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糖聚糖片段连接糖缀合物：在我们实验室中产生的突变酶可以将具有化学反应性独特官能团的糖残基转移到糖蛋白或糖脂（糖缀合物）的糖片段上。野生型多肽- α - n -乙酰半乳糖氨基转移酶（ppGalNAc-T2）在大肠杆菌中表达，并在体外从包体体中折叠，将具有化学活性的独特官能团的GalNAc片段转移到多肽链上。b4Gal-T1酶将Gal从UDP-Gal转移到存在于受体底物非还原末端的GlcNAc上，其双突变体R228K-Y289L-b4Gal-T1表现出更好的GlcNAc转移酶活性，将GlcNAc从UDP-GlcNAc转移到相同的受体底物上。在本研究中，我们发现这种双突变酶可以从udp - c2 -酮-葡萄糖转移c2 -酮-葡萄糖；然而，只对GlcNAc，而不是对其类似物c2 -酮- glc。此外，我们还发现两种野生型n -乙酰氨基葡萄糖转移酶，合成聚n -乙酰-乳糖胺的人b3GN-T2和参与合成表皮生长因子（EGF）细胞外重复区域聚糖的人MFng，可以容纳供体糖GlcNAc的n -乙酰基，也可以将c2修饰的Glc， c2 -酮-Glc转移到相应的受体。LacNAc分别作用于Asialofetuin （ASF）的n聚糖和来自Factor VII的O- focused EGF repeat。因此，我们的研究结果表明，n -乙酰氨基葡萄糖和n -乙酰半乳糖氨基转移酶的供体糖GlcNAc和GalNAc的n -乙酰基通常嵌入在一个空腔或疏水口袋中，这些空腔或疏水口袋也可以容纳n -乙酰基结合口袋中的酮基或叠氮基。突变型或野生型糖基转移酶将具有化学处理的改性糖残基转移到糖缀合物上的特定糖残基或非糖蛋白中工程多肽的特定位点上，使得通过改性糖残基连接生物活性分子成为可能。我们已经使用这里描述的几个模型系统测试了该策略，并演示了该方法的可行性。在糖缀合物或非糖蛋白的特定位点上存在具有化学柄的独特修饰的糖片段，可以将半乳糖衍生物与突变酶Tyr289Leu转移到卵清蛋白和IgG的聚糖链上的GlcNAc残基上，并将氨基生物素或氨基氟探针偶联到修饰的半乳糖残基上。我们发现突变体Tyr289Leu-Gal-T1酶可以将半乳糖的c2 -酮衍生物从其UDP衍生物转移到卵清蛋白上n -链聚糖链上的GlcNAc残基上，或者转移到没有完全成熟n -聚糖链的IgG分子上。转移后，与生物素化氨基配体偶联到半乳糖C2位置的酮基上，然后用链亲和素- hrp系统处理后用化学发光检测。野生型酶不能利用半乳糖的酮类衍生物。生物素化的氨基配体仅与卵清蛋白的n -聚糖链相连，这已经通过用PNGase F转移酮衍生物后对蛋白质进行处理得到证实，PNGase F从蛋白质上去除n -聚糖链。我们通过质谱分析与igg（如Avastin， Remicade， Rituxan和Herceptin）转移反应前后的糖链，跟踪了改性糖（如2-酮-半乳糖或2-氮化多-半乳糖）的转移，并确定了改性糖转移接近100%的条件。采用MALDI-TOF方法监测IgGs（100%）完全脱半乳糖基化为G0糖型和再半乳糖基化为G2糖型的条件。利用突变酶b4Gal-T1-Y289L，将修饰后的糖从各自的udp -衍生物转移到去半乳糖化的单克隆抗体上。通过质谱分析或化学发光或荧光方法检测，携带改性糖的单抗可以与生物素化衍生物或携带正交反应基团的氟探针完全连接。ELISA方法的使用表明，抗原-抗体相互作用没有被修饰糖转移到igg的n-链聚糖所干扰。因此，治疗性单抗的Fc n -聚糖可以在体外通过使用突变酶b4Gal-T1-Y289L从其udp衍生物中添加具有化学处理的c2修饰的半乳糖（如酮或叠氮化物）进行特异性修饰，并且这种修饰允许修饰的半乳糖偶联到携带正交反应基团的生物分子上。通过间接ELISA技术或荧光活化细胞分选（FACS）方法测定，igg的半乳糖基化或连接不会影响抗体Fab结构域的特异性。因此，通过聚糖将货物分子连接到治疗性单克隆igg的可能性可能被证明是一种宝贵的工具，可以用于(1)检测糖缀合物上的GlcNAc残基，(2)免疫治疗方法的潜在药物靶向，以及(3)开发MRI造影剂。利用多肽- α - n-乙酰半乳糖氨基转移酶（ppGalNAc-T2）对非糖蛋白进行糖偶联的方法：本文描述了一种将非糖蛋白与生物分子进行生物偶联的新方法。使用ppGalNAc-T2，我们将具有化学处理的c2修饰的半乳糖，如酮或叠氮化物，从其各自的udp糖转移到融合到非糖蛋白的受体多肽的丝氨酸/苏氨酸残基上。然后将带有改性半乳糖的蛋白质偶联到携带正交反应基团的生物分子上。作为非糖蛋白的模型系统，我们设计了谷胱甘肽- s -转移酶（GST）蛋白，在c端有一个17个氨基酸长的融合肽，该融合肽在大肠杆菌中表达为可溶性蛋白。ppGalNAc-T2蛋白，催化结构域与c端凝集素结构域，在大肠杆菌中以包涵体形式表达，并开发了一种体外折叠方法，从一升细菌培养物中产生毫克量的活性酶。这种ppGalNAc-T2酶不仅将GalNAc和具有化学柄的c2修饰的半乳糖从udp糖转移到融合肽中的丝氨酸/苏氨酸残基上。半乳糖C2的化学手柄用于偶联和组装生物纳米颗粒，以及为靶向药物输送系统制备免疫脂质体。这种新方法使人们能够用ppGalNAc-T2糖基化重要的生物非糖蛋白，如单链抗体、生长因子或细菌毒素，在分子的c端有一个工程的17个残基肽序列，用于偶联和偶联。该非糖蛋白的糖-生物偶联方法已申请专利。修饰糖核苷酸的合成：通过糖链与糖基转移酶连接各种糖蛋白和非糖蛋白需要携带正交反应基团的修饰糖。修饰糖的设计是根据糖基转移酶的催化腔的结构及其在我们实验室中产生的突变体来确定的。我们正在开发方便的化学酶法合成具有C-2修饰的功能化碳水化合物核苷酸。