The insoluble TGFBIp fraction of the cornea is covalently linked via a disulfide bond to type XII collagen

TGFBIp, also known as keratoepithelin and βig-h3, is among the most abundant proteins in the human cornea and approximately 60% is associated with the insoluble fraction following extraction in sodium dodecyl sulfate (SDS) sample buffer. TGFBIp is of particular interest because a wide range of mutations causes amyloid or fuchsinophilic crystalloid deposits in the cornea leading to visual impairment. In the present study, we show that the SDS-insoluble fraction of TGFBIp from porcine and human corneas is covalently linked to the NC3 domain of type XII collagen in a TGFBIp:type XII collagen stoichiometric ratio of 2:1 via a reducible bond. Since type XII collagen is anchored to striated collagen fibers of the extracellular matrix, its interaction with TGFBIp is likely to provide anchoring for cells to the extracellular matrix through the integrin-binding capability of TGFBIp. Furthermore, the TGFBIp-type XII collagen molecule will affect our understanding of the molecular pathogenesis of the TGFBI-linked corneal dystrophies

Composition and proteolytic processing of corneal deposits associated with mutations in the <em>TGFBI</em> gene

Different types of granular corneal dystrophy (GCD) and lattice corneal dystrophy (LCD) are associated with mutations in the transforming growth factor beta induced gene (TGFBI). These dystrophies are characterized by the formation of non-amyloid granular deposits (GCDs) and amyloid (LCD type 1 and its variants) in the cornea. Typical corneal non-amyloid deposits from GCD type 2 (R124H), amyloid from a variant of LCD type 1 (V624M) and disease-free tissue controls were procured by laser capture microdissection and analyzed by tandem mass spectrometry. Label-free quantitative comparisons of deposits and controls suggested that the non-amyloid sample (R124H) specifically accumulated transforming growth factor beta induced protein (TGFBIp/keratoepithelin/βig-h3), serum amyloid P-component, clusterin, type III collagen, keratin 3, and histone H3-like protein. The amyloid (V624M) similarly accumulated serum amyloid P-component and clusterin but also a C-terminal fragment of TGFBIp containing residues Y571-R588 derived from the fourth fasciclin-1 domain (FAS1-4), apolipoprotein E and apolipoprotein A-IV. Significantly, analyses of the amyloid sample also revealed the presence of the serine protease Htr (High-temperature requirement) A1 and a number of proteolytic cleavage sites in the FAS1-4 domain of TGFBIp. These cleavage sites were consistent with the ligand binding and proteolytic activity of HtrA1 suggesting that it plays a role in the proteolytic processing of the amyloidogenic FAS1-4 domain. Taken together, the data suggest that the amyloidogenic-prone region of the fourth FAS1 domain of TGFBIp encompasses the Y571-R588 peptide and that HtrA1 is involved in the proteolytic processing of TGFBIp-derived amyloid in vivo

Purification, crystallization and preliminary X-ray diffraction of wild-type and mutant recombinant human transforming growth factor β-induced protein (TGFBIp)

Wild-type and mutant recombinant human transforming growth factor β-induced protein (TGFBIp) were cloned, purified and crystallized. Preliminary X-ray crystallography data were obtained from wild-type TGFBIp