Ascorbate inducible N259 glycans on prolyl 4-hydroxylase subunit α1 promote hydroxylation and secretion of type I collagen

Ascorbic acid (vitamin C, VC) increases the secretion of mature collagen by promoting the activity of prolyl 4-hydroxylase subunit α 1 (P4HA1). To explore the mechanism involved, we investigated the role of N-linked glycosylation, which can regulate enzyme activity. P4HA1 has two glycosylation sites, Asn (N) 113 and N259. Our studies show that glycosylation of N259, but not N113, by STT3B and magnesium transporter 1 (MAGT1) is augmented by VC. N259 glycosylation on P4HA1 correlates with enhanced pepsin-resistant collagen 1α2 secretion. Downregulation of Stt3b and Magt1 reduces N259 glycans on P4HA1. In collagen 1α2 purified from Stt3b -silenced fibroblasts, decreased hydroxylation is found at five specific proline residues, while significantly increased hydroxylation is noted at two proline residues. Similarly, in collagen 1α1, reduced proline hydroxylation is detected at eight sites and increased proline hydroxylation is found at four sites. These results suggest that N-linked glycosylation of P4HA1 can direct hydroxylation at specific proline residues and affect collagen maturation.