O-fucosylation stabilizes the TSR3 motif in thrombospondin-1 by interacting with nearby amino acids and protecting a disulfide bond

Abstract

Thrombospondin type-1 repeats (TSRs) are small protein motifs containing six conserved cysteines forming three disulfide bonds that can be modified with an O-linked fucose. Protein O-fucosyltransferase 2 (POFUT2) catalyzes the addition of O-fucose to TSRs containing the appropriate consensus sequence, and the O-fucose modification can be elongated to a Glucose-Fucose disaccharide with the addition of glucose by β3-glucosyltransferase (B3GLCT). Elimination of Pofut2 in mice results in embryonic lethality in mice, highlighting the biological significance of O-fucose modification on TSRs. Knockout of POFUT2 in HEK293T cells has been shown to cause complete or partial loss of secretion of many proteins containing O-fucosylated TSRs. In addition, POFUT2 is localized to the endoplasmic reticulum (ER) and only modifies folded TSRs, stabilizing their structures. These observations suggest that POFUT2 is involved in an ER quality control mechanism for TSR folding and that B3GLCT also participates in quality control by providing additional stabilization to TSRs. However, the mechanisms by which addition of these sugars result in stabilization are poorly understood. Here, we conducted molecular dynamics (MD) simulations and provide crystallographic and NMR evidence that the Glucose-Fucose disaccharide interacts with specific amino acids in the TSR3 domain in thrombospondin-1 that are within proximity to the O-fucosylation modification site resulting in protection of a nearby disulfide bond. We also show that mutation of these amino acids reduces the stabilizing effect of the sugars in vitro. These data provide mechanistic details regarding the importance of O-fucosylation and how it participates in quality control mechanisms inside the ER. Thrombospondin type-1 repeats (TSRs) are small protein motifs containing six conserved cysteines forming three disulfide bonds that can be modified with an O-linked fucose. Protein O-fucosyltransferase 2 (POFUT2) catalyzes the addition of O-fucose to TSRs containing the appropriate consensus sequence, and the O-fucose modification can be elongated to a Glucose-Fucose disaccharide with the addition of glucose by β3-glucosyltransferase (B3GLCT). Elimination of Pofut2 in mice results in embryonic lethality in mice, highlighting the biological significance of O-fucose modification on TSRs. Knockout of POFUT2 in HEK293T cells has been shown to cause complete or partial loss of secretion of many proteins containing O-fucosylated TSRs. In addition, POFUT2 is localized to the endoplasmic reticulum (ER) and only modifies folded TSRs, stabilizing their structures. These observations suggest that POFUT2 is involved in an ER quality control mechanism for TSR folding and that B3GLCT also participates in quality control by providing additional stabilization to TSRs. However, the mechanisms by which addition of these sugars result in stabilization are poorly understood. Here, we conducted molecular dynamics (MD) simulations and provide crystallographic and NMR evidence that the Glucose-Fucose disaccharide interacts with specific amino acids in the TSR3 domain in thrombospondin-1 that are within proximity to the O-fucosylation modification site resulting in protection of a nearby disulfide bond. We also show that mutation of these amino acids reduces the stabilizing effect of the sugars in vitro. These data provide mechanistic details regarding the importance of O-fucosylation and how it participates in quality control mechanisms inside the ER. Thrombospondin type-1 repeats (TSRs1) are small, cysteine-rich protein motifs containing 50 to 60 amino acids. They contain six highly conserved cysteines that form three disulfide bonds which give TSRs a characteristic 3-dimensional fold and shape (1Tucker R.P. The thrombospondin type 1 repeat superfamily.Int. J. Biochem. Cell Biol. 2004; 36: 969-974Crossref PubMed Scopus (128) Google Scholar, 2Tan K. Duquette M. Liu J.H. Dong Y. Zhang R. Joachimiak A. et al.Crystal structure of the TSP-1 type 1 repeats: a novel layered fold and its biological implication.J. Cell Biol. 2002; 159: 373-382Crossref PubMed Scopus (204) Google Scholar, 3van den Bos R.M. Pearce N.M. Granneman J. Brondijk T.H.C. Gros P. Insights into enhanced complement activation by structures of properdin and its complex with the C-terminal domain of C3b.Front. Immunol. 2019; 10: 2097Crossref PubMed Scopus (22) Google Scholar, 4Sun Z. Reid K.B. Perkins S.J. The dimeric and trimeric solution structures of the multidomain complement protein properdin by X-ray scattering, analytical ultracentrifugation and constrained modelling.J. Mol. Biol. 2004; 343: 1327-1343Crossref PubMed Scopus (52) Google Scholar, 5Akiyama M. Takeda S. Kokame K. Takagi J. Miyata T. Crystal structures of the noncatalytic domains of ADAMTS13 reveal multiple discontinuous exosites for von Willebrand factor.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 19274-19279Crossref PubMed Scopus (129) Google Scholar). TSRs that contain the consensus sequence C-X-X-S/T-C (first and second conserved cysteines for group 1 TSRs, second and third cysteines for group 2 TSRs) can be modified with an O-linked fucose (6Valero-Gonzalez J. Leonhard-Melief C. Lira-Navarrete E. Jimenez-Oses G. Hernandez-Ruiz C. Pallares M.C. et al.A proactive role of water molecules in acceptor recognition by protein O-fucosyltransferase 2.Nat. Chem. 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M. et and evidence that in Pofut2 result in Biol. 2016; PubMed Scopus Google Scholar). in B3GLCT result in a of D. Keusch J.J. J. is a of and of thrombospondin type 1 repeats.J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P. R. D. et of in J. A. PubMed Scopus Google Scholar, M. is a of by a in the that modifies thrombospondin type 1 2009; PubMed Scopus Google Scholar, J. A. A. M. J. et mutation in 2009; PubMed Scopus Google Scholar, E. D. S. et in and of in a of with PubMed Scopus Google Scholar). are by of the to and can also additional and J. G. J. 2016; Google Scholar). of in mice the in B3GLCT enzyme B.C. S.J. Zhang A. et and are by loss of B3GLCT in of Mol. 2019; PubMed Scopus Google Scholar, E. S. Haltiwanger R.S. characterization of of the human B3GLCT in PubMed Scopus Google Scholar). TSRs containing the POFUT2 consensus sequence in or proteins which can contain a or multiple TSRs M. E. Haltiwanger R.S. of fucose in PubMed Scopus Google Scholar). of these proteins to the with Thrombospondin and of proteins results in of that are in Pofut2 or mice, or the importance of O-fucosylation of TSRs in proteins S. M. et and evidence that in Pofut2 result in Biol. 2016; PubMed Scopus Google Scholar, B.C. S.J. Zhang A. et and are by loss of B3GLCT in of Mol. 2019; PubMed Scopus Google Scholar). proteins containing TSRs that are POFUT2 and B3GLCT are in a of that and in biological M. E. Haltiwanger R.S. of fucose in PubMed Scopus Google properdin in den Bos R.M. Pearce N.M. Granneman J. Brondijk T.H.C. Gros P. Insights into enhanced complement activation by structures of properdin and its complex with the C-terminal domain of C3b.Front. Immunol. 2019; 10: 2097Crossref PubMed Scopus (22) Google Scholar, C. of a Immunol. PubMed Scopus Google Scholar, S. J. the of is highly Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and thrombospondin-1 involved in and ER M. C. T. et protection by the endoplasmic reticulum proteins thrombospondin 1 and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. for the of by thrombospondin-1 and PubMed Scopus Google Scholar, E. S. D. E. et human by endoplasmic reticulum PubMed Scopus Google Scholar). The of O-fucosylation on TSRs has to be understood. is that POFUT2 a folding for TSRs folding inside the ER B.C. Haltiwanger R.S. Protein O-fucosylation: structure and function.Curr. Opin. Struct. Biol. 2019; 56: 78-86Crossref PubMed Scopus (63) Google Scholar, D. E. Haltiwanger R.S. a ER Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, D. Haltiwanger R.S. for O-linked in protein J. PubMed Scopus Google Scholar). The ER is the folding for proteins the and POFUT2 and B3GLCT are localized to the ER (8Luo Y. Koles K. Vorndam W. Haltiwanger R.S. Panin V.M. Protein O-fucosyltransferase 2 adds O-fucose to thrombospondin type 1 repeats.J. Biol. Chem. 2006; 281: 9393-9399Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, K. Keusch J.J. Hegemann B. Luther K.B. Klein D. Hess D. et al.Identification and characterization of abeta1,3-glucosyltransferase that synthesizes the Glc-beta1,3-Fuc disaccharide on thrombospondin type 1 repeats.J. Biol. Chem. 2006; 281: 36742-36751Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 10Sato T. Sato M. Kiyohara K. Sogabe M. Shikanai T. Kikuchi N. et al.Molecular cloning and characterization of a novel human beta1,3-glucosyltransferase, which is localized the endoplasmic reticulum and O-linked on thrombospondin type 1 repeat 2006; PubMed Scopus Google Scholar). POFUT2 only modifies folded TSRs, that it can folded and TSRs Y. A. Haltiwanger R.S. for O-fucosylation of or thrombospondin type 1 repeats.J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). The structure of POFUT2 with a TSR also how only folded TSRs into the POFUT2 the group of the or to be modified in the site (6Valero-Gonzalez J. Leonhard-Melief C. Lira-Navarrete E. Jimenez-Oses G. Hernandez-Ruiz C. Pallares M.C. et al.A proactive role of water molecules in acceptor recognition by protein O-fucosyltransferase 2.Nat. Chem. Biol. 2016; 12: 240-246Crossref PubMed Scopus (44) Google Scholar, Keusch J.J. Klein D. Hess D. J. of human into thrombospondin type 1 repeat fold and J. PubMed Scopus Google Scholar). We also shown that addition of fucose to a TSR its folded it to in a in D. E. Haltiwanger R.S. a ER Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). These results the that O-fucosylation is involved in quality control for folding of TSRs inside the ER. In ER quality the and adds a glucose to of to with the and N. S. R. and of the endoplasmic Opin. Cell Biol. 2016; PubMed Scopus Google Scholar). In POFUT2 folded TSRs and modifies with fucose to the TSR into a folded structure for O-fucosylation by POFUT2 a quality control in the ER. further on these we that O-fucose TSRs in the folded by with of amino acids that are in proximity to the O-fucose modification the third TSR a we show that the O-fucose modification with amino acids in proximity and that these results in a in TSR3 We that the O-fucose folded TSRs by the disulfide it a mechanism for how O-fucose TSRs, a quality control that to folding and secretion of POFUT2 evidence that TSR3 modified with O-fucose or the disaccharide to TSR3 it to in D. E. Haltiwanger R.S. a ER Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). on these we that and provide by with amino acids the TSR that into proximity with the O-fucose which amino acids by the we molecular dynamics (MD) simulations on O-fucosylated on a structure K. Duquette M. Liu J.H. Dong Y. Zhang R. Joachimiak A. et al.Crystal structure of the TSP-1 type 1 repeats: a novel layered fold and its biological implication.J. Cell Biol. 2002; 159: 373-382Crossref PubMed Scopus (204) Google Scholar). the the group of the O-fucose on of the with in the the and TSR3 domains The O-fucose on TSR3 with amino which its the of the additional of the fucose with amino we the O-fucosylated form of TSRs human and the structure The for and TSR3 for we a the fucose and the cysteines and that form the disulfide the O-fucose site and a stabilizing the O-fucose and the disulfide bond. The the O-fucose and disulfide TSR3 the the disulfide and the O-fucose In the of the fucose on and TSR3 the disulfide and to the the of the O-fucose to with the disulfide bond. The of the the O-fucose and disulfide in the the O-fucose in the structure K. Duquette M. Liu J.H. Dong Y. Zhang R. Joachimiak A. et al.Crystal structure of the TSP-1 type 1 repeats: a novel layered fold and its biological implication.J. Cell Biol. 2002; 159: 373-382Crossref PubMed Scopus (204) Google also the disulfide the fucose the structure the the of the fucose in TSRs the in that the of the fucose in structure the and is a These data suggest that the O-fucose is the disulfide it by in the ER protein disulfide C. M. in and Scopus Google Scholar, T. M. A. M. K. et al.Identification of the of a Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We NMR to additional amino acids that be by O-fucose We TSR3 with and to of TSR3 modified with O-fucose and disaccharide in complete of and and complete modification of TSR3 by POFUT2 and B3GLCT on a of and NMR we complete of and of the TSR3 in TSR3 and The only of and of and only in of the and been of of TSR3 and the addition of fucose to TSR3 the is for the site of in proximity to the These and of the O-fucose and to the O-fucose site with the second conserved within the O-fucosylation consensus sequence in the in proximity to the O-fucose a with and which form the conserved disulfide to in These provide additional data that the O-fucose is the disulfide bond. of to form further enhanced for and amino acids and of the O-fucose site and and to and the C-terminal and addition of glucose in to these data by the addition of O-fucose and modification on data also evidence that in proximity to the an effect on the their is C-terminal to the conserved of and an with the group of fucose in the simulations in is in the in is an it in the in and in the of However, the of addition of the O-fucose or can be in and of the addition of fucose and that are to the O-fucose site in TSR3 of for with O-fucose the of and of and and of evidence of the within in proximity in are in in many TSRs, a importance for in the of the three TSRs and TSRs properdin with consensus for O-fucose modification a in the which is highly for and has of TSRs with a in the of the TSRs with the consensus sequence for O-fucosylation the a in the these data suggest an in the and the O-fucose modification on are highly conserved the in and of TSRs and the of the TSRs in and these TSRs, a in a The the of the TSRs in and these TSRs, a the and in the O-fucose stabilization on folded we to of these into TSR3 the TSRs in and with POFUT2 and B3GLCT to three and TSR3 shown to be for POFUT2 TSR3 modification of and with POFUT2 and B3GLCT by amino acids that addition of O-fucose to protein or of disulfide mutation amino acids that into proximity to the O-fucose site and these the of O-fucose or to we in to the TSR3 an of the TSR3 is addition of the and the and to 60 and These results the stabilizing effect O-fucosylation has on TSR3 which in D. 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In a the an and reveal that in are conserved in many TSRs with POFUT2 consensus We also evidence of an O-fucose and in the form of and the of the of O-fucose that is in of of the amino acids to the the of fucose or the disaccharide to TSR3 These results suggest that the O-fucose TSR3 by with amino resulting in protection of the results also provide additional evidence that POFUT2 and B3GLCT in a quality control for folding of TSRs in the ER B.C. Haltiwanger R.S. Protein O-fucosylation: structure and function.Curr. Opin. Struct. Biol. 2019; 56: 78-86Crossref PubMed Scopus (63) Google Scholar, D. E. Haltiwanger R.S. a ER Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). In the of the ER disulfide bonds are a quality control is for domains TSRs, in proteins are has TSRs, containing cysteines in the TSRs that to their secretion is in or HEK293T cells B.C. S.J. Zhang A. et and are by loss of B3GLCT in of Mol. 2019; PubMed Scopus Google that folding of TSRs is on the addition of fucose and We that POFUT2 the folded form of a TSR and modifies it with stabilizing it by the disulfide of glucose by the B3GLCT additional These ER which cause the TSR to a folding folding and the of disulfide to POFUT2 proteins are or secretion is HEK293T cells S. M. et and evidence that in Pofut2 result in Biol. 2016; PubMed Scopus Google Scholar, B.C. S.J. Zhang A. et and are by loss of B3GLCT in of Mol. 2019; PubMed Scopus Google Scholar, A. S.J. Leonhard-Melief C. D. Liu A. et of is for secretion and is by Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. S.J. A. Haltiwanger R.S. B.C. in is by in multiple B3GLCT in cells and PubMed Scopus Google Scholar). The is also with data that in folding of TSR3 is in the of POFUT2 and in the of POFUT2 D. E. Haltiwanger R.S. a ER Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). of the O-fucose is The that secretion of only POFUT2 are in HEK293T cells B.C. S.J. Zhang A. et and are by loss of B3GLCT in of Mol. 2019; PubMed Scopus Google Scholar, S. J. S.J. A. Haltiwanger R.S. B.C. in is by in multiple B3GLCT in cells and PubMed Scopus Google that TSRs are to fold in and the of the glucose is for and secretion of these TSRs. of TSRs has also been shown to stabilizing on TSRs A. M. J. S. B. et folding and of thrombospondin 2019; PubMed Scopus Google Scholar). However, TSRs in the the consensus sequence to the extended consensus In the extended consensus sequence, can be the in the extended consensus sequence is modified the A. B. of thrombospondin type 1 repeats by and Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). group and shown that site of TSRs containing the consensus sequence to modification O-fucosylation the stabilization on these TSRs B.C. S.J. Zhang A. et and are by loss of B3GLCT in of Mol. 2019; PubMed Scopus Google Scholar, A. S.J. Leonhard-Melief C. D. Liu A. et of is for secretion and is by Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. D. Haltiwanger R.S. et a mechanism for in 2016; PubMed Scopus Google Scholar, D. M. S.J. B. et and on of a in PubMed Scopus (44) Google Scholar). The importance of O-fucose modification on TSRs be for quality and the secretion and proteins modified with However, we the secretion of in the of mice Pofut2 has been S. S.J. et of thrombospondin type 1 repeats is for and Biol. PubMed Scopus Google loss of secretion be be to of that TSRs to fold in the of or of the that been A. B. of thrombospondin type 1 repeats by and Natl. Acad. Sci. U. S. A. PubMed Scopus Google of which is of data that O-fucosylation on TSRs is only for folding and secretion has in the that an O-fucose on TSR3 of 1 is in with its J. Y. R. Z. J. et to Full Text Full Text PDF PubMed Scopus Google Scholar). control and of the O-fucose or of POFUT2 in is highly of O-fucose on the repeats of the which is by the POFUT2 B.C. Haltiwanger R.S. Protein O-fucosylation: structure and function.Curr. Opin. Struct. Biol. 2019; 56: 78-86Crossref PubMed Scopus (63) Google Scholar). is also ER localized and only modifies folded repeats Y. and characterization of a Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We shown that TSRs, O-fucosylation by and is in HEK293T cells M. A. et the of repeats and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In addition, the O-fucose on of is in with and S. for of Scopus Google Scholar, B.C. C. S. D. M. et complex structure a in PubMed Scopus Google Scholar). of the O-fucose site on reduces S. M. et and evidence that in Pofut2 result in Biol. 2016; PubMed Scopus Google Scholar, B.C. C. S. D. M. et complex structure a in PubMed Scopus Google Scholar, S. 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Scopus Google Scholar). bonds constrained with the G. of the of of a with molecular dynamics of Scopus Google an of 2 to be The to with on the of the protein to the water molecules to for 2 with simulations for The and of human human thrombospondin 1 and (6Valero-Gonzalez J. Leonhard-Melief C. Lira-Navarrete E. Jimenez-Oses G. Hernandez-Ruiz C. Pallares M.C. et al.A proactive role of water molecules in acceptor recognition by protein O-fucosyltransferase 2.Nat. Chem. Biol. 2016; 12: 240-246Crossref PubMed Scopus (44) Google Scholar). in E. and in by and in a of The in The further by 1 The the it to the the to the The of by the of the the for the protein a of which to the addition of three fucose of the by of protein solution by the in with of solution in 60 of The by The in containing and in in the of a of The data and the W. Biol. PubMed Scopus Google and P. et of the and Biol. PubMed Scopus Google are in The structure by molecular with P. et of the and Biol. 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Haltiwanger R.S. for O-fucosylation of or thrombospondin type 1 repeats.J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). and G. W. to of their and PubMed Scopus Google Scholar). by of with or and POFUT2 (6Valero-Gonzalez J. Leonhard-Melief C. Lira-Navarrete E. Jimenez-Oses G. Hernandez-Ruiz C. Pallares M.C. et al.A proactive role of water molecules in acceptor recognition by protein O-fucosyltransferase 2.Nat. Chem. Biol. 2016; 12: 240-246Crossref PubMed Scopus (44) Google in containing 50 in a in addition to human B3GLCT A. A. R. A. N. et the and of human Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and The TSR3 and the and modification of TSR3 with fucose and glucose by a to a NMR NMR TSR3 and in and NMR of TSR3 and with with and and with or in and to and in to NMR data and or G. J. A. a on PubMed Scopus Google NMR and G. of for of NMR data with PubMed Scopus Google by with and to with and and of NMR and NMR The Scholar). of and of TSR3 with Y. W. M. S. C. et of protein NMR Mol. Biol. PubMed Scopus Google and and within on and of and of on TSR3 of and of the fucose and of and of by and of and to the and are in of and the Y. A. Haltiwanger R.S. for O-fucosylation of or thrombospondin type 1 repeats.J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google a with for mutation of of with of and and of in a of of for and for for by and modified in the in the with of in a of 50 and 2 of and TSRs and in multiple by a of D. E. Haltiwanger R.S. a ER Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, for the folding of human Biol. Chem. 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