CLONING AND EXPRESSION OF HUMAN CORNEAL CALGRANULIN-C (CO-AG)

Purpose. A host-parasite interaction is thought to be involved in the pathogenesis of Mooren's ulcer. We have identified a cornea-associated antigen (CO-Ag), which may be a target for the autoimmune process res ulting in Mooren's ulcer. This study presents the cloning, expression, and identification of a cDNA encoding human CO-Ag, Methods. Reverse t ranscription-polymerase chain reaction (RT-PCR) was performed to ampli fy a cDNA encoding CO-Ag in the human cornea. The cDNA fragment was cl oned into a prokaryotic expression vector and the resulting plasmid wa s transformed into DH5 E. coli cells. Autoantibody reactivity to the G O-A:: fusion protein in patient sera was tested by Western blots. Resu lts. A cDNA encoding human CO-Ag was amplified by RT-PCR. The entire m RNA coding region was 273 nucleotides in length, predicting a 91-amino acid protein with a molecular weight of 10,683 daltons. The cDNA sequ ence was identical to human neutrophil calgranulin C (CaGC). Human CO- Ag was expressed in E. coli carrying a plasmid in which the CO-Ag cDNA was under control of the E. coli trc promoter. The CO Ag fusion prote in, which comprised as much as 15% of the total bacterial protein, was purified to 90% homogeneity by affinity chromatography on an immobili zed metal column. The recombinant CO-Ag protein produced was recognize d by autoantibodies in the sera of 6 of 15 patients with Mooren's ulce r and none of 14 normal control sera by Western blots. Conclusion. CO- Ag is identical to calgranulin C, a neutrophil protein found on the su rface of filarial nematodes. A host-parasite interaction may cause aut oimmunity to CO-Ag (CaGC) in the cornea resulting in a Mooren's ulcer.