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The GAGOme: a cell-based library of displayed glycosaminoglycans

Abstract

Glycosaminoglycans (GAGs) are essential polysaccharides in normal physiology and disease. However, understanding of the contribution of specific GAG structures to specific biological functions is limited, largely because of the great structural heterogeneity among GAGs themselves, as well as technical limitations in the structural characterization and chemical synthesis of GAGs. Here we describe a cell-based method to produce and display distinct GAGs with a broad repertoire of modifications, a library we refer to as the GAGOme. By using precise gene editing, we engineered a large panel of Chinese hamster ovary cells with knockout or knock-in of the genes encoding most of the enzymes involved in GAG biosynthesis, to generate a library of isogenic cell lines that differentially display distinct GAG features. We show that this library can be used for cell-based binding assays, recombinant expression of proteoglycans with distinct GAG structures, and production of distinct GAG chains on metabolic primers that may be used for the assembly of GAG glycan microarrays.

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Acknowledgements

This work was supported by the Lundbeck Foundation, Læge Sofus Carl Emil Friis og hustru Olga Doris Friis’ Legat, the Novo Nordisk Foundation, Lund University, Kirsten og Freddy Johansen Fonden, the European Commission (GlycoImaging H2020-MSCA-ITN-721297; BioCapture H2020-MSCA-ITN-722171), the UCPH Excellence Programme for Interdisciplinary Research (CDO2016), and the Danish National Research Foundation (DNRF107) (all to H.C.). C.G was supported through FCT, POPH (Programa Operacional Potencial Humano) SFRH/BPD/96510/2013. A.S., T.M.C., and T.G. were supported through the ERC MalOnco Program, the Danish Cancer Society, and the Lundbeck Foundation.

Author information

Y.-H.C., Y.M., Y.N., T.M.C., H.C., and Z.Y. conceived and designed the study; Y.-H.C., Y.M., Y.N., C.G., R.K., C.S., A.M., E.P.B., and Z.Y. contributed with experimental data and data interpretation; T.M.C., C.B.S., T.G., and A.S. contributed to the VAR2CSA studies; A.P., D.W., and U.E. contributed to the xyloside-priming studies; Y.-H.C., Y.M., H.C., and Z.Y. wrote the manuscript; and all authors edited and approved the final version of the manuscript.

Competing interests

The University of Copenhagen has filed patent application EP/2017/061385 on the basis of this work. Y.N., C.S., E.P.B., H.C., and Y.Z. are named inventors on the PCT application.

Correspondence to Yang Mao or Henrik Clausen or Zhang Yang.

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Fig. 1: Graphic depiction of the GAGOme approach.
Fig. 2: Overview of the genetic regulation of GAG biosynthesis.
Fig. 3: Summary overview of effects of KO/KI engineering on HPLC disaccharide profiles of total cell lysates.
Fig. 4: Exploring the binding specificities of GAG-binding proteins by using flow cytometry with GAGOme sublibraries.