Direct cleavage and activation of gasdermin B by asthma trigger
allergens
To the Editor:
Recent fine-mapping studies have pointed to gasdermn B (GSDMB ) as
a potential asthma susceptibility gene in 17q21 locus, the strongest and
most highly replicated signal in genome-wide association
studies1. The GSDMB
protein is a member of the gasdermin family that, when cleaved, triggers
an inflammatory cell death known as pyroptosis2.
Caspase-1 and granzyme A have been shown to cut GSDMB at specific sites
to release the N-terminal fragment of the protein (GSDMB-NT) that has
the ability to induce pyroptosis in cells, including airway epithelial
cells3,4.
These findings suggest that the role of GSDMB in asthma lies in
its ability to be activated through cleavage to induce pyroptosis;
however, it remains unclear whether GSDMB cleavage and activation occur
in the context of asthma.
Common asthma trigger allergens often possess protease activities that
cause airway epithelial injury and
inflammation5,6.
We thus tested whether the allergens directly cleave GSDMB. Incubation
of extracts from house dust mite (HDM), a common asthma trigger, with
lysates from human bronchial epithelial cells, which express endogenous
GSDMB3, resulted in
GSDMB cleavage as evidenced by the appearance of a smaller protein
around 17kD (Figure 1A). Since the GSDMB antibody used in the Western
blotting targets the C-terminus of the protein, the 17kD protein band
likely represents the C-terminal GSDMB fragment. Such GSDMB cleavage was
also observed when lysates from cells expressing C-terminal-FLAG-tagged
GSDMB were mixed with HDM extract (Figure 1B). Furthermore, mold or
cockroach extract also cleaved tagged GSDMB (Figure 1C). The cleavage of
GSDMB protein by all allergen extracts resulted in a single product of
similar size (about 17 kD), suggesting a specific cutting site.
To identify the cleavage site, we incubated recombinant full-length
GSDMB with HDM extract and resolved the cleaved protein products on
SDS-PAGE (Figure 1D). We excised the putative 17 kD C-terminal fragment
(GSDMB-CT, Figure 1D) and determined the N-terminal amino acid sequence
of the fragment via Edman sequencing (Supplemental Figure S1, Figure
1E). Despite some ambiguities, the first ten amino acid residues of the
17 kD GSDMB-CT largely map to position 245 to 254 (SLGSEDSRNM) of the
full length GSDMB protein (Figure 1E). This result indicates that GSDMB
was cleaved immediately after the lysine residue at position 244 (K244).
Interestingly, granzyme A also cuts GSDMB at the same K244
site4. To confirm K244
as the site of cleavage, we mutated lysine 244 to alanine (K244A) in
GSDMB and tested whether the mutant protein can be cleaved by HDM. As
shown by Western blotting, HDM was able to cleave wild type (WT) GSDMB
but failed to cleave K244A GSDMB as evidenced by the absence of the 17
kD fragment (Figure 1F).
The cleavage of GSDMB by HDM is expected to release an N-terminal
fragment of 244 amino acids (GSDMB-NT-K244) (Figure 2A). We next tested
whether GSDMB-NT-K244 triggers pyroptosis. Transfection of GSDMB-NT-K244
induced cell morphological changes characteristic of pyroptosis,
including rounding up and detachment (Figure 2B). LDH release assay
confirmed increased toxicity in these cells (~3.4 fold)
as compared to cells transfected with the full-length GSDMB (Figure 2C).
Consistent with our previous finding on GSDMB-NT shortened by a
functional asthma-associated splice variant3,
transfection of a truncated GSDMB-NT from the variant (NT-K231var) did
not induce pyroptosis (Figure 2B,C).
While future studies are needed to identify the specific proteases
within the allergen extracts that cleave GSDMB, our current study
demonstrates that asthma triggers such as HDM can directly cleave and
activate GSDMB, thus providing biochemical evidence linking
GSDMB-mediated pyroptosis to asthma.