Castellanos-Castro Silvia, Cerda-García-Rojas Carlos M, Javier-Reyna Rosario, Pais-Morales Jonnatan, Chávez-Munguía Bibiana, Orozco Esther
Departamento de Infectómica y Patogénesis Molecular, Mexico.
Colegio de Ciencia y Tecnología, Universidad Autónoma de la Ciudad de México, Dr. García Diego 168, CP 06720, D.F. México, México.
Biochem Biophys Rep. 2015 Dec 23;5:224-236. doi: 10.1016/j.bbrep.2015.12.010. eCollection 2016 Mar.
Phospholipids are essential for vesicle fusion and fission and both are fundamental events for phagocytosis. Our aim was to identify the lysobisphosphatidic acid (LBPA) in trophozoites and investigate its cellular fate during endocytosis. LBPA was detected by TLC in a 0.5 spot of total lipids, which co-migrated with the LBPA standard. The 6C4 antibody, against LBPA recognized phospholipids extracted from this spot. Reverse phase LC-ESI-MS and MS/MS mass spectrometry revealed six LBPA species of / 772.58-802.68. LBPA was associated to pinosomes and phagosomes. Intriguingly, during pinocytosis, whole cell fluorescence quantification showed that LBPA dropped 84% after 15 min incubation with FITC-Dextran, and after 60 min, it increased at levels close to steady state conditions. Similarly, during erythrophagocytosis, after 15 min, LBPA also dropped in 36% and increased after 60 and 90 min. EhRab7A protein appeared in some vesicles with LBPA in steady state conditions, but after phagocytosis co-localization of both molecules increased and in late phases of erythrophagocytosis they were found in huge phagosomes or multivesicular bodies with many intraluminal vacuoles, and surrounding ingested erythrocytes and phagosomes. The 6C4 and anti-EhADH (EhADH is an ALIX family protein) antibodies and Lysotracker merged in about 50% of the vesicles in steady state conditions and throughout phagocytosis. LBPA and EhADH were also inside huge phagosomes. These results demonstrated that LBPA is associated to pinosomes and phagosomes during endocytosis and suggested differences of LBPA requirements during pinocytosis and phagocytosis.
磷脂对于囊泡融合和裂变至关重要，而这两者都是吞噬作用的基本事件。我们的目的是鉴定滋养体中的溶血双磷脂酸（LBPA），并研究其在胞吞作用期间的细胞命运。通过薄层层析法在总脂质的0.5斑点中检测到LBPA，其与LBPA标准品共迁移。针对LBPA的6C4抗体识别从该斑点提取的磷脂。反相液相色谱-电喷雾电离质谱和串联质谱显示有六种LBPA物种，分子量为772.58-802.68。LBPA与胞饮体和吞噬体相关。有趣的是，在胞饮作用期间，全细胞荧光定量显示，与异硫氰酸荧光素-葡聚糖孵育15分钟后，LBPA下降了84%，60分钟后，其水平接近稳态条件时有所增加。同样，在红细胞吞噬作用期间，15分钟后，LBPA也下降了36%，60分钟和90分钟后增加。在稳态条件下，EhRab7A蛋白出现在一些含有LBPA的囊泡中，但吞噬作用后，这两种分子的共定位增加，在红细胞吞噬作用的后期，它们出现在含有许多腔内空泡的巨大吞噬体或多囊泡体中，以及周围摄入的红细胞和吞噬体中。在稳态条件下以及整个吞噬过程中，6C4抗体和抗EhADH（EhADH是一种ALIX家族蛋白）抗体与溶酶体追踪染料在约50%的囊泡中合并。LBPA和EhADH也存在于巨大的吞噬体内。这些结果表明，在胞吞作用期间，LBPA与胞饮体和吞噬体相关，并提示了胞饮作用和吞噬作用期间LBPA需求的差异。
