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A. Wohabrebbi, M.M. Jablonski; The Xenopus Anti-Photoreceptor-1 (XAP-1) Antigen Is a Unique Protein and Does Not Belong to the Gamma-Crystallin Family of Proteins . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1533.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Our previous work suggested that the XAP-1 antigen, an unidentified photoreceptor-specific protein localized to the plasma membrane surrounding the inner and outer segments, was homologous to the gamma (γ)-crystallins; however this association remains controversial. We have accumulated evidence to demonstrate that the XAP-1 antigen is unique and not a member of the γ-crystallin family. Methods: Immunohistochemistry using XAP-1 and γ-crystallin antibodies was performed on whole Xenopus embryonic eyes. Retinas of several species and Xenopus lenses were collected and electrophoretically separated on 1D gels followed by Western blot analyses with the same antibodies. Outer segment-enriched fractions were obtained by mechanical isolation of outer segments from freshly dissected Xenopus retinas. These samples were then subjected to isoelectric focusing and 2D gel electrophoresis. Silver staining was performed to visualize the separated proteins. Western blot analyses were carried out to determine XAP-1 antigen localization on the 2D gel. Results: As previously reported, the XAP-1 antigen-like immunoreactivity was localized exclusively to photoreceptors. On the contrary, γ-crystallin-like immunolabeling was exclusive to the lens. Western blot analyses of Xenopus retinas and lenses separated under reduced conditions on 1D gels detected the XAP-1 antigen at relative molecular masses (Mr) of 45 and 22kDa, and at 22kDa in retinas and lenses, respectively. In contrast, γ-crystallin was detected at Mr of 22kDa in both the retina and the lens. Under non-reduced conditions, neither antibody recognized antigens of the same Mr as the other. 2D electrophoretic separation of outer segment-enriched fractions in conjunction with Western blotting identified a single XAP-1 antigen spot at an isoelectric point of 5.4 and Mr of 37kDa. Conclusions: Our evidence strongly suggests that the XAP-1 antigen is not a γ-crystallin. While both the XAP-1 and γ-crystallin antibodies recognize a band at Mr 22kDa in retina and lens samples under reduced conditions, the XAP-1 antibody alone recognizes a band at Mr 45kDa in the retina. In addition, the distinct immunohistochemical localization pattern of the proteins and the lack of cross reactivity of the antibodies under non-reduced conditions strongly suggest that the two proteins are not identical. Tryptic digestion of the XAP-1 antigen spot isolated from the 2D gel and its MALDI-TOF mass spectrum analysis are currently underway to determine the identity of the XAP-1 antigen.
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