Research

           The long term objective of this lab is to investigate the molecular mechanisms involved in fertilization. The egg envelope and extracellular matrix play important roles in sperm-egg binding, egg activation , and the block to polyspermy. Molecular mechanisms involved are lectin-ligand binding, limited proteolysis, and conformational changes. During a cortical reaction, contents of the cortical granules are released at fertilization. This exocytotic reaction is triggered by the fertilizing sperm. The macromolecular components of the cortical granules modify the molecular structure and the supramolecular architecture of the egg extracellualar matrix (ECM), in particular a substructure of the ECM termed the egg envelope. These ECM modifications maintain the diploid state of the fertilized egg chromosomes by preventing additional sperm from fusing with the egg.

               In addition, remodeling of the ECM (elevation, hardening, and permeability changes of the egg envelope) alters the barrier between the external space and internal embryonic space encapsulated by the ECM. This alteration provides mechanisms for regulating the interaction of the embryo with its external environment.

             Egg Proteases- The glycoprotein egg envelope surrounding the Xenopus laevis egg exists in three 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 gp43 to gp41 (Hardy and Hedrick, 1992). Following fertilization the envelope is converted to a sperm impenetrable form involving proteolysis of gp69 to gp66 by an egg protease cascade (Lindsay and Hedrick, 1989).
             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. Upon post-translational processing, the mature oviductin protease consists of a serine protease domain at the N-terminus, followed by two CUB domains. The remaining serine protease domain, which lacks an active site histidine, would be coupled to one CUB domain. The functional significance of the CUB domains is discussed below. Sperm-envelope binding studies showed that coelomic envelopes treated with purified oviductin to hydrolyze gp43 to gp41 exhibited a dramatic increase in the level of sperm binding. The protease trypsin was able to mimic the action of oviductin in specifically cleaving gp43 to gp41 without effecting other envelope glycoproteins, and increasing sperm binding to the envelope. Thus, proteolysis of gp43 alone appears to be responsible for increasing sperm binding. It is known that the envelope undergoes an overall conformational change in response to gp43 hydrolysis (Bakos et al., 1990). Since the Xenopus sperm binding site has been identified as gp69 (Tian et al., 1997), it appears that this site is exposed following the conformational change triggered by gp43 hydrolysis.

          The protease cascade occurring following fertilization and resulting in gp69 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 gp69, has been identified but is not yet characterized as a protein. Ovochymase cDNA has been partially sequenced (3' end complete), which indicates that the protease is translated as part of a large protein, C-terminal to at least two CUB domains. The mature protease consists of the protease domain alone.An interesting question regards the function of the CUB domains contained in both oviductin and ovochymase, and also Xenopus hatching enzyme, an astacin type protease which hydrolyzes envelope glycoproteins to allow for embryo escape (Katagiri et al., 1997). The CUB domains are composed of 110 amino acids and are common to extracellular proteins, primarily proteases, involved in developmental processes (Bork and Beckmann, 1993). Interestingly, the mammalian spermadhesin molecules, derived from the male reproductive tract, are essentially one CUB domain, and appear to mediate sperm-egg envelope interactions (Tˆpfer-Petersen et al., 1995). We propose that the CUB domains of oviductin, ovochymase, and hatching enzyme act similarly to mediate interactions with the egg envelope or extracellular matrix. In the case of oviductin and hatching enzyme, the CUB domains might act to tether the protease to the egg envelope as a way of concentrating the protease in the area of its substrate. For ovochymase, the CUB domains may serve to anchor the proenzyme in the egg extracellular matrix until the protease domain is released upon activation. Experiments to test this are underway.

            Cortical Granule Lectin- The binding of lectin (carbohydrate binding proteins) released from the cortical granule to ligand triggers modifications in the egg extracellular matrix and provides a block to polyspermy. In these studies we investigate: 1) Protein and glycan structural studies of the cortical granule lectin. The amino acid sequence of the lectin was previously determined by Edman sequencing and recombinant DNA methods. The functionally required glycan structures of the lectin were determined to be the N-linked type. 2) Cortical granule lectin functional studies include searching sequence databases for sequence homologies to infer function from other proteins/glycoproteins. 3) Ligand structural studies will determine the amino acid sequence and the glycan sequence. 4) Ligand functional studies will determine the oviductal and subcellular site of ligand biosynthesis. These studies are done with the anuran Xenopus laevis and Chinook salmon eggs.

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