The stem region of α1,6-fucosyltransferase FUT8 is required for multimer formation but not catalytic activity

Abstract

Alpha-1,6-fucosyltransferase (FUT8) synthesizes core fucose in N-glycans, which plays critical roles in various physiological processes. FUT8, as with many other glycosyltransferases, is a type-II membrane protein, and its large C-terminal catalytic domain is linked to the FUT8 stem region, which comprises two α-helices. Although the stem regions of several glycosyltransferases are involved in the regulation of Golgi localization, the functions of the FUT8 stem region have not been clarified as yet. Here, we found that the FUT8 stem region is essential for enzyme oligomerization. We expressed FUT8Δstem mutants, in which the stem region was replaced with glycine/serine linkers, in FUT8-KO HEK293 cells. Our immunoprecipitation and native-PAGE analysis showed that FUT8 WT formed a multimer but FUT8Δstem impaired multimer formation in the cells, although the mutants retained specific activity. In addition, the mutant protein had lower steady-state levels, increased endoplasmic reticulum localization, and a shorter half-life than FUT8 WT, suggesting that loss of the stem region destabilized the FUT8 protein. Furthermore, immunoprecipitation analysis of another mutant lacking a part of the stem region revealed that the first helix in the FUT8 stem region is critical for multimer formation. Our findings demonstrated that the FUT8 stem region is essential for multimer formation but not for catalytic activity, providing insights into how the FUT8 protein matures and functions in mammalian cells. Alpha-1,6-fucosyltransferase (FUT8) synthesizes core fucose in N-glycans, which plays critical roles in various physiological processes. FUT8, as with many other glycosyltransferases, is a type-II membrane protein, and its large C-terminal catalytic domain is linked to the FUT8 stem region, which comprises two α-helices. Although the stem regions of several glycosyltransferases are involved in the regulation of Golgi localization, the functions of the FUT8 stem region have not been clarified as yet. Here, we found that the FUT8 stem region is essential for enzyme oligomerization. We expressed FUT8Δstem mutants, in which the stem region was replaced with glycine/serine linkers, in FUT8-KO HEK293 cells. Our immunoprecipitation and native-PAGE analysis showed that FUT8 WT formed a multimer but FUT8Δstem impaired multimer formation in the cells, although the mutants retained specific activity. In addition, the mutant protein had lower steady-state levels, increased endoplasmic reticulum localization, and a shorter half-life than FUT8 WT, suggesting that loss of the stem region destabilized the FUT8 protein. Furthermore, immunoprecipitation analysis of another mutant lacking a part of the stem region revealed that the first helix in the FUT8 stem region is critical for multimer formation. Our findings demonstrated that the FUT8 stem region is essential for multimer formation but not for catalytic activity, providing insights into how the FUT8 protein matures and functions in mammalian cells. Protein glycosylation is a major posttranslational modification that is widely observed in all kingdoms of life (1Varki A. Biological roles of glycans.Glycobiology. 2017; 27: 3-49Crossref PubMed Scopus (1352) Google Scholar). The known functions of glycosylation include the regulation of protein folding, localization, and activity, as well as cell adhesion, cell–cell interactions, and signal transduction (2Zhao Y.-Y. Takahashi M. Gu J.-G. Miyoshi E. Matsumoto A. Kitazume S. et al.Functional roles of N-glycans in cell signaling and cell adhesion in cancer.Cancer Sci. 2008; 99: 1304-1310Crossref PubMed Scopus (324) Google Scholar). Furthermore, changes to glycan structures cause the development or improvement of various diseases, including cancer, chronic obstructive pulmonary disease (COPD), and Alzheimer’s disease (3Granovsky M. Fata J. Pawling J. Muller W.J. Khokha R. Dennis J.W. Suppression of tumor growth and metastasis in Mgat5-deficient mice.Nat. Med. 2000; 6: 306-312Crossref PubMed Scopus (479) Google Scholar, 4Wang X. Inoue S. Gu J. Miyoshi E. Noda K. Li W. et al.Dysregulation of TGF-β1 receptor activation leads to abnormal lung development and emphysema-like phenotype in core fucose-deficient mice.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15791-15796Crossref PubMed Scopus (358) Google Scholar, 5Kizuka Y. Kitazume S. Fujinawa R. Saito T. Iwata N. Saido T.C. et al.An aberrant sugar modification of BACE1 blocks its lysosomal targeting in Alzheimer’s disease.EMBO Mol. Med. 2015; 7: 175-189Crossref PubMed Scopus (138) Google Scholar), and the glycan changes are used clinically as specific disease markers (6Takahashi M. Kizuka Y. Ohtsubo K. Gu J. Taniguchi N. Disease-associated glycans on cell surface proteins.Mol. Aspects Med. 2016; 51: 56-70Crossref PubMed Scopus (58) Google Scholar). Therefore, elucidation of the biosynthetic mechanisms of glycan structures and how they are regulated in cells is extremely important for a detailed understanding of the physiological and pathological functions of glycans and provides insights into ways to control these functions. Glycans on proteins are classified into N-glycan, O-glycan, C-mannose, and glycosylphosphatidylinositol according to their core structures (1Varki A. Biological roles of glycans.Glycobiology. 2017; 27: 3-49Crossref PubMed Scopus (1352) Google Scholar, 7Moremen K.W. Tiemeyer M. Nairn A.V. Vertebrate protein glycosylation: diversity, synthesis and Mol. PubMed Scopus Google Scholar). N-glycans, the of are in the protein Taniguchi N. M. A. M. of Scholar). In the endoplasmic reticulum the N-glycan, is to in the N. S. R. glycosylation and of the endoplasmic 2016; PubMed Scopus Google Scholar). the protein is the to the the and a various N-glycans with structures are in a of cell glycosylation and glycosyltransferases have been in and their levels, localization, and the of the specific the mechanisms for the activity, and of glycosyltransferases have not been In N-glycans, fucose to core or The fucose which is to the of N-glycans, is core fucose N. S. Miyoshi E. Y. T. et and of PubMed Scopus Google Scholar, S. N. Y. Miyoshi E. N. Taniguchi N. and of PubMed Scopus Google Scholar). Alpha-1,6-fucosyltransferase (FUT8) is the enzyme for the synthesis of core fucose S. N. Y. Miyoshi E. N. Taniguchi N. and of PubMed Scopus Google Scholar), and FUT8 fucose to the in the Golgi fucose is involved in various as growth and receptor and in various diseases, including lung cancer, and the of X. Inoue S. Gu J. Miyoshi E. Noda K. Li W. et al.Dysregulation of TGF-β1 receptor activation leads to abnormal lung development and emphysema-like phenotype in core fucose-deficient mice.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15791-15796Crossref PubMed Scopus (358) Google Scholar, X. Gu J. Miyoshi E. K. Taniguchi N. growth PubMed Scopus Google Scholar, S. K. T. et on cells, for activation of receptor signaling and of in is increased in with 2016; PubMed Scopus Google Scholar, J. S. Kitazume S. Kizuka Y. Fujinawa R. et fucose is critical for receptor 2017; 27: PubMed Scopus Google Scholar, E. S. et FUT8 as a of 2017; PubMed Scopus Google Scholar, et as a of cell lung Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. W. et and with PubMed Scopus Google Scholar). In addition, core fucose a in T. K. N. E. Y. M. et of fucose but not the of or of the critical of PubMed Scopus Google Scholar), which been clinically used to findings that core fucose plays important physiological and pathological roles and is a for and In mechanisms for core fucose are FUT8 is a protein of region, region, stem region domain catalytic domain and and domain S. K. T. M. S. N. et of in of Golgi in a J. Google Scholar, Y. S. X. T. Gu J. et of mammalian PubMed Scopus Google Scholar). The functions of all and other than the catalytic for a but have the of these We that the FUT8 domain is essential for the and cell surface of FUT8 and that FUT8 with a of that with to in the in a on the FUT8 domain S. M. T. M. M. Kizuka Y. The domain in the FUT8 FUT8 and and is essential for core PubMed Google Scholar). In addition, of and analysis showed that FUT8 and core in cells. showed that the domain is involved in the and of FUT8 T. Taniguchi N. Y. of the and in the and of PubMed Scopus Google Scholar). In to and the have been on the and functions of the FUT8 stem The stem region is found in many glycosyltransferases and Golgi or with glycosyltransferases S. of and the mechanisms of Golgi PubMed Scopus Google Scholar). In addition, in the of protein the protein for the stem region functions as a domain and to core glycan to N. T. M. K. et domain of the stem region of Natl. Acad. Sci. U. S. A. 2016; PubMed Scopus Google Scholar). on these we that the FUT8 stem region functions in core fucose as the regulation of or of In to the functions of the FUT8 stem region, we the of FUT8 mutants lacking the stem region and clarified that region is for multimer formation and the of findings insights into how the and of FUT8 are regulated in cells. the functions of the FUT8 stem region, we the a FUT8 in which the stem region was replaced with a and we for other FUT8 mutants, in which the or domain was the steady-state and of these mutants in cells, HEK293 W. T. T. S. et of for core in the regulation of receptor and 2015; PubMed Scopus Google cells with FUT8 WT, or and the proteins and with showed that the steady-state of and lower than of FUT8 WT and and of the steady-state of FUT8 WT for and fucose was the specific of Y. K. Miyoshi E. J. et core the PubMed Scopus Google Scholar). in the FUT8 WT increased with in the that the core fucose FUT8 and that the biosynthetic of FUT8 in cells In to FUT8 WT, in cells and of that these mutants in the cells. The with S. M. T. M. M. Kizuka Y. The domain in the FUT8 FUT8 and and is essential for core PubMed Google Scholar). the of and we the in of FUT8 WT and its mutants and Y. S. M. K. R. et and fucose for glycan and 2016; PubMed Scopus Google Scholar, N. T. T. A. Miyoshi E. et for activity, PubMed Scopus Google Scholar). The of cells FUT8 WT or its mutants with the and the and the to and the and its core The of FUT8 and its mutants analysis to the steady-state of the in showed lower than FUT8 WT, and of was and the is with a in which a FUT8 mutant lacking the to domain was T. Taniguchi N. Y. of the and in the and of PubMed Scopus Google Scholar). In showed that FUT8 mutants or lacking the stem region all T. Taniguchi N. Y. of the and in the and of PubMed Scopus Google Scholar, A. S. R. et for and of PubMed Scopus Google Scholar, M. R. A. et of and PubMed Scopus Google Scholar), suggesting that the FUT8 stem region is not essential for the enzyme and that in cells for another the in the mutant is shorter than the FUT8 stem region we that was to the of the in its in the the of FUT8 WT and was and We used cells for the they to the and are in than HEK293 cells, which to the of FUT8 FUT8 WT with the Golgi but with the In well with suggesting that was and in the the FUT8 stem region a or we the of the FUT8 stem region showed that of two suggesting that a Therefore, is that FUT8 a stem region for its folding, and the is to FUT8 in cells. We two and which have and of which not a specific of the FUT8 stem region mutants expressed in HEK293 cells, and the proteins and showed that and formed two The of the of and lower than that of FUT8 WT and the of the lower of and increased with FUT8 WT The two not to is in FUT8, that these mutants in the cells. The of the in the was to that of FUT8 WT and the signal in the was with that in the FUT8 WT but than that in The in of and which the and to their steady-state levels, than that of these findings that the specific of the stem region is not for the FUT8 enzyme and that and are than for the functions of the FUT8 stem in the steady-state of and with FUT8 mechanisms for of the mutants, we the or of and was FUT8 was in K. T. T. T. et is in to in and a for disease and in chronic obstructive pulmonary disease PubMed Scopus Google Scholar), FUT8 is cells, many other glycosyltransferases M. M. U. S. et analysis signal and a of in Golgi glycosylation 2015; PubMed Scopus Google Scholar). Therefore, we first of the FUT8Δstem mutants is the of FUT8, proteins in the of HEK293 cells FUT8 WT, or and protein was as a as is known to into T. M. physiological functions of the protein 2017; PubMed Scopus Google Scholar). In the of FUT8 WT, the signal was in the that FUT8 WT was of the cells. The of and in the lower than that of FUT8 WT, which was to the cell that the of and is to that of FUT8 WT and that the steady-state of and in cells not a of we on the of and and lysosomal HEK293 cells FUT8 WT, or with a or a for and the of the proteins in the cells was and of and of that and functions these as K. T. Y. T. M. et protein protein to the lysosomal PubMed Scopus Google Scholar, A. J. A. R. is a for the J. PubMed Scopus Google Scholar). The of FUT8 WT, and all increased and and and In the of FUT8 WT was the for and increased and and and findings demonstrated that FUT8 WT is in and that the aberrant of and was which a in the steady-state of and these the of and and all FUT8 WT to the as the in In was to the Golgi and and was to the The of the or and the the increased of with and the of with showed that and to the than FUT8 WT, suggesting that FUT8 mutant proteins lacking the stem region are in the for and in the of and we a to cells FUT8 WT or mutants, the cells for to the of FUT8 proteins the of FUT8 WT was to of the and to to of the that the of and than that of FUT8 clarified that with the FUT8 stem of the FUT8 protein. and are than the WT protein, we on the of of these mutants, as was that FUT8 a A. S. R. et for and of PubMed Scopus Google Scholar, M. R. A. et of and PubMed Scopus Google Scholar). FUT8 a in in a with and in the of which was M. R. A. et of and PubMed Scopus Google Scholar). showed that formation of FUT8 is important for the of the catalytic domain M. R. A. et of and PubMed Scopus Google Scholar). the stem region of FUT8 a in that a for T. Y. and of PubMed Scopus Google Scholar). on these we that the stem region of FUT8 is for and that the and of and are the of a in formation. we first to the multimer other of FUT8 immunoprecipitation We expressed FUT8 and FUT8 in HEK293 cells, FUT8, and FUT8 was FUT8 or FUT8 was the signal was not in the and FUT8 a signal was that the FUT8 WT with another FUT8 to a multimer in the cells and that the multimer the multimer formation or and FUT8 WT was of or with FUT8 was the of the of the of We observed that and with FUT8 WT and and and for and In and with FUT8 WT to a multimer these that the FUT8 stem region is essential for multimer formation in cells. The multimer formation of FUT8 in cells was which to protein we the FUT8 multimer native-PAGE FUT8, which of and was a showed a of FUT8 and native-PAGE was the enzyme several which that FUT8 formed in with the analysis M. R. A. et of and PubMed Scopus Google Scholar), and that native-PAGE FUT8 We the multimer formation of FUT8 WT, and expressed in HEK293 cells In the FUT8 WT several large FUT8 In the of and and and the of the and demonstrated that of the FUT8 stem region a in multimer which have in the and of and We specific regions or the stem region are involved in multimer formation. The of the stem region that two and and In addition, which that helix with the helix in the and that in the is to in the another to on these we the mutants and and the mutant FUT8 with showed that the of core proteins in HEK293 cells or was to that in FUT8 WT In addition, the of and in to that of FUT8 WT and for and that the FUT8 stem region is not essential for FUT8 activity. multimer formation of and was The for and in the lower than that for FUT8 WT and and for and In the of was to of that of FUT8 WT FUT8 in not FUT8 that and are involved in the multimer formation of FUT8 and that is FUT8 in cells the FUT8 stem region, we the FUT8 stem region, other is for we used with which We the part the region to the stem of FUT8 WT and with a large part or part of the FUT8 stem regions in the to in cells, and to and FUT8 stem or its mutant was with its or in HEK293 cells, and of FUT8 stem and its mutant was of the cell with The of cells FUT8 with was the as that of cells, which with to cells FUT8 and FUT8 showed of suggesting that FUT8 in the cells. the of cells and was to that the part the to stem of FUT8 is to and that in the FUT8 stem region is for We the of the FUT8 stem region of the structures of the FUT8 part with a region and a region showed the and in all that are involved in multimer formation of FUT8, and in plays a critical in multimer formation on the and these findings into we a for the of the FUT8 we the FUT8 stem region is involved in with We that a of to FUT8 in a on the domain of FUT8 to FUT8 S. M. T. M. M. Kizuka Y. The domain in the FUT8 FUT8 and and is essential for core PubMed Google Scholar). on these the or and was with with the with FUT8 In and showed to suggesting that the stem region of FUT8 is for to In we revealed that the FUT8 stem region is essential for multimer and the of FUT8 in cells and their enzyme and showed the as FUT8 WT we that the stem region of FUT8 is not for enzyme have been on the functions of the FUT8 stem region or the mechanisms of FUT8 multimer insights into the mechanisms of the and of FUT8 in cells. Furthermore, as the mutants and showed to we that to the FUT8 multimer or that FUT8 to the stem to the of multimer formation the stem region, was that FUT8 a in the stem regions of to a T. Y. and of PubMed Scopus Google Scholar). The in the stem region of FUT8 is to and of the to in FUT8 was to in T. Taniguchi N. Y. of the and in the and of PubMed Scopus Google Scholar). the stem region of FUT8 to the stem region of another FUT8 to with Our showed that and of which have a region, impaired FUT8 lacking the stem region been to a in structures A. S. R. et for and of PubMed Scopus Google Scholar, M. R. A. et of and PubMed Scopus Google Scholar), suggesting that the C-terminal region of FUT8 the stem region a to a In a in which the specific in the domain or domain to demonstrated that the domain and domain are to with other T. Taniguchi N. Y. of the and in the and of PubMed Scopus Google Scholar). In addition, analysis that FUT8 a in M. R. A. et of and PubMed Scopus Google Scholar). the domain and domain to the showed that of and not impaired in the cells In the of and the C-terminal region Furthermore, showed that part of FUT8 is for in the cells of all these we that although the stem region and the C-terminal region of FUT8 to multimer the stem region plays a for multimer formation in the cells. In of the in multimer formation in the cells the FUT8 stem region, are several the stem regions of FUT8 with other to FUT8 into which in the of catalytic to the of the FUT8 as a membrane protein in cells are to than of in is that the the catalytic of FUT8 the stem the of FUT8 to the multimer formation of demonstrated that and multimer formation and to than FUT8 WT, is that to the FUT8 multimer or the multimer formation of FUT8 its stem In FUT8 WT was as a that FUT8 WT with several including and other in the cells. Although the and of the FUT8 and have not been are two roles of that as a for a protein Matsumoto K. K. Matsumoto N. Y. et a with for with PubMed Scopus Google Scholar), and to FUT8 to FUT8 in the as a is that core of N-glycans Although in analysis showed that the on the of is essential for core et and of and 2017; PubMed Scopus Google Scholar), showed that core N-glycans in the cells of in PubMed Scopus Google Scholar, M. et of and glycosylation the of the PubMed Scopus Google Scholar), suggesting for of with glycans FUT8 core glycans in the a to which FUT8 domain or region with and other are involved in the FUT8 and In to and was and that the stem region of FUT8 a In addition, and showed and multimer formation showed as a lower steady-state and findings that the of the FUT8 stem region the of the FUT8 protein. the catalytic domain of FUT8 is not the not to with the and than with to the increased and of the of the stem region important for the FUT8 and the on the that the steady-state of and lower than that of FUT8 WT, the of the of the for FUT8 insights into how the stem region of FUT8 the of the FUT8 protein. been that glycosyltransferases, as are the stem region the cells S. N. K. S. The Golgi and its PubMed Scopus Google Scholar). that the and of glycosyltransferases are regulated their and M. U. A. A. et of signal J. PubMed Scopus Google Scholar), the of of glycosyltransferases for glycan In a of FUT8 WT was in the and the of and to or lower than that of FUT8 WT in the of FUT8, the stem region is not for protein and the of FUT8 is the as that of FUT8, FUT8 is to the the region and the stem of the for FUT8 the of its and in core fucose plays important roles in the development of and metastasis X. Inoue S. Gu J. Miyoshi E. Noda K. Li W. et al.Dysregulation of TGF-β1 receptor activation leads to abnormal lung development and emphysema-like phenotype in core fucose-deficient mice.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15791-15796Crossref PubMed Scopus (358) Google Scholar, E. S. et FUT8 as a of 2017; PubMed Scopus Google Scholar), the regulation of FUT8 or is important for the of these which and multimer formation the stem region a for core Furthermore, as many glycosyltransferases have a stem region, the findings in important for the mechanisms of the and multimer formation of glycosyltransferases in cells. In to the stem region, are and regions of FUT8 that have not detailed In the of the detailed functions of all in FUT8 to a of core in cells, which to a understanding of the of and its The and was Miyoshi Y. K. Miyoshi E. J. et core the PubMed Scopus Google was was and in was and was in was and was in used for are in FUT8 with was and into of or of for of or FUT8 which was used for all in the of the stem region was replaced with a the as The the to the of FUT8 and the large the to the into of of the the domain was to the as The the to of FUT8 and the large the to and these into of the or or S. M. T. M. M. Kizuka Y. The domain in the FUT8 FUT8 and and is essential for core PubMed Google used as for The or into of of FUT8Δstem the a for was The and the two which used for of into of The for was in the as that for The for the FUT8 mutants and in the as for mutants and as FUT8 to was as Y. M. Y. K. R. K. et al.An cell and enzyme 2017; PubMed Scopus Google Scholar). the of FUT8 the the to the of FUT8 and the large the to into of the FUT8 the the to the and the large the to and these into of for FUT8 was the for FUT8 as a the according to the for FUT8 and FUT8 a FUT8 was used as a for The the to of FUT8 was into of and for and the to of FUT8 was FUT8 as a and into of or HEK293 cell and cell cells in with and Gu and the cells, which FUT8 or was to the the cells for was on or cells on a or the on a or with or of of FUT8, of the was into cells on a and in and of the cell the and protein of the a Protein according to the The cell with and to to and and the was with Protein and proteins to a the with and they with the with the with the with with or protein protein with the with the with with with the protein in the as for the The in the are of or for was according to the cells on in in the and for the and protein Protein the was of to with and and to a on the membrane with and the with The for and signal the as for The of FUT8 was as Y. M. Y. K. R. K. et al.An cell and enzyme 2017; PubMed Scopus Google Scholar). to cells on with the of the and the cells with for The and the cell was FUT8 was the a and a The for FUT8 was as Y. S. M. K. R. et and fucose for glycan and 2016; PubMed Scopus Google Scholar, N. T. T. A. Miyoshi E. et for activity, PubMed Scopus Google Scholar). of protein in a cell FUT8 was in of and for The was for to the the of of The was for and of the was to with to the and The of and including on with for with for with for the cells with they with for and with or and to the cells, for with the was and the was used to the cells, a was a of was we first the to and the or the of the or and the signal in the in and and with The cell for and the of the was used as for and the was with The with and with for to the proteins to the immunoprecipitation with the the of the was as and with protein for The with and protein and with The with for The and HEK293 cells on with the the and the cells with or cells and used for HEK293 cells with the the and the cells with for The cells and the and the cells for The for and the of and of for for the with and for The in for the used for and The of FUT8 was the The was to on the HEK293 cells on a with or and in for and the was Protein of the Protein In a of proteins in the cell in of in a and the was with of that had been with according to the signal was The of two in The of the FUT8 stem region in the R. M. A. N. A. T. et with PubMed Scopus Google Scholar). The of FUT8 in was the of FUT8 catalytic domain and the of in with analysis was the are the The that they have of with the of We and for their We for on S. T. and Y. K. S. T. and M. N. S. T. Y. K. Y. K. Y. K. Y. K. was a for to the for the of a to Y. and a to Y. a the and a the for S. T. was

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