Periostin and TGF-β-induced protein: Two peas in a pod?

Abstract

Periostin (PN) and TGF-β-induced protein (βig-h3) are paralogs that contain a single emilin and four fasciclin-1 modules and are secreted from cells. PN receives attention because of its up-regulation in cancer and degenerative and allergic diseases. βig-h3 is highly enriched in cornea and best known for harboring mutations in humans associated with corneal dystrophies. Both proteins are expressed widely, and many functions, some over-lapping, have been attributed to PN and βig-h3 based on biochemical, cell culture, and whole animal experiments. We attempt to organize this knowledge so as to facilitate research on these interesting and incompletely understood proteins. We focus particularly on whether PN and βig-h3 are modified by vitamin K-dependent γ-glutamyl carboxylation, a question of considerable importance given the profound effects of γ-carboxylation on structure and function of other proteins. We consider the roles of PN and βig-h3 in formation of extracellular matrix and as ligands for integrin receptors. We attempt to reconcile the contradictory results that have arisen concerning the role of PN, which has emerged as a marker of TH2 immunity, in murine models of allergic asthma. Finally, when possible we compare and contrast the structures and functions of the two proteins.

Keywords: Asthma; EMI module; FAS1 module; Golgi apparatus; integrin; matricellular protein; midline fasciclin; γ-carboxylation.

Fig. 1

Modular structures of of MFAS, PN, and βig-h3 and identity between mouse and human proteins. (A) The five modules are in blocks, within which are the relative locations of cysteines (C) and asparagines with potential to be glycosylated (N) and a schematic of the proposed pattern of disulfides in the EMI module. Thick lines represent tail sequences present in all variants; thin lines represent sequences that may or may not be present depending on splicing. Exons encoding sequences in the C-terminal tail of human PN are numbered. The site of the Arg-Gly-Asp (RGD) sequence in βig-h3 is indicated. (B) The total numbers of differences and non-conservative differences (in parentheses) between mouse and human proteins are given for each region as a fraction of number of residues that differed divided by total residues. For the PN C-terminus, the longest splice variant has been scored. Only the online version of this figure is in color.

Fig. 2

Sequences of EMI and FAS1 modules of MFAS (M), PN (P), and βig-h3 (β) aligned with T-Coffee as described in the text. Numbering of MFAS is based on the longest splice variant. Invariant residues and a nearly invariant phenylalanine are in red and blue, respectively. Blocks of highly conserved sequences surrounding invariant FAS1 module residues are in orange. Cysteine residues are bolded and in blue. Secondary structural elements are positioned based on the structure of FAS1-4 of βig-h3. Sequences proposed to constitute recognitions sites for γ-glutamyl carboxylase are underlined in green for three of the FAS1 modules. Arrows point to the locations of Tyr-His (YH) and Asp-Ile (DI) sequences implicated as being important for integrin binding. Only the online version of this figure is in color.

Fig. 3

Structure of FAS1-4 of βig-h3 as determined by NMR. Cartoon and surface representations of βig-h3 FAS1-4 NMR structure (PDB 2LTB (Underhaug et al., 2013)). Highlighted in orange are the two most conserved regions among the FAS1 modules of human βig-h3 and PN and Drosophila fasciclin 1 and MFAS. Residues that are conserved in all 16 modules, Thr538, Pro542 and His572, are in hot pink. Phe540, which in some modules is replaced by another large hydrophobic residue, is in blue. Sequences proposed to constitute recognitions sites for γ-glutamyl carboxylase are underlined in green. The two views are related by a 90° rotation around a vertical axis and have been chosen to illustrate how the four conserved residues are buried in the core of the FAS1 domain. Only the online version of this figure is in color.

Fig. 4

Immuno-staining of PN in MG63 human osteosarcoma cells. Cells were incubated for 18 h on coverslips in medium containing 2% calf serum without or with 10 μg/ml Vitamin K and then fixed with formaldehyde, rendered permeable with 0.5% Triton, and stained with rabbit anti-PN followed by Alexa-Fluor-555-conjugated goat anti-rabbit-IgG antibody. Images were obtained with an Olympus BX60 fluorescence microscope using a 100X/1.30 oil immersion objective lens. Arrows point to PN in a patchy peri-nuclear pattern that was the same without and with vitamin K.

Fig. 5

Interactions of PN and βig-h3 inside and outside cells. The cartoon depicts collagen (COL), βig-h3 (β), fibronectin (FN), PN, tenascin-C (TN), LOX, and BMP1 being led into endoplasmic reticulum (ER) and trafficking through endoplasmic reticulum and the Golgi apparatus during secretion. Components close to one another have been localized together by immuno-fluorescence microscopy along with a Golgi marker or surmised to interact during secretion. Fibrils of fibronectin and collagen are shown outside the cell along with tenascin-C, which patterns the fibrils, and BMP-1, which activates LOX. LOX, in turn, cross-links collagen. The relationship of the two fibril systems is purposely left vague, and COL could be type I, VI, or XII collagen as described in the text. Only the online version of this figure is in color.

Fig. 6

Neutrophil adhesion and motility on PN. (A) Adhesion of blood neutrophils incubated at 10⁵ cells/ml for 1 hr in the presence or absence of GM-CSF, 10 ng/ml, in wells of microtiter plates. Wells were coated with PN, 10 μg/ml, and post-coated with fetal bovine serum or coated with fetal bovine serum alone. Adhesion was assayed by cell content of myeloperoxidase and is expressed as percentage of input cells (Stark et al., 1996). (B) Migration of blood neutrophils at 10⁴ cells/ml in the presence or absence of GM-CSF, 10 ng/ml. Monolayers of polystyrene beads were made in wells coated with PN at different concentrations and post-coated with fetal bovine serum (diagram below). Migration was determined by morphometric analysis of percentage of the bead coating cleared after 20 hr, as described (Johansson et al., 2013). (C) Migration of blood neutrophils preincubated for 5 min with blocking monoclonal antibody to integrin subunits or isotype control (mIgG1) before application to wells coated with PN, 5 μg/ml, and a monolayer of beads as in (B). GM-CSF, 10 ng/ml, was present, and percentage of bead coating cleared after 20 hr was determined. The cartoons underneath panels A and B/C depict the experimental set-ups with neutrophils in red and latex beads in green. Only the online version of this figure is in color.