These tools will no longer be maintained as of December 31, 2024. Archived website can be found here. PubMed4Hh GitHub repository can be found here. Contact NLM Customer Service if you have questions.


Pubmed for Handhelds

PUBMED FOR HANDHELDS

Search MEDLINE/PubMed

  • Title: 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.
    Author: Yang CQ, Zhan X, Hu X, Kondepudi A, Perdue JF.
    Journal: Endocrinology; 1996 Jul; 137(7):2766-73. PubMed ID: 8770896.
    Abstract:
    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.
    [Abstract] [Full Text] [Related] [New Search]