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

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Abstract

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.

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Acknowledgements

We thank the Core Facility and Technical Support, Wuhan Institute of Virology for assistance with confocal microscope (Dr. Ding Gao) and flow cytometry (Ms. Juan Min).

Funding

This work was supported in part by National Institutes of Health, National Heart, Lung, and Blood Institute (Grant R01-HL41178 to LMG), by MOST (2018YFA0507201 to CL), by the Strategic Priority Research Program A of the Chinese Academy of Sciences (XDA12010309 to CL), by the National Basic Research Priorities Program of China (2013CB911102 to CL), and by National Science Foundation of China (31670170 to CL).

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Authors and Affiliations

  1. State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, 44 Xiao Hong Shan Zhong Qu, Wuhan, 430071, Hubei, China

    Run Shi, Yan Zhang, Shanshan Gao, Lili Cai & Chaoyang Li

  2. University of Chinese Academy of Sciences, Beijing, China

    Run Shi

  3. Affiliated Cancer Hospital and Institute of Guangzhou Medical University, 78 Heng Zhi Gang Road, Guangzhou, 510095, China

    Run Shi, Weimin Hu & Chaoyang Li

  4. Department of Biomedical Engineering, Lerner Research Institute, Cleveland, OH, USA

    Andrew H. Smith & Linda M. Graham

  5. Department of Vascular Surgery, Cleveland Clinic, Cleveland, OH, USA

    Andrew H. Smith & Linda M. Graham

  6. Department of Statistics, University of Akron, Akron, OH, USA

    Jun Ye

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  1. Run Shi
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  2. Weimin Hu
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  3. Yan Zhang
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  8. Linda M. Graham
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Contributions

RS, LMG, and CL designed the experiments; RS and YZ performed the experiments; RS, WH, YZ, LC, SSG, AHS, JY, LMG, and CL analyzed the data; RS, LMG, and CL wrote the paper.

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Correspondence to Linda M. Graham or Chaoyang Li.

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Shi, R., Hu, W., Zhang, Y. et al. Ascorbate inducible N259 glycans on prolyl 4-hydroxylase subunit α1 promote hydroxylation and secretion of type I collagen. Cell. Mol. Life Sci. 76, 3449–3464 (2019). https://doi.org/10.1007/s00018-019-03081-w

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  • DOI: https://doi.org/10.1007/s00018-019-03081-w

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