Abstract
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.
Highlights
Glycan antigens recognized by monoclonal antibodies have served as stem cell markers
A Close Comparison of Glycans Recognized by mAbs R-10G, anti-i P1A ELL, FC10.2, TRA-1– 60 and TRA-1– 81 with Sequence-defined Glycans Based on T1-T2 and T2-T2 Backbones—Initial analyses were performed with R-10G, anti-i P1A ELL, FC10.2, TRA-1– 60, and TRA-1– 81 using a microarray of sequence-defined glycans based on T1-T2 and T2-T2 backbones
Differences were revealed among these four antibodies: FC10.2, anti-LNT and TRA-1– 60 but not TRA-1– 81 gave binding albeit weak to the structure with a fucose (Fuc) 3-linked to the inner GlcNAc, and the binding was abolished in the presence of 2-linked Fuc at the nonreducing end
Summary
R-10G antigen of human iPS and ES cells has been assigned as a unique mono-sulfated glycan. With the advent of hybridoma technology, there followed the demonstration that the 8-cell stagespecific embryonic antigen of mouse recognized by mAb antiSSEA-1 is expressed on the ␣-3 fucosylated forms of the I and i antigens (termed LewisX), structures 4 and 5, respectively [5] (Table I) These antigens were detected predominantly on high molecular weight glycoproteins [7, 8] the common feature being the abundance of repeated T2 backbone sequences. 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 (Gal-3GlcNAc) -T2 backbone sequence, structure 6 (Table I); this was detected on a glycoprotein with an apparent molecular weight, ϳ200 kDa, [9, 10] similar to that of podocalyxin [11]. 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
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