Bovine Corneal Protein Research Articles

Induction of autoantibodies to rat corneal protein 54.

Many studies have described the presence of circulating antibodies against corneal components in patients with corneal disease or uveitis, and in patients with skin or systemic disease with or without ocular involvement. The role of such antibodies in the underlying immunopathological process remains obscure. Here we describe the induction of autoantibodies against the rat cornea. Our attempts to induce corneal autoantibodies by various forms of keratitis and corneal trauma failed. However, circulating corneal autoantibodies could be detected by Western blotting after immunization of BN rats and Lewis rats with bovine corneal protein 54 (BCP 54). Rats immunized with rat corneal extracts (RaCE) or human serum albumin (HSA) as (auto) antigen did not develop corneal autoantibodies. During the study period (greater than 4 months), it was observed that the presence of circulating corneal autoantibodies did not elicit corneal inflammation. Severe keratitis did develop when BCP 54-immunized rats were challenged intracorneally with BCP 54, but the clinical signs were not significantly different from HSA-immunized rats after an intracorneal HSA challenge. Injection of corneal autoantibodies into the corneal stroma did not provoke keratitis. To the best of our knowledge this is the first study demonstrating corneal autoantibodies in rats without actual manipulation of the eye. This model may provide further insights in the role and significance of corneal autoantibodies in disease.

Kinetic studies on class 3 aldehyde dehydrogenase from bovine cornea.

In 1973 Holt and Kinoshita first demonstrated the occurence of a bovine corneal protein with a mass of 54 kDa (BCP 54). This protein has subsequently been shown to be a class 3 aldehyde dehydrogenase which is 84% identical to rat tumor-associated AlDH at the amino acid level (Cooper et al., 1991). Abedinia et al. (1990) purified the bovine corneal AlDH and demonstrated that it was the major soluble protein in bovine cornea, representing 20 to 40% of the soluble protein.

In Vitro Evaluation of Ocular Irritants Using Tissue Isoelectric Focusing Protein Profiles from Human, Rabbit, and Bovine Corneal Specimens

Tissue isoelectric focusing (TIF) was used to establish a reference bovine corneal protein profile and to compare the human, rabbit, and bovine corneal response to in vitro chemical exposure. TIF provided a direct, semiquantitative comparison of tissue-irritant interactions without the potential limitations of standard homogenization and extraction procedures. The in vitro exposure of corneal sections to acetic acid (2%) and ammonium hydroxide (1%) solutions resulted in selective modifications of tissue protein profiles, which were evaluated by scanning densitometry. Comparison of the human and rabbit corneal protein changes associated with in vitro chemical exposure revealed a similar acid/base effect on a common rabbit and human corneal protein band (pl = 8.1). The pl = 8.1 protein band did not occur in the bovine protein profile; however, a similar acid/base effect occurred with a protein band having a pl = 7.2.

Changes in the immune response against BCP 54 following corneal transplantation in man.

Bovine corneal protein (BCP) 54 is the major soluble protein of the cornea. Immune responses against this protein can be observed in patients with corneal disease or inflammatory disease of the anterior chamber of the eye. We wanted to determine whether an immune response against this corneal protein plays a role in corneal transplantation. A cell-mediated immune response against BCP 54 was therefore determined in 46 patients prior to and on several occasions during the first year following corneal transplantation. The presence of an anti-BCP 54 response before transplantation was not associated with any clinical parameters or with rejection following transplantation. The highest frequency of positive responses was observed three months after transplantation. Transplantation may have stimulated a temporary immune response against a previously sequestered antigen, but this tissue-specific immune response did not necessarily lead to rejection. Therefore, no prognostic value can be attributed to the anti-BCP 54 response.

Kinetic properties of the bovine corneal aldehyde dehydrogenase (BCP 54)

The major soluble protein of bovine cornea (BCP 54: bovine corneal protein 54 kDa) was isolated successively by gel filtration, anion-exchange chromatography and chromatofocusing. The amino acid sequence of a fragment of the purified BCP 54 obtained by lysyl-endopeptidase digestion showed marked homology with tumor-associated and 2,3,7,8-tetrachloro-dibenzo-p-dioxin-inducible aldehyde dehydrogenase (AIDH). From the high similarity of BCP 54 with tumor-associated AIDH in structural form, it is suggested that BCP 54 has AIDH activity. We confirmed a high AIDH activity of BCP 54 by immunoprecipitation using a mouse anti-BCP 54 monoclonal antibody followed by a spectrophotometric assay for AIDH activity. Next we demonstrated the unique properties of the purified BCP 54 as AIDH. The major isoelectric point is 6.41. BCP 54 preferentially oxidizes aromatic aldehyde such as benzaldehyde with NAD as coenzyme, but cannot oxidize phenylacetaldehyde. After heat treatment the AIDH activity is more stable with propionaldehyde-NAD than with benzaldehyde-NADP. With propionaldehyde-NAD the pH profile shows a broad plateau from pH 6-9 followed by a sharp rise up to pH 10. In contrast, with benzaldehyde-NADP there is a sharp optimum at pH 9.0. The activity with only benzaldehyde-NADP is inhibited by p-hydroxymercuribenzoate, but is not affected by disulfiram and diethylstilbestrol. So we suggested that BCP 54 is an AIDH with kinetic properties different from the rat tumor-associated AIDH.

Degenerate oligonucleotide sequence-directed cross-species PCR cloning of the BCP 54/ALDH 3 cDNA: priming from inverted repeats and formation of tandem primer arrays.

Bovine corneal protein 54 (BCP 54) is the major soluble protein of the bovine cornea, and immunoreactive forms of this protein have been described in a wide range of mammals. Dideoxy sequence determination of a previously synthesized 420-bp cDNA to BCP 54 generated by the novel mixed oligonucleotide primer amplification of cDNA (MOPAC) procedure revealed extensive similarity to the cDNA encoding tumor-associated rat liver (class 3) aldehyde dehydrogenase (RATALD). PCR amplification with additional pairs of degenerate oligonucleotide sequence (DOS) primers derived from both BCP 54-amino-acid sequence and amino acid and nucleotide sequence data from RATALD produced three PCR products that were cloned and subsequently sequenced. The major product was 716-bp BCP 54 cDNA clone encompassing the BCP 54 carboxy-terminal amino acid sequence for which the DOS pair was designed. Sequence alignment of the BCP 54 cDNA and its translation product with RATALD demonstrated 81% and 85% identity at the nucleotide and amino acid levels, respectively. Analysis of the additional two clones established that they were the results of PCR artifactual processes. The first of these was a 552-bp product occurring at elevated primer concentrations that formed through bidirectional amplification from a single DOS annealing to an inverted repeat located in the BCP 54 coding sequence. The second artifactual product was a 212-bp sequence that contained several unreported amplification anomalies, including the formation of a tandem primer array.

Bovine corneal protein 54K (BCP54) is a homologue of the tumor-associated (class 3) rat aldehyde dehydrogenase (RATALD)

Amino acid (aa) sequence data from Staphylococcus aureus V8 protease-digested bovine corneal 54-kDa protein (BCP54) fragments were utilized to derive mixed oligodeoxyribonucleotide (oligo) primers complementary to the reverse translation products of these sequences. These degenerate oligo primers were used to prime the amplification of BCP54 sequence from bovine corneal epithelial cell cDNA. The cDNA probe generated by this mixed oligo-primed amplification of cDNA was cloned and dideoxy-sequenced. A search of the GenBank database (version 63.0) revealed extensive sequence similarity to the cDNA encoding tumor-associated rat liver (class 3) aldehyde dehydrogenase (RATALD). Nucleotide (nt) and aa sequence alignment of the BCP54 translation product reveals it is 78% and 84% homologous with RATALD at the nt and aa levels, respectively. Conservation of aa sequence elements common to the aldehyde dehydrogenase family thought to be of structural/functional significance is further substantiated by this analysis. Included in the discussion is the likelihood that gene sharing (genes encoding metabolic enzymes and other stable proteins) may extend to the cornea.

Partial amino acid sequence determination of Bovine Corneal Protein 54 K (BCP 54)

The most abundant soluble protein of bovine cornea, BCP 54 (Bovine Corneal Protein, molecular weight 54 kD) was isolated and digested under both limited and complete digestion conditions with Staphylococcus aureus V8 protease. The fragments resulting from limited digestion were separated by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, visualized by Coomassie Blue staining, cut out, and submitted to N-terminal protein sequence analysis. Complete digestion fragments were separated on a Vydac C18 reverse-phase HPLC, collected, and concentrated prior to sequencing. Using this method, we obtained amino acid sequence data from three internal V8 protease derived fragments of BCP 54 and a number of HPLC fragments. Comparison of these amino acid sequences, corresponding to 30% of the BCP 54 molecule, to those sequences contained within release 22 of the National Biomedical Research Foundation Protein Identification Resource revealed no extended sequence similarity of known proteins to BCP54.

Behcet's Syndrome with Antibody to a Corneal Antigen

A patient with Behcet's syndrome has been followed for more than 6 years. He had aphthous ulcers, thrombophlebitis, multiple lesions of pyoderma increasingly in the genital area as he entered puberty, and uveitis. When he developed anterior uveitis manifested by iritis, his serum was tested for antibodies against a highly purified bovine corneal protein that shares antigenic determinants with human cornea. His high antibody levels to this protein suggest that iritis and anterior uveitis are manifestations of autoimmune phenomena that are a part of Behcet's syndrome.

Longitudinal study of serum antibody response to bovine corneal protein (BCP 54) in Behçet's disease.

Serum samples from a patient diagnosed as having active Behçet's syndrome with iritis were evaluated by the enzyme-linked immunosorbent assay for antibodies against a purified bovine corneal protein, molecular weight 54,000 daltons (BCP 54). Significantly elevated antibody levels to BCP 54 were found in all serum samples collected over a 2-year period. Anti-BCP 54 antibody levels remained within a very narrow range, at least twofold higher than those of controls. In contrast, no antibody to retinal S antigen was detected. These results indicate the presence of specific anti-BCP 54 autoantibodies in sera from a Behçet's patient and suggest that autoantibodies may play a role in the etiopathogenesis of uveitis.

Tissue and species specificity of BCP 54, the major soluble protein of bovine cornea

The isolation and biochemical characterization of the most abundant intrinsic soluble protein of bovine cornea, BCP 54 (Bovine Corneal Protein, mol. wt 54000 daltons), have recently been described. We now report results of immunochemical experiments on the distribution of BCP 54 in bovine tissues. Antisera directed against BCP 54 were raised in rabbits and used in immunodiffusion analyses of soluble extracts of bovine tissues. BCP 54 was a significant constituent of all three corneal layers-epithelium, stroma and endothelium. This protein was also present in lens epithelium but it was absent from other bovine ocular tissues. Furthermore, BCP 54 was absent from all bovine non-ocular tissues tested. Proteins immunologically related to BCP 54 were present in human cornea and the corneas of the other mammalian species tested, swine and guinea pig. However, proteins analogous to BCP 54 were not detected in the three non-mammalian corneas tested: chicken, toad and fish. The absence of BCP 54 from bovine non-ocular tissues, in addition to the finding that similar proteins exist in the corneas of other mammalian species, suggests an important, although unknown, function for BCP 54 in the cornea.

Isolation and characterization of BCP 54, the major soluble protein of bovine cornea

The most abundant soluble protein of bovine cornea, which we have named “BCP 54” (Bovine Corneal Protein, molecular weight 54000 daltons), has been isolated and biochemically characterized. This protein constitutes approximately 30% of the total soluble protein of whole cornea. BCP 54 is present in both epithelium and stroma; however, its presence in endothelium has not been demonstrated conclusively. Immunochemical analysis indicates that BCP 54 is of corneal, but not of serum, origin. Polyacrylamide gel electrophoresis in SDS of either unreduced or reduced BCP 54 yields a single protein band with a molecular weight of 54000 daltons. By gel filtration the molecular weight of the native molecule was also shown to be about 54000 daltons. Isoelectric focusing under denaturing conditions yields four bands clustered between pH 6·6 and pH 6·9. Thus BCP 54 appears to be a protein composed of a single polypeptide chain, but with some degree of microheterogeneity. The amino acid composition of BCP 54 demonstrates that it is not a collagenous or actin-like protein; furthermore, it is not a glycoprotein. The abundance and distribution of BCP 54 suggest that this protein fulfills an important, but as yet unknown, function in the cornea.