Mutations in the TGFBI (BIGH3) gene that encodes for transforming growth factor beta induced protein (TGFBIp) are the cause of several phenotypically different corneal dystrophies. While the genetics of these protein misfolding diseases are well documented, relatively little is known about this extracellular matrix protein itself. In this study, we have purified TGFBIp from normal human and porcine corneas using nondenaturing conditions and standard chromatography techniques. The two homologues were shown to be monomers, and we did not find evidence for posttranslational additions. The C-terminal of both human and porcine TGFBIp is truncated predominantly after the integrin binding sequence Arg(642)-Gly(643)-Asp(644) (RGD). However, using an antibody against the C-terminal fragment (residues 648-683), we also detected a small amount of full-length TGFBIp in corneal extracts. Approximately 60% of TGFBIp was covalently associated with insoluble components of the extracellular matrix in both human and porcine corneas through a disulfide bridge.

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The overlapping yaaG and yaaF genes from Bacillus subtilis were cloned and overexpressed in Escherichia coli. Purification of the gene products showed that yaaG encoded a homodimeric deoxyguanosine kinase (dGK) and that yaaF encoded a homodimeric deoxynucleoside kinase capable of phosphorylating both deoxyadenosine and deoxycytidine. The latter was identical to a previously characterized dAdo/dCyd kinase (Møllgaard, H. (1980) J. Biol. Chem. 255, 8216 -8220). The purified recombinant dGK was highly specific toward 6-oxopurine 2-deoxyribonucleosides as phosphate acceptors showing only marginal activities with Guo, dAdo, and 2,3-dideoxyguanosine. UTP was the preferred phosphate donor with a K m value of 6 M compared with 36 M for ATP. In addition, the K m for dGuo was 0.6 M with UTP but 6.5 M with ATP as phosphate donor. The combination of these two effects makes UTP over 50 times more efficient than ATP. Initial velocity and product inhibition studies indicated that the reaction with dGuo and UTP as substrates followed an Ordered Bi Bi reaction mechanism with UTP as the leading substrate and UDP the last product to leave. dGTP was a potent competitive inhibitor with respect to UTP. Above 30 M of dGuo, substrate inhibition was observed, but only with UTP as phosphate donor.

Chicken beta 2-microglobulin (beta 2m) and class I (B-F19 alpha chain) cDNA clones were isolated and the sequences compared to those of B-F Ag isolated from chicken E. These clones represent the major expressed class I molecules on E, with B-F alpha size variants evidently due to alternative use of small exons in the cytoplasmic region. The cDNA sequences were compared to turkey beta 2m, the apparent allele B-F12 alpha and other vertebrate homologs, using the 2.6 A structure of the human HLA-A2 molecule as a model. Both chicken alpha 1 and alpha 2 domains resemble mammalian classical class I molecules and the MHC-encoded nonclassical molecules more than CD1 or the class I-like FcR. In contrast, the chicken alpha 3 domain is equally homologous to all alpha 3 domains, to beta 2m and to class II beta 2 domains. For each pair of extracellular domains (alpha 1 vs alpha 2, alpha 3 vs beta 2m), the level of sequence homology between mammalian and avian molecules is quite different. This suggests that the structurally homologous domains have been under different selective pressures during evolution. There is a very strong G + C bias in alpha 3 and beta 2m, leading to an overall change in amino acid composition in B-F compared to class I molecules from other taxa. Many of the surface residues are quite diverged, particularly in alpha 3 and beta 2m. There are fewer changes in intra- and interdomain contact sites. Some residues with important functions are invariant, including seven residues that bind the ends of the peptide, two residues that bind CD8, and three residues that are phosphorylated. The positions of the allelic residues are conserved. There are other patches of invariant residues on alpha 1, alpha 2, and beta 2m; these might bind TCR or other molecules involved in class I function.