Carbohydrate Binding Specificity of a Fucose-specific Lectin from Aspergillus oryzae

The α1,6-fucosyl residue (core fucose) of glycoproteins is widely distributed in mammalian tissues and is altered under pathological conditions. A probe that specifically detects core fucose is important for understanding the role of this oligosaccharide structure. Aleuria aurantia lectin (AAL) and Lens culimaris agglutinin-A (LCA) have been often used as carbohydrate probes for core fucose in glycoproteins. Here we show, by using surface plasmon resonance (SPR) analysis, that Aspergillus oryzae l-fucose-specific lectin (AOL) has strongest preference for the α1,6-fucosylated chain among α1,2-, α1,3-, α1,4-, and α1,6-fucosylated pyridylaminated (PA)-sugar chains. These results suggest that AOL is a novel probe for detecting core fucose in glycoproteins on the surface of animal cells. A comparison of the carbohydrate-binding specificity of AOL, AAL, and LCA by SPR showed that the irreversible binding of AOL to the α1,2-fucosylated PA-sugar chain (H antigen) relative to the α1,6-fucosylated chain was weaker than that of AAL, and that the interactions of AOL and AAL with α1,6-fucosylated glycopeptide (FGP), which is considered more similar to in vivo glycoproteins than PA-sugar chains, were similar to their interactions with the α1,6-fucosylated PA-sugar chain. Furthermore, positive staining of AOL, but not AAL, was completely abolished in the cultured embryo fibroblast (MEF) cells obtained from α1,6-fucosyltransferase (Fut8) knock-out mice, as assessed by cytological staining. Taken together, these results suggest that AOL is more suitable for detecting core fucose than AAL or LCA. The α1,6-fucosyl residue (core fucose) of glycoproteins is widely distributed in mammalian tissues and is altered under pathological conditions. A probe that specifically detects core fucose is important for understanding the role of this oligosaccharide structure. Aleuria aurantia lectin (AAL) and Lens culimaris agglutinin-A (LCA) have been often used as carbohydrate probes for core fucose in glycoproteins. Here we show, by using surface plasmon resonance (SPR) analysis, that Aspergillus oryzae l-fucose-specific lectin (AOL) has strongest preference for the α1,6-fucosylated chain among α1,2-, α1,3-, α1,4-, and α1,6-fucosylated pyridylaminated (PA)-sugar chains. These results suggest that AOL is a novel probe for detecting core fucose in glycoproteins on the surface of animal cells. A comparison of the carbohydrate-binding specificity of AOL, AAL, and LCA by SPR showed that the irreversible binding of AOL to the α1,2-fucosylated PA-sugar chain (H antigen) relative to the α1,6-fucosylated chain was weaker than that of AAL, and that the interactions of AOL and AAL with α1,6-fucosylated glycopeptide (FGP), which is considered more similar to in vivo glycoproteins than PA-sugar chains, were similar to their interactions with the α1,6-fucosylated PA-sugar chain. Furthermore, positive staining of AOL, but not AAL, was completely abolished in the cultured embryo fibroblast (MEF) cells obtained from α1,6-fucosyltransferase (Fut8) knock-out mice, as assessed by cytological staining. Taken together, these results suggest that AOL is more suitable for detecting core fucose than AAL or LCA. Lectins are specific carbohydrate-binding or carbohydrate-cross-linking proteins. Many studies have isolated and investigated lectins from a wide range of species including plants, animals and microorganisms. The cell surfaces of organisms are covered with abundant and diverse carbohydrates. Because of structural diversity, the set of carbohydrates that is expressed on a cell surface has a role in various biological recognition phenomena, including cell-cell interactions, cell-substratum interactions, and metastasis of tumor cells, among others (1Drickamer K. Taylor M.E. Annu. Rev. Cell Biol. 1993; 9: 237-264Crossref PubMed Google Scholar). Therefore, some lectins have particular value as specific probes for investigating the distribution, structure and biological function of carbohydrate chains on the cell surface of animal, plant, and microorganism because of their specificity for defined carbohydrate structures (2Vijayan M. Chandra N. Curr. Opin. Struct. Biol. 1999; 9: 707-714Crossref PubMed Scopus (232) Google Scholar). α-l-Fucopyranosyl residues are widely distributed in cell-surface sugar chains and often play important roles in biological phenomena. These residues constitute a part of important antigens, such as the blood group antigen H (3Pereira M.E.A. Kabat E.A. Biochemistry. 1974; 13: 3184-3192Crossref PubMed Google Scholar) and stage-specific embryonic antigens (4Stelck S. Robitzki A. Willbold E. Layer P.G. Glycobiology. 1999; 9: 1171-1179Crossref PubMed Google Scholar). Increased levels of fucosyl residues and changes in fucosylation patterns, as a result of different expression levels of various fucosyltransferases, act as specific markers for developmental antigens, particularly in inflammatory processes and in various cancers (5Kolanus W. Bevilacqua M. Seed B. Science. 1990; 250: 1132-1135Crossref PubMed Scopus (887) Google Scholar, 6Noda K. Miyoshi E. Gu J. Gao C. Nakahara S Kitada T. Honke K. Suzuki K. Yoshihara H. Yoshikawa K. Kawano K. Tonetti M. Kasahara A. Hori M. Hayashi N. Taniguchi N. Cancer Res. 2003; 63: 6282-6289PubMed Google Scholar). Furthermore, the α1,6-fucosylated oligosaccharide content of both liver and serum glycoproteins is elevated during the development of malignant liver diseases because the activity of Fut8 is increased (7Hutchinson W.L. Du M.Q. Johnson P.J. Williams R. Hepatology. 1991; 13: 683-688Crossref PubMed Google Scholar). In particular, the sugar chains of α-fetoprotein in serum, a well established tumor marker that is produced by hepatocellular carcinomas, have an abundance of core fucose (8Ohno M. Nishikawa A. Kouketsu M. Taga H. Endo Y. Hada T. Higashino K. Taniguchi N. Int. J. Cancer. 1992; 51: 315-317Crossref PubMed Scopus (55) Google Scholar). To date, some lectins have been identified as fucose-specific including Lotus tetragonolobus (3Pereira M.E.A. Kabat E.A. Biochemistry. 1974; 13: 3184-3192Crossref PubMed Google Scholar) and Ulex europaeus (9Matsumoto I. Osawa T. Biochim. Biophys. Acta. 1969; 194: 180-189Crossref PubMed Google Scholar) lectins from plants, Anguilla lectin from eel (10Watkins W.M. Morgan W.T.J. Nature. 1952; 169: 825-826Crossref PubMed Scopus (114) Google Scholar), Aleuria aurantia lectin (AAL) 2The abbreviations used are: AAL, Aleuria aurantica lectin; LCA, Lens culinaris agglutinin; AOL, Aspergillus oryzae lectin; PA, pyridylaminated; SPR, surface plasmon resonance; FGP, α1,6-fucosylated glycopeptide; MEF, mouse embryo fibroblasts; FUT8, α1,6-fucosyltransferase; SGP, sialylglycopeptide; PBS, phosphate-buffered saline; RU, resonance units; GlcNAc, N-acetylglucosamine; MES, 4-morpholineethanesulfonic acid. from mushroom (11Kochibe N. Furukawa K. Biochemistry. 1980; 19: 2841-2846Crossref PubMed Google Scholar), Rhizopus stolonifer lectin from fungi (12Oda Y. Senaha T. Matsuno Y. Nakajima K. Naka R. Kinoshita M. Honda E. Furuta I. Kakehi K. J. Biol. Chem. 2003; 278: 32439-32447Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), and Ralstonia solanacearum lectin from bacteria (13Sudakevitz D. Imbertyu A. Gilboa-Garber N. J. Biochem. (Tokyo). 2002; 132: 353-358Crossref PubMed Google Scholar). Among these lectins, AAL and R. stolonifer lectin preferentially bind to α1,6-fucosylated oligosaccharides, whereas Ulex europaeus and Lotus tetragonolobus lectins prefer α1,2-linked fucose residues (14Stephan E.B. Juergen T. Young-Ok P. Franz-George H. Jacques B. Robert F. Glycoconj. J. 1996; 13: 585-590Crossref PubMed Scopus (63) Google Scholar). AAL is a commercially available lectin that is known for its high affinity for α1,6-fucosylated oligosaccharides (15Fukumori F. Takeuchi N. Hagiwara T. Ohbayashi H. Endo T. Kochibe N. Nagata Y. Kobata A. J. Biochem. 1990; 107: 190-196Crossref PubMed Google Scholar), and it is widely used to estimate the extent of α1,6-fucosylation (core fucosylation) on glycoproteins and to fractionate glycoproteins (16Yamashita K. Kochibe N. Ohkura T. Ueda I. Kobata A. J. Biol. Chem. 1985; 260: 4688-4693Abstract Full Text PDF PubMed Google Scholar). Another lectin that recognizes oligosaccharides containing core fucose would be a valuable tool in glycobiological research because only a few lectins have been identified as specific for core fucose, and AAL itself exhibits broad specificity for α1,2-, α1,3-, and α1,4-fucose-containing oligosaccharides (17Wimmerova M. Mitchell E. Sanchez J.F. Gautier C. Imberty A. J. Biol. Chem. 2003; 278: 27059-27067Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). We previously identified a novel lectin, AOL, in iron-deficient cultures of the filamentous fungus A. oryzae; this lectin turned out to be l-fucose-specific from a hemagglutination inhibition assay using several monosaccharides and the encoding gene, fleA, was found to share 26% homology with AAL in primary structure (18Ishida H. Moritani T. Hata Y. Kawato A. Suginami K. Abe Y. S. Biochem. 2002; PubMed Google Scholar). to AOL be a valuable tool in glycobiological studies and for we have the carbohydrate binding specificity of AOL by using SPR analysis, lectin affinity lectin analysis, and staining as with results that AOL to the specific probe for core fucose identified and LCA were from The PA-sugar chains were from The was from The sugar was pyridylaminated with and was to a to PA-sugar chain. of the oligosaccharides used are in and and α1,6-fucosyltransferase were from and of PA-sugar chains and in a of of oligosaccharides by AOL and AAL lectin affinity α1,6-fucosylated and PA-sugar chains were to an AOL or AAL with containing of were and PA-sugar chain was by and by of were to the and a of fucose in the was by a of fucose, a of fucose, and of α1,6-fucosylated PA-sugar of was from a obtained from as by A. M. M. Y. M. T. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). In the was in of and with for The was in a for and and the was to a to the was in of and with for with the the glycopeptide was in of and with α1,6-fucosyltransferase for The were with and by as previously K. Endo T. Gu J. I. Y. S. H. H. Miyoshi E. Honke K. Taniguchi N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The structure of the was by of was by the encoding gene, fleA, in the of A. oryzae as we previously (18Ishida H. Moritani T. Hata Y. Kawato A. Suginami K. Abe Y. S. Biochem. 2002; PubMed Google Scholar). The A. oryzae was used to A the under the of the was cultured in of with for the a was by with in containing The was and the was used for were with the of the was in The was the and to a of with AOL was with a in the The with activity were and the AOL was because of the SPR and the were obtained from The surface of a research was a of with of and for lectin, a of in was for and the were by the of for AOL, AAL, and LCA were to cells on the and were out on of which were AOL, AAL, and LCA, and the was the The was for the of were in containing a of The of PA-sugar chains and and in of The were for and a of the surface was with a of binding were and similar results were of by AOL or AAL AOL and AAL were in containing and to an to the The of was by the of in the affinity was The or was with containing α1,6-fucosylated and PA-sugar chains were to The was with of The was with a of fucose in and the was with a of fucose, a of fucose, and of of were To PA-sugar chains, a of was to using containing PA-sugar chain was by of AAL, and LCA were using a from The in was to a of lectin in phosphate-buffered with a for with the lectin was by and were as previously E. Y. Hayashi N. H. T. Taniguchi N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In of was to the were The were with serum in and for with of AOL lectins or of AAL in containing with the were with for and with was with with cell cultures were as previously S. Gu J. Miyoshi E. K. W. Y. M. M. M. N. S. Y. Y. Y. Y. J. Suzuki K. A. C. T. M. Honke K. Taniguchi N. PubMed Scopus Google Scholar). In cultures of from or Fut8 knock-out S. Gu J. Miyoshi E. K. W. Y. M. M. M. N. S. Y. Y. Y. Y. J. Suzuki K. A. C. T. M. Honke K. Taniguchi N. PubMed Scopus Google Scholar) were by of The cells were in in a with in The cell was an of on and in The cells were for and were with The cells were in containing for and in for the were by with of serum in for the was the were with of AOL or or AAL or in for of lectin was by an The cultured cells were with for and with were with an and of the function of AOL, its encoding was in A. in a of of The AOL was to such an extent that it in the of blood cells a of of lectin in as under The AOL a on we AOL 26% homology with AAL in primary structure (18Ishida H. Moritani T. Hata Y. Kawato A. Suginami K. Abe Y. S. Biochem. 2002; PubMed Google Scholar). in the of AOL on was a than that of LCA as to of and studies have the of AAL as of of (15Fukumori F. Takeuchi N. Hagiwara T. Ohbayashi H. Endo T. Kochibe N. Nagata Y. Kobata A. J. Biochem. 1990; 107: 190-196Crossref PubMed Google Scholar), and that of LCA as of of and of S. Osawa T. A. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). In SPR analysis, a cell with AOL in a in resonance to the of not which that AOL of In to the AOL and AAL were found to have the not These results suggest that AOL is similar to AAL not only in but in The of oligosaccharide binding has that AAL is specific for α1,6-fucosylated oligosaccharides (16Yamashita K. Kochibe N. Ohkura T. Ueda I. Kobata A. J. Biol. Chem. 1985; 260: 4688-4693Abstract Full Text PDF PubMed Google Scholar). To AOL is specific for α1,6-fucosylated oligosaccharides, we investigated the of PA-sugar chains with AOL to a The of PA-sugar chain that with AOL be from the in the of an of the which are to the of PA-sugar chain that with AOL AOL has the strongest preference for the α1,6-fucosylated PA-sugar chain among the α1,2-, α1,3-, α1,4-, and α1,6-fucosylated chains These results suggest AAL, AOL is specific for α1,6-fucosylated of PA-sugar chain that with AOL or The of PA-sugar chain that with AOL or AAL was from the in the of an of the and which are to binding in the the of and the of irreversible binding the of and the and been by a of the of AOL, AAL, and and LCA are widely used to estimate the extent of core fucosylation on glycoproteins and to fractionate glycoproteins. To the carbohydrate binding specificity of AOL with that of AAL and LCA, we investigated the of the PA-sugar chains with these lectins to the cells by using in AOL and AAL showed binding to the PA-sugar chains whereas LCA showed binding to not the of PA-sugar chain that with lectin both AOL and AAL similar for the α1,6-fucosylated PA-sugar chain. in the of the of the in the value the and the the that the binding to AAL in a in a of and interactions, the binding to AOL Furthermore, the value during the in the of and as and (H antigen) PA-sugar chains to AAL as to These results suggest that the irreversible binding of these PA-sugar chains is to AAL than to we the carbohydrate-binding specificity of AOL with that of AAL for different binding binding and irreversible as in and of for of AOL with of which is the more specific lectin for core we AOL or AAL showed binding to the PA-sugar chains relative to α1,6-fucosylated PA-sugar chains. the results of binding in AAL binding to fucosyl PA-sugar chains relative to the α1,6-fucosylated PA-sugar chain than the results of the irreversible binding in the of AAL binding to (H antigen) relative to PA-sugar chains is than that of Taken together, we that AOL is more specific for core fucosylation than is AAL in of irreversible of the of AOL for with of AAL and we investigated the interactions of lectins with PA-sugar chains to their for PA-sugar chains are oligosaccharides and not be of in vivo oligosaccharides to the of the of these sugar chains with to the interactions of the lectins with an oligosaccharide of in vivo we from as under has with α1,6-fucosyl residue to the core to as in The chain has residues and the an of of these were with not in the of the in the value the and the of the the that binding to AAL in a in a of and interactions, and the value during the that as to AAL as to These results were similar to the for binding to the PA-sugar chains in the irreversible binding the interactions of with AOL and AAL were weaker than of the PA-sugar chains, weaker binding result from a from than from the PA-sugar chains because both AOL and AAL have positive under the used for SPR and has more positive than the PA-sugar chains. In the PA-sugar chains, showed binding to LCA The of LCA and the PA-sugar chains results from of the with Taken together, these results suggest that AOL and AAL bind to in the as bind to the PA-sugar chains or not the oligosaccharides have of the of of by AOL with by the specificity of AOL for core fucose in we the of α1,6-fucosylated and PA-sugar chains using an with that using an The PA-sugar chains were to as under and chain was in the of the The PA-sugar chains that to the lectins were with fucose as a binding the was with fucose, both PA-sugar chains in more or the and not be by the lectins not Because AAL has range affinity for fucose (11Kochibe N. Furukawa K. Biochemistry. 1980; 19: 2841-2846Crossref PubMed Google Scholar), we the with a of fucose of which is the value for AAL binding of in α1,6-fucosylated PA-sugar chains to the AOL showed a in as with to the AAL In PA-sugar chains to the AOL than to the AAL a an of PA-sugar chains was obtained from these lectin Furthermore, from the AAL by fucose, more PA-sugar chain than from the AOL that AAL with part of the PA-sugar chain than Taken together, these results that AOL has specificity for core fucose than of by AOL and the that AOL be a valuable tool in glycobiological we out lectin of glycoproteins from the serum and of and Fut8 knock-out using AOL or in the of glycoproteins was similar the AOL and AAL with the that the of AAL binding was than that of AOL the serum and glycoproteins from Fut8 knock-out mice, the which are by an showed in the AAL as with the AOL The staining for and which in and embryonic cells, but not serum, from Fut8 knock-out to be because are by the in the of lectins not These results that serum and glycoproteins fucose residues than core fucose because the expression of is and that AAL lectin recognizes these residues as with of by AOL and in lectin showed that AOL is more specific for core fucosylation than AAL, in glycoproteins. To the specificity of AOL for core we the staining of cultured cells from and Fut8 knock-out using AOL or in positive staining for AOL and AAL was in cultured cells from AOL but not AAL was completely abolished in cultured cells from Fut8 knock-out the of AOL staining of the cells from was weaker than that of AAL, and we AOL staining with AAL staining with a of lectin, AOL staining of cultured cells from Fut8 knock-out to be weaker than AAL that AOL, as with AAL, binding to oligosaccharides relative to α1,6-fucosylated Taken together, these results that AOL is a more specific probe for core fucose than We have that AOL is specific for core fucose and that the binding of this lectin are more suitable for detecting core fucosylation than of AAL and LCA. in AOL and AAL share similar We that AOL, AAL, would specificity for core AAL has a structure and as from its structure by (17Wimmerova M. Mitchell E. Sanchez J.F. Gautier C. Imberty A. J. Biol. Chem. 2003; 278: 27059-27067Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) and M. N. Nagata Y. K. Biochemistry. 2003; PubMed Scopus Google Scholar). is a that is found in lectins of and it carbohydrate recognition that has been to be in the recognition of several including the R. solanacearum (17Wimmerova M. Mitchell E. Sanchez J.F. Gautier C. Imberty A. J. Biol. Chem. 2003; 278: 27059-27067Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, N. Mitchell E. H. S. M. Gilboa-Garber N. M. Imberty A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). AOL that are similar to of AAL, and it has been that AOL has a structure (17Wimmerova M. Mitchell E. Sanchez J.F. Gautier C. Imberty A. J. Biol. Chem. 2003; 278: 27059-27067Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The specificity of AOL for core fucosylation found in this would a in the structures of these of AOL and homology to of of of and binding of (18Ishida H. Moritani T. Hata Y. Kawato A. Suginami K. Abe Y. S. Biochem. 2002; PubMed Google (15Fukumori F. Takeuchi N. Hagiwara T. Ohbayashi H. Endo T. Kochibe N. Nagata Y. Kobata A. J. Biochem. 1990; 107: 190-196Crossref PubMed Google (16Yamashita K. Kochibe N. Ohkura T. Ueda I. Kobata A. J. Biol. Chem. 1985; 260: 4688-4693Abstract Full Text PDF PubMed Google (17Wimmerova M. Mitchell E. Sanchez J.F. Gautier C. Imberty A. J. Biol. Chem. 2003; 278: 27059-27067Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) in a In to their similar specificity for core fucose in SPR analysis, the of results that AOL and AAL bind to oligosaccharides in a different from the of and binding of the PA-sugar chain and to AAL in a their binding to AOL and We that these interactions of AAL the different affinity of the the structure of AAL has been by (17Wimmerova M. Mitchell E. Sanchez J.F. Gautier C. Imberty A. J. Biol. Chem. 2003; 278: 27059-27067Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) and M. N. Nagata Y. K. Biochemistry. 2003; PubMed Scopus Google Scholar). In the structures by these the structures to the fucose are found in of the in a by but in only by to fucose residues are in the binding in the structure of and are the binding which are from in the of the to the binding of AAL have different for and are binding be in and are and is not a the of AAL have the interactions not be in a suggest that the binding of AAL have different for AOL showed weaker irreversible to and (H antigen) PA-sugar chains and than AAL as in We that the weaker irreversible binding of AOL with AAL was from the of available on AOL, as well as binding affinity for The of AOL and AAL that the of the and the of the that are to fucose in AAL are in and in AOL, whereas and are in the and in AOL (17Wimmerova M. Mitchell E. Sanchez J.F. Gautier C. Imberty A. J. Biol. Chem. 2003; 278: 27059-27067Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). it is not the binding of this lectin such as carbohydrate are in the fucose binding In this we which lectin is more specific for core fucosylation by which lectins binding to PA-sugar chains relative to the α1,6-fucosylated PA-sugar chain. We that a comparison of the of AOL and AAL is for a of such such as the and be from the which was to a affinity by using In that the PA-sugar chain was various a cell containing lectin not The and were to be and for AOL, and and for These results suggest that the and of AOL are more than of The of the PA-sugar chain to AOL and AAL were as and the for AAL by SPR was not in with the value obtained by because a of PA-sugar chain of the value is to Therefore, we not the of the α1,6-fucosylated PA-sugar which showed the strongest binding to lectin of Furthermore, AAL irreversible binding to PA-sugar chains than we the carbohydrate-binding specificity of AOL with that of AAL different binding binding and irreversible the irreversible binding of AOL to core fucose is weaker than that of AAL, AOL is more specific for core fucosylation than AAL in of irreversible and staining of showed that AOL is more for detecting core fucosylation than In this the of lectin or staining to the of lectin that during SPR analysis, because are used in the or staining similar to the of irreversible binding in SPR of results the the affinity of AOL for fucose is weaker than that of SPR showed that the irreversible binding of AOL to oligosaccharides was weaker than that of AAL In as with AAL, AOL the showed weaker staining in cells from as with AAL, AOL showed binding to oligosaccharides relative to α1,6-fucosylated PA-sugar chains. SPR showed that irreversible binding of AOL to the α1,2-fucosylated PA-sugar chain (H antigen) relative to the α1,6-fucosylated chain was weaker than that of AAL as with AAL, AOL staining of cultured cells from Fut8 knock-out to be of AOL were used The staining of and from Fut8 knock-out by AAL lectin, was in the of of not The in that AAL has to fucose residues than core fucose in of irreversible Furthermore, in the of PA-sugar chains by lectin AAL showed with part of the PA-sugar chain than AOL These results suggest that some of AAL have affinity for oligosaccharides not α1,6-fucosylated as with Taken together, staining with AAL fucose residues than core fucose, whereas staining with AOL the extent of core fucosylation on glycoproteins more The filamentous fungus A. oryzae is a used to in the of because it has high levels of The has been in the of A. oryzae (18Ishida H. Moritani T. Hata Y. Kawato A. Suginami K. Abe Y. S. Biochem. 2002; PubMed Google Scholar), of and In to its binding this of AOL it to be used widely as a novel probe for the of core fucose, as well as the of its biological We and Ueda of of for their and and of of for for SPR with