Glycosylation stabilizes IGF-1Ea pro hormone and regulates its secretion

Insulin-like growth factor-1 (IGF-1) is a growth factor with multiple roles in various aspects of normal and pathological growth and differentiation. The translation of the IGF-1 gene gives rise to an immature IGF-1 peptide, which has a signal peptide at the 5’ end of the gene, a core region and an Ea-peptide at the 3’ end. The signal peptide is removed after facilitating the passage of the polypeptide into the endoplasmic reticulum and give rise to the IGF-1 prohormone (proIGF-1Ea), retaining C-terminal Ea peptide. Recent studies demonstrated that intracellular IGF-1 is mainly expressed as prohormone, not mature IGF-1. Moreover, we recently demonstrated that the Ea peptide is an intrinsically disordered region (IDR) enriched in regulatory elements including a highly conserved N-glycoylation site. In this study we investigate the role of Ea peptide glycosylation on proIGF-1Ea stability and secretion. After transient transfection of Hek293 cells with IGF-1Ea transgene two IGF-1 prohormones were produced intracellularly: glycosylated (~17kDa) and non-glycosylated forms (~11-12kDa). Subsequently, we wondered whether glucose withdrawal or direct inhibition of N-glycosylation by tunicamycin (Tun) might interfere with IGF-1Ea production. To achieve this aim, we overexpressed proIGF-1Ea in Hek293 cells cultured in glucose depleted medium or treated with Tun. Notably, the band corresponding to glycosylated proIGF-1Ea completely disappeared in the absence of glucose or after treatment with Tun. Moreover, the analysis of culture media of IGF-1Ea-transfected Hek293 cells showed that the inhibition of glycosylation by glucose deprivation or Tun completely abrogated the glycosylated proIGF-1Ea secretion and markedly reduced the mature IGF-1 secretion. After that, using the protein synthesis inhibitor cycloheximide, we demonstrated that the turnover rate for non-glycosylated IGF-1Ea was faster than glycosylated IGF-1Ea. To test the involvement of 26S proteasome machinery, we subsequently treated IGF-1Ea-transfected Hek293 cells with proteasome inhibitor MG132 and we found an increase of non-glycosylated IGF-1Ea, while IGF-1Ea glycosylated was marginally affected by proteasome inhibitor. In conclusion, these results show that glycosylation of Ea peptide enhances the export efficiency of proIGF-1Ea and it is necessary for IGF-1 secretion. We hypothesize that proIGF-1Ea glycosylation ensures proper prohormone folding and secretion preventing its entry into the ER-associated degradation (ERAD) pathway.