Egg yolk proteins in grey mullet (Mugil cephalus): purification and classification of multiple lipovitellins and other vitellogenin‐derived yolk proteins and molecular cloning of the parent vitellogenin genes

Abstract

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 grey mullet (Mugil cephalus) by combinations of hydroxylapatite, ion exchange, immunoadsorbent, and gel filtration chromatography. The molecular masses of native YP1, 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 ( approximately 110, approximately 99, and approximately 97 kDa, respectively) and a light chain ( approximately 30, approximately 29, and approximately 21.5 kDa, respectively), while YP4 exhibited a heavy chain ( approximately 110 kDa) and two more polypeptide bands ( approximately 70 and approximately 54 kDa). 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.

The reproductive hormone cycle of adult female American alligators from a barrier island population

“…Fractions, which contained Lv as a dominant yolk protein, were collected, pooled, and subjected to the second gel filtration on a Superose 6 column equilibrated with Tris-HCl buffer. Elution of the Lv sample was performed using an FPLC system (GE Healthcare UK Ltd) under conditions described previously (Amano et al 2007). A single peak was observed at the position corresponding to w410 kDa; this peak fraction was collected as purified Lv.…”

Biochemical and Immunochemical Characterization of Two Discrete Vitellogenin Proteins and Their Derived Lipovitellins in the Inshore Hagfish (Eptatretus burgeri)

“…Purification of Vtg-related YPs has been conducted in various teleost species and the procedures typically include separations by water precipitation, ammonium sulfate precipitation, HA chromatography, gel filtration and ion exchange chromatography (Hara et al, 1993;Hiramatsu and Hara, 1996;Matsubara and Sawano, 1995;Hiramatsu et al, 2002 a, c;Fujiwara et al, 2005;Amano et al, 2007b). In the present study, it was difficult to use ion-exchange chromatography, as the putative Lv appeared to precipitate using a buffer with a low NaCl concentration.…”

“…In SDS-PAGE of the YE, two major bands (116LvH and 106LvH) were observed and expected to represent heavy (H) chains of Lv on the basis of the following characteristics reported for teleost Lvs: (1) relatively large molecules (~300 kDa to 400 kDa), and (2) made up of at least two polypeptides, i.e., a larger heavy chain (~94 to ~110 kDa) and (a) smaller light chains (~28 to ~54 kDa), in SDS-PAGE (Hiramatsu and Hara, 1996;Matsubara et al, 1999;Hiramatsu et al, 2002a;Amano et al, 2007b). Following purification, the apparent intact mass of the purified hagfish Lv (> 669 kDa) was found to be much higher than those reported for fish Lvs (~300 to ~400 kDa) (Hiramatsu and Hara, 1996;Matsubara et al, 1999;Hiramatsu et al, 2002a;Amano et al, 2007b). In contrast, such a large-mass Lv (> 669 kDa) has also been reported for catshark Lv (Yamane et al, 2013).…”

“…Electrophoresis and N-terminal amino acid sequencing were performed following the method described in Amano et al (2007b). Simply, after trans-blotting following SDS-PAGE, peptide bands were visualized on the polyvinylidene difluoride (PVDF) membrane (Immobilon-PSQ; Millipore, Bedford, MA, USA) by staining with Coomassie Brilliant Blue (CBB), cut out from the membrane and subjected to N-terminal amino acid sequencing on a Model 492 Procise Sequencing System (Applied Biosystems, Foster City, CA, USA).…”

“…Prior to its deposition in yolk granules, Vtg undergoes limited proteolytic cleavage, giving rise to several classes of YPs. In teleosts, Vtg-derived YPs are typically designated as lipovitellin (Lv), phosvitin (Pv) and β′-component (β′-c) (Matsubara and Sawano, 1995;Hiramatsu and Hara, 1996;Hiramatsu et al, 2002b, c), while some other YP variants (e.g., Cterminal peptide and Lv-Pv complexes) are also evident Sawaguchi et al, 2005;Amano et al, 2007b).…”