Suvi Natunen

Publications (8)27.1 Total impact

• Article: Nanoscale reversed-phase liquid chromatography-mass spectrometry of permethylated N-glycans.

  Ilja Ritamo, Jarkko Räbinä, Suvi Natunen, Leena Valmu
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  ABSTRACT: Reversed-phase liquid chromatography on the nanoscale coupled to electrospray tandem mass spectrometry was used to analyse a mixture of four commercial glycan standards, and the method was further adapted to N-glycans enzymatically released from alpha-1-acid glycoprotein and immunoglobulin gamma. Glycans were permethylated to enable their separation by reversed-phase chromatography and to facilitate interpretation of fragmentation data. Prior to derivatization of glycans by permethylation, they were reduced to cancel anomerism because, although feasible, it was not desired to separate α- and β-anomers. The effect of supplementing chromatographic solvent with sodium hydroxide to guide adduct formation was investigated. Raising the temperature in which the separation was performed improved chromatographic resolution and affected retention times as expected. It was shown by using the tetrasaccharides sialyl Lewis X and sialyl Lewis A that reversed-phase chromatography could achieve the separation of methylated isobaric glycan analytes. Isobaric glycans were detected among the N-glycans of immunoglobulin gamma and further analysed by tandem mass spectrometry.
  Analytical and Bioanalytical Chemistry 01/2013; · 3.78 Impact Factor
• Article: Cell Surface Structures Influence Lung Clearance Rate of Systemically Infused Mesenchymal Stromal Cells.

  Johanna Nystedt, Heidi Anderson, Jonne Tikkanen, Mika Pietilä, Tia Hirvonen, Reijo Takalo, Annamari Heiskanen, Tero Satomaa, Suvi Natunen, Siri Lehtonen, Tanja Hakkarainen, Matti Korhonen, Saara Laitinen, Leena Valmu, Petri Lehenkari
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  ABSTRACT: The promising clinical effects of mesenchymal stromal/stem cells (MSCs) rely especially on paracrine and non-immunogenic mechanisms. Delivery routes are essential for the efficacy of cell therapy and systemic delivery by infusion is the obvious goal for many forms of MSC therapy. Lung adhesion of MSCs might, however, be a major obstacle yet to overcome. Current knowledge does not allow us to make sound conclusions whether MSC lung entrapment is harmful or beneficial and thus we wanted to explore MSC lung adhesion in greater detail. We found a striking difference in the lung clearance rate of systemically infused MSCs derived from two different clinical sources, namely bone marrow (BM-MSCs) and umbilical cord blood (UCB-MSCs). The BM-MSCs and UCB-MSCs used in this study differed in cell size, but our results also indicated other mechanisms behind the lung adherence. A detailed analysis of the cell surface profiles revealed differences in the expression of relevant adhesion molecules. The UCB-MSCs had higher expression levels of α4 integrin (CD49d, VLA-4), α6 integrin (CD49f, VLA-6) and the hepatocyte growth factor receptor (c-Met) and a higher general fucosylation level. Strikingly, the level of CD49d and CD49f expression could be functionally linked with the lung clearance rate. Additionally, we saw a possible link between MSC lung adherence and higher fibronectin expression and we show that the expression of fibronectin increases with MSC culture confluency. Future studies should aim at developing methods of transiently modifying the cell surface structures in order to improve the delivery of therapeutic cells.
  Stem Cells 11/2012; · 7.78 Impact Factor
• Article: Novel data analysis tool for semiquantitative LC-MS-MS(2) profiling of N-glycans.

  Hannu Peltoniemi, Suvi Natunen, Ilja Ritamo, Leena Valmu, Jarkko Räbinä
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  ABSTRACT: Despite recent technical advances in glycan analysis, the rapidly growing field of glycomics still lacks methods that are high throughput and robust, and yet allow detailed and reliable identification of different glycans. LC-MS-MS(2) methods have a large potential for glycan analysis as they enable separation and identification of different glycans, including structural isomers. The major drawback is the complexity of the data with different charge states and adduct combinations. In practice, manual data analysis, still largely used for MALDI-TOF data, is no more achievable for LC-MS-MS(2) data. To solve the problem, we developed a glycan analysis software GlycanID for the analysis of LC-MS-MS(2) data to identify and profile glycan compositions in combination with existing proteomic software. IgG was used as an example of an individual glycoprotein and extracted cell surface proteins of human fibroblasts as a more complex sample to demonstrate the power of the novel data analysis approach. N-glycans were isolated from the samples and analyzed as permethylated sugar alditols by LC-MS-MS(2), permitting semiquantitative glycan profiling. The data analysis consisted of five steps: 1) extraction of LC-MS features and MS(2) spectra, 2) mapping potential glycans based on feature distribution, 3) matching the feature masses with a glycan composition database and de novo generated compositions, 4) scoring MS(2) spectra with theoretical glycan fragments, and 5) composing the glycan profile for the identified glycan compositions. The resulting N-glycan profile of IgG revealed 28 glycan compositions and was in good correlation with the published IgG profile. More than 50 glycan compositions were reliably identified from the cell surface N-glycan profile of human fibroblasts. Use of the GlycanID software made relatively rapid analysis of complex glycan LC-MS-MS(2) data feasible. The results demonstrate that the complexity of glycan LC-MS-MS(2) data can be used as an asset to increase the reliability of the identifications.
  Glycoconjugate Journal 06/2012; · 2.12 Impact Factor
• Article: The i blood group antigen as a marker for umbilical cord blood-derived mesenchymal stem cells.

  Tia Hirvonen, Heli Suila, Annika Kotovuori, Ilja Ritamo, Annamari Heiskanen, Pertti Sistonen, Heidi Anderson, Tero Satomaa, Juhani Saarinen, Sari Tiitinen, Jarkko Räbinä, Saara Laitinen, Suvi Natunen, Leena Valmu
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  ABSTRACT: Multipotent mesenchymal stem cells (MSCs) offer great promise for future regenerative and anti-inflammatory therapies. However, there is a lack of methods to quickly and efficiently isolate, characterize, and ex vivo expand desired cell populations for therapeutic purposes. Single markers to identify cell populations have not been characterized; instead, all characterizations rely on panels of functional and phenotypical properties. Glycan epitopes can be used for identifying and isolating specific cell types from heterogeneous populations, on the basis of their cell-type specific expression and prominent cell surface localization. We have now studied in detail the cell surface expression of the blood group i epitope (linear poly-N-acetyllactosamine chain) in umbilical cord blood (UCB)-derived MSCs. We used flow cytometry and mass spectrometric glycan analysis and discovered that linear poly-N-acetyllactosamine structures are expressed in UCB-derived MSCs, but not in cells differentiated from them. We further verified the findings by mass spectrometric glycan analysis. Gene expression analysis indicated that the stem-cell specific expression of the i antigen is determined by β3-N-acetylglucosaminyltransferase 5. The i antigen is a ligand for the galectin family of soluble lectins. We found concomitant cell surface expression of galectin-3, which has been reported to mediate the immunosuppressive effects exerted by MSCs. The i antigen may serve as an endogenous ligand for this immunosuppressive agent in the MSC microenvironment. Based on these findings, we suggest that linear poly-N-acetyllactosamine could be used as a novel UCB-MSC marker either alone or within an array of MSC markers.
  Stem cells and development 09/2011; 21(3):455-64. · 4.15 Impact Factor
• Article: The binding specificity of the marker antibodies Tra-1-60 and Tra-1-81 reveals a novel pluripotency-associated type 1 lactosamine epitope.

  Suvi Natunen, Tero Satomaa, Virve Pitkänen, Hanna Salo, Milla Mikkola, Jari Natunen, Timo Otonkoski, Leena Valmu
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  ABSTRACT: The expression of the epitopes recognized by the monoclonal antibodies Tra-1-60 and Tra-1-81 is routinely used to assess the pluripotency status of human embryonic stem cells (hESCs) and induced pluripotent stem (iPS) cells. Although it is known that the epitopes recognized by Tra-1-60 and Tra-1-81 are carbohydrates, the exact molecular identity of these epitopes has been unclear. Glycan array analysis with more than 500 oligosaccharide structures revealed specific binding of Tra-1-60 and Tra-1-81 to two molecules containing terminal type 1 lactosamine: Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc and Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6(Galβ1-3GlcNAcβ1-3)Galβ1-4Glc. The type 1 disaccharide in itself was not sufficient for binding, indicating that the complete epitope requires an extended tetrasaccharide structure where the type 1 disaccharide is β1,3-linked to type 2 lactosamine. Our mass spectrometric analysis complemented with glycosidase digestions of hESC O-glycans indicated the presence of the extended tetrasaccharide epitope on an O-glycan with the likely structure Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAc. Thus, the present data indicate that the pluripotency marker antibodies Tra-1-60 and Tra-1-81 recognize the minimal epitope Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc, which is present in hESCs as a part of a mucin-type O-glycan structure. The exact molecular identity of Tra-1-60 and Tra-1-81 is important for the development of improved tools to characterize the pluripotent phenotype.
  Glycobiology 09/2011; 21(9):1125-30. · 3.58 Impact Factor
• Article: Are globoseries glycosphingolipids SSEA-3 and -4 markers for stem cells derived from human umbilical cord blood?

  Heli Suila, Virve Pitkänen, Tia Hirvonen, Annamari Heiskanen, Heidi Anderson, Anita Laitinen, Suvi Natunen, Halina Miller-Podraza, Tero Satomaa, Jari Natunen, Saara Laitinen, Leena Valmu
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  ABSTRACT: Umbilical cord blood (UCB) is an efficient and valuable source of hematopoietic stem cells (HSCs) for transplantation. In addition to HSCs it harbours low amounts of mesenchymal stem cells (MSCs). No single marker to identify cord blood-derived stem cells, or to indicate their multipotent phenotype, has been characterized so far. SSEA-3 and -4 are cell surface globoseries glycosphingolipid epitopes that are commonly used as markers for human embryonic stem cells, where SSEA-3 rapidly disappears when the cells start to differentiate. Lately SSEA-3 and -4 have also been observed in MSCs. As there is an ongoing discussion and variation of stem-cell markers between laboratories, we have now comprehensively characterized the expression of these epitopes in both the multipotent stem-cell types derived from UCB. We have performed complementary analysis using gene expression analysis, mass spectrometry and immunochemical methods, including both flow cytometry and immunofluoresence microscopy. SSEA-4, but not SSEA-3, was expressed on MSCs but absent from HSCs. Our findings indicate that SSEA-3 and/or -4 may not be optimal markers for multipotency in the case of stem cells derived from cord blood, as their expression may be altered by cell-culture conditions.
  Journal of Molecular Cell Biology 12/2010; 3(2):99-107.
• Article: Glycomics of bone marrow-derived mesenchymal stem cells can be used to evaluate their cellular differentiation stage.

  Annamari Heiskanen, Tia Hirvonen, Hanna Salo, Ulla Impola, Anne Olonen, Anita Laitinen, Sari Tiitinen, Suvi Natunen, Olli Aitio, Halina Miller-Podraza, Manfred Wuhrer, André M Deelder, Jari Natunen, Jarmo Laine, Petri Lehenkari, Juhani Saarinen, Tero Satomaa, Leena Valmu
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  ABSTRACT: Human mesenchymal stem cells (MSCs) are adult multipotent progenitor cells. They hold an enormous therapeutic potential, but at the moment there is little information on the properties of MSCs, including their surface structures. In the present study, we analyzed the mesenchymal stem cell glycome by using mass spectrometric profiling as well as a panel of glycan binding proteins. Structural verifications were obtained by nuclear magnetic resonance spectroscopy, mass spectrometric fragmentation, and glycosidase digestions. The MSC glycome was compared to the glycome of corresponding osteogenically differentiated cells. More than one hundred glycan signals were detected in mesenchymal stem cells and osteoblasts differentiated from them. The glycan profiles of MSCs and osteoblasts were consistently different in biological replicates, indicating that stem cells and osteoblasts have characteristic glycosylation features. Glycosylation features associated with MSCs rather than differentiated cells included high-mannose type N-glycans, linear poly-N-acetyllactosamine chains and alpha2-3-sialylation. Mesenchymal stem cells expressed SSEA-4 and sialyl Lewis x epitopes. Characteristic glycosylation features that appeared in differentiated osteoblasts included abundant sulfate ester modifications. The results show that glycosylation analysis can be used to evaluate MSC differentiation state.
  Glycoconjugate Journal 12/2008; 26(3):367-84. · 2.12 Impact Factor
• Article: PSGL-1 from the murine leukocytic cell line WEHI-3 is enriched for core 2-based O-glycans with sialyl Lewis x antigen.

  Ziad S Kawar, Thomas K Johnson, Suvi Natunen, John B Lowe, Richard D Cummings
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  ABSTRACT: Leukocyte trafficking involves specific recognition between P-selectin and L-selectin and PSGL-1 containing core 2-based O-glycans expressing sialyl Lewis x (SLe(x)) antigen. However, the structural identity of the glycan component(s) displayed by murine neutrophil PSGL-1 that contributes to its P-selectin counter-receptor activity has been uncertain, since these cells express little if any SLe(x) antigen, and because there have been no direct studies to examine murine PSGL-1 glycosylation. To address this uncertainty, we studied PSGL-1 glycosylation in the murine cell line WEHI-3 using metabolic-radiolabeling with (3)H-monosaccharide precursors to detect low-abundance O-glycan structures. We report that PSGL-1 from WEHI-3 cells expresses a di-sialylated core 2 O-glycan containing the SLe(x) antigen. This fucosylated O-glycan is scarce on PSGL-1 and essentially undetectable in total leukocyte glycoproteins from WEHI-3 cells. These results demonstrate that WEHI-3 cells selectively fucosylate PSGL-1 to generate functionally important core 2-based O-glycans containing the SLe(x) antigen.
  Glycobiology 07/2008; 18(6):441-6. · 3.58 Impact Factor