Regulatory Mechanism of SNAP23 in Phagosome Formation and Maturation

Abstract

Synaptosomal associated protein of 23 kDa (SNAP23), a plasma membrane-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), is a ubiquitously expressed protein that is generally involved in fusion of the plasma membrane and secretory or endosomal recycling vesicles during several types of exocytosis. SNAP23 is expressed in phagocytes, such as neutrophils, macrophages, and dendritic cells, and functions in both exocytosis and phagocytosis. This review focuses on the function of SNAP23 in immunoglobulin G Fc receptor-mediated phagocytosis by macrophages. SNAP23 and its partner SNAREs mediate fusion of the plasma membrane with intracellular organelles or vesicles to form phagosomes as well as the fusion of phagosomes with endosomes or lysosomes to induce phagosome maturation, characterized by reactive oxygen species production and acidification. During these processes, SNAP23 function is regulated by phosphorylation. In addition, microtubule-associated protein 1A/1B light chain 3 (LC3)-associated phagocytosis, which tightly promotes or suppresses phagosome maturation depending on the foreign target, requires SNAP23 function. SNAP23 that is enriched on the phagosome membrane during LC3-associated phagocytosis may be phosphorylated or dephosphorylated, thereby enhancing or inhibiting subsequent phagosome maturation, respectively. These findings have increased our understanding of the SNAP23-associated membrane trafficking mechanism in phagocytes, which has important implications for microbial pathogenesis and innate and adaptive immune responses.

Keywords: MAP1LC3A; SNAP23 protein; SNARE proteins; membrane fusion; phagocytosis.

Fig. 1.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and the SNARE complex. (A) Schematic representation of the SNARE family based on the central amino acid residue of the SNARE motif and the domain structure. (B) SNARE-mediated membrane fusion of target and vesicle membranes. VAMP, vesicle-associated membrane protein; TM, transmembrane domain.

Fig. 2.

Analysis of an intramolecular FRET probe of synaptosomal-associated protein of 23 kDa (SNAP23). (A) Schematic representation of SNAP23 and its FRET probe. The predicted conformation of the SNAP23-based FRET construct upon structural alteration by forming adequate SNARE complexes during phagocytosis. (B) FRET imaging analyses using J774 cells transiently expressing the SNAP23 probe. TagGFP and TagRFP images were captured on a confocal microscope using excitation-emission wavelengths of 488‒522 and 561‒632 nm, respectively. FRET ratio signals were determined on a pixel-by-pixel basis by division of the TagRFP images by the TagGFP2 images, and are shown as pseudo-color images. High FRET ratios were observed in the presence of phorbol 12-myristate 13-acetate (PMA) at a final concentration of 100 ng/mL. Bar, 10 μm.

Fig. 3.

Function and regulation of synaptosomal-associated protein of 23 kDa (SNAP23) in canonical phagocytosis and LC3-associated phagocytosis (LAP). During Fc receptor (FcR)-mediated phagocytosis in macrophages, phagosome formation and maturation are promoted by SNAP23, but suppressed by SNAP23 phosphorylation at Ser95. Membrane occupation and recognition nexus repeat-containing-2 (MORN2) enhances the recruitment of SNAP23 to the formed phagosome containing foreign particles and subsequent LAP. SNAP23 promotes phagosomal reactive oxygen species (ROS) production by assembling NOX2 components. Phagosomal ROS induce the recruitment of LC3 to phagosomes (LAPosome formation). LC3-II may maintain SNAP23 on the phagosomes to enhance or suppress the fusion of LAPosomes with endocytic compartments. Precisely, enriched SNAP23 may be able to tightly regulate LAPosome maturation by its phosphorylation status depending on foreign particles. IKK2, IκB kinase 2.