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LeAnn L. Lindsay, Ph. D.Assistant Research BiochemistPhone: (530)752-6789
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The long-term goal of our laboratory is to understand the functions of the egg envelope which is found surrounding all vertebrate eggs. This extracellular matrix structure plays a crucial role in regulating sperm-egg interactions both before and after fertilization, and is thus a major subject of interest in developmental biology. We use the model system Xenopus laevis to study the egg envelope in its several functionally distinct forms. Conversion between forms involves proteolysis of specific envelope glycoproteins. When the egg is released from the ovary, the envelope cannot be penetrated by sperm. Conversion to a penetrable state occurs during passage through the pars recta portion of the oviduct, where a secreted protease, oviductin, hydrolyzes envelope glycoprotein ZPC. Following fertilization the envelope is converted to a sperm impenetrable form involving proteolysis of envelope component ZPA by an egg protease cascade. We have determined the sequence of oviductin cDNA, and found that the protease is translated as part of a large mosaic protein containing two protease domains and three CUB domains (Lindsay et al., 1999). Upon post-translational processing, the mature oviductin protease consists of a serine protease domain at the N-terminus, followed by two CUB domains. Sperm-envelope binding studies showed that coelomic envelopes treated with purified oviductin to hydrolyze ZPC exhibited a dramatic increase in the level of sperm binding. The protease trypsin was able to mimic the action of oviductin in specifically cleaving ZPC without affecting other envelope glycoproteins, and increasing sperm binding to the envelope (Lindsay and Hedrick, 1998). Thus, proteolysis of ZPC alone appears to be responsible for increasing sperm binding. The protease cascade occurring following fertilization and resulting in ZPA hydrolysis appears to involve at least three egg proteases. An extracellularly anchored serine protease with chymotrypsin-like substrate specificity (Phe-X), termed ovochymase, is activated by a trypsin-like protease (Arg-X)(Lindsay and Hedrick, 1989; Lindsay et al., 1992). The current hypothesis is that ovochymase is an intermediate in the protease cascade. A third protease, which hydrolyzes ZPA, has been identified and characterized as a metalloprotease; current work is focused on purifying this enzyme for functional studies. Ovochymase cDNA was sequenced to show that the protease is translated as part of a large polyprotein containing two other proteases and five CUB domains (Lindsay et al., 1999). The mature protease consists of the protease domain alone. (This material is based upon work supported by the National Science Foundation under Grant No. 9723667 and 9728447. Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.)
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