Structural characterizations of human periostin dimerization and cysteinylation

Abstract

Human periostin plays a multifaceted role in remodeling the extracellular matrix milieu by interacting with other proteins and itself in both a heterophilic and homophilic manner. However, the structural mechanism for its extensive interactions has remained elusive. Here, we report the crystal structures of human periostin (EMI-Fas1 ) and its Cys60Ala mutant. In combination with multi-angle light-scattering analysis and biochemical assays, the crystal structures reveal that periostin mainly exists as a dimer in solution and its homophilic interaction is mainly mediated by the EMI domain. Furthermore, Cys60 undergoes cysteinylation as confirmed by mass spectroscopy, and this site hardly affects the homophilic interaction. Also, the structures yield insights into how periostin forms heterophilic interactions with other proteins under physiological or pathological conditions.

Development of an engineered peptide antagonist against periostin to overcome doxorubicin resistance in breast cancer

“…The physical properties of this peptide would be determined by an online tool (https://www.thermofisher.com) (Thermo Fisher Scientific). Structure of this peptide and the binding properties to PN would be predicted by PEP-FOLD3 [34] and pepATTRACT [35] tools from RPBS Web Portal (https://bioserv.rpbs.univ-paris-diderot.fr) using PN 3D-structure from RCSB PDB database (https:// www.rcsb.org/structure/5YJG) [36]. Binding affinity of anti-PN peptide was determined with isothermal titration calorimetry [37] using the MicroCal PEAQ-ITC Machine (Malvern Panalytical Ltd., Malvern, UK) in which 700 nM of rPN (or BSA as negative control) and either 70 nM of commercial goat anti-PN polyclonal antibody or 100 nM of plain anti-PN peptide in 50 mM Tris and 150 mM NaCl (pH 7.5) buffer were added into syringe and cell compartments of the machine.…”

Biochemical mechanisms of aggregation in TGFBI-linked corneal dystrophies

“…The main difference between the two proteins is in the C-terminal region, which lacks the RGD motif and undergoes alternative splicing in periostin (Horiuchi et al, 1999). The structure of periostin was recently published, and the protein is similar to TGFBIp folded into a banana shape with the FAS1 domains arranged like beads on a string (Liu et al, 2018). The CROPT domain is connected to the FAS1-2 domain through a disulfide bridge, with the free C60 of periostin exposed on the surface.…”

Generation of a Fully Human scFv that binds Tumor-Specific Glycoforms

“…The functions of the glycosylation present on periostin are not known; however, this site is highly conserved in sequence implying its potential importance, and this site is present in all known isoforms of periostin. The conservation and location of the N-glycosylation site in an unstructured, solvent exposed region 34,35 (Fig. 1B) led us to the hypothesis that we could use the perostin protein as a scaffold to display different glycoforms of periostin allowing subtraction and enrichment of specific scFv antibodies to glycoforms of periostin.…”

“…( B ) NMR structure (PDB 5WT7) of the FAS4 domain showing the unstructured loop where asparagine 599 is located 35 . Crystal structure (PDB 5YJG) of the FAS1-FAS4 domains for human periostin with the N599 solvent exposed 34 . ( C ) Western blot analysis of periostin protein purified from culture supernatant on anti-Flag resin.…”