Abstract
HSP-3 is a member of the cysteine-rich secretory protein (CRISP) family from stallion seminal plasma. We report a large-scale purification protocol for native HSP-3. This protein is a non-glycosylated polypeptide chain with a p I of 8–9 and an isotope-averaged molecular mass of 24 987±3 Da. The molecular mass of HSP-3, determined by equilibrium sedimentation, is 26 kDa, showing that the protein exists in solution as a monomer. The concentration of HSP-3 in the seminal plasma of different stallions ranged from 0.3 to 1.3 mg/ml. On average, 0.9–9 million HSP-3 molecules/cell coat the postacrosomal and mid-piece regions of an ejaculated, washed stallion spermatozoon, suggesting a role in sperm physiology. Conformational characterisation of purified HSP-3 was assessed by combination of circular dichroism and Fourier-transform infrared spectroscopies and differential scanning microcalorimetry. Based on secondary structure assignment, HSP-3 may belong to the α+β class of proteins. Thermal denaturation of HSP-3 is irreversible and follows a non-two state transition characterised by a T m of 64°C, an enthalpy change of 75 kcal/mol, and a van 't Hoff enthalpy of 184 kcal/mol. Analysis of the spectroscopic and calorimetric data indicates the occurrence of aggregation of denatured HSP-3 molecules and suggests the monomer as the cooperative unfolding unit.
Highlights
Mammalian fertilisation involves a sequence of speci¢c and well orchestrated interactions between homologous gametes
Isolation and molecular characterisation of HSP-3 HSP-3 is an abundant component of stallion seminal plasma [18] and a novel member of the androgen-dependent cysteine-rich secretory protein (CRISP) family originally described in the mouse reproductive tract, and more recently found predominantly in the salivary glands of several species [15]
Quantitation of HSP-3 in the seminal plasma of di¡erent stallions by competitive ELISA showed a large variation of its concentration, ranging from 0.3 to 1.3 mg/ml
Summary
Mammalian fertilisation involves a sequence of speci¢c and well orchestrated interactions between homologous gametes (reviewed in [1]). After getting round the cumulus cell mass surrounding the egg at ovulation, capacitated spermatozoa bind to glycoconjugates of the oocyte's extracellular coat, the zona pellucida This glycoprotein matrix induces the acrosome reaction, an exocytotic event by which proteolytic enzymes (e.g. acrosin) are liberated aiding acrosome-reacted spermatozoa to penetrate the zona pellucida. Protein DE, originally localised on the dorsal region of the sperm head, migrates to the equatorial segment concomitantly with the occurrence of the acrosome reaction [13] and is believed to participate in sperm-egg fusion by binding to complementary sites on the egg plasma membrane [8,14]. In stallion seminal plasma we have identi¢ed a 27 kDa protein (called horse seminal plasma protein 3, or HSP-3), which displays extensive amino acid sequence similarity with CRISPs [18]. We report the biochemical and conformational characterisation of HSP-3
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