Glycan Markers of Human Stem Cells Assigned with Beam Search Arrays*[S]
2019; Elsevier BV; Volume: 18; Issue: 10 Linguagem: Inglês
10.1074/mcp.ra119.001309
ISSN1535-9484
AutoresNian Wu, Lisete M. Silva, Yan Liu, Yi-Bing Zhang, Chao Gao, Fuming Zhang, Li Fu, Yanfei Peng, Robert J. Linhardt, Toshisuke Kawasaki, Barbara Mulloy, Wengang Chai, Ten Feizi,
Tópico(s)Viral Infectious Diseases and Gene Expression in Insects
ResumoGlycan antigens recognized by monoclonal antibodies have served as stem cell markers. To understand regulation of their biosynthesis and their roles in stem cell behavior precise assignments are required. We have applied state-of-the-art glycan array technologies to compare the glycans bound by five antibodies that recognize carbohydrates on human stem cells. These are: FC10.2, TRA-1–60, TRA-1–81, anti-i and R-10G. Microarray analyses with a panel of sequence-defined glycans corroborate that FC10.2, TRA-1–60, TRA-1–81 recognize the type 1-(Galβ-3GlcNAc)-terminating backbone sequence, Galβ-3GlcNAcβ-3Galβ-4GlcNAcβ-3Galβ-4GlcNAc, and anti-i, the type 2-(Galβ-4GlcNAc) analog, Galβ-4GlcNAcβ-3Galβ-4GlcNAcβ-3Galβ-4GlcNAc, and we determine substituents they can accommodate. They differ from R-10G, which requires sulfate. By Beam Search approach, starting with an antigen-positive keratan sulfate polysaccharide, followed by targeted iterative microarray analyses of glycan populations released with keratanases and mass spectrometric monitoring, R-10G is assigned as a mono-sulfated type 2 chain with 6-sulfation at the penultimate N-acetylglucosamine, Galβ-4GlcNAc(6S)β-3Galβ-4GlcNAcβ-3Galβ-4GlcNAc. Microarray analyses using newly synthesized glycans corroborate the assignment of this unique determinant raising questions regarding involvement as a ligand in the stem cell niche. Glycan antigens recognized by monoclonal antibodies have served as stem cell markers. To understand regulation of their biosynthesis and their roles in stem cell behavior precise assignments are required. We have applied state-of-the-art glycan array technologies to compare the glycans bound by five antibodies that recognize carbohydrates on human stem cells. These are: FC10.2, TRA-1–60, TRA-1–81, anti-i and R-10G. Microarray analyses with a panel of sequence-defined glycans corroborate that FC10.2, TRA-1–60, TRA-1–81 recognize the type 1-(Galβ-3GlcNAc)-terminating backbone sequence, Galβ-3GlcNAcβ-3Galβ-4GlcNAcβ-3Galβ-4GlcNAc, and anti-i, the type 2-(Galβ-4GlcNAc) analog, Galβ-4GlcNAcβ-3Galβ-4GlcNAcβ-3Galβ-4GlcNAc, and we determine substituents they can accommodate. They differ from R-10G, which requires sulfate. By Beam Search approach, starting with an antigen-positive keratan sulfate polysaccharide, followed by targeted iterative microarray analyses of glycan populations released with keratanases and mass spectrometric monitoring, R-10G is assigned as a mono-sulfated type 2 chain with 6-sulfation at the penultimate N-acetylglucosamine, Galβ-4GlcNAc(6S)β-3Galβ-4GlcNAcβ-3Galβ-4GlcNAc. Microarray analyses using newly synthesized glycans corroborate the assignment of this unique determinant raising questions regarding involvement as a ligand in the stem cell niche. Cellular glycans were historically among the earliest biochemical and immunochemical markers in embryonic developments (1Wright A. Andrews P. Surface marker antigens in the characterization of human embryonic stem cells.Stem Cell Res. 2009; 3: 3-11Crossref PubMed Scopus (74) Google Scholar, 2Muramatsu T. Embryoglycan: a highly branched poly-N-acetyllactosamine in pluripotent stem cells and early embryonic cells.Glycoconjugate J. 2016; : 1-12PubMed Google Scholar, 3Feizi T. Demonstration by monoclonal antibodies that carbohydrate structures of glycoproteins and glycolipids are onco-developmental antigens.Nature. 1985; 314: 53-57Crossref PubMed Scopus (1022) Google Scholar). Using as reagents glycan sequence-specific monoclonal antibodies (initially, natural human antibodies followed by murine hybridoma-derived) remarkable insights were gained into the expression and polarization of glycans in post-implantation embryos and teratocarcinomas of mouse associated with cell differentiation (4Kapadia A. Feizi T. Evans M. Changes in the expression and polarization of blood group I and i antigens in post-implantation embryos and teratocarcinomas of mouse associated with cell differentiation.Exp. Cell Res. 1981; 131: 185-195Crossref PubMed Scopus (132) Google Scholar, 5Gooi H. Feizi T. Kapadia A. Knowles B. Solter D. Evans M. Stage-specific embryonic antigen involves α1–3 fucosylated type 2 blood group chains.Nature. 1981; 292: 156-158Crossref PubMed Scopus (494) Google Scholar). We overview here below the glycan antigens on murine and human stem cells that have been reported over the years. These antigens have been used as markers of stem cells (1Wright A. Andrews P. Surface marker antigens in the characterization of human embryonic stem cells.Stem Cell Res. 2009; 3: 3-11Crossref PubMed Scopus (74) Google Scholar) although little is known about their roles in stem cell behavior. Undifferentiated murine embryonal carcinoma cells were observed to be rich in surface-associated and cytoplasmic I antigen (4Kapadia A. Feizi T. Evans M. Changes in the expression and polarization of blood group I and i antigens in post-implantation embryos and teratocarcinomas of mouse associated with cell differentiation.Exp. Cell Res. 1981; 131: 185-195Crossref PubMed Scopus (132) Google Scholar). This antigen is recognized by human monoclonal anti-I Ma and anti-I Step; these being designations of monoclonal antibodies in sera of cases of cold agglutinin disease (6Feizi T. The blood group Ii system: a carbohydrate antigen system defined by naturally monoclonal or oligoclonal autoantibodies of man.Immunol. Commun. 1981; 10: 127-156Crossref PubMed Scopus (97) Google Scholar). These antibodies are directed at different domains on the branched structures of the poly-N-acetyllactosamine series as indicated in structures 1 and 2 (Table I). These are glycan sequences consisting of repeated type 2 (T2, Galβ-4GlcNAc) on glycoproteins and glycolipids, where Gal is galactose and GlcNAc is N-acetylglucosamine. Differentiation into primary endoderm was observed to be associated with the appearance of i antigen (4Kapadia A. Feizi T. Evans M. Changes in the expression and polarization of blood group I and i antigens in post-implantation embryos and teratocarcinomas of mouse associated with cell differentiation.Exp. Cell Res. 1981; 131: 185-195Crossref PubMed Scopus (132) Google Scholar) recognized by the human anti-i Den, which is directed at the unbranched repeated T2 sequence as in structure 3 (Table I). With the advent of hybridoma technology, there followed the demonstration that the 8-cell stage-specific embryonic antigen of mouse recognized by mAb anti-SSEA-1 is expressed on the α-3 fucosylated forms of the I and i antigens (termed LewisX), structures 4 and 5, respectively (5Gooi H. Feizi T. Kapadia A. Knowles B. Solter D. Evans M. Stage-specific embryonic antigen involves α1–3 fucosylated type 2 blood group chains.Nature. 1981; 292: 156-158Crossref PubMed Scopus (494) Google Scholar) (Table I). These antigens were detected predominantly on high molecular weight glycoproteins (7Childs R. Pennington J. Uemura K. Scudder P. Goodfellow P. Evans M. Feizi T. High-molecular-weight glycoproteins are the major carriers of the carbohydrate differentiation antigens I, i and SSEA-1 of mouse teratocarcinoma cells.Biochem. J. 1983; 215: 491-503Crossref PubMed Scopus (82) Google Scholar, 8Ozawa M. Muramatsu T. Solter D. SSEA-1, a stage-specific embryonic antigen of the mouse, is carried by the glycoprotein-bound large carbohydrate in embryonal carcinoma cells.Cell Differentiation. 1985; 16: 169-173Crossref PubMed Scopus (60) Google Scholar) the common feature being the abundance of repeated T2 backbone sequences.Table IMurine and human stem cell-associated carbohydrate markers and carrier moleculesNumbers in columns 4 are designations of the sequences cited in the text. Glycan sequences recognized by anti-I Ma and Step, anti-i Den, mAbs FC10.2, TRA-1–60 and TRA-1–81 were originally determined using glycolipids or glycans with Glc at the reducing ends. On stem cells they occur predominantly on glycoproteins. An unnamed human serum. Numbers in columns 4 are designations of the sequences cited in the text. Glycan sequences recognized by anti-I Ma and Step, anti-i Den, mAbs FC10.2, TRA-1–60 and TRA-1–81 were originally determined using glycolipids or glycans with Glc at the reducing ends. On stem cells they occur predominantly on glycoproteins. An unnamed human serum. In contrast with the murine embryonic endoderm and embryonal carcinoma-associated antigens, the human embryonic endoderm- and embryonal carcinoma-associated antigen recognized by the monoclonal mAb FC10.2 was assigned as the lacto-N-tetraose sequence, with T1 (Galβ-3GlcNAc) -T2 backbone sequence, structure 6 (Table I); this was detected on a glycoprotein with an apparent molecular weight, ∼200 kDa, (9Williams L. Sullivan A. McIlhinney R. Neville A. A monoclonal antibody marker of human primitive endoderm.Int. J. Cancer. 1982; 30: 731-738Crossref PubMed Scopus (13) Google Scholar, 10Gooi H. Williams L. Uemura K. Hounsell E. McIlhinney R. Feizi T. A marker of human foetal endoderm defined by a monoclonal antibody involves type 1 blood group chains.Mol. Immunol. 1983; 20: 607-613Crossref PubMed Scopus (32) Google Scholar) similar to that of podocalyxin (11Schopperle W. DeWolf W. The TRA-1–60 and TRA-1–81 Human pluripotent stem cell markers are expressed on podocalyxin in embryonal carcinoma.Stem Cells. 2007; 25: 723-730Crossref PubMed Scopus (110) Google Scholar). This glycoprotein is the carrier of the antigen(s) recognized by more recently generated mAbs, TRA-1–60 and TRA-1–81, which recognize human stem cells (12Andrews P. Banting G. Damjanov I. Arnaud D. Avner P. Three monoclonal antibodies defining distinct differentiation antigens associated with different high molecular weight polypeptides on the surface of human embryonal carcinoma cells.Hybridoma. 1984; 3: 347-361Crossref PubMed Scopus (197) Google Scholar), and, like FC10.2, bind to the T1-T2 sequence, structures 6 and 7 (Table I). The expression of this antigenic activity is reported to be common to various types of human stem cells: embryonic stem cell (ES) 1The abbreviations used are:ESembryonic stemAOPEN-aminooxyacetyl-1,2-dihexadecyl-sn-glycero-3-phosphoethanolamineBMPbone morphogenetic proteinsCSchondroitin sulfatesECembryonal carcinomaFGFfibroblast growth factorGAGglycosaminoglycanGalgalactoseGalNAcN-acetylgalactosmineGlcglucoseGlcNAcN-acetylglucosamineiPSinduced pluripotentKSkeratan sulfateMIRAGEminimum information required for a glycomics experimentNGLneoglycolipidRIrefractive indexSAXstrong anion-exchangeShhsonic hedgehogsulfate(6S)sulfate at position 6 of GlcNAcWntint/wingless.1The abbreviations used are:ESembryonic stemAOPEN-aminooxyacetyl-1,2-dihexadecyl-sn-glycero-3-phosphoethanolamineBMPbone morphogenetic proteinsCSchondroitin sulfatesECembryonal carcinomaFGFfibroblast growth factorGAGglycosaminoglycanGalgalactoseGalNAcN-acetylgalactosmineGlcglucoseGlcNAcN-acetylglucosamineiPSinduced pluripotentKSkeratan sulfateMIRAGEminimum information required for a glycomics experimentNGLneoglycolipidRIrefractive indexSAXstrong anion-exchangeShhsonic hedgehogsulfate(6S)sulfate at position 6 of GlcNAcWntint/wingless., induced pluripotent stem cell (iPS), and embryonal carcinoma-associated stem cell (EC) (12Andrews P. Banting G. Damjanov I. Arnaud D. Avner P. Three monoclonal antibodies defining distinct differentiation antigens associated with different high molecular weight polypeptides on the surface of human embryonal carcinoma cells.Hybridoma. 1984; 3: 347-361Crossref PubMed Scopus (197) Google Scholar, 13Qiu D. Xiang J. Li Z. Krishnamoorthy A. Chen L. Wang R. Profiling TRA-1–81 antigen distribution on a human embryonic stem cell.Biochem. Biophys. Res. Commun. 2008; 369: 735-740Crossref PubMed Scopus (13) Google Scholar). embryonic stem N-aminooxyacetyl-1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine bone morphogenetic proteins chondroitin sulfates embryonal carcinoma fibroblast growth factor glycosaminoglycan galactose N-acetylgalactosmine glucose N-acetylglucosamine induced pluripotent keratan sulfate minimum information required for a glycomics experiment neoglycolipid refractive index strong anion-exchange sonic hedgehog sulfate at position 6 of GlcNAc int/wingless. embryonic stem N-aminooxyacetyl-1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine bone morphogenetic proteins chondroitin sulfates embryonal carcinoma fibroblast growth factor glycosaminoglycan galactose N-acetylgalactosmine glucose N-acetylglucosamine induced pluripotent keratan sulfate minimum information required for a glycomics experiment neoglycolipid refractive index strong anion-exchange sonic hedgehog sulfate at position 6 of GlcNAc int/wingless. Three other T1-related antigens common to human ES and iPS have been identified: two are T1-based blood group H recognized by mAbs anti-SSEA-5 and R-17F, structures 8 and 9 (Table I). The third is branched sequence structure 10 (Table I), containing blood group H based on the T3 (Galβ-3GalNAc) backbone (where GalNAc is N-acetylgalactosamine). This antigen is bound by the lectin rBC2LCN, and reported to be cross-reactive with T1-based blood group H, and expressed on podocalyxin. The R-17F antigen is additionally borne on a glycolipid. Three other glycolipid-borne antigens on human ES and EC recognized by mAbs anti-SSEA-3, anti-SSEA-4 and anti-Sialyl lactotetra antibodies are structures 11, 12 and 13 respectively (Table I), of which the first two are of the globo series based on Galβ-3GalNAcβ-3Galα-4Galβ-4Glc (14Kannagi R. Cochran N. Ishigami F. Hakomori S. Andrews P. Knowles B. Solter D. Stage-specific embryonic antigens (SSEA-3 and-4) are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma cells.EMBO J. 1983; 2: 2355Crossref PubMed Scopus (439) Google Scholar) and the third is based on the type 1 lacto series, Galβ-3GlcNAcβ-3Galβ-4Glc (15Barone A. Saljo K. Benktander J. Blomqvist M. Mansson J. Johansson B. Molne J. Aspegren A. Bjorquist P. Breimer M. Teneberg S. Sialyl-lactotetra, a novel cell surface marker of undifferentiated human pluripotent stem cells.J. Biol. Chem. 2014; 289: 18846-18859Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Other studies have shown that not only the terminal epitopes but also the core structures are differentially expressed in stem versus differentiated cells (16Liang Y. Kuo H. Lin C. Chen Y. Yang B. Cheng Y. Yu A. Khoo K. Yu J. Switching of the core structures of glycosphingolipids from globo- and lacto- to ganglio-series upon human embryonic stem cell differentiation.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 22564-22569Crossref PubMed Scopus (89) Google Scholar, 17Säljö K. Barone A. Vizlinhodzic D. Johansson B. Breimer M. Funa K. Teneberg S. Comparison of the glycosphingolipids of human-induced pluripotent stem cells and human embryonic stem cells.Glycobiology. 2017; 27: 291-305PubMed Google Scholar). T2-based human stem cell antigens have been two in number. The first is a human mesenchymal stem cell-associated antigen expressed on the repeated T2-T2 based backbone, namely, i antigen (18Hirvonen T. Suila H. Kotovuori A. Ritamo I. Heiskanen A. Sistonen P. Anderson H. Satomaa T. Saarinen J. Tiitinen S. The i blood group antigen as a marker for umbilical cord blood-derived mesenchymal stem cells.Stem Cells Dev. 2011; 21: 455-464Crossref PubMed Scopus (10) Google Scholar). The second is recognized by mAb R-10G, which distinguishes human ES and iPS from human EC cells (19Kawabe K. Tateyama D. Toyoda H. Kawasaki N. Hashii N. Nakao H. Matsumoto S. Nonaka M. Matsumura H. Hirose Y. A novel antibody for human induced pluripotent stem cells and embryonic stem cells recognizes a type of keratan sulfate lacking oversulfated structures.Glycobiology. 2013; 23: 322-336Crossref PubMed Scopus (48) Google Scholar). It was shown that binding of mAb R-10G to an antigen-positive glycoprotein was unaffected by its treatment with neuraminidases and fucosidases. The antibody was shown to bind to the polysaccharide keratan sulfate (KS), which is based on long repeated T2-T2 sequences (19Kawabe K. Tateyama D. Toyoda H. Kawasaki N. Hashii N. Nakao H. Matsumoto S. Nonaka M. Matsumura H. Hirose Y. A novel antibody for human induced pluripotent stem cells and embryonic stem cells recognizes a type of keratan sulfate lacking oversulfated structures.Glycobiology. 2013; 23: 322-336Crossref PubMed Scopus (48) Google Scholar). A highly sulfated KS from bovine articular cartilage (with sulfation on many of the GlcNAc and Gal residues) was lacking in R-10G antigen activity, whereas bovine corneal KS, which contains some regions with sulfated GlcNAc and nonsulfated Gal residues (20Funderburgh J. MINI REVIEW Keratan sulfate: structure, biosynthesis, and function.Glycobiology. 2000; 10: 951-958Crossref PubMed Scopus (337) Google Scholar), was antigen-positive (19Kawabe K. Tateyama D. Toyoda H. Kawasaki N. Hashii N. Nakao H. Matsumoto S. Nonaka M. Matsumura H. Hirose Y. A novel antibody for human induced pluripotent stem cells and embryonic stem cells recognizes a type of keratan sulfate lacking oversulfated structures.Glycobiology. 2013; 23: 322-336Crossref PubMed Scopus (48) Google Scholar). For these reasons, the R-10G antigen was deduced to be expressed on an under-sulfated form of KS. While the present study was under way a KS-related tetrasaccharide, structure 14, which has sulfate at position 6 of each of two GlcNAc residues (Table I) was chemically synthesized and when biotinylated was bound by mAb R-10G (21Nakao H. Nagai Y. Kojima A. Toyoda H. Kawasaki N. Kawasaki T. Binding specificity of R-10G and TRA-1–60/81, and substrate specificity of keratanase II studied with chemically synthesized oligosaccharides.Glycoconjugate J. 2017; 34: 789-795Crossref PubMed Scopus (14) Google Scholar). Knowledge of the glycan markers has not yet been matched by knowledge of the regulation of their biosynthesis and their functions in the stem cell niche. There also remain some ambiguities in reports of the binding specificities, for example those of the TRA-1–60 and -81 toward KS-like saccharides (19Kawabe K. Tateyama D. Toyoda H. Kawasaki N. Hashii N. Nakao H. Matsumoto S. Nonaka M. Matsumura H. Hirose Y. A novel antibody for human induced pluripotent stem cells and embryonic stem cells recognizes a type of keratan sulfate lacking oversulfated structures.Glycobiology. 2013; 23: 322-336Crossref PubMed Scopus (48) Google Scholar, 22Badcock G. Pigott C. Goepel J. Andrews P. The human embryonal carcinoma marker antigen TRA-1–60 is a sialylated keratan sulfate proteoglycan.Cancer Res. 1999; 59: 4715-4719PubMed Google Scholar, 23Natunen S. Satomaa T. Pitkänen V. Salo H. Mikkola M. Natunen J. Otonkoski T. Valmu L. The binding specificity of the marker antibodies Tra-1–60 and Tra-1–81 reveals a novel pluripotency-associated type 1 lactosamine epitope.Glycobiology. 2011; 21: 1125-1130Crossref PubMed Scopus (61) Google Scholar). Glycan arrays, since their inception (24Fukui S. Feizi T. Galustian C. Lawson A. Chai W. Oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions.Nat. Biotechnol. 2002; 20: 1011-1017Crossref PubMed Scopus (527) Google Scholar) have been a powerful means of analyzing glycan-binding specificities of diverse carbohydrate-recognition systems (25Palma A. Feizi T. Childs R. Chai W. Liu Y. The neoglycolipid (NGL)-based oligosaccharide microarray system poised to decipher the meta-glycome.Current Opinion Chem. Biol. 2014; 18: 87-94Crossref PubMed Scopus (71) Google Scholar, 26Rillahan C.D. Paulson J.C. Glycan microarrays for decoding the glycome.Annu. Rev. Biochem. 2011; 80: 797-823Crossref PubMed Scopus (348) Google Scholar). The continued expansion of libraries of sequence-defined glycan probes offers increasing opportunities to keep under review the repertoires of glycans recognized by the anti-stem cell antibodies. There are moreover technological advances that enable the generation of "bespoke" glycome probes, which we have termed Designer arrays (27Gao C. Liu Y. Zhang H. Zhang Y. Fukuda M. Palma A. Kozak R. Childs R. Nonaka M. Li Z. Carbohydrate sequence of the prostate cancer-associated antigen F77 assigned by a mucin O-glycome designer array.J. Biol. Chem. 2014; 289: 16462-16477Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) and Beam Search arrays (28Li Z. Gao C. Zhang Y. Palma A. Childs R. Silva L. Liu Y. Jiang X. Chai W. Feizi T. O-glycome beam search arrays for carbohydrate ligand discovery.Mol. Cell. Proteomics. 2018; 17: 121-133Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) from antigen positive macromolecules to detect, isolate and characterize natural determinants. Here we use an array of sequence-defined glycans to make for the first time a close comparison of the glycan-binding specificities of the five antibodies that recognize human stem cells: mAbs TRA-1–60, TRA-1–81 and FC10.2 that broadly recognize ES, iPS and EC cells, an anti-i P1A ELL directed at linear poly-N-acetyllactosamine sequences (29Gao C. Zhang Y. Liu Y. Feizi T. Chai W. Negative-ion electrospray tandem mass spectrometry and microarray analyses of developmentally regulated antigens based on type 1 and type 2 backbone sequences.Anal. Chem. 2015; 87: 11871-11878Crossref PubMed Scopus (12) Google Scholar), which according to ref (30Hirvonen T. Suila H. Tiitinen S. Natunen S. Laukkanen M. Kotovuori A. Reinman M. Satomaa T. Alfthan K. Laitinen S. Production of a recombinant antibody specific for i blood group antigen, a mesenchymal stem cell marker.Bioresearch Open Access. 2013; 2: 336-345Crossref PubMed Google Scholar) is predicted to recognize mesenchymal stem cells, and R-10G that recognizes ES and iPS cells. To assign the determinant of mAb R-10G, which is clearly different from those of the four other antibodies, we use the Beam Search microarray approach (28Li Z. Gao C. Zhang Y. Palma A. Childs R. Silva L. Liu Y. Jiang X. Chai W. Feizi T. O-glycome beam search arrays for carbohydrate ligand discovery.Mol. Cell. Proteomics. 2018; 17: 121-133Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) using an antigen-positive KS polysaccharide as the macromolecular start-point. This is followed by a targeted, iterative microarray analyses and concomitant mass spectrometry of glycan populations derived from the polysaccharide after partial depolymerization by keratanase I or keratanase II or acid hydrolysis. Microarray analyses with newly synthesized glycans corroborate the assignment we have made for the unique sequence of R-10G antigen associated with ES and iPS cells. The glycan array-based data are reported in accordance with MIRAGE (Minimum Information Required for A Glycomics Experiment) Glycan Microarray Guidelines (31Liu Y. McBride R. Stoll M. Palma A. Silva L. Agravat S. Aoki-Kinoshita K. Campbell M. Costello C. Dell A. Haslam S. Karlsson N. Khoo K. Kolarich D. Novotny M. Packer N. Ranzinger R. Rapp E. Rudd P. Struwe W. The minimum information required for a glycomics experiment (MIRAGE) project: improving the standards for reporting glycan microarray-based data.Glycobiology. 2017; 27: 280-284PubMed Google Scholar). These include details of the glycan binding samples investigated (antibodies, growth factors and morphogens) and their detection systems, the saccharides and the neoglycolipids (NGLs) in the glycan libraries for different array sets, the conditions used for array construction, as well as information on image and data analyses: these are summarized in the Supplemental Glycan Microarray document based on MIRAGE Guidelines (supplemental Table S2). The experimental data not covered are given below. Keratanase I from Pseudomonas sp. (EC 3.2.1.103) was from Seikagaku (Joetsu, Japan). Recombinant Keratanase II from Bacillus circulans. (EC 3.2.1) was as prepared according to (32Wang H. He W. Jiang P. Yu Y. Lin L. Sun X. Koffas M. Zhang F. Linhardt R. Construction and functional characterization of truncated versions of recombinant keratanase II from Bacillus circulans.Glycoconjugate J. 2017; 35: 643-649Crossref Scopus (9) Google Scholar). Please note that to avoid confusion in the names of the two KS specific enzymes, the term "keratanase I" was used throughout the paper instead of the commonly used "keratanase." The conditions for partial digestion of KS with keratanase I were adapted from those described by Nakazawa and Suzuki (33Nakazawa K. Suzuki S. Purification of Keratan Sulfate-endogalactosidase and its action on keratan sulfates of different origin.J. Biol. Chem. 1975; 250: 912-917Abstract Full Text PDF PubMed Google Scholar). In brief, KS (10 mg) was incubated with 0.5 unit of keratanase I in 0.5 ml Tris-HCl buffer (20 mm, pH 7.4) at 37 °C. Partial digestion of KS with keratanase II was performed as described by Brown et al. (34Brown G. Huckerby T. Morris H. Abram B. Nieduszynski I. Oligosaccharides derived from bovine articular cartilage keratan sulfates after keratanase II digestion: implications for keratan sulfate structural fingerprinting.Biochemistry. 1994; 33: 4836-4846Crossref PubMed Scopus (54) Google Scholar). In brief, KS (20 mg) was incubated with 0.96 unit of keratanase II in 2 ml NH4OAc buffer (50 mm, pH 7.4) at 37 °C. For partial acid hydrolysis, KS (1.4 mg) was incubated with 0.1 m HCl at 80 °C. The partial depolymerization of bovine corneal KS using keratanase I, keratanase II and acid hydrolysis was monitored by Superdex Peptide column (1 × 30 cm, GE Healthcare, Fairfield, Connecticut) and ESI-MS, and stopped at 23 h, 7.5 h and 4 h, respectively, at the disappearance of the peak at void volume (supplemental Fig. S1). The keratanase digestion reactions were stopped by heating the solutions in a water bath at 100 °C for 1 min, whereas the acid hydrolysis was stopped at the indicated times by neutralizing with 0.1 m NaOH. The reaction mixtures were lyophilized. Bio-Gel P-6 (1.6 × 90 cm, Bio-Rad Laboratories, Hercules, California) was used for size fractionation of glycans after keratanase treatment of KS, and elution was with 200 mm NH4Cl at a flow rate of 15 ml/h. Eluates were monitored by a refractive index (RI) detector, fractions were pooled as indicated (supplemental Fig. S2A and S2B) and lyophilized. Sephadex G10 column (1.6 × 35 cm, GE Healthcare) was used for desalting of fractions collected from Bio-Gel P-6 chromatography. Deionized water was used for elution at a flow rate of 20 ml/h and eluates were monitored with a RI detector. Superdex Peptide column (GE Healthcare, 1 × 30 cm) was used to fractionate the products obtained after acid hydrolysis of KS. Elution was with 50 mm NH4OAc at a flow rate of 0.3 ml/min and monitored by RI. Fractions were pooled as indicated and lyophilized and were coevaporated repeatedly with Deionized water by freeze-drying to remove the volatile buffer salt NH4OAc (supplemental Fig. S2C). Selected fractions were analyzed by ESI-MS to determine chain lengths and sulfate contents (supplemental Table S6). The molar quantities of the glycans in the KS fractions were estimated based on the galactose content and molecular mass determined by MS analysis. The galactose contents were determined by established dot orcinol-sulfuric acid method on TLC plates, using galactose as standard (35Chai W. Stoll M. Galustian C. Lawson A. Feizi T. Neoglycolipid technology: deciphering information content of glycome.Methods Enzymol. 2003; 362: 160-195Crossref PubMed Scopus (53) Google Scholar). Strong anion-exchange (SAX) column (4.6 × 250 mm, Waters, Milford, Massachusetts) or HiTrap ion exchange cartridge (7 × 25 mm, GE Healthcare) or hypercarb porous graphitized carbon (PGC) column (30 × 4.6 mm, Thermo Scientific, Waltham, Massachusetts) were used for fractionation of glycans by HPLC (Gilson, Madison, Wisconsin). Elution was carried out by a gradient of LiClO4 for both SAX-HPLC and HiTrap cartridge (Solvent 1: 10 mm LiClO4 and Solvent 2: 500 mm LiClO4). Chromatographies by SAX and HiTrap were performed in 40 min at a flow rate of 1 ml/min with detection by UV at 206 nm. For PGC-HPLC the elution was by a gradient of H2O/ACN (Solvent 1: H2O; Solvent 2: ACN/H2O: 80:20; both containing 0.05% trifluoroacetic acid) in 40 min at a flow rate of 0.5 ml/min with detection by UV at 206 nm. The gradients used are given in the figures. Fractions were pooled as indicated in the respective figures. The fractions obtained from SAX-HPLC and HiTrap were lyophilized and desalted with a Superdex Peptide column and lyophilized as described above. To fractions obtained from PGC-HPLC were added 50 mm NH4HCO3 before removal of the ACN under N2 stream and the TFA by repeated lyophilization. Desulfation of KS glycans was performed as described (36Tang P. Scudder P. Mehmet H. Hounsell E. Feizi T. Sulphate groups are involved in the antigenicity of keratan sulphate and mask i antigen expression on their poly-N-acetyllactosamine backbones. An immunochemical and chromatographic study of keratan sulphate oligosaccharides after desulphation or nitrosation.Eur. J. Biochem. 1986; 160: 9PubMed Google Scholar). After several preliminary experiments, the conditions selected were as follows: freeze-dried hexasaccharides substituted with 4 to 6 sulfates (∼100 nmol) were added to 150 μl of 20 mm methanolic HCl for incubation at room temperature for 17 h. The reactions were stopped by immediate removal of the methanolic HCl reagent after addition of 50 μl of 50 mm NH4HCO3. The reaction product was dried by lyophilization. NGLs were prepared from reducing KS glycans by oxime-ligation using the lipid reagent N-aminooxyacetyl-1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine, AOPE, essentially as described (37Liu Y. Feizi T. Campanero-Rhodes M. Childs R. Zhang Y. Mulloy B. Evans P. Osborn H. Otto D. Crocker P. Chai W. Neoglycolipid probes prepared via oxime ligation for microarray analysis of oligosaccharide-protein interactions.Chem. Biol. 2007; 14: 847-859Abstract Fu
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