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

^a Departments of Cell Biology and ^b Obstetrics & Gynecology, University of Virginia Health System, Charlottesville, Virginia 22908 ^c Ludwig Institute for Cancer Research, University College Branch, London, United Kingdom W1P 8BT 91

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.

FOOTNOTES

First decision: 24 January 2000.

¹ Supported by NIH grants HD U54 29099, P30 28934, T32 DK 07642, T32 HD 07382, U54 HD 28934, and D43 TW/HD 00654; and by the Fogarty International Center and the Andrew W. Mellon Foundation.

² Correspondence: John C. Herr, Department of Cell Biology, Health Sciences Center, Box 800732, University of Virginia, Charlottesville, VA 22908. FAX: 804 982 3912; jch7k{at}virginia.edu

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