SPRASA, a novel protein: an investigation into the expression profile, evolutionary conservation and the association with infertility

Abstract

SPRASA (SPACA3, SLLP1) is a newly identified protein that belongs to the lysozyme super family. Lysozymes are a family of enzymes that are universally expressed in animals, insects and plants and are ubiquitously expressed in a number of tissues and secretions in these organisms. In contrast, SPRASA expression appears to be restricted to the equatorial region and the inner acrosomal membrane of sperm. Antibodies reactive with SPRASA have been identified in some infertile men and an antiserum reactive with recombinant SPRASA prevented human sperm binding to hamster oocytes in vitro, indicating an important role in sperm oocyte recognition. The aim of this study was to investigate the role of SPRASA in human infertility. Quantitative real time RT-PCR analysis confirmed the expression of SPRASA in the testes, however, in contrast to previous reports, SPRASA expression was also identified in the ovaries and heart of both males and females. In silico analysis identified two putative promoter regions within the SPRASA gene. As expected, the first promoter is located 5’ to exon one, while the second promoter is located 5’ to exon two. Further investigations by luciferase promoter constructs identified that both promoters were capable of supporting transcriptional activity, however promoter 2 was the more effective promoter. Mutation screening of 102 infertile and 104 fertile couples identified three variants in SPRASA. Orthologue sequence analysis indicates that SPRASA is a mammalian-only gene. This is similar to another member of the lysozyme family, alpha-lactalbumin. However, unlike alpha-lactalbumin, which expresses one variant in all mammals, the SPRASA gene’s organisation in humans and simians is different to that found in prosimians and non-primates. This suggests that SPRASA has a unique function in simians compared to other mammals. Amino acid sequence alignment of the SPRASA orthologues revealed a unique motif that is not seen in other lysozyme family members. Computer modelling showed that this SPRASA motif is located in a region analogous to the substrate binding region of c-type lysozyme/alpha-lactalbumin. In conclusion, SPRASA appears to have a role in both male and female fertility and may be important in species-specific differences in mammalian fertilisation.