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Analyzing the Effects of O-Fucosylation on Secretion of ADAMTS Proteins Using Cell-Based Assays

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ADAMTS Proteases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2043))

Abstract

Metalloproteinases of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin type 1 repeats) superfamily are extensively modified with glycan moieties. Glycosylation occurs as these enzymes are trafficked through the endoplasmic reticulum (ER) and Golgi apparatus on their way to the extracellular space and includes N-linked glycosylation, O-linked fucosylation and C-linked mannosylation. This chapter focuses on O-linked fucose, which is added to properly folded thrombospondin type 1 repeats (TSRs) in the ER by protein O-fucosyltransferase 2 (POFUT2) and elongated to a Glucoseβ1–3Fucose disaccharide by β3-glucosyltransferase (B3GLCT). Knockout of POFUT2 results in embryonic lethality in mice, and inactivating mutations in B3GLCT cause Peters plus syndrome, a congenital disorder of glycosylation in humans. Addition of the disaccharide by POFUT2 and B3GLCT stabilizes folded TSRs, enhancing folding in the ER and secretion efficiency of several ADAMTS proteins from cells. Thus, POFUT2 and B3GLCT both function as an ER quality control pathway for folding of TSRs in ADAMTS proteins. In this chapter we describe in detail the methods developed to analyze secretion defects of ADAMTS proteins upon loss of either POFUT2 or B3GLCT. The methods described include creation of CRISPR/Cas9-mediated knockout cell lines of POFUT2 and B3GLCT and use of these cell lines to analyze and quantify secretion defects of ADAMTS proteins.

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Acknowledgments

We thank Dr. Hideyuki Takeuchi for his assistance and guidance in generating CRISPR/Cas9 knockouts of POFUT2 and B3GLCT, and for his comments on this manuscript. We also thank Rachel Lopilato, Ao Zhang, and Amanda Natasha Iyam Perumal for their assistance in creating knockouts as well. Lastly, we thank Julie Nelson at Center for Tropical and Emerging Global Diseases, Cytometry Shared Resource Laboratory at University of Georgia for assistance with FACS cell sorting. This research is supported by NIH grant R01HD090156.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, and Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA

    Steven John Berardinelli & Robert S. Haltiwanger

Authors
  1. Steven John Berardinelli
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  2. Robert S. Haltiwanger
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Correspondence to Robert S. Haltiwanger .

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Editors and Affiliations

  1. Department of Biomedical Engineering-ND20, Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA

    Suneel S. Apte

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Berardinelli, S.J., Haltiwanger, R.S. (2020). Analyzing the Effects of O-Fucosylation on Secretion of ADAMTS Proteins Using Cell-Based Assays. In: Apte, S. (eds) ADAMTS Proteases. Methods in Molecular Biology, vol 2043. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9698-8_3

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  • DOI: https://doi.org/10.1007/978-1-4939-9698-8_3

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9697-1

  • Online ISBN: 978-1-4939-9698-8

  • eBook Packages: Springer Protocols

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