title: The binding specificity of the marker antibodies Tra-1-60 and Tra-1-81 reveals a novel pluripotency-associated type 1 lactosamine epitope.
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.
authors.affiliation: Finnish Red Cross Blood Service, Kivihaantie 7, 00310 Helsinki, Finland.
authors.forename: Suvi
authors.initials: S
authors.lastname: Natunen
authors.forename: Tero
authors.initials: T
authors.lastname: Satomaa
authors.forename: Virve
authors.initials: V
authors.lastname: Pitkänen
authors.forename: Hanna
authors.initials: H
authors.lastname: Salo
authors.forename: Milla
authors.initials: M
authors.lastname: Mikkola
authors.forename: Jari
authors.initials: J
authors.lastname: Natunen
authors.forename: Timo
authors.initials: T
authors.lastname: Otonkoski
authors.forename: Leena
authors.initials: L
authors.lastname: Valmu
mesh_terms: D000606:Amino Sugars / Q000737:chemistry / Q000276:immunology; D000906:Antibodies / Q000276:immunology / Q000378:metabolism*; D000918:Antibody Specificity; D001665:Binding Sites; D015415:Biomarkers / Q000032:analysis; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D053595:Embryonic Stem Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D005434:Flow Cytometry; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000276:immunology; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D000606:Amino Sugars; D000906:Antibodies; D015415:Biomarkers; D000939:Epitopes; D009844:Oligosaccharides; C032233:lactosamine; D006026:Glycoside Hydrolases