Differential Nuclear Localization of the Cancer/Testis-Associated Protein, SPAN-X/CTp11, in Transfected Cells and in 50% of Human Spermatozoa1

Abstract

Cancer-testis antigens (CTAs) represent potential targets for cancer immunotherapy because these proteins are widely distributed in tumors but not in normal tissues, except testes. In this paper, we identify homology of the CTA CTp11 with SPAN-X (sperm protein associated with the nucleus mapped to the X chromosome). On two-dimensional Western blots of human sperm extracts, SPAN-X antibodies recognized 19 spots ranging from 20 to 23 kDa with isoelectric points from 5.0 to 5.5. Differential extraction of spermatozoa demonstrated 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 number of X- and Y-bearing spermatozoa exhibited SPAN-X staining. In transfected mammalian CV1 cells, the SPAN-Xa and SPAN-Xb proteins were localized to the nucleus and cytoplasm, respectively, by indirect immunofluorescence. On immunoblots of CV1 cells, the SPAN-Xa protein migrated at 15-20 kDa, whereas 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 specifically localized to these poorly characterized structures of the mammalian sperm nucleus and provides a unique biochemical marker for investigation of their function in spermatozoa as well as the role of SPAN-X/CTp11 in human tumors.

Genomic Organization, Incidence, and Localization of the SPAN-X Family of Cancer-Testis Antigens in Melanoma Tumors and Cell Lines

“…[7][8][9]. Ultrastructurally, the proteins are associated with the nuclear envelope of ejaculated spermatozoa.…”

Dynamic structure of the SPANX gene cluster mapped to the prostate cancer susceptibility locus HPCX at Xq27

“…SPANX genes encode small unfolded proteins (∼100 amino acid residues), to some extent resembling the high mobility group A (HMGA) proteins involved in the formation of various nucleoprotein complexes. Similar to these proteins, SPANXs can form dimers and complexes with other proteins (Westbrook et al 2000(Westbrook et al , 2001. In transformed mammalian cells, SPANX proteins are associated with the nuclear envelope, a location similar to the one in human spermatozoa (Zendman et al 1999;Westbrook et al 2001Westbrook et al , 2004.…”

The SPANX gene family of cancer/testis-specific antigens: Rapid evolution and amplification in African great apes and hominids

“…he sperm protein associated with the nucleus on the X chromosome (SPANX) multigene family encodes proteins whose expression is restricted to the normal testis and certain tumors (1,2). These postmeiotically transcribed genes comprise one of the few examples of haploid expression from X-linked genes (3).…”

“…In spermatozoa, SPANX proteins are found in the form of dimers or complexes with other proteins (1,3). The SPANX cluster on chromosome X consists of five genes.…”

“…Expression profile analysis showed that at least four of the family members (SPANX-A1, -A2, -B, and -D) are expressed in cancer cells, including highly metastatic cell lines from melanomas, bladder carcinomas, and myelomas (1,(9)(10)(11). In transformed mammalian cells, SPANX proteins are associated with the nuclear envelope, a location similar to that in human spermatids and spermatozoa (1,9,10). Therefore SPANX represent a specific subgroup of cancer͞testis-associated antigens (8), which are considered to be prime candidates for tumor vaccines.…”