Ogata M, Fletcher M F, Kloczewiak M, Loiselle P M, Zanzot E M, Vermeulen M W, Warren H S
Department of Pediatrics, Massachusetts General Hospital and Harvard Medical School, Boston, USA.
Infect Immun. 1997 Jun;65(6):2160-7. doi: 10.1128/iai.65.6.2160-2167.1997.
The 18-kDa cationic protein CAP18 is an antimicrobial protein isolated from rabbit granulocytes that binds lipopolysaccharide (LPS) and inhibits many of its biological activities. We covalently coupled a synthetic peptide representing amino acids 106 to 138 of CAP18 to human immunoglobulin G (IgG) by using the heterobifunctional linker N-succinimidyl-3-(2-pyridyidithio)propionate. The ability of CAP18(106-138)-IgG to bind and neutralize LPS in whole blood in the presence and absence of anticoagulants was studied. Both CAP18(106-138) and CAP18(106-138)-IgG significantly suppressed LPS-induced tumor necrosis factor (TNF) production in whole blood in the absence of anticoagulants. EDTA potentiated the ability of CAP18(106-138) and CAP18(106-138)-IgG to decrease LPS-induced TNF production in a dose-dependent manner. In contrast, heparin inhibited the ability of CAP18(106-138) and CAP18(106-138)-IgG to suppress LPS-induced TNF production. EDTA also enhanced LPS capture in a fluid-phase binding assay that utilizes magnetic anti-IgG beads to capture CAP18(106-138)-IgG (and bound [3H]LPS) in whole blood. In contrast, heparin inhibited the binding dose dependently. We conclude that CAP18(106-138)-IgG binds to and neutralizes LPS in whole blood in the absence of anticoagulants. Further studies of its protective efficacy in animal models are warranted. Caution should be used in interpreting assays that measure the binding and neutralization of LPS in whole blood in the presence of calcium-binding anticoagulants or heparin.
18 kDa阳离子蛋白CAP18是一种从兔粒细胞中分离出的抗菌蛋白，它能结合脂多糖（LPS）并抑制其多种生物学活性。我们通过使用异双功能连接剂N-琥珀酰亚胺基-3-（2-吡啶基二硫代）丙酸酯，将代表CAP18第106至138位氨基酸的合成肽与人类免疫球蛋白G（IgG）共价偶联。研究了CAP18（106 - 138）-IgG在有无抗凝剂存在的情况下结合并中和全血中LPS的能力。在无抗凝剂时，CAP18（106 - 138）和CAP18（106 - 138）-IgG均能显著抑制全血中LPS诱导的肿瘤坏死因子（TNF）产生。乙二胺四乙酸（EDTA）以剂量依赖方式增强了CAP18（106 - 138）和CAP18（106 - 138）-IgG降低LPS诱导的TNF产生的能力。相反，肝素抑制了CAP18（106 - 138）和CAP18（106 - 138）-IgG抑制LPS诱导的TNF产生的能力。在利用磁珠抗IgG捕获全血中CAP18（106 - 138）-IgG（以及结合的[³H]LPS）的液相结合试验中，EDTA也增强了LPS捕获。相反，肝素剂量依赖性地抑制结合。我们得出结论，在无抗凝剂时，CAP18（106 - 138）-IgG能结合并中和全血中的LPS。有必要在动物模型中对其保护效果进行进一步研究。在解释存在钙结合抗凝剂或肝素时全血中LPS结合和中和的测定结果时应谨慎。
