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.

Fig. 1.

Undifferentiated hESCs are stained by Tra-1-60 and Tra-1-81 in a β1,3-galactosidase sensitive manner. hESCs were treated with β1,3-galactosidase (B and E), β1,4-galactosidase (C and F) or buffer alone (A and D), stained with Tra-1-60 (A–C) and Tra-1-81 (D–F) and analyzed by flow cytometry. Filled red histograms represent positively stained cells. Filled gray histograms represent cells stained with secondary antibody alone.

Fig. 2.

Probing a glycan array with the monoclonal antibodies Tra-1-60 (A) and Tra-1-81 (B). The array was analyzed at 25 µg/mL of the antibodies. Structures of the ligands that give the strongest signals are indicated using symbol nomenclature. A complete list of all the glycans on the array and the results of Tra-1-60 and Tra-1-81 binding are provided in Supplementary data, Table SI.

Fig. 3.

Analysis of hESC O-glycosylation by MS. (A) Partial MALDI-TOF mass spectrum of O-glycans released from hESC. (B) Mass spectrum after the endo-β-galactosidase digestion of the O-glycans, (C) after β1,4-galactosidase digestion and (D) after β1,3-galactosidase digestion. The signal intensity of m/z 1136 is compared with those of m/z 1079 and 1095, which arise from contaminating N-glycans not digested by endo-β-galactosidase, β1,4-galactosidase or β1,3-galactosidase. Symbol nomenclature: green circle, mannose; red triangle, fucose; blue square, N-acetylglucosamine; yellow circle, galactose; yellow square, N-acetylgalactosamine.