Purification of P26h: a hamster sperm protein

Abstract

P26h is a 26 kDa glycoprotein, located on the acrosome cap of hamster spermatozoa, involved in the species specificity of gamete interaction. We have purified this protein from hamster spermatozoa collected from the distal cauda region of the epididymis. Its purification was realized following a three-step procedure: detergent extraction, ion-exchange chromatography, and chromatofocusing. Protein partitioning using Triton X-114 (the first step) showed a ratio of 5:1 between the resulting aqueous and detergent phase. P26h was found almost exclusively in the aqueous phase where it represented about 10-12% of the total protein content. When the desalted aqueous phase was loaded on a carboxymethyl column for cation-exchange chromatography, about 80% of the proteins did not bind to the matrix and were eliminated. P26h was eluted from the column with a linear gradient of salt. At this point, P26h had a rate of purification estimated at 45-55%; three other major proteins of <21, 45, and 63 kDa remained in the sample. These undesired proteins were eliminated by submitting these samples to chromatofocusing using a PBE 94 polybuffer exchanger column. Indeed, P26h was collected almost in the dead volume of the column while the other proteins were eluted much later. Two-dimensional gel electrophoresis and Western blotting were performed to determine the purity of P26h. Only one major spot was detected, confirming the purity of P26h. Usefulness of this purified sperm antigen in the understanding of the physiology of mammalian fertilization is discussed.

Identification of a Hamster Sperm 26-Kilodalton Dehydrogenase/Reductase That Is Exclusively Localized to the Mitochondria of the Flagellum1

“…Immunoblots of total sperm lysate analyzed by two-dimensional PAGE showed the presence of one isoform of MP26 exhibiting pI ;9.0 (Fig. 3b), which is similar to the reported pI (;9.0) of P26h [25]. No spot was seen when an identical blot was stained with nonimmune guinea pig IgG (data not shown).…”

“…Epididymal fluid P26h accumulates on the acrosomal cap of hamster spermatozoa occurs as they travel through the epididymis [25,[29][30]. It was proposed that P26h is anchored to the membrane through a phosphaditylinositol linkage and that prostasome-like particles may be involved in the transfer of P26h from the epididymal fluid to the sperm plasma membrane [25].…”

“…Epididymal fluid P26h accumulates on the acrosomal cap of hamster spermatozoa occurs as they travel through the epididymis [25,[29][30]. It was proposed that P26h is anchored to the membrane through a phosphaditylinositol linkage and that prostasome-like particles may be involved in the transfer of P26h from the epididymal fluid to the sperm plasma membrane [25]. Later, the same group of investigators cloned a cDNA from a hamster testicular cDNA library and reported that P26h is a member of the shortchain dehydrogenase/reductase superfamily [31].…”

Testicular descent, sperm maturation and capacitation. Lessons from our most distant relatives, the monotremes

“…Of particular interest are the proteins that are secreted by the epididymis and interact with spermatozoa to modify their function. A number have been identified in eutherian mammals, including SPAM1 (formerly PH20; Chen et al 2006), SED1 (Ensslin and Shur 2003), α-d-mannosidase (Jin et al 1999), CRISP1 (Eberspaecher et al 1995), CD52 (Kirchhoff et al 1993), ASF (Thomas et al 1984), HE1 (Kirchhoff et al 1996) and p26H (Coutu et al 1996). However, whilst knockout mice for SPAM1 (Baba et al 2002) and SED1 (Ensslin and Shur 2003) have reduced fertility compared with wild-type, none of the knockouts is infertile.…”

Differential extraction and enrichment of human sperm surface proteins in a proteome: Identification of immunocontraceptive candidates

“…In the present study, we have exploited this differential solubilization in TX-114 to separate the hydrophobic proteins, which could include integral membrane or amphipathic proteins, from hydrophilic proteins. In previous reports TX-114 extraction was used to characterize sperm-associated proteins [16,[34][35][36][37][38][39][40]. We chose SAGA-1, a hydrophobic, GPI-anchored human sperm membrane protein [16] as a positive control in order to ensure the quality of our preparation.…”