Galectin binding to cells and glycoproteins with genetically modified glycosylation reveals galectin–glycan specificities in a natural context

Mathias Ingemann Nielsen; John Stegmayr; Oliver C. Grant; Zhang Yang; Ulf J. Nilsson; Irene Boos; Michael C. Carlsson; Robert J. Woods; Carlo Unverzagt; Hakon Leffler; Hans H. Wandall

doi:10.1074/jbc.ra118.004636

Galectin binding to cells and glycoproteins with genetically modified glycosylation reveals galectin–glycan specificities in a natural context

Mathias Ingemann Nielsen, John Stegmayr, Oliver C. Grant, Zhang Yang, Ulf J. Nilsson, Irene Boos, Michael C. Carlsson, Robert J. Woods, Carlo Unverzagt, Hakon Leffler, Hans H. Wandall^*

^*Corresponding author af dette arbejde

Glycomics Program

19 Citationer (Scopus)

Abstract

Galectins compose a protein family defined by a conserved sequence motif conferring affinity for -galactose– containing glycans. Moreover, galectins gain higher affinity and fine-tune specificity by glycan interactions at sites adjacent to their -galactoside– binding site, as revealed by extensive testing against panels of purified glycans. However, in cells, galectins bind glycans on glycoproteins and glycolipids in the context of other cellular components, such as at the cell surface. Because of difficulties in characterizing natural cellular environments, we currently lack a detailed understanding of galectin-binding specificities in the cellular context. To address this challenge, we used a panel of genetically stable glycosylation mutated CHO cells that express defined glycans to evaluate the binding affinities of 10 different carbohydrate-recognition domains in galectins to N-glycans and mucin-type O-glycans. Using flow cytometry, we measured the cell-surface binding of the galectins. Moreover, we used fluorescence anisotropy to determine the galectin affinities to recombinant erythropoietin used as a reporter glycoprotein produced by the glycoengineered cells and to synthetic N-glycans with defined branch structures. We found that all galectins, apart from galectin-8N, require complex N-glycans for high-affinity binding. Galectin-8N targeted both N- and O-linked glycans with high affinity, preferring 2,3-sialylated N-acetyllactosamine (LacNAc) structures. Furthermore, we found that 2,3-sialylation suppresses high-affinity binding of select galectins, including galectin-2, -3, -4N, and -7. Structural modeling provided a basis for interpreting the observed binding preferences. These results underscore the power of a glycoengineered platform to dissect the glycan-binding specificities of carbohydrate-binding proteins.

Originalsprog	Engelsk
Tidsskrift	Journal of Biological Chemistry
Vol/bind	293
Udgave nummer	52
Sider (fra-til)	20249-20262
Antal sider	14
ISSN	0021-9258
DOI	https://doi.org/10.1074/jbc.ra118.004636
Status	Udgivet - 28 dec. 2018

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10.1074/jbc.ra118.004636

http://www.jbc.org/content/293/52/20249.full.pdfLicens: CC BY

Citationsformater

Nielsen, M. I., Stegmayr, J., Grant, O. C., Yang, Z., Nilsson, U. J., Boos, I., Carlsson, M. C., Woods, R. J., Unverzagt, C., Leffler, H., & Wandall, H. H. (2018). Galectin binding to cells and glycoproteins with genetically modified glycosylation reveals galectin–glycan specificities in a natural context. Journal of Biological Chemistry, 293(52), 20249-20262. https://doi.org/10.1074/jbc.ra118.004636

Galectin binding to cells and glycoproteins with genetically modified glycosylation reveals galectin–glycan specificities in a natural context. / Nielsen, Mathias Ingemann; Stegmayr, John; Grant, Oliver C. et al.

I: Journal of Biological Chemistry, Bind 293, Nr. 52, 28.12.2018, s. 20249-20262.

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

Nielsen, MI, Stegmayr, J, Grant, OC, Yang, Z, Nilsson, UJ, Boos, I, Carlsson, MC, Woods, RJ, Unverzagt, C, Leffler, H & Wandall, HH 2018, 'Galectin binding to cells and glycoproteins with genetically modified glycosylation reveals galectin–glycan specificities in a natural context', Journal of Biological Chemistry, bind 293, nr. 52, s. 20249-20262. https://doi.org/10.1074/jbc.ra118.004636

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title = "Galectin binding to cells and glycoproteins with genetically modified glycosylation reveals galectin–glycan specificities in a natural context",

abstract = "Galectins compose a protein family defined by a conserved sequence motif conferring affinity for -galactose– containing glycans. Moreover, galectins gain higher affinity and fine-tune specificity by glycan interactions at sites adjacent to their -galactoside– binding site, as revealed by extensive testing against panels of purified glycans. However, in cells, galectins bind glycans on glycoproteins and glycolipids in the context of other cellular components, such as at the cell surface. Because of difficulties in characterizing natural cellular environments, we currently lack a detailed understanding of galectin-binding specificities in the cellular context. To address this challenge, we used a panel of genetically stable glycosylation mutated CHO cells that express defined glycans to evaluate the binding affinities of 10 different carbohydrate-recognition domains in galectins to N-glycans and mucin-type O-glycans. Using flow cytometry, we measured the cell-surface binding of the galectins. Moreover, we used fluorescence anisotropy to determine the galectin affinities to recombinant erythropoietin used as a reporter glycoprotein produced by the glycoengineered cells and to synthetic N-glycans with defined branch structures. We found that all galectins, apart from galectin-8N, require complex N-glycans for high-affinity binding. Galectin-8N targeted both N- and O-linked glycans with high affinity, preferring 2,3-sialylated N-acetyllactosamine (LacNAc) structures. Furthermore, we found that 2,3-sialylation suppresses high-affinity binding of select galectins, including galectin-2, -3, -4N, and -7. Structural modeling provided a basis for interpreting the observed binding preferences. These results underscore the power of a glycoengineered platform to dissect the glycan-binding specificities of carbohydrate-binding proteins.",

author = "Nielsen, {Mathias Ingemann} and John Stegmayr and Grant, {Oliver C.} and Zhang Yang and Nilsson, {Ulf J.} and Irene Boos and Carlsson, {Michael C.} and Woods, {Robert J.} and Carlo Unverzagt and Hakon Leffler and Wandall, {Hans H.}",

year = "2018",

month = dec,

day = "28",

doi = "10.1074/jbc.ra118.004636",

language = "English",

volume = "293",

pages = "20249--20262",

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T1 - Galectin binding to cells and glycoproteins with genetically modified glycosylation reveals galectin–glycan specificities in a natural context

AU - Nielsen, Mathias Ingemann

AU - Stegmayr, John

AU - Grant, Oliver C.

AU - Yang, Zhang

AU - Nilsson, Ulf J.

AU - Boos, Irene

AU - Carlsson, Michael C.

AU - Woods, Robert J.

AU - Unverzagt, Carlo

AU - Leffler, Hakon

AU - Wandall, Hans H.

PY - 2018/12/28

Y1 - 2018/12/28

N2 - Galectins compose a protein family defined by a conserved sequence motif conferring affinity for -galactose– containing glycans. Moreover, galectins gain higher affinity and fine-tune specificity by glycan interactions at sites adjacent to their -galactoside– binding site, as revealed by extensive testing against panels of purified glycans. However, in cells, galectins bind glycans on glycoproteins and glycolipids in the context of other cellular components, such as at the cell surface. Because of difficulties in characterizing natural cellular environments, we currently lack a detailed understanding of galectin-binding specificities in the cellular context. To address this challenge, we used a panel of genetically stable glycosylation mutated CHO cells that express defined glycans to evaluate the binding affinities of 10 different carbohydrate-recognition domains in galectins to N-glycans and mucin-type O-glycans. Using flow cytometry, we measured the cell-surface binding of the galectins. Moreover, we used fluorescence anisotropy to determine the galectin affinities to recombinant erythropoietin used as a reporter glycoprotein produced by the glycoengineered cells and to synthetic N-glycans with defined branch structures. We found that all galectins, apart from galectin-8N, require complex N-glycans for high-affinity binding. Galectin-8N targeted both N- and O-linked glycans with high affinity, preferring 2,3-sialylated N-acetyllactosamine (LacNAc) structures. Furthermore, we found that 2,3-sialylation suppresses high-affinity binding of select galectins, including galectin-2, -3, -4N, and -7. Structural modeling provided a basis for interpreting the observed binding preferences. These results underscore the power of a glycoengineered platform to dissect the glycan-binding specificities of carbohydrate-binding proteins.

AB - Galectins compose a protein family defined by a conserved sequence motif conferring affinity for -galactose– containing glycans. Moreover, galectins gain higher affinity and fine-tune specificity by glycan interactions at sites adjacent to their -galactoside– binding site, as revealed by extensive testing against panels of purified glycans. However, in cells, galectins bind glycans on glycoproteins and glycolipids in the context of other cellular components, such as at the cell surface. Because of difficulties in characterizing natural cellular environments, we currently lack a detailed understanding of galectin-binding specificities in the cellular context. To address this challenge, we used a panel of genetically stable glycosylation mutated CHO cells that express defined glycans to evaluate the binding affinities of 10 different carbohydrate-recognition domains in galectins to N-glycans and mucin-type O-glycans. Using flow cytometry, we measured the cell-surface binding of the galectins. Moreover, we used fluorescence anisotropy to determine the galectin affinities to recombinant erythropoietin used as a reporter glycoprotein produced by the glycoengineered cells and to synthetic N-glycans with defined branch structures. We found that all galectins, apart from galectin-8N, require complex N-glycans for high-affinity binding. Galectin-8N targeted both N- and O-linked glycans with high affinity, preferring 2,3-sialylated N-acetyllactosamine (LacNAc) structures. Furthermore, we found that 2,3-sialylation suppresses high-affinity binding of select galectins, including galectin-2, -3, -4N, and -7. Structural modeling provided a basis for interpreting the observed binding preferences. These results underscore the power of a glycoengineered platform to dissect the glycan-binding specificities of carbohydrate-binding proteins.

U2 - 10.1074/jbc.ra118.004636

DO - 10.1074/jbc.ra118.004636

M3 - Journal article

C2 - 30385505

AN - SCOPUS:85059244147

SN - 0021-9258

VL - 293

SP - 20249

EP - 20262

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 52

ER -

Galectin binding to cells and glycoproteins with genetically modified glycosylation reveals galectin–glycan specificities in a natural context

Abstract

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