Supplementary file 1_Conformational stability of hemocyanins regulates their lysosomal and proteasomal degradation, influencing their pro-inflammatory effects on mammalian antigen-presenting cells.docx

IntroductionMollusk hemocyanins are known for their immunomodulatory properties in mammals. Their applications include serving as carrier glycoproteins, functioning as protein-based adjuvants, and acting as non-specific immunostimulants in cancer vaccine strategies. Their immunomodulatory effects are attributed to their xenogenicity, structural complexity, high molecular mass, and glycosylations. Recent studies have begun to clarify the immunological mechanisms by which hemocyanins induce: multiligand properties arising from interactions with C-type lectins and Toll-like receptors, and the promotion of a Th1 immune response. However, the subsequent effects of hemocyanins, particularly their intracellular targeting and degradation kinetics, remain poorly understood. The present study is the first to comprehensively examine the processing of two well-characterized hemocyanins, known for their conformational stability and clinical significance: KLH from Megathura crenulata and CCH from Concholepas concholepas.
MethodsWe correlated their degradation with the kinetics of the proinflammatory response they induce and their subcellular localization using the JAWS II cell line and bone marrow-derived dendritic cells (BMDCs). We utilize OVA to highlight the differences between this protein and the hemocyanins.
ResultsThe results showed that KLH and CCH induced significant TNF levels after 24 hours and also promoted the secretion of IL-6 and IL-12p40 after 96 hours, along with the upregulation of CD80 and CD86. This delayed response corresponds with their slow intracellular degradation. Colocalization studies using LAMP-1 demonstrated that hemocyanins were localized to lysosomes only after prolonged stimulation, suggesting that they are likely stored in intracellular depots. Furthermore, hemocyanins were shown to colocalize with LMP-2 and a4, indicating that they undergo processing in the proteasome. In contrast, OVA displayed faster degradation with mild pro-inflammatory effects within 24 hours. Pharmacological inhibition of lysosomal cathepsins or the proteasome reduced the hemocyanin-dependent secretion of IL-6 and IL-12p40. Additionally, fragmented hemocyanins led to lower, less sustained cytokine levels compared to their native form.
ConclusionsThese findings emphasize that hemocyanins, owing to their complex oligomeric structure and high stability, are slowly processed by APCs, thereby contributing to their immunogenicity. This property is particularly relevant when hemocyanins are used as carriers for vaccine antigens because their delayed kinetics can enhance the magnitude, quality, and persistence of responses.

引言：软体动物血蓝蛋白（Mollusk hemocyanins）以其在哺乳动物体内的免疫调节特性而广受认可。其应用场景包括作为载体糖蛋白、基于蛋白质的佐剂，以及在癌症疫苗策略中充当非特异性免疫刺激剂。该类蛋白的免疫调节效应源于其异种源性、结构复杂性、高分子量以及糖基化修饰。近期研究已逐步阐明血蓝蛋白诱导免疫应答的免疫学机制：其与C型凝集素（C-type lectins）和Toll样受体（Toll-like receptors）相互作用所产生的多配体特性，以及对Th1型免疫应答的促进作用。然而，血蓝蛋白的后续作用效应，尤其是其细胞内靶向与降解动力学特征，仍未得到充分解析。本研究首次全面探究了两种特性明确的血蓝蛋白的加工过程——这两种蛋白以构象稳定性与临床应用价值见长：分别为源自大螯石鳖（Megathura crenulata）的KLH以及源自智利海螺（Concholepas concholepas）的CCH。
方法：本研究利用JAWS II细胞系与骨髓源树突状细胞（bone marrow-derived dendritic cells, BMDCs），将血蓝蛋白的降解与其诱导的促炎应答动力学以及亚细胞定位相关联。我们以卵清蛋白（OVA）作为对照，以凸显该蛋白与血蓝蛋白之间的实验差异。
结果：实验结果显示，KLH与CCH在24小时后即可诱导显著的TNF分泌，并在96小时后促进IL-6与IL-12p40的分泌，同时上调CD80与CD86的表达。这种延迟应答与其缓慢的细胞内降解过程高度契合。使用LAMP-1进行的共定位研究表明，血蓝蛋白仅在长时间刺激后才会定位于溶酶体，提示它们可能被储存于细胞内贮库中。此外，研究证实血蓝蛋白可与LMP-2及α4发生共定位，表明其在蛋白酶体中完成加工过程。与之形成鲜明对比的是，OVA的降解速度更快，并在24小时内仅表现出轻度的促炎效应。对溶酶体组织蛋白酶或蛋白酶体进行药理学抑制，可显著降低血蓝蛋白依赖的IL-6与IL-12p40分泌。此外，与天然形式的血蓝蛋白相比，裂解后的血蓝蛋白所诱导的细胞因子水平更低、持续时间更短。
结论：本研究结果表明，血蓝蛋白凭借其复杂的寡聚体结构与高稳定性，可被抗原呈递细胞（antigen-presenting cells, APCs）缓慢加工，进而增强其免疫原性。这一特性在血蓝蛋白被用作疫苗抗原载体时尤为关键，因为其延迟的加工动力学可提升免疫应答的强度、质量与持久性。