely linked to biotinylated derivatives or fluoroprobe probes carrying an orthogonal reactive group as monitored by MS analysis or chemiluminescence or florescence methods. Use of ELISA methodology shows that antigen-antibody interactions have not been disturbed by the transfer of modified sugars to the N-linked glycans of the IgGs.Thus, the Fc N-glycans of therapeutic MAb can be specifically modified in vitro by the addition of C2-modified galactose having a chemical handle, such as ketone or azide, from its UDP-derivative using the mutant enzyme b4Gal-T1-Y289L, and that this modification permits the coupling of the modified galactose to a bio-molecule that carries an orthogonal reactive group. Re-galactosylation or linking of the IgGs does not affect the specificity of the Fab domain of the antibodies measured by indirect ELISA techniques or fluorescence activated cell sorting (FACS) methods. Thus, the possibility of linking cargo molecules to therapeutic monoclonal IgGs via glycans could prove to be an invaluable tool for (1) detection of GlcNAc residues on glycoconjugates, (2) potential drug targeting by immunotherapeutic methods, and (3) for developing contrast agents for MRI.Method to use polypeptide-alpha-N-acetylgalactosaminyltransferase (ppGalNAc-T2) for the glycoconjugation of non-glycoproteins: Here we describe a new method for bioconjugation of a non-glycoprotein with bio-molecules. Using ppGalNAc-T2, we transfer a C2-modified galactose that has a chemical handle, such as ketone or azide, from its respective UDP-sugars to the Ser/Thr residue(s) of an acceptor polypeptide fused to the non-glycoprotein. The protein with the modified galactose is then coupled to a bio-molecule that carries an orthogonal reactive group. As a model system for the non-glycoprotein, we engineered glutathione-S-transferase (GST) protein with a 17-amino-acid-long fusion peptide at the C-terminal end that was expressed as a soluble protein in E. coli. The ppGalNAc-T2 protein, the catalytic domain with the C-terminal lectin domain, was expressed as inclusion bodies in E. coli, and an in vitro folding method was developed to produce milligram quantities of the active enzyme from a liter of bacterial culture. This ppGalNAc-T2 enzyme transfers from the UDP-sugars, not only GalNAc, but also C2-modified galactose that has a chemical handle, to the Ser/Thr residue(s) in the fusion peptide. The chemical handle at the C2 of galactose is used for conjugation and assembly of bionanoparticles and preparation of immuno-liposomes for a targeted drug delivery system. This novel method enables one to glycosylate with ppGalNAc-T2 the important biological non-glycoproteins, such as single-chain antibodies, growth factors, or bacterial toxins, with an engineered 17-residue peptide sequence at the C-terminus of the molecule, for conjugation and coupling. Patent has been filled on this glyco-bioconjugation method of non-glycoproteins.Synthesis of Modified Sugar Nucleotides: Linking of various glycoproteins and non-glycoproteins via glycan chains with glycosyltransferases requires modified sugars that carry orthogonal reactive groups. The design of the modified sugars is determined based on the structure of the catalytic cavity of the glycosyltransferase and their mutants that are being generated in our laboratory. We are developing convenient chemoenzymatic methods of synthesis of functionalize carbohydrate nucleotides with C-2 modifications.

利用野生型和突变型糖基转移酶通过聚糖部分将糖缀合物连接起来：我们实验室中生成的突变酶可以将具有化学反应性独特功能基团的糖残基转移到糖蛋白或糖蛋白Orcopolipids的糖中的糖残留物（果糖）（果胶）（果糖）。野生型多肽 - α-N-乙酰乳糖酰二氨基转移酶（PPGALNAC-T2）已在大肠杆菌中表达，并从包含体中折叠的体外折叠将GalNAC部分转移到GalNAC中，用化学反应性的独特功能组转移到B4Gal-T1 Enzyzeme Tressn eNZeme Tress.glc galc galcer。受体底物的非还原端及其双突变体R228K-Y289L-B4GAL-T1具有更好的GlcNAC转移酶活性，其中它将GlcNAC从UDP-GLCNAC转移到同一受体底物。在本研究中，我们发现该双突变酶可以从UDP-C2-keto-GLC转移C2-Keto-GLC。但是，仅针对GlcNAC，而不是与其类似物C2-Keto-GLC。此外，我们还表明，合成多聚-N-乙酰基酰胺胺的两个野生型N-乙酰葡萄糖氨基转移酶，人B3GN-T2，以及与表皮生长因子（EGF）重复的notacellulth domains of notac of notac of notacellulch domers of notac of notacellulch domer的人类MFNG相关的人类MFNG。供体糖GLCNAC还可以将C2改性的GLC C2-Keto-GLC转移到其相应的受体中，分别从Asialofetuin（ASF）的N-聚糖中将LACNAC和O-cucosylated EGF从因子VII中重复分别转移到FIANSE VII中。因此，我们的结果表明，供体糖GlcNAC的N-乙酰基组和N-乙酰基葡萄氨酰和N-乙酰基乳糖酰乳糖胺基转移酶的N-乙酰基通常被嵌入一个腔或疏水性袋中，这些口袋也可以容纳酮基组或a唑酮组中的n-核心组。通过糖缀合物或非糖蛋白在多肽中的特定位点上的特定糖残基的化学手柄的改性糖残留物转移到非糖蛋白中设计的特定位点，从而使通过修饰的糖残基在非糖蛋白中工程的特定位点可以链接生物活性分子。我们已经使用此处描述的一些模型系统测试了该策略，并证明了这种方法的可行性。在糖缀合物或非糖蛋白上，在特定地点的特定地点存在一个独特的修改糖部分，可以将半乳糖衍生物转移到glcnac残留物上的glcnac残留物，并与Ovalbumin ovant cofant and croutance and cotant couty coty couty congg，cofant and cofant and cotant和im iggggly couounaC残留物。 the aminooxy-biotin or aminooxy- fluoroprobes to the modified galactose residue: We showed that the mutant Tyr289Leu-Gal-T1 enzyme can transfer the C2-ketone derivative of galactose from its UDP derivative to the GlcNAc residue on the N-linked glycan chain on Ovalbumin or to an IgG molecule which does not have a fully matured n-Glycan链。转移之后是在半乳糖的C2位置与生物素化氨基配体的酮基偶联，然后在用链霉亲和素HRP系统处理后，通过化学发光检测到酮基。 野生型酶不能利用半乳糖的酮衍生物。生物素化的aminooxy配体仅与N-聚糖链相关，已通过使用PNGASE F转移酮衍生物后通过处理蛋白质来证实，从而去除蛋白质的N-聚糖链。我们遵循了通过MS对糖链的分析，在与IgGs转移反应之前和之后的MS分析（例如Avastin，Remicade，Remicade，Rituxan和Herceptin）的转移，例如2-酮 - 半乳糖或2-齐多半乳糖，并确定了修饰糖转移的条件几乎为100％。 MALDI-TOF方法用于监测IgGS（100％）对G0糖型的完全去乳糖基化的条件，并将重新乳糖基化对G2糖型。使用突变酶B4GAL-T1-Y289L，将修饰的糖从相应的UDP衍生物转移到去乳糖基化的mAb。携带改性糖的mAb可以完全连接到通过MS分析或化学发光或荧光方法监测的携带正交反应性基团的生物素化衍生物或氟探针探针。 ELISA方法论的使用表明，抗原 - 抗体相互作用并未被修饰的糖转移到IgG的N连接的糖基因的N-连接的糖基因上，因此，可以通过添加具有C2固定的Galactose（例如，从乳胶中添加化学物质或a z）来在体外特异性地修饰治疗MAB的FC N-聚糖。 B4GAL-T1-Y289L，并且这种修饰允许将修饰的半乳糖与带有正交反应组的生物分子偶联。 IgGS的重新乳糖基化或连接不影响通过间接ELISA技术或荧光激活细胞排序（FACS）方法测量的抗体的FAB结构域的特异性。因此，将货物分子与通过聚糖连接到治疗性的单克隆IgG的可能性可能是（1）在糖节上检测GlcNAC残留物的宝贵工具，（2）通过免疫治疗方法靶向潜在的药物来靶向免疫治疗方法，以及（3）对MRI的使用MRI。非糖蛋白的糖结合的多肽-Alpha-N-乙酰乳糖酰乳糖苷转移酶（PPGALNAC-T2）：在这里，我们描述了一种与生物分子的非糖蛋白生物结合的新方法。使用PPGALNAC-T2，我们将具有化学手柄（例如酮或叠氮化物）的C2改性半乳糖从其各自的UDP-糖从融合到非糖蛋白融合的受体多肽的SER/THR残基（S）。然后将带有修饰半乳糖的蛋白质耦合到携带正交反应基的生物分子。作为非糖蛋白的模型系统，我们在C末端设计了具有17-氨基酸的长融合肽的谷胱甘肽-S-转移酶（GST）蛋白，该蛋白在C末端以大肠杆菌中的可溶性蛋白表示。 pPGALNAC-T2蛋白是与C末端凝集素结构域的催化结构域，表示为大肠杆菌中的包含体，并开发了一种体外折叠方法来产生来自细菌培养物的毫克活性酶。这种PPGALNAC-T2酶从UDP糖中不仅从galnac转移，而且还从融合肽中的Ser/Thr残基（S）转移到具有化学手柄的C2修饰的半乳糖。半乳糖C2处的化学手柄用于偶联和组装二酮粒子，并为靶向药物递送系统制备免疫脂质体。这种新型方法使人们可以使用PPGALNAC-T2糖基化的一种重要的生物学非糖蛋白，例如单链抗体，生长因子或细菌毒素，并具有17个沉积的肽序列在分子的C-末端，用于分子的C-末端，用于分子和偶数。在这种非糖蛋白的这种糖生物结合方法上已填写了专利。修饰的糖核苷酸的结合：通过聚糖链通过聚糖链与糖基转移酶与需要修改的糖需要携带正交反应性组的糖。改性糖的设计是根据在我们实验室中生成的糖基转移酶及其突变体的催化腔的结构来确定的。我们正在开发具有C-2修饰的碳水化合物核苷酸官能化的合成化学化学方法。