The expression and characterization of human recombinant proinsulin-like growth factor II and a mutant that is defective in the O-glycosylation of its E domain.
In humans, newly synthesized proinsulin-like growth factor II (pro-IGF-II), i.e. IGF-II with an E domain extension of 89 amino acids, is 0-glycosylated on Thr75. As an approach to define the role that glycosylation of the E domain serves in the processing, secretion, and biological activities of IGF-II and to identify the sites of endoproteolytic processing, we constructed a mutant that encodes carbohydrate-free prepro-IGF-II. The mutant and wild-type prepro-IGF-II were expressed in NIH-3T3 cells, and the protein products were analyzed by SDS-PAGE followed by immunoblots with antipeptide antibodies to human and homologous rat E domain sequences. Transfectants that express glycosylated pro-IGF-II, i.e. xz97 and G11 cells, have intracellular forms of the growth factor with apparent Mr (appMr) of 21, 23, and 27K. NIH-3T3 xz95 cells, i.e. transfected with DNA that is missing the 0-glycosylation sequence, could also synthesize pro-IGF-II with an appMr of 21K. However, they did not accumulate the 23K and 27K forms of presumably glycosylated growth factor. None of the transfected NIH 3T3 cells processed much pro-IGF-II intracellularly, as the appMr 21K, 23K, and 27K forms had terminal E domain amino acid sequences that were recognized by antibodies to the homologous rat peptide sequence Met117 to Gln156. Subsequent to their secretion, the IGF-II in xz97 and G11 cells accumulated in the conditioned medium mostly as two partially processed species with appMr, of 17K and 14K, respectively. The IGF-II that accumulated in the conditioned medium of the xz95 cells had an appMr of 11K. As evidenced by a decrease in mass after treatment with neuraminidase and 0-glycosidase, the 17-kDa form of pro-IGF-II secreted by the NIH-3T3 xz97 cells was 0-glycosylated, whereas that secreted by the xz95 cells was oligosaccharide free. All of the pro-IGF-II forms have E domain amino acid sequences that reacted with antipeptide Ab to the Asp69 to Lys88 sequence. However, appMr 17K IGF-II, but not 14K IGF-II, also contained a larger E domain that was recognized by Ab to the sequence Phe89 to Arg101. The final step in the processing of 11- to 17-kDa IGF-II at Arg68 and the generation of mature IGF-II did not occur in the NIH-3T3 transfectants and is similar to what has been observed in human embryonic cells and mesenchymal tumors. The failure to remove the glycosylated E domain peptide from appMr, 14K and 17K IGF-II did not affect their binding to IGF-II/cation-independent mannose-6 phosphate receptors or presumably to IGF-I receptors, because in in vitro mitogenic assays they were equipotent with mature IGF-II. Unglycosylated pro-IGF-II from the NIH-3T3 xz95 cells also bound to these receptors. However, it was about 10 times more potent than IGF-II in stimulating thymidine incorporation into NIH-3T3 i24 IGF-IR cells, possibly because of the absence of negatively charged sialic acid and/or steric occlusion.
Yang
C Q
CQ
Department of Molecular Biology, Holland Laboratory, American Red Cross, Rockville, Maryland 20855, USA.
Zhan
X
X
Hu
X
X
Kondepudi
A
A
Perdue
J F
JF
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006031:Glycosylation; D006133:Growth Substances / Q000096:biosynthesis; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000096:biosynthesis* / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D010920:Placenta / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011498:Protein Precursors / Q000096:biosynthesis* / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D006133:Growth Substances; D011498:Protein Precursors; D017527:Receptor, IGF Type 2; D011994:Recombinant Proteins; C042532:preproinsulin-like growth factor II; D007335:Insulin-Like Growth Factor II; D004247:DNA