Structure and function of epididymal protein cysteine-rich secretory protein-1.

Cysteine-rich secretory protein-1 (CRISP-1) is a glycoprotein secreted by the epididymal epithelium. It is a member of a large family of proteins characterized by two conserved domains and a set of 16 conserved cysteine residues. In mammals, CRISP-1 inhibits sperm-egg fusion and can suppress sperm capacitation. The molecular mechanism of action of the mammalian CRISP proteins remains unknown, but certain non-mammalian CRISP proteins can block ion channels. In the rat, CRISP-1 comprises two forms referred to as Proteins D and E. Recent work in our laboratory demonstrates that the D form of CRISP-1 associates transiently with the sperm surface, whereas the E form binds tightly. When the spermatozoa are washed, the E form of CRISP-1 persists on the sperm surface after all D form has dissociated. Cross-linking studies demonstrate different protein-protein interaction patterns for D and E, although no binding partners for either protein have yet been identified. Mass spectrometric analyses revealed a potential post-translational modification on the E form that is not present on the D form. This is the only discernable difference between Proteins D and E, and presumably is responsible for the difference in behavior of these two forms of rat CRISP-1. These studies demonstrate that the more abundant D form interacts with spermatozoa transiently, possibly with a specific receptor on the sperm surface, consistent with a capacitation-suppressing function during sperm transit and storage in the epididymis, and also confirm a tightly bound population of the E form that could act in the female reproductive tract.

Epididymal secreted protein Crisp-1 and sperm function.

Crisp-1 is a member of the cysteine-rich secretory protein family. This family of proteins is characterized by the presence of 16 conserved cysteine residues, the characteristic from which the family name is derived. Members of the Crisp protein family are found in the secretions of the reproductive tract and salivary glands, including venom toxins from several species of snakes and lizards. The Crisp proteins are modular, each containing an amino terminal pathogenesis-related (PR)-like domain and a carboxyl terminal cysteine-rich domain (CRD) connected by a hinge region. Sequence and structural similarities to proteins with known functions suggest that the Crisp family of proteins may act by regulating cellular ion channels. Rat Crisp-1 is synthesized as two distinct isoforms (referred to as Proteins D and E) by the epididymal epithelium and both are secreted into the luminal fluid where they interact with spermatozoa. Our laboratory has correlated Crisp-1 binding to sperm with inhibiting the signaling cascades that initiate capacitation while others have shown that blocking Crisp-1 binding sites on oocytes interferes with sperm-egg fusion. We hypothesize that the D and E populations of rat Crisp-1 have different interactions with sperm that modulate these distinct biological activities. Through tandem mass spectrometry (MS/MS) and monosaccharide composition analyses, we have identified at least one difference between the D and E forms as an additional single O-linked N-acetyl galactosamine on an amino terminal threonine residue in Protein E. This post-translational modification appears to account for the unique 'E' epitope bound by monoclonal antibody 4E9 developed in our laboratory, and may also lead to differential processing and localization of Protein E on sperm, when compared to Protein D. These findings are the first step in distinguishing the molecular basis of the biological activities of the D and E forms of rat Crisp-1. The epididymal-specific expression of Crisp-1, combined with its role in regulation of sperm capacitation and oocyte interaction, make it an attractive target for post-testicular contraceptive development.