The Disulfide Bond Pattern of Transforming Growth Factor Beta-Induced protein

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Transforming growth factor beta-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 to 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 intra-molecule disulfide bonds leaving a single cysteine residue available for the collagen cross-link. The structures of TGFBIp and its homologs are unknown. We here present the disulfide bridge pattern of TGFBIp, which was solved by generating specific peptides. These were separated by ion exchange followed by reverse phase HPLC and analyzed by mass spectrometry and Edman degradation. The NH2-terminal CRD contains six cysteine residues, 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 intra-domain disulfide bridges (Cys49-Cys85 and Cys84-Cys97) and one inter-domain 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 intra-domain disulfide bridge. Finally, an inter-domain disulfide bridge between FAS1-1 and FAS1-2 (Cys214-Cys317) was identified. The inter-domain disulfide bonds indicate that the NH2-terminal of TGFBIp (CRD, FAS1-1 and FAS1-2) adopts a compact globular fold leaving FAS1-3 and FAS1-4 exposed.

Original languageEnglish
JournalBiochemistry
Volume55
Issue39
Pages (from-to)5610–5621
ISSN0006-2960
DOIs
Publication statusPublished - 8 Sep 2016

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