L L Lindsay

University of California, Davis, Davis, CA, United States

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Publications (16)46.83 Total impact

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    ABSTRACT: The vitelline coats (VCs) of Bufo japonicus and Xenopus laevis undergo coelomic egg (CEVC)- to uterine egg (UEVC)-type conversions, after passage through the pars recta (PR) portion of oviduct or treatment of eggs with secretory granules from PR (PRGs), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of glycoproteins. These conversions include for Bufo the loss of 40K–52K components from the CEVC, concomitant with increasing stainability of a 39K component and the appearance of a 36K component in the UEVC. For Xenopus, a 43K glycoprotein in the CEVC is transformed to a 41K glycoprotein in the UEVC. When coelomic eggs of both Bufo and Xenopus were treated with PRGs from the heterologous species, the macromolecular components of the VCs were processed in exactly the same way as with homologous PRGs. The PRGs from both species showed strikingly similar hydrolytic activities against Boc-Val-Leu-Lys-MCA and several peptidyl-Arg-MCAs. Bufo coelomic eggs treated with Xenopus PRGs were not fertilizable by Bufo sperm, whereas treatment with Bufo PRGs rendered the eggs fertilizable. We conclude that the oviductal enzymes and VC substrates in these species are evolutionarily conserved and that macromolecular alterations of VC glycoproteins as detected by SDS-PAGE are necessary, but not sufficient, for making coelomic eggs fertilizable.
    Journal of Experimental Zoology 04/2005; 244(1):145 - 150.
  • Leann L Lindsay, Jerry L Hedrick
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    ABSTRACT: The egg envelope of most animal eggs is modified following fertilization, resulting in the prevention of polyspermy and hardening of the egg envelope. In frogs and mammals a prominent feature of envelope modification is N-terminal proteolysis of the envelope glycoprotein ZPA. We have purified the ZPA protease from Xenopus laevis eggs and characterized it as a zinc metalloprotease. Proteolysis of isolated egg envelopes by the isolated protease resulted in envelope hardening. The N-terminal peptide fragment of ZPA remained disulfide bond linked to the ZPA glycoprotein moiety following proteolysis. We propose a mechanism for egg envelope hardening involving ZPA proteolysis by an egg metalloprotease as a triggering event followed by induction of global conformational changes in egg envelope glycoproteins.
    Biochemical and Biophysical Research Communications 12/2004; 324(2):648-54. · 2.28 Impact Factor
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    ABSTRACT: A strategy combining accurate mass determination, tandem mass spectrometry, structure homology, and exoglycosidases is described that allows the structural characterization of mucin-type O-linked oligosaccharides. The method is used to profile with quantitation the O-linked oligosaccharide (both neutral and anionic) components of the only diploid Xenopus frog, Xenopus tropicalis. Collision-induced dissociation was used to determine connectivity, to identify previously characterized oligosaccharides, and to determine the presence of structural motifs in unknown oligosaccharides. Exoglycosidase digestion was used to identify the individual residues along with the linkages. The enzymes were also used to cleave larger oligosaccharides to smaller units that are similar to previously elucidated components. By using CID, isomeric structures were compared to determine whether they were identical. In this way, the exoglycosidases were more effectively used, and their use was minimized. A total of 35 oligosaccharides including neutral, sialylated, and sulfated were characterized in this way. The relative abundances of all components were also determined based on HPLC.
    Analytical Chemistry 11/2004; 76(20):5990-6001. · 5.70 Impact Factor
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    ABSTRACT: While the anuran amphibian Xenopus laevis is a widely used vertebrate model system, it is not optimal for genetic manipulations due to its tetraploid genome and long generation time. A current alternative amphibian model system, Xenopus tropicalis, has the advantages of a diploid genome and a much shorter generation time. We undertook a comparative investigation of X. tropicalis egg extracellular matrix glycoproteins in relation to those already characterized in X. laevis. Fertilization methods and isolation of egg extracellular molecules were directly transferable from X. laevis to X. tropicalis. Cross-fertilizations were successful in both directions, indicating similar molecules involved in sperm-egg interactions. Egg envelopes analyzed by SDS-PAGE were found to have almost identical gel patterns, whereas jelly component profiles were similar only for the larger macromolecules (>90 kDa). The cDNA sequences for egg envelope glycoproteins ZPA, ZPB, ZPC, ZPD and ZPAX, and also egg cortical granule lectin involved in the block to polyspermy, were cloned for X. tropicalis and showed a consistent approximately 85% amino acid identity to the X. laevis sequences. Thus, homologous egg extracellular matrix molecules perform the same functions, and the molecular and cellular mechanisms of fertilization in these two species are probably equivalent.
    Comparative Biochemistry and Physiology - Part A Molecular & Integrative Physiology 11/2003; 136(2):343-52. · 2.17 Impact Factor
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    LeAnn L Lindsay, Joy C Yang, Jerry L Hedrick
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    ABSTRACT: We report the identification of a previously undetected Xenopus laevis egg envelope component discovered through cloning experiments. A cDNA sequence was found that represented a mature protein of 32 kDa. Peptide antibodies were generated to probe for the protein in egg envelope samples and reactivity was found to a glycoprotein of approximately 80 kDa. When deglycosylated egg envelope samples were probed, a 32 kDa protein was labeled, confirming the size of the translated cDNA sequence. A BLAST analysis showed that it is most closely related (34% amino acid identity) to the ZP domains of mammalian tectorin, uromodulin and ZPA. From a dendrogram of known egg envelope glycoproteins, the new glycoprotein was shown to be unique among egg envelope components and was designated ZPD. A similar glycoprotein was identified by immunocrossreactivity in Xenopus tropicalis and Xenopus borealis egg envelopes.
    Embryologia 07/2002; 44(3):205-12. · 2.40 Impact Factor
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    L L Lindsay, M A Wallace, J L Hedrick
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    ABSTRACT: The Xenopus laevis egg envelope is composed of six or more glycoproteins, three of which have been cloned and identified as the mammalian homologs ZPA (ZP2), ZPB (ZP1) and ZPC (ZP3). The remaining glycoproteins are a triplet of high molecular weight components that are selectively hydrolyzed by the hatching enzyme. We have isolated one of these proteins and cloned its cDNA. The mRNA for the protein was found to be expressed only in early stage oocytes, as are other envelope components. From the deduced amino acid sequence, it was indicated to be a secreted glycoprotein with a characteristic ZP domain in the C-terminal half of the molecule. The N-terminal half was unrelated to any known glycoprotein. Comparative sequence analysis of the ZP domain indicated that it was derived from an ancestor of ZPA and ZPB, with the greatest identity to ZPA. This envelope component has been designated ZPAX.
    Embryologia 07/2001; 43(3):305-13. · 2.40 Impact Factor
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    L L Lindsay, J C Yang, J L Hedrick
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    ABSTRACT: Ovochymase, an extracellular Xenopus laevis egg serine active-site protease with chymotrypsin-like (Phe-X) substrate specificity, is released during egg activation. Molecular cloning results revealed that ovochymase is translated as part of an unusual polyprotein proenzyme. In addition to the ovochymase protease domain at the C terminus of the deduced amino acid sequence, two unrelated serine protease domains were present, each with apparent trypsin-like (Arg/Lys-X) substrate specificity, and thus, they were designated ovotryptase1 (at the N terminus) and ovotryptase2 (a mid domain). Also, a total of five CUB domains were interspersed between the protease domains. The presence of a hydrophobic signal sequence indicated that the polyprotein was secreted. Immunolocalization and Western blot studies of all three proteases showed that they are all present in the perivitelline space of unactivated eggs, apparently as proenzymes processed away from the original polyprotein. Western blot analysis also showed that the vast majority of the proteases in ovary, eggs, and embryos were present as the proenzyme forms, suggesting that the functions of these proteases depend on very limited levels of activation.
    Proceedings of the National Academy of Sciences 10/1999; 96(20):11253-8. · 9.81 Impact Factor
  • L L Lindsay, M J Wieduwilt, J L Hedrick
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    ABSTRACT: The glycoprotein envelope surrounding the Xenopus laevis egg is converted from an unfertilizable to a fertilizable form during transit through the pars recta portion of the oviduct. Envelope conversion involves the pars recta protease oviductin, which selectively hydrolyzes envelope glycoprotein gp43 to gp41. Oviductin cDNA was cloned, and sequence analysis revealed that the protease is translated as the N terminus of an unusual mosaic protein. In addition to the oviductin protease domain, a protease domain with low identity to oviductin was present, possessing an apparent nonfunctional catalytic site. Three CUB domains were also present, which are related to the mammalian spermadhesin molecules implicated in mediating sperm-envelope interactions. We propose that during post-translational proteolytic processing of the mosaic oviductin glycoprotein, the processed N-terminal protease domain is released coupled to two C-terminal CUB domains and constitutes the enzymatically active protease molecule. In functional studies, isolated coelomic egg envelopes treated with oviductin purified from the oviduct showed a dramatic increase in sperm binding. This observation established that oviductin alone was the oviductal factor responsible for converting the egg envelope to a sperm-penetrable form, via an increase in sperm binding. Trypsin mimicked oviductin's effect on envelope hydrolysis and sperm binding, demonstrating that gp43 processing is the only requirement for envelope conversion.
    Biology of Reproduction 05/1999; 60(4):989-95. · 4.03 Impact Factor
  • Leann L. Lindsay, Jerry L. Hedrick
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    ABSTRACT: Xenopus laevis coelomic (body cavity) eggs are not fertilizable until they pass through the pars recta oviduct. A secreted pars recta oviductal protease with trypsin-like activity, oviductin, selectively hydrolyzes egg envelope glycoprotein gp43 to gp41; this limited proteolysis is believed to render the egg fertilizable. The effects of trypsin as a substitute for oviductin in modifying envelope structure and function were examined. Trypsinolysis (5 mIU for 30 min at room temperature) selectively converted gp43 to gp41 without hydrolysis of other envelope glycoproteins, and rendered coelomic eggs fertilizable in the presence of a jelly water preparation. Chymotrypsin had no effect on the acquisition of fertilizability, indicating that the reaction was dependent on trypsin-like specificity. This was confirmed by the use of p-aminobenzamindine and leupeptin to inhibit the ability of trypsin preparations to induce fertilizability. A sperm binding assay revealed that trypsin treatment dramatically increased sperm binding to egg envelopes derived from both coelomic eggs and ovarian eggs. Jelly water was not required for sperm binding. Therefore, trypsin can mimic the biological action of oviductin, selectively cleaving egg envelope gp43 to generate or expose sperm binding sites, rendering the envelope penetrable by sperm and permitting fertilization. J. Exp. Zool. 281:132–138, 1998. © 1998 Wiley-Liss, Inc.
    Journal of Experimental Zoology 12/1998; 281(2):132 - 138.
  • Leann L. Lindsay, Jerry L. Hedrick
    Journal of Experimental Zoology - J EXP ZOOL. 01/1998; 281(2):132-138.
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    ABSTRACT: Neutral oligosaccharides were released by alkaline sodium borohydride reduction of the jelly coating from the South African clawed toad, Xenopus laevis. The oligosaccharides were isolated by HPLC and analyzed by matrix-assisted laser desorption ionization (MALDI)-Fourier transform mass spectrometry (FTMS). The mass spectrometry analysis allowed confirmation of 12 structures first proposed by Strecker et al. using nuclear magnetic resonance. In addition, seven new oligosaccharides with weak abundances were found and characterized by mass spectrometry. A method for discriminating metastable fragments from quasimolecular ions is described. It involves doping the sample with cesium chloride. Cesium-coordinated oligosaccharides do not fragment as readily as those coordinated to sodium. Tandem MS experiments are performed on an unknown oligosaccharide illustrating the potential of MALDI-collision-induced dissociation-FTMS.
    Analytical Biochemistry 08/1997; 250(1):18-28. · 2.58 Impact Factor
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    L L Lindsay, J L Hedrick
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    ABSTRACT: A chymotrypsin-like protease contained in the perivitelline space of unactivated Xenopus eggs is released during egg activation and appears to participate in vitelline envelope conversion. This 30-kDa protease, which we have termed ovochymase, was isolated from the exudate of activated eggs using a soy bean trypsin inhibitor-agarose affinity column. The column eluant contained only two proteins, the 30-kDa ovochymase plus a 78-kDa chymotrypsin-like proteolytic activity. The 78-kDa protease was not usually observed in fresh egg exudate samples and thus was activated during the purification process and may represent the proposed precursor of the 30-kDa protease. The 30- and 78-kDa proteases were separated by gel filtration HPLC or by SDS-PAGE. The N-terminal amino acid sequence of SDS-PAGE-isolated ovochymase was determined to be VVGGQQAAPR. This conserved amino acid sequence, plus active site specific inhibition and substrate specificity studies, places ovochymase in the serine protease I family of enzymes. A two-dimensional protease activity gel revealed that ovochymase is present as several isozymes with a wide range of pI's.
    Developmental Biology 03/1995; 167(2):513-6. · 3.87 Impact Factor
  • L L Lindsay, C A Larabell, J L Hedrick
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    ABSTRACT: A chymotrypsin-like protease is released from Xenopus laevis eggs at activation and is involved in conversion of the vitelline envelope to the fertilization envelope. To localize this enzyme in unactivated and activated eggs, we used the synthetic peptide substrate succinylalanylalanylprolylphenylalanyl-4-methoxy-2-naphthylamide whose product can be visualized using transmission electron microscopy. Protease product was localized within the perivitelline space of unactivated eggs, appearing as strings of beads. No protease activity was detected in activated eggs, which is consistent with the observation that the protease is released from the egg at activation.
    Developmental Biology 01/1993; 154(2):433-6. · 3.87 Impact Factor
  • L L Lindsay, J L Hedrick
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    ABSTRACT: During fertilization of the Xenopus laevis egg, the egg envelope is converted so that further sperm contact with the egg is prevented. In this study two envelope conversion reactions were investigated, envelope hardening and limited hydrolysis of two structurally related envelope glycoproteins. Both of these reactions were shown to be sensitive to protease inhibitors. In an attempt to identify egg proteases involved in envelope conversion, the medium around activated dejellied eggs was collected and analyzed. The exudate was able to convert isolated envelopes and, when the exudate was analyzed using peptide substrates, two major activities were found, one with a preference for cleavage after argininyl peptide bonds and one with a preference for phenylalaninyl peptide bonds. Analysis of exudate using SDS-polyacrylamide gel electrophoresis with gelatin cast into the gel showed two bands of proteolytic activity, one at Mr 45,000 that was identified as the trypsin-like activity and one at Mr 30,000 that was identified as the chymotrypsin-like activity. When cortical granule exocytosis was suppressed using ammonium chloride, release of the two exudate proteases was also suppressed. Studies of the envelope conversion reactions using protease inhibitors indicated that the chymotrysin-like protease was involved in envelope conversion once it had been activated by the trypsin-like protease.
    Developmental Biology 10/1989; 135(1):202-11. · 3.87 Impact Factor
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    ABSTRACT: The envelope of the Bufo japonicus egg becomes impenetrable to sperm following fertilization. Electrophoretic analysis of envelopes showed that two glycoprotein components with apparent molecular weights of 65,000 and 61,000 were hydrolyzed during fertilization to 62,000 and 58,000, respectively. These two envelope components were structurally related as shown by peptide mapping and deglycosylation studies. Hardening of the envelope following egg activation was also observed, as detected by an increase in the envelope melting temperature. The involvement of proteolytic activities in the envelope hydrolysis and hardening reactions was demonstrated using protease inhibitors, and was verified for the hydrolysis reaction by observing a loss of mass in deglycosylated envelope components obtained before and after fertilization. A low ionic strength medium (less than 50 mM) was required for both the hardening and hydrolysis reactions. Envelopes from eggs activated in a high ionic strength medium were resistant to lysin from sperm, indicating that neither hydrolysis nor hardening was necessary to block lysin activity on the envelope. Both envelope hydrolysis and hardening could be effected in the absence of sperm (i.e., when eggs were activated by electric shock) and after egg jelly had been removed, indicating that neither sperm nor jelly factors were required for the envelope modifications. In addition, when eggs were activated in the presence of NH4Cl to suppress cortical granule exocytosis, envelope hardening and hydrolysis were still observed, indicating that a cortical granule-derived factor may not be involved.
    Developmental Biology 12/1988; 130(1):37-44. · 3.87 Impact Factor
  • L L Lindsay, J L Hedrick
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    ABSTRACT: Interacting egg envelope and sperm surface components were identified for Xenopus laevis using blotting methods. Sperm were extracted with sodium dodecyl sulfate (SDS), the extracted proteins separated by gel electrophoresis and blotted, and the blots treated with 125I-labeled heat solubilized envelopes. The converse experiment was also performed where envelope components were separated by gel electrophoresis, blotted, and the blots treated with 125I-labeled sperm components. Blotted sperm components with apparent molecular weights of 14K, 19K, 25K, and 35K selectively bound the solubilized envelopes. All of the envelope binding components were found to be localized on the sperm surface by radioiodinating intact sperm using Iodo-Gen. The blotted egg envelope component with an apparent molecular weight of 37K selectively bound to solubilized sperm components, and this binding was due to the protein moiety of the glycoprotein. 125I-labeled heat solubilized envelopes from unfertilized and fertilized eggs showed the same pattern of binding to blotted sperm components. Selected sulfated carbohydrates (fucoidan, dextran sulfate, and heparin, but not chondroitin sulfate) inhibited fertilization and binding of 125I-labeled heat solubilized envelopes to blotted sperm extract. Thus, the binding of heat solubilized envelopes to electrophoretically separated and blotted sperm proteins may reflect cellular interactions.
    Journal of Experimental Zoology 04/1988; 245(3):286-93.