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\begin{document}

\title{Direct cleavage and activation of gasdermin B by asthma trigger
allergens}



\author[1]{Quan Lu}%
\author[1]{Ronald Allan M. Panganiban}%
\author[1]{Jin-Ah  Park}%
\affil[1]{Harvard University Department of Environmental Health}%


\vspace{-1em}



  
  \date{May 21, 2024}


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\textbf{Direct cleavage and activation of gasdermin B by asthma trigger
allergens}

To the Editor:

Recent fine-mapping studies have pointed to gasdermn B (\emph{GSDMB} )
as a potential asthma susceptibility gene in 17q21 locus, the strongest
and most highly replicated signal in genome-wide association
studies\textsuperscript{1}. The GSDMB protein is a member of the
gasdermin family that, when cleaved, triggers an inflammatory cell death
known as pyroptosis\textsuperscript{2}. 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
cells\textsuperscript{3,4}. These findings suggest that the role of
\emph{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 inflammation\textsuperscript{5,6}. 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
GSDMB\textsuperscript{3}, 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
site\textsuperscript{4}. 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 (\textasciitilde{}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 variant\textsuperscript{3},
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.

\textbf{Figure Legends:}

\textbf{Figure 1} . Asthma triggers cleave GSDMB. (A) Western blot
showing cleaved GSDMB from differentiated airway epithelial cell lysates
mixed with HDM extracts or 293T cell lysates (transfected with GSDMB)
mixed with increasing concentrations of (B) HDM extracts and (C) mold or
cockroach extracts. (D) Cleavage of recombinant GSDMB by HDM allergen.
Box marks the fragment subjected to sequencing. (E) Upper panel: Results
of the Edman Sequencing of the first 10 amino acids. Residues that were
identified with low degree of certainty are in parentheses. Lowe panel:
Diagram depicting the position of the identified amino acids in the
GSDMB protein. (F) \emph{In vitro} cleavage assay in 293T lysates
transfected with WT-GSDMB or K244A and incubated with HDM extracts.
Anti-GSDMB (A) or anti-FLAG antibody (B,C,F) was used to detect
full-length and cleaved GSDMB.

\textbf{Figure 2.} GSDMB-NT generated by HDM cleavage induces
pyroptosis. (A) Schematic diagrams showing the WT GSDMB and the
resulting GSDMB-NT fragments (NT-K244 and NT-K231var) after HDM
cleavage. (B) Representative images of cells transfected with indicated
plasmids. Blue arrowheads mark pyroptotic cells. (C) LDH release assay
from 293T cells transfected with the indicated plasmid constructs.
Results indicate mean +/- SEM of n=6 from a representative experiment.
*\emph{p} \textless{}0.05.

\textbf{References:}

1. Schoettler, N.\emph{, et al.} New Insights Relating Gasdermin B to
the Onset of Childhood Asthma. \emph{Am J Respir Cell Mol Biol}
\textbf{67} , 430-437 (2022).

2. Broz, P., Pelegrin, P. \& Shao, F. The gasdermins, a protein family
executing cell death and inflammation.\emph{Nat Rev Immunol} \textbf{20}
, 143-157 (2020).

3. Panganiban, R.A.\emph{, et al.} A functional splice variant
associated with decreased asthma risk abolishes the ability of gasdermin
B to induce epithelial cell pyroptosis. \emph{J Allergy Clin Immunol}
\textbf{142} , 1469-1478 e1462 (2018).

4. Zhou, Z.\emph{, et al.} Granzyme A from cytotoxic lymphocytes cleaves
GSDMB to trigger pyroptosis in target cells. \emph{Science} \textbf{368}
(2020).

5. Holgate, S.T.\emph{, et al.}Asthma. \emph{Nat Rev Dis Primers}
\textbf{1} , 15025 (2015).

6. Lambrecht, B.N. \& Hammad, H. Allergens and the airway epithelium
response: gateway to allergic sensitization. \emph{J Allergy Clin
Immunol} \textbf{134} , 499-507 (2014).

Ronald Allan M. Panganiban, Jin-Ah Park, and Quan Lu\textsuperscript{*}

Program in Molecular and Integrative Physiological Sciences

Department of Environmental Health

Harvard T.H. Chan School of Public Health

Boston, Massachusetts 02115 USA

\textsuperscript{*}Corresponding author: Harvard T.H. Chan School of
Public Health, Building 1, Room 309, 665 Huntington Ave, Boston, MA
02115. E-mail: qlu@hsph.harvard.edu

Ronald Allan M. Panganiban (ORCID: 0000-0003-0894-1318)

Jin-Ah Park (ORCID: 0000-0002-8930-9287)

Quan Lu (ORCID: 0000-0003-3915-0762)

\textbf{Funding Sources}

This work was supported in part by a scholar award from the American
Asthma Foundation and by the National Institutes of Health grant
R01HL139496 to Q.L.

\textbf{Disclosure of Potential Conflict of Interest}

Q.L. a co-founder, scientific advisor and shareholder of Vesigen
Therapeutics. All other co-authors declare no competing interests.

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