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Total Cellular Glycomics Allows Characterizing Cells and Streamlining the Discovery Process for Cellular Biomarkers

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Total cellular glycomics allows characterizing cells and streamlining the discovery process for cellular biomarkers Naoki Fujitania,1, Jun-ichi Furukawaa,1, Kayo Arakia,1, Tsuyoshi Fujiokab, Yasuhiro Takegawaa, Jinhua Piaoa, Taiki Nishiokac, Tomohiro Tamurac, Toshio Nikaidod, Makoto Itoe, Yukio Nakamurab, and Yasuro Shinoharaa,2 aLaboratory of Medical and Functional Glycomics, Graduate School of Advanced Life Science, and Frontier Research Center for Post-Genome Science and Technology, Hokkaido University, Sapporo 001-0021, Japan; bCell Engineering Division, RIKEN BioResource Center, Tsukuba 305-0074, Japan; cBioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo 062-8517, Japan; dDepartment of Regenerative Medicine, Faculty of Medicine and Graduate School of Medicine and Pharmaceutical Sciencies, University of Toyama, Toyama 930-0194, Japan; and eLaboratory of Marine Resource Chemistry, Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581, Japan Edited by Chi-Huey Wong, Academia Sinica, Taipei, Taiwan, and approved December 26, 2012 (received for review August 20, 2012) Although many of the frequently used pluripotency biomarkers extraordinarily complex N-andO-linked glycans [e.g., glycans de- are glycoconjugates, a glycoconjugate-based exploration of novel rived from glycoproteins, glycosaminoglycans (GAGs), and gly- cellular biomarkers has proven difficult due to technical difficul- cosphingolipids (GSLs); reviewed in refs. 5 and 6]. Considering ties. This study reports a unique approach for the systematic over- that cell surfaces are coated with a variety of intricately arranged x view of all major classes of oligosaccharides in the cellular glycome. glycoconjugates, and some carbohydrate epitopes (e.g., Lewis , The proposed method enabled mass spectrometry–based structur- also known as SSEA-1) may be constituents of different glyco- ally intensive analyses, both qualitatively and quantitatively, of conjugates (5), a systematic overview of all major classes of oligo- N O saccharides within the cellular glycome would be most opportune cellular - and -linked glycans derived from glycoproteins, glyco- fi saminoglycans, and glycosphingolipids, as well as free oligosacchar- for the identi cation of novel stem cell biomarkers. ides of human embryonic stem cells (hESCs), induced pluripotent To date, total cellular glycosylation analyses are scarce. Wearne et al. (7) used fluorescently labeled lectins to classify structural stem cells (hiPSCs), and various human cells derived from normal glycosylation motifs on the stem cell surface, and the utility of lectin and carcinoma cells. Cellular total glycomes were found to be highly arrays for cell characterization has recently been illustrated (8, 9). cell specific, demonstrating their utility as unique cellular descriptors. fi fi However, only a limited number of lectins and glycan-speci c Structures of glycans of all classes speci cally observed in hESCs and antibodies are in fact available, and it is often difficult to discrim- hiPSCs tended to be immature in general, suggesting the presence of inate between different glycoconjugate species by these methods. stem cell–specific glycosylation spectra. The current analysis revealed Furthermore, although analysis of the total cellular glycome has the high similarity of the total cellular glycome between hESCs and been addressed in pioneering mass spectrometric work (10, 11), no hiPSCs, although it was suggested that hESCs are more homoge- attempts have yet been made to determine all major classes of neous than hiPSCs from a glycomic standpoint. Notably, this study glycans within a cell or the relative concentration of each type of enabled a priori identification of known pluripotency biomarkers glycan present. such as SSEA-3, -4, and -5 and Tra-1–60/81, as well as a panel of This study now provides an integrated analytical technique to glycans specifically expressed by hESCs and hiPSCs. visualize the entire complement of sugars in the cellular glycome, including N-glycans, O-glycans, glycosaminoglycans, GSL-asso- omics-based biomarker discovery | stemness | interglycomic correlations | ciated glycans, and free oligosaccharides (FOSs). The usefulness glycoblotting | β-elimination in the presence of pyrazolone of this approach was validated by clarifying intra- and intergly- comic correlations (correlations within and between each in- uman embryonic stem cells (hESCs) derived from the inner dividual glycome of various glycoconjugates) under conditions of a perturbed glycan synthetic pathway. This approach was finally Hcell mass of the blastocyst and, more recently, induced plu- fi ripotent stem cells (hiPSCs) reprogrammed from somatic cells applied to delineate the glycomic pro les of hESCs and hiPSCs, share the property of indefinite growth while maintaining pluri- as well as various human cells derived from normal and carci- noma cells. The cellular glycomic profiles were revealed in their potency (1, 2). Extensive research has been conducted with these fi cells to develop disease models, methods for drug screening, and entirety, allowing the accurate identi cation of known pluri- ultimately, regenerative therapies. Because stem cells are cur- potency biomarkers and the discovery of unique pluripotency rently defined by a combination of physical, phenotypic, and func- biomarker candidates. fi tional properties, the identi cation of novel cell surface markers is Results highly advantageous for the rapid characterization and isolation of different stem cell populations. Systematic Survey of Cellular Glycomes. The general analytical scheme SI Appendix Many of the frequently used pluripotency biomarkers, including that was used in this study is shown in ,Fig.S1.Gly- stage-specific embryonic antigens (SSEA-3, -4, and -5) and tumor- cans can be regarded as a class of organic compounds that have rejection antigens (Tra-1–60 and Tra-1–81), are glycoconjugates (3, 4). These glycomarkers have been identified following the rather fortuitous development of specific anti-glycoconjugate antibodies. Author contributions: T. Nikaido, Y.N., and Y.S. designed research; N.F., J.-i.F., K.A., J.P., BIOCHEMISTRY A glycoconjugate-based approach would therefore be expected to and Y.S. performed research; N.F., J.-i.F., T.F., Y.T., T. Nishioka, T.T., M.I., Y.N., and Y.S. streamline the discovery process for novel cellular biomarkers. contributed new reagents/analytic tools; N.F., J.-i.F., K.A., and Y.S. analyzed data; and N.F. and Y.S. wrote the paper. However, such an approach has proven difficult due to technical fl difficulties associated with the analysis of various types of cellular The authors declare no con ict of interest. glycomes, given the entire repertoire of glycoconjugate-associated This article is a PNAS Direct Submission. sugars in cells and tissues. Glycoconjugates represent the most 1N.F., J.-i.F., and K.A. contributed equally to this work. structurally and functionally diverse class of molecules in nature, 2To whom correspondence should be addressed. E-mail: [email protected]. ranging from relatively simple nuclear or cytosolic glycoproteins This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. with dynamic monosaccharide modifications (O-GlcNAcylation) to 1073/pnas.1214233110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1214233110 PNAS | February 5, 2013 | vol. 110 | no. 6 | 2105–2110 Downloaded by guest on September 24, 2021 a hemiacetal group at the reducing terminal following their en- can potentially interfere with glycomic analyses for all types of zymatic release from proteins and lipids. GSL glycans were re- glycoconjugates. The structures of FOSs are identical or very leased by digestion with EGCases I and II (12), and N-glycans similar to those of N-glycans because FOSs are generated as the were released by digestion with PNGase F. GAGs are highly result of N-glycoproteins catabolism. The latter occurs via two complicated molecules, and therefore their constituent repeating distinct metabolic pathways: the endoplasmic reticulum–asso- disaccharides were analyzed. These repeating disaccharides can be ciated degradation (ERAD) of misfolded, newly synthesized generated on enzymatic depolymerization with chondroitinase, N-glycoproteins and the mature N-glycoprotein turnover path- heparitinase, hyaluronidase, etc. To maximize the deglycosylation/ way. Therefore, it is indispensable to separate FOSs from glyco- depolymerization efficiency, conditions for sample preparation, conjugates before analysis or to subtract the amount of background including extraction of glycoconjugates and enzymatic liberation FOSs from the final analytical readout. of glycans, were optimized (SI Appendix,Fig.S2). Apart from the technical issues, analysis of FOSs may help to Hemiacetal is a masked aldehyde, and because aldehydes are characterize/describe cells. FOSs reportedly increase under rare functionalities in cells, GSL/N-glycans and GAGs disaccha- conditions of endoplasmic reticulum (ER) stress (18), which is in rides can be selectively enriched by chemoselective ligation on turn linked to various disease states (19). As shown in SI Ap- reaction of the aldehyde with reagents bearing hydrazide func- pendix, Fig. S4, FOSs were effectively separated from glyco- tional groups as we previously described (13). Any impurities can proteins by ethanol precipitation before PNGase F digestion
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