Studies on the spermatogenic sulfogalactolipid binding protein SLIP 1

Abstract

We have purified the testicular sulfogalactolipid binding protein SLIP 1 and shown by photoaffinity labeling that it contains an ATP binding site. Purified SLIP 1 was fluorescently labeled and shown to retain specific sulfogalactolipid binding function. This probe was used to investigate the topology of SLIP 1 binding sites on testicular germ cells. The binding pattern precisely coincided with the previously demonstrated asymmetric surface domains of sulfogalactoglycerolipid (SGG). Occasionally these SGG-containing, SLIP 1-binding cell surface domains exactly coincided with structural features on the cell surface as detected by differential interference contrast microscopy. These results demonstrate that SLIP 1/SGG interactions could provide an effective intercellular communication network between testicular germ cells within the seminiferous tubule.

Isolation of antiSLIP1-reactive boar sperm P68/62 and its binding to mammalian zona pellucida

“…The pelleted sperm were washed twice in phosphatebuffered saline (PBS), pH 7.4, at 37°C by gentle resuspension and centrifugation. Based on the previously described method for SLIP1 isolation from rat testicular cells (Lingwood and Nuttika, 1991), a crude extract of SLIP1 was prepared by treating the sperm pellet from two boars with nine volumes of 20 mM Tris HCl, pH 7.6, 1 mM ATP, 0.34 M sucrose, 1 mM EDTA, and 0.2 mM N-⑀-p-tosyl-L-lysine chloromethylketone hydrochloride (TLCK) (ATP-EDTA-sucrose solution) at 4°C for 1 hr with constant agitation and then subsequently pelleted by centrifugation (500 ϫ g, 25 min, 4°C). The supernatant was ultracentrifuged (100,000 ϫ g, 1 hr, 4°C), and the final supernatant containing SLIP1 was then dialyzed extensively against Milli-Q water (Millipore Canada, Nepean, Ontario) at 4°C and lyophilized.…”

“…SLIP1 also was purified from the ATP-EDTA-sucrose extract of rat testis cells by DEAE cellulose column chromatography, following the method described previously (Lingwood and Nuttika, 1991).…”

“…A method to purify SLIP1 from rat testicular ATP-EDTA-sucrose extracts using DEAE cellulose column chromatography has been described (Lingwood and Nuttika, 1991). Although this method produced a major silver-stained 68-kDa protein band by SDS-PAGE (12% polyacrylamide gel) (Lingwood and Nuttika 1991;Tanphaitchitr et al, 1993), our attempts to sequence the purified protein by repetitive Edman degradation revealed that SLIP1 was copurified with rat serum albumin, which has the same molecular mass of 68 kDa.…”

“…A method to purify SLIP1 from rat testicular ATP-EDTA-sucrose extracts using DEAE cellulose column chromatography has been described (Lingwood and Nuttika, 1991). Although this method produced a major silver-stained 68-kDa protein band by SDS-PAGE (12% polyacrylamide gel) (Lingwood and Nuttika 1991;Tanphaitchitr et al, 1993), our attempts to sequence the purified protein by repetitive Edman degradation revealed that SLIP1 was copurified with rat serum albumin, which has the same molecular mass of 68 kDa. Taking advantage of the fact that SLIP1 is conserved across various vertebrate species (Law et al, 1988) and that it exists in not only spermatogenic cells (Law et al, 1988;Lingwood, 1985) but also in mature spermatozoa (Lingwood, 1986;Tanphaitchitr et al, 1993), we used ejaculated boar spermatozoa as a biological source for ATP-EDTA-sucrose extraction.…”

“…SLIP1 appears to be evolutionally conserved, since it is present in the testes of various vertebrates (i.e., rat, mouse, hamster, guinea pig, rabbit, dog, pig, bull, rooster, frog, and fish) (Law et al, 1988). Based on its ATP binding property (Lingwood and Nuttika, 1991), SLIP1 can be extracted from testicular germ cells and ejaculated sperm by a sucrose solution containing low concentrations of ATP and EDTA, suggesting that it is a peripheral plasma membrane protein (Law et al, 1988). We have demonstrated by electron microscopy, using immunoprotein A-gold labeling, that SLIP1 is localized on the plasma membrane of the mouse sperm head (Tanphaichitr et al, 1993).…”