Impaired myoblast differentiation and muscle IGF-1 receptor signaling pathway activation after N-glycosylation inhibition.

The role of N-glycosylation in the myogenic process remains poorly understood. Here, we evaluated the impact of N-glycosylation inhibition by Tunicamycin (TUN) or by phosphomannomutase 2 (PMM2) gene knockdown, which encodes an enzyme essential for catalyzing an early step of the N-glycosylation pathway, on C2C12 myoblast differentiation. The effect of chronic treatment with TUN on tibialis anterior (TA) and extensor digitorum longus (EDL) muscles of WT and MLC/mIgf-1 transgenic mice, which overexpress muscle Igf-1Ea mRNA isoform, was also investigated. TUN-treated and PMM2 knockdown C2C12 cells showed reduced ConA, PHA-L, and AAL lectin binding and increased ER-stress-related gene expression (Chop and Hspa5 mRNAs and s/uXbp1 ratio) compared to controls. Myogenic markers (MyoD, myogenin, and Mrf4 mRNAs and MF20 protein) and myotube formation were reduced in both TUN-treated and PMM2 knockdown C2C12 cells. Body and TA weight of WT and MLC/mIgf-1 mice were not modified by TUN treatment, while lectin binding slightly decreased in the TA muscle of WT (ConA and AAL) and MLC/mIgf-1 (ConA) mice. The ER-stress-related gene expression did not change in the TA muscle of WT and MLC/mIgf-1 mice after TUN treatment. TUN treatment decreased myogenin mRNA and increased atrogen-1 mRNA, particularly in the TA muscle of WT mice. Finally, the IGF-1 production and IGF1R signaling pathways activation were reduced due to N-glycosylation inhibition in TA and EDL muscles. Decreased IGF1R expression was found in TUN-treated C2C12 myoblasts which was associated with lower IGF-1-induced IGF1R, AKT, and ERK1/2 phosphorylation compared to CTR cells. Chronic TUN-challenge models can help to elucidate the molecular mechanisms through which diseases associated with aberrant N-glycosylation, such as Congenital Disorders of Glycosylation (CDG), affect muscle and other tissue functions.

Defective IGF-1 prohormone N-glycosylation and reduced IGF-1 receptor signaling activation in congenital disorders of glycosylation.

The insulin-like growth factor-1 (IGF-1) signaling pathway is crucial for the regulation of growth and development. The correct processing of the IGF-1Ea prohormone (proIGF-1Ea) and the IGF-1 receptor (IGF-1R) peptide precursor requires proper N-glycosylation. Deficiencies of N-linked glycosylation lead to a clinically heterogeneous group of inherited diseases called Congenital Disorders of Glycosylation (CDG). The impact of N-glycosylation defects on IGF-1/IGF-1R signaling components is largely unknown. In this study, using dermal fibroblasts from patients with different CDG [PMM2-CDG (n = 7); ALG3-CDG (n = 2); ALG8-CDG (n = 1); GMPPB-CDG (n = 1)], we analyzed the glycosylation pattern of the proIGF-1Ea, IGF-1 secretion efficiency and IGF-1R signaling activity. ALG3-CDG, ALG8-CDG, GMPPB-CDG and some PMM2-CDG fibroblasts showed hypoglycosylation of the proIGF-1Ea and lower IGF-1 secretion when compared with control (CTR). Lower IGF-1 serum concentration was observed in ALG3-CDG, ALG8-CDG and in some patients with PMM2-CDG, supporting our in vitro data. Furthermore, reduced IGF-1R expression level was observed in ALG3-CDG, ALG8-CDG and in some PMM2-CDG fibroblasts. IGF-1-induced IGF-1R activation was lower in most PMM2-CDG fibroblasts and was associated with decreased ERK1/2 phosphorylation as compared to CTR. In general, CDG fibroblasts showed a slight upregulation of Endoplasmic Reticulum (ER) stress genes compared with CTR, uncovering mild ER stress in CDG cells. ER-stress-related gene expression negatively correlated with fibroblasts IGF-1 secretion. This study provides new evidence of a direct link between N-glycosylation defects found in CDG and the impairment of IGF-1/IGF-1R signaling components. Further studies are warranted to determine the clinical consequences of reduced systemic IGF-1 availability and local activity in patients with CDG.

The intrinsically disordered E-domains regulate the IGF-1 prohormones stability, subcellular localisation and secretion.

Insulin-like growth factor-1 (IGF-1) is synthesised as a prohormone (proIGF-1) requiring enzymatic activity to yield the mature IGF-1. Three proIGF-1s are encoded by alternatively spliced IGF-1 mRNAs: proIGF-1Ea, proIGF-1Eb and proIGF-1Ec. These proIGF-1s have a common IGF-1 mature sequence but different E-domains. The structure of the E-domains has not been resolved, and their molecular functions are still unclear. Here, we show that E-domains are Intrinsically Disordered Regions that have distinct regulatory functions on proIGF-1s production. In particular, we identified a highly conserved N-glycosylation site in the Ea-domain, which regulated intracellular proIGF-1Ea level preventing its proteasome-mediated degradation. The inhibition of N-glycosylation by tunicamycin or glucose starvation markedly reduced proIGF-1Ea and mature IGF-1 production. Interestingly, 2-deoxyglucose, a glucose and mannose analogue, increased proIGF-1Ea and mature IGF-1 levels, probably leading to an accumulation of an under-glycosylated proIGF-1Ea that was still stable and efficiently secreted. The proIGF-1Eb and proIGF-1Ec were devoid of N-glycosylation sites, and hence their production was unaffected by N-glycosylation inhibitors. Moreover, we demonstrated that alternative Eb- and Ec-domains controlled the subcellular localisation of proIGF-1s, leading to the nuclear accumulation of both proIGF-1Eb and proIGF-1Ec. Our results demonstrated that E-domains are regulatory elements that control IGF-1 production and secretion.