Differential nuclear localization of the cancer/testis-associated protein,SPAN-X/CTp11, in transfected cells and in 50% of human spermatozoa

Cancer-testis antigens (CTAs) represent potential targets for cancer immuno therapy because these proteins are widely distributed in tumors but not in normal tissues, except testes. in this paper, we identify homology of the C TA CTp11 with SPAN-X (sperm protein associated with the nucleus mapped to t he X chromosome). On two-dimensional Western blots of human sperm extracts, SPAN-X antibodies recognized 19 spots ranging from 20 to 23 kDa with isoel ectric points from 5.0 to 5.5. Differential extraction of spermatozoa demon strated that the SPAN-X protein is highly insoluble. Only 50% of ejaculated spermatozoa exhibited SPAN-X immunofluorescent staining. Dual localization of the sex chromosomes and the SPAN-X protein demonstrated that an equal n umber of X- and Y-bearing spermatozoa exhibited SPAN-X staining. In transfe cted mammalian CV1 cells, the SPAN-Xa and SPAN-Xb proteins were localized t o the nucleus and cytoplasm, respectively, by indirect immunofluorescence. On immunoblots of CV1 cells, the SPAN-Xa protein migrated at 15-20 kDa, whe reas the SPAN-Xb protein migrated at a higher molecular weight of 21-22 kDa . The SPAN-X protein was ultrastructurally associated with nuclear vacuoles and the redundant nuclear envelope. SPAN-X is the first protein specifical ly localized to these poorly characterized structures of the mammalian sper m nucleus and provides a unique biochemical marker for investigation of the ir function in spermatozoa as well as the role of SPAN-X/CTp11 in human tum ors.