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 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.
Amano
Haruna
H
Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan.
Fujita
Toshiaki
T
Hiramatsu
Naoshi
N
Shimizu
Munetaka
M
Sawaguchi
Sayumi
S
Matsubara
Takahiro
T
Kagawa
Hirohiko
H
Nagae
Masaki
M
Sullivan
Craig V
CV
Hara
Akihiko
A
D000595:Amino Acid Sequence; D000818:Animals; D002845:Chromatography; D003001:Cloning, Molecular; D004527:Egg Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D005260:Female; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D023701:Smegmamorpha / Q000235:genetics* / Q000378:metabolism; D014819:Vitellogenins / Q000235:genetics*
D004527:Egg Proteins; D014819:Vitellogenins; C001549:lipovitellin