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* Centro de Investigación del Cáncer, Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Cientificas-Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, Spain;
Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands; and
Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi, Japan
The correct mobilization of cytoplasmic granules is essential for the proper functioning of human neutrophils in host defense and inflammation. In this study, we have found that human peripheral blood neutrophils expressed high levels of Rab27a, whereas Rab27b expression was much lower. This indicates that Rab27a is the predominant Rab27 isoform present in human neutrophils. Rab27a was up-regulated during neutrophil differentiation of HL-60 cells. Subcellular fractionation and immunoelectron microscopy studies of resting human neutrophils showed that Rab27a was mainly located in the membranes of specific and gelatinase-enriched tertiary granules, with a minor localization in azurophil granules. Rab27a was largely absent from CD35-enriched secretory vesicles. Tertiary and specific granule-located Rab27a population was translocated to the cell surface upon neutrophil activation with PMA that induced exocytosis of both tertiary and specific granules. Specific Abs against Rab27a inhibited Ca2+ and GTP-
-S activation and PMA-induced exocytosis of CD66b-enriched tertiary and specific granules in electropermeabilized neutrophils, whereas secretion of CD63-enriched azurophil granules was scarcely affected. Human neutrophils lacked or expressed low levels of most Slp/Slac2 proteins, putative Rab27 effectors, suggesting that additional proteins should act as Rab27a effectors in human neutrophils. Our data indicate that Rab27a is a major component of the exocytic machinery of human neutrophils, modulating the secretion of tertiary and specific granules that are readily mobilized upon neutrophil activation.
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1 This work was supported in part by Grants SAF2005-04293 and SAF2008-00251 from Ministerio de Educación y Ciencia of Spain, Fundación de Investigación Médica Mutua Madrileña (FMM), Grant BM05-30-0) from Fundación "la Caixa", Grants CSI04A05, CSI01A08, and SAN196/SA07/07 from Junta de Castilla y León, Grant FIS-FEDER 04/0843 from Fondo de Investigación Sanitaria and European Commission, and by Grant RD06/0020/1037 from Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III, Ministerio de Sanidad of Spain (to F.M.). This work is also supported by Grants 18022048, 18050038, 18057026, 18207015, and 19044003 from the Ministry of Education, Culture, Sports, and Technology of Japan, Naito Foundation, Takeda Science Foundation, Gushinkai Foundation, and Uehara Memorial Foundation (to M. F.).
2 Address correspondence and reprint requests to Dr. Faustino Mollinedo, Centro de Investigación del Cáncer, Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Cientificas-Universidad de Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain. E-mail address: fmollin{at}usal.es
3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophil; VAMP, vesicle-associated membrane protein.
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