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Comparative Study
. 2017 Feb 14;114(7):E1128-E1137.
doi: 10.1073/pnas.1616783114. Epub 2017 Feb 1.

Gene polymorphism linked to increased asthma and IBD risk alters gasdermin-B structure, a sulfatide and phosphoinositide binding protein

Kinlin L Chao  1 , Liudmila Kulakova  1 , Osnat Herzberg  2   3
Affiliations
Comparative Study

Gene polymorphism linked to increased asthma and IBD risk alters gasdermin-B structure, a sulfatide and phosphoinositide binding protein

Kinlin L Chao et al. Proc Natl Acad Sci U S A. .

Abstract

The exact function of human gasdermin-B (GSDMB), which regulates differentiation and growth of epithelial cells, is yet to be elucidated. In human epidermal growth factor receptor 2 (HER2)-positive breast cancer, GSDMB gene amplification and protein overexpression indicate a poor response to HER2-targeted therapy. Genome-wide association studies revealed a correlation between GSDMB SNPs and an increased susceptibility to Crohn's disease, ulcerative colitis, and asthma. The N- and C-terminal domains of all gasdermins possess lipid-binding and regulatory activities, respectively. Inflammatory caspases cleave gasdermin-D in the interdomain linker but not GSDMB. The cleaved N-terminal domain binds phosphoinositides and cardiolipin, forms membrane-disrupting pores, and executes pyroptosis VSports手机版. We show that both full-length GSDMB and the N-terminal domain bind to nitrocellulose membranes immobilized with phosphoinositides or sulfatide, but not with cardiolipin. In addition, the GSDMB N-terminal domain binds liposomes containing sulfatide. The crystal structure of the GSDMB C-terminal domain reveals the structural impact of the amino acids encoded by SNPs that are linked to asthma and inflammatory bowel disease (IBD). A loop that carries the polymorphism amino acids corresponding to healthy individuals (Gly299:Pro306) exhibits high conformational flexibility, whereas the loop carrying amino acids found in individuals with increased disease risk (Arg299:Ser306) exhibits a well-defined conformation and higher positive surface charge. Apoptotic executioner caspase-3, -6, and -7, but not the inflammatory caspases, cleave GSDMB at 88DNVD91 within the N-terminal domain. Selective sulfatide binding may indicate possible function for GSDMB in the cellular sulfatide transport. .

Keywords: GSDMB; X-ray crystallography; complex trait inflammatory disease; disease risk polymorphism; lipid binding. V体育安卓版.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Structure-based sequence alignment of four human gasdermins (GSDMA to -D) and mouse Gsdma3. Secondary structure elements of the GSDMB C-terminal domain determined in the current study are shown above the GSDMB sequence, and those of Gsdma3 are shown below the Gsdma3 sequence (25). The GSDMB α-helices that form the helix bundle are labeled sequentially, beginning with α5, consistent with the α-helix numbering scheme assigned in the structure of Gsdma3. However, the Gsdma3 α11 is missing in GSDMB; thus, the α11 of GSDMB corresponds to Gsdma3 α12. The short α-helix within the GSDMB polymorphism loop is labeled α′. Invariant residues are highlighted by a red background. Conservatively replaced amino acids are blocked. The two GSDMB polymorphism residues, 299 and 306, are highlighted by an orange background. The alignment was performed using the program T-Coffee (55), and the figure was prepared with ESPript 3 (espript.ibcp.fr/ESPript/ESPript/).
Fig. 2.
Fig. 2.
Structure of human MBP-GSDMB_C. (A) Representation of two MBP-GSDMB_C Arg299:Ser306 molecules in the asymmetric unit. The cylinders depict α-helices. MBP is colored gray, and GSDMB_C colors are ramped from blue at the N terminus to red at the C terminus. Core α-helices that form the α-helix bundle are numbered α5 to α11. A short α-helix within the polymorphism loop connecting the α7 and α8 helices is labeled α′. An additional unlabeled 1-turn α-helix within the polymorphism loop is present only in structures of Ser306-containing variants. (B) Stereoscopic representation of the GSDMB_C Arg299:Ser306 variant, in the same coloring scheme as in A. Two disordered regions, the interdomain linker before α5 and the loop between α10 and α11, are depicted in dotted lines.
Fig. 3.
Fig. 3.
Structural comparison of GSDMB_C and the cytoplasmic domain of a putative membrane protein from C. difficile 630 (PDB ID code 3KMI). Helices are shown as cylinders. Colors are ramped from blue at the N terminus to red at the C terminus, except that the first GSDMB_C helix, α5, missing in the 3KMI structure, is colored gray. (Top) Side-by-side view of the two structures. (Bottom) Stereoscopic view of the superposed structures.
Fig. 4.
Fig. 4.
Alternative conformations of the GSDMB polymorphism loop. The superposed loops are shown in different colors. The superpositions were obtained from the least-squared alignment of the flanking helices. The polymorphism residues are as indicated. (A) MBP-GSDMB_C Gly299:Pro306; Gly299 is shown as a sphere. (B) MBP-GSDMB_C Arg299:Ser306 (disease-associated variant) and Gly299:Ser306. (C) The environment of Ser306 in the structure of MBP-GSDMB_C Arg299:Ser306 and a model of a proline, built in the context of that environment. The proline side chain clashes with the carbonyl oxygen of Met303, and thus a proline cannot be accommodated while maintaining the helical conformation.
Fig. 5.
Fig. 5.
GSDMB is a substrate of apoptotic, executioner caspase 3, 6, and 7. In vitro cleavage of (A) His6-GSDMB by recombinant human caspases 1 to 3 and 6 to 10 and (B) MBP-GSDMB_N by recombinant human caspases 3, 6, and 7. The cleavage products are separated on 14% (wt/vol) SDS/PAGE (Top) and immunoblotted with anti-GSDMB mAb and goat anti-Mouse HRP conjugate (A, Bottom) and with anti-MBP conjugated to HRP mAb (B, Bottom).
Fig. 6.
Fig. 6.
Protein-lipid overlay assay shows the binding of phosphatidylinositides and sulfatide to His6-GSDMB and MBP-GSDMB_N. (A) His6-GSDMB or MBP-GSDMB_N were incubated with phospholipid (Left) or membrane lipid (Right) strips. Then, 100 pmol of each lipid was immobilized on nitrocellulose membrane. Schematics of lipid strip templates are shown with lipid labels. CHOL, cholesterol; DAG, diacylglycerol; GT, triglyceride; LPA, lysophosphatidic acid; LPC, lysophosphocholine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PtdIns, phosphatidylinositol; PtdIns(3)P, phosphatidylinositol-3-phosphate; PtdIns(4)P, phosphatidylinositol-4-phosphate; PtdIns(5)P, phosphatidylinositol-5-phosphate; PtdIns(3,4)P2, phosphatidylinositol-3,4-bisphosphate; PtdIns(3,5)P2, phosphatidylinositol-3,5-bisphosphate; PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate; PtdIns(3,4,5)P3, phosphatidylinositol-3,4,5-triphosphate; SM, sphingomyelin; S1P, sphingosine-1-phosphate; sulfatide, 3-sulfogalactosylceramide. (B) His6-GSDMB (2 μg/mL) and MBP-GSDMB_N (2.6 μg/mL) were incubated with varying amounts of immobilized phosphoinositides, phosphatidic acid (PA), phosphatidylglycerol (PG), cardiolipin (CL), and sulfatide on nitrocellulose membrane. Phosphoinositides, PA, and PG concentrations are 100, 50, 25, 12.5, 6.25, 3.75, and 1.56 pmol. Cardiolipin and sulfatide concentrations are 1,000, 800, 600, 400, 200, 100, and 50 pmol.
Fig. 7.
Fig. 7.
Comparison of GSDMB_C and Gsdma3 structures. Stereoscopic view of the superposed structures. GSDMB_C is colored in yellow and Gsdma3 is colored in cyan. The α7 to α8 GSDMB loop carrying the polymorphism residues is colored in red. The Gsdma3 region corresponding to the flexible GSDMB loop connecting α11 and α12 is colored in blue. Regions of the Gsdma3 N-terminal domain that interact with the C-terminal domain are colored in magenta.
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References

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