Ectocytosis caused by sublytic autologous complement attack on human neutrophils. The sorting of endogenous plasma-membrane proteins and lipids into shed vesicles

Abstract

During sublytic complement attack on human neutrophils, plasma-membrane vesicles are shed from the cell surface as a cell-protection mechanism. By using surface-iodinated neutrophils it was found that less than 2% of surface label was recovered in shed vesicles under conditions where 40% of complement component C9 was shed. SDS/PAGE of 125I-labelled shed vesicles and plasma membranes showed differences in iodination pattern, demonstrating the sorting of membrane proteins into the shed vesicles. Analysis of 32P-labelled phospholipids after labeling of neutrophils with [32P]Pi before sublytic complement attack showed the presence of phosphatidic acid, phosphatidylcholine, phosphatidyl-ethanolamine, phosphatidylinositol and polyphosphoinositides in shed vesicles. Quantitative analysis using [3H]acetic anhydride-labelling method showed that the molar proportions of phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and sphingomyelin were the same in shed vesicles as in plasma membranes. In contrast, the molar proportions of cholesterol and diacylglycerol relative to sphingomyelin were almost twice those found in plasma membranes. The data demonstrate the existence of protein and lipid sorting mechanisms during the formation of shed vesicles when neutrophils are subject to sublytic complement attack. The term ‘ectocytosis’ is proposed to describe triggered shedding of right-side-out membrane vesicles from the surface of eukaryotic cells.