Glycosylation at Asn254 Is Required for the Activation of the PDGF-C Protein.

Wenjie Hu,Ruting Zhang,Dongyue Lin,Kun Wei,Xuri Li,Jiahui Li,Wei Chen,Bo Zhang,Zhongshu Tang

doi:10.3389/fmolb.2021.665552

Abstract

Platelet-derived growth factor C (PDGF-C) is a member of the PDGF/VEGF (vascular endothelial growth factor) family, which includes proteins that are well known for their mitogenic effects on multiple cell types. Glycosylation is one of the most important forms of posttranslational modification that has a significant impact on secreted and membrane proteins. Glycosylation has many well-characterized roles in facilitating protein processing and contributes to appropriate folding, conformation, distribution, and stability of proteins that are synthesized intracellularly in the endoplasmic reticulum (ER) and Golgi apparatus. Although the general process and functions of glycosylation are well documented, there are most likely others yet to be discovered, as the glycosylation of many potential substrates has not been characterized. In this study, we report that the PDGF-C protein is glycosylated at three sites, including Asn25, Asn55, and Asn254. However, we found that mutations at any of these sites do not affect the protein expression or secretion. Similarly, disruption of PDGF-C glycosylation had no impact on its progression through the ER and Golgi apparatus. However, the introduction of a mutation at Asn254 (N254 A) prevents the activation of full-length PDGF-C and its capacity for signaling via the PDGF receptor. Our findings reveal that glycosylation affects PDGF-C activation rather than the protein synthesis or processing. This study characterizes a crucial modification of the PDGF-C protein, and may shed new light on the process and function of glycosylation.

Highlights

Platelet-derived growth factor C (PDGF-C) is a member of the PDGF/VEGF family
After 5 min of exposure to the Conditioned medium (CM), NIH 3T3 cells were collected, lysed, and analyzed by Western blot. We found that both PDGF receptors (PDGFRs)-α and PDGFR-β were phosphorylated in response to CMs from all HEK293 A cultures transfected with plasmids that maintained an intact Asn254 residue
This result indicates that disruption of PDGF-C glycosylation sites does not affect its progression through the endoplasmic reticulum (ER)

Summary

Introduction

Platelet-derived growth factor C (PDGF-C) is a member of the PDGF/VEGF (vascular endothelial growth factor) family. Proteins in this family include eight Cys residues and a cystine knot (Li et al, 2000). Unlike other members of this family, full-length PDGF-C (PC-FL) protein is composed of two domains, including an N-terminal complement C1r/C1s, Uegf, Bmp (CUB) domain, and a C-terminal core domain (PC core) (Fredriksson et al, 2004). PDGF-D is the only other member of this family that has a CUB domain (LaRochelle et al, 2001). Most other family members have the core domain only. The PC core can interact with PDGF receptors (PDGFRs) α and β (Gilbertson et al, 2001; Fredriksson et al, 2005; Hurst et al, 2012; Lee and Li, 2018)

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