具有IgG糖基化修饰活性的糖苷内切酶Endo-SB的分子改造

Chemoenzymatic glycan remodeling approach by endo-β-N-acetylglucosaminidase-catalyzed deglycosylation and endo-β-N-aetylglucosaminidase mutant-catalyzed transglycosylation is emerging as a highly efficient strategy for synthesizing structurally well-defined, homogeneous N-glycoproteins including intact antibodies in structure-function relationship study and in therapeutic drug research of glycoproteins, and the realization of increasingly board application of this method relies on the continuous identification and characterization of efficient endo-β-N-acetylglucosaminidases and their glycosynthase mutants to expand the availability of the synthetic toolkit. ..Although many ENGases have been characterized from various organisms, so far no fucose-containing oligosaccharides-specific ENGase has been identified in any organism. In previous study, we screened soil samples, using dansyl chloride (Dns)-labeled sialylglycopeptide (Dns-SG) as a substrate, and a strain, that exhibits ENGase activity in the culture supernatant, was discovered; this strain, named as strain HMA12, was identified as a Sphingobacterium species by 16S ribosomal RNA gene analysis. By draft genome sequencing, five candidate ENGase encoding genes were identified in the genome of this strain. The recombinant protein, purified from Escherichia coli expressing candidate gene ORF1188 exhibited fucose-containing oligosaccharides-specific ENGase activity. This ENGase, named Endo-SB, exhibited optimum activity at very acidic pH (pH 2.5). Recombinant Endo-SB released the fucose-containing oligosaccharides residues from rituximab (immunoglobulin G) but not the high-mannose-containing oligosaccharides residues from RNase B, a result that not only confirmed the substrate specificity of this novel ENGase but also suggested that natural glycoproteins could be their substrates. ..In this study, the transglycosylation activity of Endo-SB on human IgG will be studied. Glycosynthase mutants from Endo-SB will be generated by molecular engineering, such as single or double amino acid substitution. Our finding will identify new glycosynthases with transglycosylation activity for antibody glycosylation remodeling, which is very useful for the convergent synthesis of optimised antibody therapeutics with improved pharmacokinetic properties.

由于糖蛋白糖链的不均一性，阻碍了糖链结构和功能的研究，也阻碍了治疗性糖蛋白药物的开发利用。糖苷内切酶法合成均一糖链的糖蛋白，是目前发展最快且最有发展前景的方法。然而，该方法的广泛和深入的应用依赖于不断的发掘，不同底物特异性的糖苷内切酶和由其突变得到的糖苷合成酶。前期研究通过对土壤微生物进行筛选，我们发掘到了新型的糖苷内切酶Endo-SB（来源于鞘氨醇杆菌）。该酶能够特异性作用于含有核心岩藻糖结构的二天线复杂型N-糖链，并且能够水解人类免疫球蛋白IgG的Fc糖链。本申请拟研究，通过对糖苷内切酶Endo-SB的分子改造，筛选出具有高效转糖基活性的糖苷合成酶突变体，为合成带有均一糖链糖蛋白的糖苷内切酶法，添加工具酶。该工具酶有望替代昂贵的Endo-S，应用到对IgG的糖基化改造。另外，对Endo-SB的底物特异性分子机制进行研究，将为ENGase催化机制研究提供证据和有用信息。

肿瘤免疫治疗策略的探索在很大程度上依赖于肿瘤免疫逃逸分子机制的发现。根据不同的机制，可能需要不同的治疗策略。主要组织相容性复合体I类分子(MHC-I)的肿瘤细胞表面表达缺陷是肿瘤免疫逃逸的最重要原因之一。因此,揭示肿瘤细胞低表达MHC-I的新机制,将为肿瘤免疫治疗提供新思路。.MHC-I的N糖基化修饰对其细胞膜的表达水平有显著影响。我们深入研究了特定的某一种N糖基化修饰的糖型（待文章发表后将公开此特定糖型，在此以“基因A”代称控制此糖型合成的基因）对MHC-I细胞膜表达水平的影响，并对更上游的基因调控机制进行了探索，找到了“基因A”新的上游分子“基因B7” （待文章发表后将公开此基因，在此以“基因B7”代称此基因）。.大数据分析发现，“基因A”在乳腺癌和肺腺癌中低表达，其高表达预后好。构建过表达“基因A”的小鼠肺癌和乳腺癌细胞系荷瘤小鼠发现，过表达“基因A”抑制肿瘤生长。但是，体外实验显示，过表达“基因A”不抑制肿瘤细胞体外增殖，这说明“基因A”是通过免疫系统抑制了肿瘤生长。流式细胞术检测发现，过表达“基因A”导致肿瘤组织中T细胞数量增多和功能增强。肿瘤细胞和T细胞共孵育实验证实，过表达“基因A”增强肿瘤细胞对CD8 T细胞杀伤的敏感性。蛋白组学测序揭示出，过表达“基因A”提高肿瘤细胞MHC-I在细胞膜的含量。分子机制上发现，过表达“基因A”抑制MHC-I的溶酶体降解，增强其细胞膜表面稳定性。对更上游的基因调控机制进行探索，发现“基因B7”通过“基因A”调控MHC-I表达。“基因A”的上游调控分子“基因B7”可以作为药理学的切入点，通过靶向“基因B7”，上调“基因A”，进而上调MHC-I，进而控制肿瘤生长。低浓度的“基因B7”抑制剂联合PD-1抗体治疗，能更好的控制肿瘤生长。该研究成果具有一定的临床医学转化潜力。

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