Table_2_Establishment of a human cell line with a surface display system for screening and optimizing Na+-taurocholate cotransporting polypeptide-binding peptides.XLSX

One of the most desirable targets for HBV medications is the sodium taurocholate cotransporting polypeptide (NTCP), an entry receptor for the hepatitis B virus (HBV). N-myristoylated preS1 2–48 (Myrcludex B or Hepcludex), an NTCP-binding peptide from the large surface protein of HBV, has been developed as the first-in-class entry inhibitor. However, its relatively large molecular weight contributes to increased immunogenicity and antibody production. As a result, it is preferable to look for an NTCP-binding peptide with a smaller size. To do this, we developed a human cell surface display strategy and screened peptides based on preS1-21. PreS1-21 (genotype D) was extended by 7 random amino acids and fused with mCherry and FasL transmembrane domain. The pooled constructs were transfected into HEK293 cells by using the transposon/transposase system to create a library displaying various peptides on the cell surface with red fluorescence. On the other hand, we expressed NTCP protein fused with EGFP on HEK293 and used the membrane lysate containing NTCP-GFP as the bait protein to select peptides with increased NTCP affinity. After 7 cycles of selection, the deep sequencing results revealed that some polypeptides were more than 1,000 times enriched. Further screening of the mostly enriched 10 peptides yields the peptide preS1-21-pep3. Replacing the preS1-21 sequence of preS1-21-pep3 with those from different genotypes demonstrated that the consensus sequence of genotype A–F had the best performance. The peptide (Myr-preS1-21-pep3) was synthesized and tested on the HepG2-NTCP cell model. The results showed that Myr-preS1-21-pep3 is approximately 10 times more potent than the initial peptide Myr-preS1-21 in preventing HBV infection. In conclusion, we developed a new strategy for screening peptides binding to membrane proteins and identified a new NTCP-binding peptide with a much smaller size than Hepcludex.

HBV（乙型肝炎病毒）药物的理想靶点之一为钠牛磺胆酸共转运多肽（NTCP），其为HBV的进入受体。N-肉豆蔻酰化前S1 2–48（Myrcludex B或Hepcludex），一种源自HBV大型表面蛋白的与NTCP结合的肽段，已被开发为首个进入抑制剂。然而，其相对较大的分子量导致免疫原性增强和抗体产生增加。因此，寻找一种尺寸更小的NTCP结合肽段显得尤为重要。为此，我们开发了一种人类细胞表面展示策略，并基于前S1-21筛选肽段。前S1-21（基因型D）通过7个随机氨基酸的延伸并与mCherry和FasL跨膜结构域融合。将这些构建体通过转座子/转座酶系统转染至HEK293细胞，从而创建了一个在细胞表面展示多种肽段并具有红色荧光的文库。另一方面，我们在HEK293细胞中表达与EGFP融合的NTCP蛋白，利用含有NTCP-GFP的膜裂解物作为诱饵蛋白，以选择具有增强NTCP亲和力的肽段。经过7轮筛选后，深度测序结果表明，某些多肽的富集程度超过1000倍。进一步筛选最富集的10个肽段，得到肽段preS1-21-pep3。将preS1-21-pep3中的preS1-21序列替换为不同基因型的序列，表明基因型A-F的共识序列具有最佳性能。该肽段（Myr-preS1-21-pep3）被合成并测试于HepG2-NTCP细胞模型。结果表明，Myr-preS1-21-pep3在预防HBV感染方面的活性大约是初始肽段Myr-preS1-21的10倍。总之，我们开发了一种新的筛选结合膜蛋白肽段的方法，并鉴定出一种比Hepcludex尺寸小得多的新型NTCP结合肽段。