Egg yolk proteins in gray mullet (Mugil cephalus):: Purification and classification of multiple lipovitellins and other vitellogenin-derived yolk proteins and molecular cloning of the parent vitellogenin genes

Seven yolk proteins (YPs), four large lipoproteins (YPs1-4) and three minor yolk components (YPs5-7) including one phosphoprotein (YP7), were purified from extracts of vitellogenic ovaries of gray mullet (Mugil cephalus) by combinations of hydroxylapatite, ion exchange, immunoadsorbent, and gel filtration chromatography. The molecular masses of native Y-P1, YP2, YP3, and YP4 were estimated to be 330, 325, 335, and 570 kDa, respectively. The tertiary structures of YP1, YP2, and YP3 revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis were typical of teleost lipovitellins (Lvs), consisting of a heavy chain (similar to 110, similar to 99, and similar to 97 kDa, respectively) and a light chain (similar to 30, similar to 29, and similar to 21.5 kDa, respectively), while YP4 exhibited a heavy chain (similar to 110 kDa) and two more polypeptide bands (similar to 70 and similar to 54kDa). Mapping of N-terminal peptide sequences of the purified YPs to the primary structure of multiple mullet vitellogenins (Vgs) deduced from their respective complete cDNAs, which were cloned and sequenced, conclusively identified YP1, YP2, and YP3 as Lvs derived from mullet VgA, VgB, and VgC, respectively. The fourth YP (YP4) appeared to be a proteolytic variant consisting of Lv and phosvitin components of VgA. Two other YPs (YP5 and YP6) were identified as beta'-components derived from VgA and VgB based on their structures and common, but not identical, antigenicity to salmonid beta'-component, while purified YP7, a phosphoprotein with a high content of serine residues, was identified as a phosvitin derived from VgB. This is the first report, of which we are aware, on purification and molecular classification of three distinct forms of Lv from any oviparous vertebrate.