Immunochemical recognition of A2E, a pigment in the lipofuscin of retinal pigment epithelial cells
- Chandima Abeywickrama*,
- Hiroko Matsuda*,
- Steffen Jockusch*,
- Jilin Zhou†,
- Young P. Jang†,
- Bi-Xing Chen‡,
- Yasuhiro Itagaki*,
- Bernard F. Erlanger‡,
- Koji Nakanishi*,§,
- Nicholas J. Turro*,§, and
- Janet R. Sparrow†,¶,‖
- *Department of Chemistry, Columbia University, New York, NY 10027; and
- †Departments of Ophthalmology,
- ¶Pathology and Cell Biology, and
- ‡Microbiology, Columbia University, New York, NY 10032
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Contributed by Nicholas J. Turro, July 19, 2007 (received for review May 15, 2007)
Abstract
The autofluorescent lipofuscin pigment A2E accumulates in retinal pigment epithelial cells with age and is particularly abundant in some retinal disorders. To generate a polyclonal antibody that recognizes this pyridinium bisretinoid molecule, we immunized rabbits with bovine serum albumin (BSA) conjugates in which the protein was linked to the A2E molecule via its pyridinium ethanolamine moiety. Analysis by matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) of the A2E–BSA conjugate indicated the presence of five intact A2E molecules covalently linked to BSA, thus deeming it a suitable antigen for immunization. By immunocytochemical staining, the rabbit polyclonal antibody recognized A2E that had accumulated in cultured cells, whereas dot-blot analysis revealed binding to both A2E and A2E-rabbit serum albumin (A2E–RSA) conjugate but no cross-reactivity with various retinoids. Preimmune serum was nonreactive. In fluorescence spectroscopy studies, antibody-A2E binding was evidenced by a fluorescence increase and by a blue-shift in the emission maximum consistent with a change in A2E milieu upon antibody binding. The changes in fluorescence emission upon antibody binding could reflect several processes including restrictions on trans-cis isomerization and intersystem crossing of photo-excited A2E.
Footnotes
- §To whom correspondence may be addressed at: Department of Chemistry, Columbia University, 3000 Broadway, New York, NY 10027. E-mail: njt3{at}columbia.edu or kn5{at}columbia.edu
- ‖To whom correspondence may be addressed at: Departments of Ophthalmology, Pathology, and Cell Biology, Columbia University, 630 West 168th Street, New York, NY 10032. E-mail: jrs88{at}columbia.edu
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Author contributions: C.A., S.J., B.F.E., K.N., N.J.T., and J.R.S. designed research; C.A., H.M., S.J., J.Z., Y.P.J., B.-X.C., and Y.I. performed research; K.N., N.J.T., and J.R.S. contributed new reagents/analytic tools; C.A., S.J., B.F.E., and J.R.S. analyzed data; and C.A., S.J., and J.R.S. wrote the paper.
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The authors declare no conflict of interest.
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↵ ** An induced CD was not observed in the 330–440 nm region of the A2E maxima (data not shown), presumably because of weak intensity of the induced CD.
- Abbreviations:
- RSA,
- rabbit serum albumin;
- RPE,
- retinal pigment epithelium.
- © 2007 by The National Academy of Sciences of the USA
