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Tyrosine Phosphorylation Generates Multiple Isoforms of the Mitochondrial Capsule Protein, Phospholipid Hydroperoxide Glutathione Peroxidase (PHGPx), During Hamster Sperm Capacitation¹

Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Sperm capacitation is a maturation process, occurring in the female reproductive tract, that produces fertilization-competent spermatozoa. Protein tyrosine phosphorylation represents an important event in capacitation. The present study demonstrates the capacitation-dependent tyrosine-phosphorylation of phospholipid hydroperoxide glutathione peroxidase (PHGPx), the disulfide cross-linked, major structural protein of the sperm mitochondrial capsule. Immunofluorescence microscopy using an antiphosphotyrosine monoclonal antibody (anti-pY20) demonstrated the presence of capacitation-associated tyrosine phosphorylated proteins in the flagellum of hamster spermatozoa. Among the tyrosine-phosphorylated polypeptides (M_r 19 000– 99 000), a 19-kDa polypeptide was the only one that can be solubilized completely by Triton X-100-dithiothreitol (DTT). The 19-kDa polypeptide was purified by anion-exchange chromatography and by immunoaffinity chromatography. Proteomic identification of the 19-kDa polypeptide by nano-electrospray tandem mass spectrometry yielded six peptides that matched the National Center for Biotechnology Information (NCBI) database sequences of bovine PHGPx. Indirect immunofluorescence localized PHGPx to the midpiece of the flagellum and the immunoblot analysis demonstrated its DTT-dependent release from purified flagella. DTT extracts of noncapacitated spermatozoa exhibited a charge train of four major PHGPx isoforms (pIs 7.5– 9.0) by two-dimensional PAGE, whereas capacitated spermatozoa revealed the generation of new acidic PHGPx isoforms with isoelectric points ranging between pH 6.0–7.0 and 4.0–5.0, indicating that it is posttranslationally modified during capacitation. These data suggest that the tyrosine-phosphorylation of PHGPx may represent an important event in the signaling pathway(s) associated with capacitation and could potentially affect mitochondrial function.

¹ Supported by HD044863.

² Correspondence. FAX: 615 343 4539; subir.k.nag.das{at}vanderbilt.edu

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