A novel retinal pigment epithelial protein suppresses neutrophil superoxide generation. II. Purification and...

In the preceding communication, a novel protein was reported. This protein is secreted by retinal pigment epithelial (RPE) cells, and inhibits generation of superoxide by activated neutrophils in vitro. This protein is synthesized endogenously by RPE cells. The mechanism of inhibition is not by scavenging of the released superoxide; rather the protein intervenes the activation sequence of the neutrophils and, therefore, the consequential release of superoxide. In this study, the novelty of this RPE protein is further established by structural characterization. The supernatant proteins from rabbit RPE cultures were initially separated by a conventional, high capacity, DEAE Sepharose CL-6B anion exchange chromatography. The inhibitory activity on neutrophil superoxide generation was displayed in one fraction. This fraction was further purified either by microseparation anion exchange-high performance liquid chromatography or by microcapillary reversed phase-high performance liquid chromatography. Sodium dodecyl sulfate gel electrophoresis was used in every step of the purification to monitor the homogeneity of the protein. The final purification gave a doublet of 69/75 kDa without other contaminating bands. The microsequencing analysis was carried out by the Edman degradation. Both N-termini were found to be blocked, and the 'on membrane' internal trypsin digestion was carried out. Secondary ion mass spectrometry was used to assess the purity of the tryptic fragments before sequencing analysis. From both the 69 and 75 kDa bands, a total of eight polypeptide sequences were obtained. Three of these sequences share some degree of homology with transferrin family proteins; and the other five sequences did not match any sequence in the database. Therefore, this RPE secretory protein appears to be novel, both in the primary amino acid sequence and in its function. No RPE secretory protein has been reported to display the superoxide inhibitory capability. If this function is also operative in vivo, it could suppress neutrophil-mediated tissue damage in inflammation.