Identification of sperm equatorial segment protein 1 in the acrosome as the primary binding target of peanut agglutinin (PNA) in the mouse testis.

Peanut agglutinin (PNA), a plant lectin protein that recognizes the galactose β (1 -> 3) N-acetylgalactosamine carbohydrate sequence, has been widely used as a sperm acrosome-specific marker; however, the acrosomal glycoproteins that specifically bind to PNA have yet to be identified. We herein purified and identified PNA-binding glycoproteins in the mouse testis using biotinylated PNA and streptavidin-coupled magnetic beads, and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. In six repeated experiments, sperm equatorial segment protein 1 (SPESP1) was detected most frequently as a PNA-binding glycoprotein, followed by dipeptidase 3, proacrosin-binding protein, and acrosin prepropeptide. The identification of SPEPS1 in the testis lysate and its PNA-bound fraction was verified with lectin and Western blot analyses, and the co-localization of PNA and SPEPS1 in acrosomes was confirmed with lectin- and immunohistochemistry. Since the PNA reactivity of sperm acrosomes was observed not only in normal mice, but also in SPESP1-deficient mice, although at lower levels, PNA was also considered to bind to other candidate glycoproteins. The present study identified SPESP1 in the acrosome as the primary binding target of PNA in the mouse testis. Further defining the specific lectin-glycoprotein relationships in individual cells will enhance the value of lectin histochemistry.