Disulfide Bond Pattern of Transforming Growth Factor β-Induced Protein

Abstract

Transforming growth factor β-induced protein (TGFBIp) is an extracellular matrix protein composed of an NH-terminal cysteine-rich domain (CRD) annotated as an emilin (EMI) domain and four fasciclin-1 (FAS1-1-FAS1-4) domains. Mutations in the gene cause corneal dystrophies, a group of debilitating protein misfolding diseases that lead to severe visual impairment. Previous studies have shown that TGFBIp in the cornea is cross-linked to type XII collagen through a reducible bond. TGFBIp contains 11 cysteine residues and is thus able to form five intramolecule disulfide bonds, leaving a single cysteine residue available for the collagen cross-link. The structures of TGFBIp and its homologues are unknown. We here present the disulfide bridge pattern of TGFBIp, which was determined by generating specific peptides. These were separated by ion exchange followed by reversed-phase high-performance liquid chromatography and analyzed by mass spectrometry and Edman degradation. The NH-terminal CRD contains six cysteine residues, and one of these (Cys65) was identified as the candidate for the reducible cross-link between TGFBIp and type XII collagen. In addition, the CRD contained two intradomain disulfide bridges (Cys49-Cys85 and Cys84-Cys97) and one interdomain disulfide bridge to FAS1-2 (Cys74-Cys339). Significantly, this arrangement violates the predicted disulfide bridge pattern of an EMI domain. The cysteine residues in FAS1-3 (Cys473 and Cys478) were shown to form an intradomain disulfide bridge. Finally, an interdomain disulfide bridge between FAS1-1 and FAS1-2 (Cys214-Cys317) was identified. The interdomain disulfide bonds indicate that the NH terminus of TGFBIp (CRD, FAS1-1, and FAS1-2) adopts a compact globular fold, leaving FAS1-3 and FAS1-4 exposed.

Structural characterizations of human periostin dimerization and cysteinylation

“…S7). However, human TGFBIp was monomeric in crystal and did not see cysteinylation at Cys65 (equivalent to Cys60 of human periostin) that has been previously reported . As mentioned above, both the Fas1 III /Fas1 IV pair and the EMI domain of TGFBIp has a large movement relative to periostin (Fig.…”

“…To our best knowledge, it is first time to observe cysteinylation in periostin and provide structural evidence for cysteinylation in the EMI family of proteins (including TGFBIp). Likely similar to the case of human TGFBIp Cys65 [16][17][18], cysteinylation at Cys60 of human periostin would mediate its heterophilic interaction with the extracellular matrix proteins. In order to obtain insights into whether cysteinylation affects homophilic interaction of human periostin, we examined the Cys60Ala mutant through structural and biophysical characterizations.…”

“…Meanwhile, the free Cys65 in the EMI domain of human TGFBIp, a homologous protein of human periostin with 48% sequence identity, was observed to undergo cysteinylation, and this residue may form a disulfide bond linkage with collagens . Does human periostin have the same chemical modification?…”

Control of cytokine-driven eosinophil migratory behavior by TGF-beta-induced protein (TGFBI) and periostin

“…Transforming growth factor (TGF) β-induced protein (TGFBI, βig-h3) is a paralog of periostin and, like periostin, contains an amino (N)-terminal cysteine-rich sequence and four fasciclin-1 (FAS1) modules [ 1 ]. The disulfide pattern of TGFBI was recently determined [ 8 ]. Inter-module bridges between the cysteine-rich sequence and the second FAS1 module and between the first two FAS1 modules indicate that the N-terminal cysteine-rich sequence and the first two FAS1 modules adopt a complicated fold, possibly leaving the third and fourth FAS1 modules exposed [ 8 ].…”

“…The disulfide pattern of TGFBI was recently determined [ 8 ]. Inter-module bridges between the cysteine-rich sequence and the second FAS1 module and between the first two FAS1 modules indicate that the N-terminal cysteine-rich sequence and the first two FAS1 modules adopt a complicated fold, possibly leaving the third and fourth FAS1 modules exposed [ 8 ]. This model should be applicable to periostin, which contains the same set of cysteine residues [ 1 ].…”