Proteomic profiling of TGFBI‐null mouse corneas reveals only minor changes in matrix composition supportive of TGFBI knockdown as therapy against TGFBI‐linked corneal dystrophies

Abstract

TGFBIp is a constituent of the extracellular matrix in many human tissues including the cornea, where it is one of the most abundant proteins expressed. TGFBIp interacts with Type I, II, IV, VI, and XII collagens as well as several members of the integrin family, suggesting it plays an important role in maintaining structural integrity and possibly corneal transparency as well. Significantly, more than 60 point mutations within the TGFBI gene have been reported to result in aberrant TGFBIp folding and aggregation in the cornea, resulting in severe visual impairment and blindness. Several studies have focused on targeting TGFBIp in the cornea as a therapeutic approach to treat TGFBI-linked corneal dystrophies, but the effect of this approach on corneal homeostasis and matrix integrity remained unknown. In the current study, we evaluated the histological and proteomic profiles of corneas from TGFBI-deficient mice as well as potential redundant functions of the paralogous protein POSTN. The absence of TGFBIp in mouse corneas did not grossly affect the collagen scaffold, and POSTN is unable to compensate for loss of TGFBIp. Proteomic comparison of wild-type and TGFBI À/À mice revealed 11 proteins were differentially regulated, including Type VI and XII collagens. However, as these alterations did not manifest at the macroscopic and behavioral levels, these data support partial or complete TGFBI knockdown as a potential therapy against TGFBI-linked corneal dystrophies. Lastly, in situ hybridization verified TGFBI mRNA in the epithelial cells but not in other cell types, supportive of a therapy directed specifically at this lineage.Abbreviations ECM, extracellular matrix; iTRAQ, isobaric tags for relative and absolute quantitation; LC-MS/MS, liquid chromatography tandem mass spectrometry; POSTN, periostin gene; SRM, selected reaction monitoring; TEM, transmission electron microscopy; TGFBI, transforming growth factor beta-induced protein.

The serine protease HtrA1 cleaves misfolded transforming growth factor β–induced protein (TGFBIp) and induces amyloid formation

“…We have previously shown that the major fraction of corneal mouse TGFBIp originated from the corneal epithelium (6). Furthermore, quantification showed abundant HtrA1 protein in the corneal proteome of the endothelial layer, including Descemet's membrane (1).…”

“…In addition, TGFBIp binds covalently to type XII collagen via a reducible bond (5). Knocking out TGFBIp in mice showed little effect on corneal integrity and mouse behavior in general (6). TGFBIp is involved in the pathophysiology of the amyloid corneal disease lattice corneal dystrophy (LCD) 3 and amorphous granular corneal dystrophy (GCD).…”

Protein Analysis of the TGFBIR124H Mouse Model Gives Insight into Phenotype Development of Granular Corneal Dystrophy

“…which is supported by the lower molar percentage in our results compared to the human corneal proteome. [9] The disease mechanism of TGFBI-linked corneal dystrophies has in several studies been linked to altered stability and aberrant proteolytic processing of the disease-causing TGFBIp mutants. [4,8,10] We performed 2DE of the proteins in the right cornea of the different genotypes and subsequently immunoblotted against TGFBIp.…”

Fascinating Fasciclins: A Surprisingly Widespread Family of Proteins that Mediate Interactions between the Cell Exterior and the Cell Surface

“…This is due to the thermal and pH instability of the protein and to its high abundance in the cornea [ 85 ]; however, the condition might not help us to understand HsTgfbi function. A complete Tgfbi gene knockout in mice caused neither corneal abnormalities nor any other major defects [ 86 ], suggesting compensation by other ECM molecules. Although Pn knock-out mice showed that this locus is essential for normal osteogenesis and dental development [ 87 , 88 ], the two closely-related Pn and Tgfbi loci function identically in many ways [ 43 ].…”