PMID: 1569071

 

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Title : The identification of O-glycosylated precursors of insulin-like growth factor II

Abstract :
  1. A procedure that combined ion exchange, gel permeation, and insulin-like growth factor-binding protein 3 3 (IGF-BP-3 ) affinity chromatography with chromatofocusing and reversed-phase high pressure liquid chromatography was used to isolate high molecular weight precursors of human insulin-like growth factor II (IGF-II ) from acetic acid extracts of Cohn fraction IV1
  2. Two precursors had isoelectric points (pI) of 5.1 and 5.4 and apparent Mr values of 15,000 and 11,500, respectively
  3. An apparent Mr = 16,000 RLPG/Ser29 variant of IGF-II variant of IGF-II was also identified in the acetic acid extracts
  4. Amino-terminal amino acid sequencing of the major E domain-containing peptide that had been isolated from apparent Mr = 15,000 IGF-II ( pI 5 .1), following its digestion with the endoprotease Lys-C, indicated the carboxyl terminus of this precursor was near or at Lys88
  5. During the sequencing of this peptide, a sharply reduced yield of derivatized amino acid occurred at cycle 10, indicating that Thr75 had been posttranslationally modified, possibly by O-glycosylation
  6. To evaluate this possibility, the 125I-labeled high molecular weight IGF- IIs and their endoprotease-generated peptides were treated with glycosidases, and their effects were determined from the change in relative mobilities of the polypeptide and peptides during sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  7. Neuraminidase treatment of apparent Mr = 15,000 and 11,500 IGF-II reduced their Mr values to a common value of 10,500
  8. When the desialylated precursors of IGF-II were treated with O-glycosidase, but not when treated with N-glycosidase, the Mr values were reduced further to about 10,000
  9. This was the Mr value that would be predicted for an unglycosylated form of precursor IGF-II that had a carboxyl-terminal end at or near Lys88
  10. When the Ser66-Lys88 endoprotease-generated E domain peptides from pI5 .1 and 5.4 high Mr IGF-II were treated with the glycosidases, they had relative mobility changes during sodium dodecyl sulfate-polyacrylamide gel electrophoresis that were similar to those of the intact precursors
  11. Finally, the association of O-linked oligosaccharide with the E domain peptide of IGF-II was confirmed by demonstrating the specificity of binding of the Ser66-Lys88 asialoglycopeptide to jackfruit lectin