PARENT SESSION
GAMETE BIOLOGY AND GAMETOGENESIS - A

Monday, August 2, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(143) SLLP2, A NOVEL NON-BACTERIOLYTIC LYSOZYME-LIKE PROTEIN OF HUMAN SPERMATOZOA LINKED TO THE X CHROMOSOME.

Mandal, Arabinda¹, Klotz, Kenneth ¹, Shetty, Jagathpala¹, Vemuganti, Soumya¹, Coppola, Michael¹, Haystead, Timothy², Flickinger, Charles¹, Herr, John¹, ¹ University of Virginia, Charlottesville, VA² Duke University Medical Center, Durham, NC

ABSTRACT- Here we report SLLP2, a new member of the Sperm Lysozyme-Like Protein family, distinct from SLLP1. Micro-sequencing of human sperm proteins resolved by 2-D IEF/SDS-PAGE analyses identified novel C (chicken type) lysozyme-like peptides from a spot with a molecular weight of ∼16 kDa and a pI of ∼5.8 to 5.9. A full length cDNA encoding SLLP2 was amplified from human testicular cDNA by RACE PCR using inosine containing degenerate primers designed from a sequenced peptide. The cloned cDNA encodes a full length protein of 159 amino acids with a predicted MW of 18 kDa and a pI of 5.9 with a putative protease cleavage site between A21 and K22 after the N-terminal signal sequence. Anti-sera to the mature protein (138 amino acids) identified only a ∼15 kDa processed protein in human sperm extracts and localized SLLP2 to the acrosomal matrix by immuno-electron microscopy. Northern analysis and a multiple tissue expression array using a ³²P-labelled cDNA probe corresponding to the mature protein identified a SLLP2 mRNA of ∼0.8 kb only in testis. Quantitative RT-PCR revealed SLLP2 mRNA levels to be ∼15 times less abundant than SLLP1. The gene encoding SLLP2 is composed of four exons and is located on the X chromosome at p11.3, in contrast to SLLP1 which is encoded by the gene SPACA3 at 17q12. The mature SLLP2 showed high homology to C lysozymes (identity: human 44%, chicken 48%) and 91.3% conservation of 23 invariant residues of vertebrate C lysozymes. The modified non-conserved residues were N44D and D52E, the latter being one of the two catalytic residues of C lysozymes. As predicted from partial conservation of critical catalytic residues, expression of a soluble mature human SLLP2 in yeast showed no bacteriolytic activity. Furthermore, when both the non-conserved invariant residues of SLLP2 were restored by site-directed mutagenesis in an E. coli expression system, along with parallel expression of human lysozyme, no bacteriolytic activity was detected in the mutated SLLP2, although the recombinant human lysozyme was active. The results suggest that SLLP2 may have undergone evolutionary changes adapting to a modified role in sperm. This work was supported by Fogarty International Center D43 TW/HD 00645, NIH HD U54 29099, P30 28934, the Andrew W. Mellon Foundation and Schering AG.

KEY WORDS: sperm, lysozyme, human, sllp