title: Lipovitellin constitutes the protein backbone of glycoproteins involved in sperm-egg interaction in the amphibian Discoglossus pictus.
abstract: Our knowledge of the molecules that interact with sperm at the egg membrane is restricted to a short list. In the eggs of Discoglossus pictus, fusion with sperm is limited to a differentiated structure, the dimple, offering several advantages for detecting molecules involved in fertilization. Previous studies have identified fucosylated glycoproteins of 200, 260, and 270 kDa located at the surface of the dimple that are able to bind sperm in vitro. Here, we show that dimple glycoproteins and a protein represented by a 120-kDa band released following gel-into-gel SDS-PAGE of both glycoproteins share the same N-terminal amino acid sequence, which itself is similar to the N-termini of Xenopus liver-synthesized vitellogenin (VTG) and the lipovitellin 1. MALDI/MS mass spectrometry indicated that the 120-kDa band is part of both gps 200 and 270/260. A 117-kDa major protein of the egg lysate exhibits the same MALDI/MS spectrum, and LC-MSMS indicates that this is a lipovitellin 1 (DpLIV) that coincides with the 120-kDa band and is responsible for the formation of the 200-270-kDa dimers. Therefore, lipovitellin 1 constitutes the protein backbone of the dimple glycoconjugates. In vitro assays using polystyrene beads coated with DpLIV or with its dimers indicate that significant sperm binding occurs only with DpLIV dimers. In amphibians, VTG is taken up by the oocyte, where it releases lipovitellins destined to form yolk. In Discoglossus, our data suggest that yolk proteins are also synthesized by the oocyte. The dimple forms in the ovulated oocyte following the exocytosis of vesicles that likely expose DpLIVs at their membrane. Indeed, in whole mounts of immunostained eggs, anti-vitellogenin antibodies label only the surface of the dimple.
authors.affiliation: Department of Structural and Functional Biology, University of Naples Federico II, MSA, Naples, Italy. chiara.campanella@unina.it
authors.forename: C
authors.initials: C
authors.lastname: Campanella
authors.forename: M
authors.initials: M
authors.lastname: Caputo
authors.forename: M C
authors.initials: MC
authors.lastname: Vaccaro
authors.forename: N
authors.initials: N
authors.lastname: De Marco
authors.forename: L
authors.initials: L
authors.lastname: Tretola
authors.forename: M
authors.initials: M
authors.lastname: Romano
authors.forename: M
authors.initials: M
authors.lastname: Prisco
authors.forename: L
authors.initials: L
authors.lastname: Camardella
authors.forename: A
authors.initials: A
authors.lastname: Flagiello
authors.forename: R
authors.initials: R
authors.lastname: Carotenuto
authors.forename: E
authors.initials: E
authors.lastname: Limatola
authors.forename: A
authors.initials: A
authors.lastname: Polzonetti-Magni
authors.forename: V
authors.initials: V
authors.lastname: Infante
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001001:Anura / Q000378:metabolism* / Q000502:physiology; D015153:Blotting, Western; D002853:Chromatography, Liquid; D019281:Dimerization; D004527:Egg Proteins / Q000235:genetics* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D008297:Male; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009865:Oocytes / Q000648:ultrastructure; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013084:Sperm-Ovum Interactions / Q000502:physiology*; D053719:Tandem Mass Spectrometry
chemical_list: D004527:Egg Proteins; C001549:lipovitellin