Characterization of latent recombinant TGF-beta 2 produced by Chinese hamster ovary cells.
Latent recombinant transforming growth factor-beta 2 (LrTGF-beta 2) complex has been purified from serum-free media conditioned by Chinese hamster ovary cells transfected with a plasmid encoding the TGF-beta 2 (414) precursor. Under neutral conditions, LrTGF-beta 2 had an apparent molecular weight of 130 kDa. The complex contained both mature and pro-region sequences. Acidification of LrTGF-beta 2 resulted in the release of mature 24 kDa TGF-beta 2 from the high molecular weight pro-region-containing complex, suggesting that TGF-beta 2 was non-covalently associated with this complex. These results were confirmed by crosslinking experiments performed on partially purified LrTGF-beta 2. Protein sequence analysis of the purified TGF-beta 2 pro-region indicated that signal peptide cleavage occurred between ser(20) and leu(21). The pro-region, which previously was found to contain mannose-6-phosphate, bound to the mannose-6-phosphate receptor. Proteolytic cleavage of mature TGF-beta 2 from pro-TGF-beta 2 was inhibited by monensin and chloroquine suggesting that binding to this receptor and subsequent transport to acidic vesicles may be involved in the processing of rTGF-beta 2 precursor.
Lioubin
M N
MN
Oncogen, Seattle, Washington 98121.
Madisen
L
L
Marquardt
H
H
Roth
R
R
Kovacina
K S
KS
Purchio
A F
AF
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology; D002850:Chromatography, Gel; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007700:Kinetics; D008360:Mannosephosphates / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D008985:Monensin / Q000494:pharmacology; D010053:Ovary; D021382:Protein Sorting Signals / Q000378:metabolism; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D008360:Mannosephosphates; D021382:Protein Sorting Signals; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D002738:Chloroquine; D008985:Monensin