Keizer Mischa P, Kamp Angela, van Mierlo Gerard, Kuijpers Taco W, Wouters Diana
Department of Immunopathology, Sanquin Blood Supply, Division Research and Landsteiner Laboratory of the Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands.
Department of Pediatric Hematology, Immunology and Infectious Diseases, Emma Children's Hospital, AMC, University of Amsterdam, Amsterdam, Netherlands.
Front Immunol. 2018 Jun 27;9:1406. doi: 10.3389/fimmu.2018.01406. eCollection 2018.
The lectin pathway (LP) of complement activation depends on the activation of the MBL-associated serine proteases (MASPs) circulating in complex with mannan-binding lectin (MBL). MBL deficiency is the most common complement deficiency and has been associated with several pathological conditions. As we had previously shown, plasma-derived MBL (pdMBL) contains pre-activated MASPs that upon pdMBL substitution results in restoration of MBL concentrations but no LP functionality due to immediate inactivation of pdMBL-MASP complexes upon infusion. In this study, we analyzed MBL-sufficient and -deficient serum by size-exclusion chromatography for complexes of LP activation. In both sera, we identified non-bound free forms of MASP-2 and to lesser extent MASP-1/3. After addition of recombinant MBL (rMBL) to MBL-deficient serum, these free MASPs were much less abundantly present, which is highly suggestive for the formation of high-molecular complexes that could still become activated upon subsequent ligand binding as shown by a restoration of C4-deposition of MBL-deficient serum. Ficolin (FCN)-associated MASPs have been described to redistribute to ligand-bound MBL, hereby forming new MBL/MASP complexes. However, reconstitution of MBL-deficient serum with rMBL did not change the relative size of the FCN molecules suggestive for a limited redistribution in fluid phase of already formed complexes. Our findings demonstrate that rMBL can associate with free non-bound MASPs in fluid phase while preserving full restoration of LP functionality. In contrast to pdMBL products containing pre-activated MASPs which become inactivated almost immediately, these current data provide a rationale for substitution studies using rMBL instead.
补体激活的凝集素途径（LP）依赖于与甘露聚糖结合凝集素（MBL）形成复合物循环的MBL相关丝氨酸蛋白酶（MASP）的激活。MBL缺乏是最常见的补体缺乏症，并与多种病理状况相关。正如我们之前所表明的，血浆来源的MBL（pdMBL）含有预激活的MASP，在pdMBL替代后，MBL浓度得以恢复，但由于输注后pdMBL-MASP复合物立即失活，LP功能并未恢复。在本研究中，我们通过尺寸排阻色谱法分析了MBL充足和缺乏的血清中LP激活复合物的情况。在两种血清中，我们都鉴定出了未结合的游离形式MASP-2以及较少程度的MASP-1/3。向MBL缺乏的血清中添加重组MBL（rMBL）后，这些游离MASP的含量大大减少，这强烈提示形成了高分子复合物，如MBL缺乏血清的C4沉积恢复所示，这些复合物在随后的配体结合时仍可被激活。已描述纤维胶凝蛋白（FCN）相关的MASP会重新分布到与配体结合的MBL上，从而形成新的MBL/MASP复合物。然而，用rMBL重建MBL缺乏的血清并没有改变FCN分子的相对大小，这表明已形成的复合物在液相中的重新分布有限。我们的研究结果表明，rMBL可以在液相中与游离的未结合MASP结合，同时保持LP功能的完全恢复。与含有预激活MASP且几乎立即失活的pdMBL产品不同，这些现有数据为使用rMBL进行替代研究提供了理论依据。
