Cloning and expression of human corneal calgranulin C (CO-Ag).
OBJECTIVE
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 resulting in Mooren's ulcer. This study presents the cloning, expression, and identification of a cDNA encoding human CO-Ag.


METHODS
Reverse transcription-polymerase chain reaction (RT-PCR) was performed to amplify a cDNA encoding CO-Ag in the human cornea. The cDNA fragment was cloned into a prokaryotic expression vector and the resulting plasmid was transformed into DH5 E. coli cells. Autoantibody reactivity to the CO-Ag fusion protein in patient sera was tested by Western blots.


RESULTS
A cDNA encoding human CO-Ag was amplified by RT-PCR. The entire mRNA coding region was 273 nucleotides in length, predicting a 91-amino acid protein with a molecular weight of 10,683 daltons. The cDNA sequence 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 protein, which comprised as much as 15% of the total bacterial protein, was purified to 90% homogeneity by affinity chromatography on an immobilized metal column. The recombinant CO-Ag protein produced was recognized by autoantibodies in the sera of 6 of 15 patients with Mooren's ulcer and none of 14 normal control sera by Western blots.


CONCLUSIONS
CO-Ag is identical to calgranulin C, a neutrophil protein found on the surface of filarial nematodes. A host-parasite interaction may cause autoimmunity to CO-Ag (CaGC) in the cornea resulting in a Mooren's ulcer.
Gottsch
J D
JD
Wilmer Ophthalmological Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Liu
S H
SH
D000595:Amino Acid Sequence; D001323:Autoantibodies / Q000032:analysis; D001324:Autoantigens / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003315:Cornea / Q000276:immunology*; D003320:Corneal Ulcer / Q000276:immunology; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005136:Eye Proteins / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D009418:S100 Proteins; D000067616:S100A12 Protein
D001323:Autoantibodies; D001324:Autoantigens; D002135:Calcium-Binding Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D005136:Eye Proteins; D011993:Recombinant Fusion Proteins; D009418:S100 Proteins; D000067616:S100A12 Protein; C090441:S100A12 protein, human