Isolation and characterization of vascular smooth muscle cell growth promoting factor from bovine ovarian follicular fluid and its cDNA cloning from bovine and human ovary.
A protein possessing vascular smooth muscle cell (SMC) growth-promoting activity (VSGP) was purified from bovine ovarian follicular fluid. The purified protein showed a broad band on SDS-PAGE with an apparent molecular mass of 90-100 kDa. The purified protein was characterized by amino acid sequence analysis of its N-terminal and internal peptides. Based on the information of the peptide sequences, bovine ovarian cDNA library was screened and cDNA clones encoding the protein were isolated. Human homolog of the protein was also cloned from human ovarian cDNA library. Nucleotide sequence analysis revealed that bovine VSGP transcript has a 2421-bp open reading frame, which encodes a protein of 807 amino acid residues. A homology search indicated that bovine and human VSGP are counterparts of rat F-spondin, which has been previously identified as a promoter molecule of neurite extension in rat fetal floor plate. RNA blot analysis showed wide distribution of VSGP/F-spondin transcripts in fetal and adult human tissues. Especially the expression was highest in the adult human ovary. The purified bovine VSGP/F-spondin showed vascular SMC growth promoting activity with an ED(50) value of 10(-8) M. Together with these findings, we demonstrated here that VSGP/F-spondin is a major factor for vascular SMC proliferation in the ovary. In conclusion, our present study provides a distinct and important function of VSGP/F-spondin as a strong VSMC proliferation promoting factor, in addition to the previously proposed function in neuronal system, and also provides insight into mechanisms underlying vascular SMC proliferation during ovarian folliculogenesis.
Miyamoto
K
K
Department of Biochemistry, Fukui Medical University, Shimoaizuki, Matsuoka, Fukui 910-1193, Japan. kmiyamot@fmsrsa.fukui-med.ac.jp
Morishita
Y
Y
Yamazaki
M
M
Minamino
N
N
Kangawa
K
K
Matsuo
H
H
Mizutani
T
T
Yamada
K
K
Minegishi
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D016326:Extracellular Matrix Proteins; D005260:Female; D015571:Follicular Fluid / Q000737:chemistry*; D006133:Growth Substances / Q000235:genetics* / Q000302:isolation & purification* / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D010053:Ovary / Q000737:chemistry; D010455:Peptides; D012333:RNA, Messenger / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D016326:Extracellular Matrix Proteins; D006133:Growth Substances; D010455:Peptides; D012333:RNA, Messenger; C429104:SPON1 protein, human