A synthetic degenerate oligonucleotide probe based on the major peptide sequence was used to screen a filter-based cDNA library prepared from whole rat epididymis in p-SPORT1 vector. Twenty-seven independent clones were selected that hybridized to the oligonucleotide probe. A single clone containing a plasmid with the largest insert was chosen for further analysis. The insert encoded a 653 nucleotide mRNA, representing a 111-amino-acid open reading frame (Fig. 13A), that included the two polypeptide sequences determined by the mass spectrometry analysis (amino acids 40-51 and 60-64 as highlighted in bold text). A Kyte Doolittle analysis of the amino acid sequence (Fig. 13B) indicated that the protein had a hydrophobic N-ter-minus suggestive of a signal peptide. Further analysis of this region using the program SignalP supported this conclusion and predicted the signal peptide to be 20 amino acids in length (underlined in Fig. 13A). The presence of a putative signal peptide is consistent with its being a secreted protein. The hydrophobicity plot also indicates the presence of a further hydrophobic region on either side of residue 60. As this region of hydrophobicity is too short to represent a transmembrane domain, it likely to be buried in the middle of a globular region of the protein. in detail
Comparison of the protein sequence of the 24-kDa subunit with other proteins in the NCBI databases (Fig. 14) using the computer program BLAST revealed 100% identity with rat epididymal protein E3 recently described by Rao et al.. Significant similarity was also found to epididymal protein ESP13.2, which has been cloned from both human and the macaque monkey. The sequence similarity between the rat 24-kDa protein and macaque ESP13.2 is not sufficiently high for the two proteins to be homologues. All three proteins, however, show weak similarity to other members of the p-defensin family (Fig. 14), especially in the central core region, which retains a group of six conserved cysteine residues. Particularly noteworthy among these sequences is that of Bin1b, which has been cloned from rat epididymis and reported to have antimicrobial activity on the basis of an anti-sense RNA assay.
FIG. 13. A) Nucleotide and deduced amino acid sequence of the 24-kDa antigen from the sperm’s plasma membrane. The amino acids underlined constitute the predicted signal sequence, and the peptides highlighted in bold type were identified and automated by MALDITOF-MS/MS sequencing. B) Kyte Doolittle hydropho-bicity plot of the amino acid sequence shown in A.
FIG. 14. Multiple sequence alignment of members of the p-defensin family. A small group of the large p-defensin family was aligned to highlight the conserved central core of six cysteines (shaded in gray). The protein sequences included have the following accession numbers: bovine p-defensin 12, AAD43032; human EP2, AAG21882; mouse p-defensin 1, P56386; rat p-defensin 2, O88514; mouse p-defensin 3, Q9WTL0; mouse p-defensin 4, P82019; human ESP13.2, CAC27121; macaca ESP13.2, CAC27133; rat 2D6, CAC36191;rat Bin-1b, AAL55636.