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Biochemistry. 2016 Oct 4;55(39):5610-5621. Epub 2016 Sep 23.

Disulfide Bond Pattern of Transforming Growth Factor β-Induced Protein.

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Interdisciplinary Nanoscience Center (iNANO) and ‡Department of Molecular Biology and Genetics, Aarhus University , DK-8000 Aarhus, Denmark.


Transforming growth factor β-induced protein (TGFBIp) is an extracellular matrix protein composed of an NH2-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 NH2-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 NH2 terminus of TGFBIp (CRD, FAS1-1, and FAS1-2) adopts a compact globular fold, leaving FAS1-3 and FAS1-4 exposed.

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