Cloning and sequence analysis of human and bovine corneal antigen (CO-Ag) cDNA: identification of host-parasite protein calgranulin C.

PURPOSE

The primary structure of a cornea-associated antigen (CO-Ag) has been identified and has been implicated in the pathogenesis of Mooren's ulcer. The study designs were to isolate full-length clones encoding CO-Ag from a bovine and a human corneal cDNA library so that complete sequence analyses might further define the possible role of this protein in Mooren's ulcer.

METHODS

DNA fragments of bovine and human CO-Ag were generated using unique oligonucleotide primers and reverse transcription polymerase chain reaction. These fragments were used as probes to obtain cDNA clones from a bovine and a human corneal cDNA libraries. The clones with the longest cDNA inserts were selected for sequence analyses. Human cDNA fragment was digested with Stu I and Hind III and cloned into a expression vector, pPROEXHT, at the same restriction enzyme sites. The plasmid was transformed into E. coli cells. Correct cloning and the full-length sequence of human CO-Ag were determined by sequencing the insert cDNA.

RESULTS

The bovine cDNA insert sequence was 273 nucleotides in length for the entire mRNA coding region, 212 nucleotides in the 5' untranslated region, 83 nucleotides in the 3' untranslated region and a poly(A) tail. The DNA base sequence of this clone also contained a standard initiation codon, termination codon, and the polyadenylation signal. This cDNA predicts a protein which contains 91 amino acids with a molecular weight of 10,584 daltons. Plasmid expression vector, pPROEXHT-CO-Ag, was constructed that direct the synthesis of human CO-Ag in E. coli as fusion protein. Human CO-Ag fusion protein was purified to 90% pure with a yield of 17.2 mg per liter of the bacterial cell lysate. The nucleotide sequence of the CO-Ag cDNA insert was completely identical to human neutrophil calgranulin C. The deduced amino acid sequence was completely identical to a Ca(2+)-binding protein isolated on the surface of filarial nematodes.

CONCLUSIONS

The isolation and analysis of cDNA clones containing the complete coding sequence of bovine and human CO-Ag proteins is reported. The proteins identified by deduced amino acid sequences demonstrate 100% sequence homology with human and bovine calgranulin C. Immune recognition of calgranulin C to a filarial nematode may lead to a hyperactive autoimmune response to CO-Ag in the cornea leading to a Mooren's ulcer.