N-glycosylation as a regulatory process in the IGF-1 system: from mechanisms to clinical implications

The introductory chapter of the Thesis provides a detailed description of glycosylation, as one of the most common and complex protein post-translation modifications critical for physiological and pathological cellular functions. Particular attention is focused on the importance of the N-glycosylation in the insulin-like growth factor-1 (IGF-1) system. IGF-1 is a polypeptide growth factor with essential roles in normal tissue development and differentiation. More than 90% of IGF-1 in the bloodstream is bound to IGF binding protein- 3 (IGFBP-3) and acid labile subunit (ALS). This ternary complex of 150 kDa protects IGF-1 from proteolytic degradation, and regulates its interaction with the IGF-1 receptor (IGF-1R). In the first chapter, we provide novel evidence for structural features of IGF-1 prohormone domains and their role as regulators of IGF-1 production. Our study highlights that IGF-1 prohormones are composed of both protein structural domain, i.e. the mature IGF-1, and intrinsically disordered regions, i.e. the C-terminal E-domains. We document that disordered E-domains have distinct regulatory functions on IGF-1 prohormones production. In particular, N-glycosylation status of Ea-domain regulates the stability and secretion of IGF- 1Ea prohormone and mature IGF-1. Accordingly, the interference with IGF-1Ea prohormone N-glycosylation by tunicamycin (TUN), glucose starvation or 2-deoxyglucose directly affects protein IGF-1 level. Furthermore, we demonstrate that the alternative IGF-1Eb and IGF-1Ec prohormones are devoid of N-glycosylation sites, and hence are insensitive to modulation of glycosylation. Notably, the Eb- and Ec- disordered tails promote the nuclear accumulation of IGF-1Eb and IGF-1Ec prohormones. Thus, disordered E-domains are regulatory elements that control the structure, production, regulation, functioning and secretion of IGF- 1. The second chapter describes the effects of N-glycosylation inhibition on C2C12 myoblast differentiation. TUN treatment or genetic knockdown of PMM2 gene, which encodes a key enzyme in N-glycosylation, inhibits myoblast fusion and interferes with the early steps of the myogenic program. In addition, we find that N-glycosylation inhibition by TUN or PMM2 downregulation also affects the IGF-1R signaling pathway reducing the IGF-1R expression, inhibiting the AKT activation and deregulating IGF-1 mRNA expression and protein secretion. In the third chapter of the Thesis, we evaluate the IGF-1 system components in fibroblasts obtained from patients with different types of Congenital Disorders of Glycosylation (CDG). We observe that primary fibroblasts from CDG patients have reduced levels of IGF-1R, associated with an impairment in the IGF-1R signaling pathway activation. Moreover, the IGF-1Ea prohormone is underglycosylated in CDG fibroblasts and it is also associated with lower IGF-1 secretion in the cell culture media. In conclusion, our results provide new insights into the importance of N-glycosylation for the regulation of the IGF-1 system. These findings may open new diagnostic options and therapeutic strategies for disorders characterized by altered N-glycosylation.