Fertilization and Early Embryogenesis
(M289) SPERM ACROSOMAL SLLP2 IS INVOLVED IN BINDING TO EGG PLASMA MEMBRANE AND PERIVITELLINE MATRIX.
Mandal, Arabinda1, Digilio, Laura1, Klotz, Kenneth1, Flickinger, Charles1, Herr, John1, 1 University of Virginia, Charlottesville, VA
ABSTRACT- Sperm Lysozyme Like Protein 2 (SLLP2), encoded by the gene SPACA5, was identified in a human sperm proteome by microsequencing. The full length cDNA encodes a 18 kDa protein with a N-terminal signal sequence followed by a protease cleavage site between A21 and K22. The SPACA5 transcript is 0.8 kb and appears to be testis specific as determined by 8 tissue Northern analysis and multiple tissue (n = 76) expression array. Both polyclonal and monoclonal antibodies generated against recombinant SLLP2 identified a 15 kDa protein in human sperm extracts. In capacitated spermatozoa, SLLP2 was detected in the acrosome by indirect immunofluoresence including anterior acrosome and equatorial segment. The antigen was distributed throughout the acrosomal matrix and equatorial segment as indicated by immunoelectron microscopy. The mature human SLLP2 without the signal sequence is 48 % identical to chicken egg white lysozyme and 44 % identical to human chicken type lysozyme. Both human SLLP2 and its mouse orthologue are devoid of one of the two catalytic residues essential for bacteriolytic activity of chicken type lysozymes. Soluble recombinant mature human SLLP2 expressed in yeast or in E. coli showed no bacteriolytic activity when tested against the substrate, Micrococcus lysodeikticus while control recombinant human lysozyme was active. Mature recombinant mouse SLLP2 was purified from E. coli and incubated with zona-intact and zona-free metaphase II mouse eggs at various concentrations and probed with anti-SLLP2 monoclonal antibody. Mouse SLLP2 showed specific binding to the microvillar region of zona-free eggs and to the perivitelline matrix of zona-intact mouse eggs. Recombinant human SLLP2 showed an identical pattern of binding to mouse eggs. More importantly, in mouse in vitro fertilization tests, human SLLP2 showed significant inhibition (at 30 and 15 microgram/ml) of mouse sperm-egg binding in a dose dependent manner. The initial results suggest a role of SLLP2 in mediating sperm-egg binding event during fertilization, possibly through binding to receptor(s) across the perivitelline matrix and microvilli of egg plasma membrane. 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: Perivitelline matrix, Egg plasma membrane, Acrosome, SLLP2