Transketolase Is a Major Protein in the Mouse Cornea

Abstract

Earlier experiments in this laboratory identified a highly expressed 65-68-kDa protein in both mouse and human corneas (Cuthbertson, R. A., Tomarev, S. I., and Piatigorsky J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4004 -4008). Here, we demonstrate that this protein is transketolase (TKT; EC 2.2.1.1), an enzyme in the nonoxidative branch of the pentose-phosphate pathway, based on peptide and cDNA isolation and sequence analysis of mouse cornea protein and RNA samples, respectively. While expressed at low levels in a number of tissues, the 2.1-kilobase TKT mRNA was expressed at a 50-fold higher level in the adult mouse cornea. The area of most abundant expression was localized to the cornea epithelial cell layer by in situ hybridization. Western blot analysis confirmed TKT protein abundance in the cornea and indicated that TKT may comprise as much as 10% of the total soluble protein of the adult mouse cornea. Soluble cornea extracts exhibited a correspondingly high level of TKT enzymatic activity. TKT expression increased progressively through cornea maturation, as shown by Northern blot, in situ hybridization, Western blot, and enzymatic analyses. TKT mRNA and protein were expressed at low levels in the cornea prior to eye opening, while markedly increased levels were observed after eye opening. Taken together, these observations suggest that TKT may be a cornea enzymecrystallin, and suggest that the crystallin paradigm and concept of gene sharing, once thought to be restricted to the lens, apply to other transparent ocular tissues.

In Situ Localization of Transketolase Activity in Epithelial Cells of Different Rat Tissues and Subcellularly in Liver Parenchymal Cells

“…TK is a moonlighting protein, showing different functions depending on its localization, and has been implicated in different pathologies such as cancer, diabetes, and neurological disorders (McCool et al 1993;Sax et al 1996;Paoletti et al 1997;Hammes et al 2003). The method that we developed was shown to be specific for TK activity when the proper control reaction-an incubation in the absence of the substrate mixture and in the presence of 10 mM ADP-is subtracted from the test reaction, which is an incubation in the presence of the substrate mixture.…”

“…Because TK is a thiamine-dependent enzyme, TK activity has been used to assess the thiamine status in man (Sauberlich 1984). Besides its role as enzyme that generates ribose-5-phosphate and shuttles carbons generated by the oxidative PPP back into glycolysis, TK has also been demonstrated to be a structural protein in cornea, being important for eye transparency (Sax et al 1996).…”

The Crystal Structure of Human Transketolase and New Insights into Its Mode of Action

“…Mammalian TKT is expressed in all tissues highlighting its central metabolic function (22,23). Interestingly, the highest expression level for TKT in mouse was found in cornea, where TKT amounts for ϳ10% of the total soluble protein, suggesting that it functions as an enzyme-crystallin (23).…”

Evidence for Gelsolin as a Corneal Crystallin in Zebrafish

“…For example, a major protein in most mammalian corneal epithelial cells is aldehyde dehydrogenase class 3 (ALDH3), 1 which represents 20 -40% of the water-soluble protein of the bovine (20,21), rodent, and marsupial (22,23) corneal epithelial cells. Transketolase (TKT) comprises at least 10% of the water-soluble protein of the mouse corneal epithelial cells (24) and NADP ϩ -dependent isocitrate dehydrogenase comprises approximately 13% of the water-soluble proteins of the bovine corneal epithelial cells (25). An additional similarity with the enzyme-crystallins of the lens is that the abundant proteins in the corneal epithelial cells are taxon-specific.…”