SFE1, a constituent of the fertilization envelope in the sea urchin is made by oocytes and contains low-density lipoprotein-receptor-like repeats.
At fertilization in most animals, cortical granules of the egg or oocyte secrete their contents, whose function it is to modify the extracellular matrix. This modified matrix then participates in the block to polyspermy and protection for early embryonic development. In the sea urchin, contents of the cortical granules are secreted within 30 sec of insemination. Several of these content proteins then bind to the nascent vitelline layer of the egg and lift off the cell surface to form a stable, impervious, fertilization envelope. At least six major proteins are present in the envelope, and recently we have identified cDNA clones of two, ovoperoxidase, and SFE9. Here we report on the identification and characterization of SFE1, a constituent of the fertilization envelope of the sea urchin Strongylocentrotus purpuratus, that has revealing characteristics of how the envelope might form and what protein interaction domains might predominate. We present the largest cDNA sequence we were able to identify representing approximately two thirds of the predicted protein coding region. The C-terminal half of the cognate SFE1 protein contains two different amino acid repeat motifs: a cysteine-rich (15%) motif of 40 amino acids that is tandemly repeated 22 times and is followed by a serine/threonine-rich (38%) repeat of 63 amino acids that is tandemly repeated 3.5 times. Surprisingly, just N-terminal to the cysteine-rich repeat region is a sequence of five repeats with similarity to repeats in another cortical granule protein, SFE9, and to the motif originally identified in the receptor of low-density lipoproteins, the LDLr motif. The amino acid composition deduced from the partial SFE1 cDNA is similar also to the composition of proteoliaisin, a protein thought to tether the ovoperoxidase to the vitelline layer of the egg and thereby sequester the crosslinking activity of the ovoperoxidase to a limited population of proteins in the fertilization envelope. However, by use of monoclonal and polyclonal antibodies to SFE1 and proteoliaisin, we show here that they are distinct gene products. We also show that SFE1 is packed selectively into the cortical granules and then is crosslinked into the fertilization envelope following fertilization. In situ RNA hybridization analysis shows that the mRNA of SFE1 (9 kilobases) is present in oocytes selectively and is turned over rapidly in the oocyte following germinal vesicle breakdown. Our findings suggest that the gene encoding this major product of the egg is activated concomitantly with the other cortical granule-specific products already identified, and that a common LDLr-like motif of the fertilization envelope may reveal a structural mechanism for protein interactions in its construction.
Wessel
G M
GM
Department of Molecular and Cell Biology, Brown University, Providence, Rhode Island 02912, USA. rhet@brown.edu
Conner
S
S
Laidlaw
M
M
Harrison
J
J
LaFleur
G J
GJ
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D005306:Fertilization / Q000502:physiology*; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism* / Q000648:ultrastructure; D010063:Ovum / Q000378:metabolism* / Q000648:ultrastructure; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011973:Receptors, LDL / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D012617:Sea Urchins; D012795:Sialoglycoproteins / Q000096:biosynthesis*
D018076:DNA, Complementary; D012333:RNA, Messenger; D011973:Receptors, LDL; D011994:Recombinant Proteins; D012795:Sialoglycoproteins; C057821:apopolysialoglycoprotein, salmonid fish eggs