Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata 700054, India.
Department of Pharmacy, University of Copenhagen, DK-2100 Copenhagen, Denmark.
Bioconjug Chem. 2021 Aug 18;32(8):1729-1741. doi: 10.1021/acs.bioconjchem.1c00259. Epub 2021 Jul 20.
Conjugation with poly(ethylene glycol) ("PEGylation") is a widely used approach for improving the therapeutic propensities of peptide and protein drugs through prolonging bloodstream circulation, reducing toxicity and immunogenicity, and improving proteolytic stability. In the present study, we investigate how PEGylation affects the interaction of host defense peptides (HDPs) with bacterial lipopolysaccharide (LPS) as well as HDP suppression of LPS-induced cell activation. In particular, we investigate the effects of PEGylation site for KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR), a peptide displaying potent anti-inflammatory effects, primarily provided by its N-terminal part. PEGylation was performed either in the N-terminus, the C-terminus, or in both termini, keeping the total number of ethylene groups ( = 48) constant. Ellipsometry showed KYE28 to exhibit pronounced affinity to both LPS and its hydrophobic lipid A moiety. The PEGylated peptide variants displayed lower, but comparable, affinity for both LPS and lipid A, irrespective of the PEGylation site. Furthermore, both KYE28 and its PEGylated variants triggered LPS aggregate disruption. To investigate the peptide structure in such LPS complexes, a battery of nuclear magnetic resonance (NMR) methods was employed. From this, it was found that KYE28 formed a well-folded structure after LPS binding, stabilized by hydrophobic domains involving aromatic amino acids as well as by electrostatic interactions. In contrast, the PEGylated peptide variants displayed a less well-defined secondary structure, suggesting weaker LPS interactions in line with the ellipsometry findings. Nevertheless, the N-terminal part of KYE28 retained helix formation after PEGylation, irrespective of the conjugation site. For THP1-Xblue-CD14 reporter cells, KYE28 displayed potent suppression of LPS activation at simultaneously low cell toxicity. Interestingly, the PEGylated KYE28 variants displayed similar or improved suppression of LPS-induced cell activation, implying the underlying key role of the largely retained helical structure close to the N-terminus, irrespective of PEGylation site. Taken together, the results show that PEGylation of HDPs can be done insensitively to the conjugation site without losing anti-inflammatory effects, even for peptides inducing such effects through one of its termini.
聚乙二醇（"PEGylation"）缀合是一种广泛应用的方法，通过延长血液循环、降低毒性和免疫原性以及提高蛋白水解稳定性，来改善肽和蛋白质药物的治疗倾向。在本研究中，我们研究了 PEGylation 如何影响宿主防御肽（HDPs）与细菌脂多糖（LPS）的相互作用，以及 HDP 对 LPS 诱导的细胞激活的抑制作用。特别是，我们研究了 PEGylation 位点对 KYE28（KYEITTIHNLFRKLTHRLFRRNFGYTLR）的影响，该肽显示出强大的抗炎作用，主要来自其 N 端部分。PEGylation 分别在 N 端、C 端或两端进行，保持乙烯基总数（=48）不变。椭圆测量显示 KYE28 对 LPS 及其疏水性脂质 A 部分都表现出明显的亲和力。PEG 化肽变体对 LPS 和脂质 A 的亲和力较低，但相当，无论 PEG 化位点如何。此外，KYE28 及其 PEG 化变体都能触发 LPS 聚集体的破坏。为了研究肽在这种 LPS 复合物中的结构，我们采用了一系列核磁共振（NMR）方法。由此发现，KYE28 与 LPS 结合后形成了一个折叠良好的结构，由涉及芳香族氨基酸的疏水性结构域和静电相互作用稳定。相比之下，PEG 化肽变体显示出不太明确的二级结构，表明与椭圆测量结果一致的 LPS 相互作用较弱。然而，KYE28 的 N 端在 PEG 化后仍然保持螺旋形成，无论结合位点如何。对于 THP1-Xblue-CD14 报告细胞，KYE28 在同时低细胞毒性的情况下，对 LPS 激活具有强大的抑制作用。有趣的是，PEG 化的 KYE28 变体对 LPS 诱导的细胞激活显示出相似或改善的抑制作用，这表明靠近 N 端的保留的螺旋结构在很大程度上发挥了关键作用，无论 PEG 化位点如何。总的来说，结果表明，PEGylation 可以在不失去抗炎作用的情况下，不敏感地进行 HDP 的缀合，即使是通过其一端诱导这种作用的肽也是如此。
