Identification of glyceraldehyde-3-phosphate dehydrogenase as a Ca2+-dependent fusogen in human neutrophil cytosol

Abstract

The membrane fusion events observed during neutrophil degranulation are important aspects of the immunoregulatory system. In an attempt to understand the regulation of granuleplasma membrane fusion, we have begun characterizing human neutrophil cytosol for fusion activity, finding that 50% of the fusogenic activity could be attributed to members of the annexin family of proteins. The major non-annexin fusion activity (25% of the total cytosolic activity) was enriched by ion exchange chromatography after depletion of annexins by Ca 2؉ -dependent phospholipid affinity chromatography. The fusion activity co-purified with a 10,14-kDa dimer identified as leukocyte L1 (which was non-fusogenic), along with an approximately 36-kDa protein. This protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by amino-terminal sequencing, and the fusion activity was verified using commercially available GAPDH. GAPDH may play an important role in degranulation because it is as potent as annexin I on a mass basis and may constitute up to 25% of the total cytosolic fusion activity of the neutrophil. J.

Glyceraldehyde-3-phosphate Dehydrogenase Is Phosphorylated by Protein Kinase Cι/λ and Plays a Role in Microtubule Dynamics in the Early Secretory Pathway

“…Lastly, it has been reported that GAPDH undergoes autophosphorylation as well as phosphatidylserine (PS)-dependent phosphorylation by PKC (18 -20). Although GAPDH is commonly known as a key enzyme in glycolysis (GAPDH catalyzes the NAD-mediated oxidative phosphorylation of glyceraldehyde 3-phosphate to 1,3-diphosphoglycerate), a number of intriguing intracellular roles have been reported including modulation of the cytoskeleton, kinase activity, and the promotion of vesicle fusion (18,(21)(22)(23)(24). These activities are all essential to the maintenance of mem-brane trafficking.…”

Membrane tubulovesicular extensions (cytonemes)

“…49 A growing amount of evidence has shown that the glycolytic enzymes GAPDH and enolase also play a role in membrane fusion. [52][53][54][55][56] The capacity of NO and agents that disrupt the actin cytoskeleton to induce TVE formation could be realized via the L-plastin is a single cytosolic protein in neutrophils that binds BPB, an inducer of TVE formation. 74 In murine macrophages, L-plastin is one of the major S-nitrosylation targets for NO, a natural inducer of TVE formation.…”

“…84 Annexin 1 and GAPDH appear to be the major fusion proteins of neutrophils shown to promote aggregation and fusion neutrophil granules. 53,85,86 The S100A8/A9 protein binds and transports arachidonic acid, 87 an essential component of annexin-mediated membrane fusion.…”

Participation of a Fusogenic Protein, Glyceraldehyde-3-phosphate Dehydrogenase, in Nuclear Membrane Assembly

“…It is known that GAPDH can cause the fusion of reconstituted phospholipid vesicles in vitro (49). We recently reported that rabbit GAPDH binds to phosphatidylserine in lipid bilayers and stimulates membrane fusion of reconstituted phospholipid vesicles (28).…”