Crucial role of the hydrophobic pocket region of Munc18 protein in mast cell degranulation

Abstract

The function of the Munc18-1 protein hydrophobic pocket, which interacts with the syntaxin-1 N-terminal peptide, has been highly controversial in neurosecretion. Recent analysis of patients with familial hemophagocytic lymphohistiocytosis type 5 has identified the E132A mutation in the hydrophobic pocket of Munc18-2, prompting us to examine the role of this region in the context of immune cell secretion. Double knockdown of Munc18-1 and Munc18-2 in RBL-2H3 mast cells eliminates both IgE-dependent and ionomycin-induced degranulation and causes a significant reduction in syntaxin-11 without altering expressions of the other syntaxin isoforms examined. These phenotypes were effectively rescued on reexpression of wild-type Munc18-1 or Munc18-2 but not the mutants (F115E, E132A, and F115E/E132A) in the hydrophobic pocket of Munc18. In addition, these mutants show that they are unable to directly interact with syntaxin-11, as tested through protein interaction experiments. Our results demonstrate the crucial roles of the hydrophobic pocket of Munc18 in mast cell degranulation, which include the regulation of syntaxin-11. We also suggest that the functional importance of this region is significantly different between neuronal and immune cell exocytosis.

Fig. 1.

KD of both Munc18-1 and Munc18-2 results in dramatic reductions of syntaxin-11 expressions and striking degranulation defects in RBL-2H3 cells. (A) Expression profiles of Munc18-1 and Munc18-2, as well as various syntaxin proteins present in wild-type RBL-2H3 and PC12 cells. Twenty micrograms of homogenates from wild-type RBL-2H3 and PC12 cells were analyzed by SDS/PAGE and immunoblotting, using the antibodies indicated. (B) Stable Munc18-1 KD, Munc18-2 KD, and Munc18-1/2 DKD RBL-2H3 cells were generated by lentivirus-mediated shRNA. Twenty micrograms of cell homogenates were analyzed by SDS/PAGE and immunoblotting, using the antibodies indicated. (C and D) β-hexosaminidase release was stimulated by applying 0.01 μg/mL DNP-IgE and 50 ng/mL dinitrophenyl-human serum albumin (DNP-HSA) (C) and 0.5 μM and 2.5 μM ionomycin (D) for 1 h from control, Munc18-1 KD, Munc18-2 KD, and Munc18-1/2 DKD cells. Error bars, SEM (n = 5). The statistical significance of the differences in β-hexosaminidase release between control and Munc18-1 KD (C), Munc18-2 KD, and Munc18-1/2 DKD (D) is indicated. *P < 0.05 (Student t test). *Nonspecific band observed with rabbit polyclonal anti-Munc18-2 and anti-syntaxin-8 antibodies.

Fig. 2.

Confocal immunofluorescence microscopy revealing that the subcellular localization of syntaxin-11 is unaltered on Munc18-1/2 DKD. Control and stable Munc18-1/2 DKD RBL-2H3 cells were permeabilized and stained with either GST or GST-syntaxin-11–absorbed rabbit polyclonal anti-syntaxin-11 antibody followed by Alexa488-conjugated goat anti-rabbit antibody (Invitrogen) and DAPI (see SI Materials and Methods for more detail). Green indicates syntaxin-11 and blue indicates DAPI. (A) Control and (B) Munc18-1/2 DKD cells. Note that there is substantially diminished green intensity (Right) where GST-syntaxin-11 absorbed the anti-syntaxin-11 antibody was used to stain. (Scale bar, 10 μm.)

Fig. 3.

Stable reexpression of wild-type Munc18-1 or Munc18-2 restores the syntaxin-11 expressions and the degranulation defects of Munc18-1/2 DKD RBL-2H3 cells while functional specificities exist in PC12 cells. (A) Twenty micrograms of homogenates of Munc18-1/2 DKD PC12 cells rescued with EmGFP, wild-type Munc18-1, and Munc18-2 (without tag, with myc, and with EmGFP) were analyzed by SDS/PAGE and immunoblotting, using the antibodies indicated. (B) NA release was stimulated by 70 mM KCl for 15 min in the rescued cells. Error bars, SEM (n = 9). The statistical significance of the differences in NA release between wild-type Munc18-1 and Munc18-2 rescued PC12 cells in each tag configuration are indicated. *P < 0.05 (paired t test). (C) Munc18-1/2 DKD RBL-2H3 cells were infected with lentiviruses that express EmGFP, wild-type Munc18-1-EmGFP, and wild-type Munc18-2-EmGFP. Twenty micrograms of cell homogenates were analyzed by SDS/PAGE and immunoblotting, using the antibodies indicated. (D) β-Hexosaminidase release was stimulated by applying 0.01 μg/mL DNP-IgE and 50 ng/mL DNP-HSA, as well as 0.5 μM and 2.5 μM ionomycin for 1 h from wild-type RBL-2H3 cells and rescued cells. Error bars, SEM (n = 9). *Nonspecific band observed with rabbit polyclonal anti-Munc18-2 antibody that comigrated with Munc18-2-EmGFP.

Fig. 4.

Hydrophobic pocket mutants of either Munc18-1 or Munc18-2 do not restore the syntaxin-11 expressions or the degranulation defects of Munc18-1/2 DKD RBL-2H3 cells. (A) Sequence alignment among Munc18-1, Munc18-2, and Munc18-3, indicating that F115 and E132 are conserved residues in Munc18 isoforms. (B and D) Munc18-1/2 DKD RBL-2H3 cells were infected with lentiviruses that express EmGFP, wild-type Munc18-EmGFP, and hydrophobic pocket mutant Munc18-EmGFP (F115E, E132A, and F115E/E132A) (B) Munc18-2 and (D) Munc18-1. Infected cells were selected with blasticidin (20 μg/mL). Twenty micrograms of homogenates of surviving cells were analyzed by SDS/PAGE and immunoblotting, using the antibodies indicated. *Nonspecific band observed with rabbit polyclonal anti-Munc18-2 antibody that comigrated with Munc18-2-EmGFP. (C and E) β-hexosaminidase release was stimulated by applying 0.01 μg/mL DNP-IgE and 50 ng/mL DNP-HSA, as well as 0.5 μM and 2.5 μM ionomycin for 1 h from rescued cells (C) Munc18-2 and (E) Munc18-1. Error bars, SEM (n = 5 for C, n = 6 for E).

Fig. 5.

Hydrophobic pocket mutants of Munc18-2 abolish the direct interaction with syntaxin-11. (A, Upper) Solubilized lysates of HEK-293FT cells that were transfected with EmGFP, wild-type Munc18-2-EmGFP, F115E, E132A, and F115E/E132A of Munc18-2-EmGFP were incubated with 10 μg GST or GST-syntaxin-11 attached to glutathione agarose beads. The mixtures were washed extensively, and the proteins were analyzed on SDS/PAGE and immunoblotting, using mouse monoclonal anti-EmGFP antibody. Solubilized lysates of HEK-293FT cells were loaded in lysate lanes. (A, Lower) Ponceau S staining of the above membrane showing similar loadings between lanes. *Wild-type Munc18-2-EmGFP band that was pulled down by GST-syntaxin-11. (B) The binding mixture between solubilized lysates of HEK-293FT cells that were transfected with wild-type Munc18-2-EmGFP, F115E, E132A, F115E/E132A, and 10 μg of GST-syntaxin-11 was analyzed on SDS/PAGE, followed by Coomassie Blue staining. Note that only wild-type Munc18-2-EmGFP was pulled down by GST-syntaxin-11, not any of the mutants.