Binding specificity of R-10G and TRA-1-60/81, and substrate specificity of keratanase II studied with chemically synthesized oligosaccharides

Abstract

Recently, we established a mouse monoclonal antibody specific to hiPS/ hES cells, R-10G, which recognizes a type of keratan sulfate. Keratan sulfates (KS) comprise a family of glycosaminoglycans consisting of the repeating unit of [Gal-GlcNAc(6S)]. However, there is a diversity in the degree of sulfation at Gal and GlcNAc residues, and also in the mode of linkage, Galβ1 - 3GlcNAc (type 1) or Galβ1 - 4GlcNAc (type 2). To gain more insight into the binding specificity of R-10G, we carried out an ELISA test on avidin-coated plates using polyethylene glycol (PEG)-biotinylated derivatives of a series of N-acetyllactosamine tetrasaccharides (keratan sulfates (KSs)). The results suggested that the minimum epitope structure is Galβ1 - 4GlcNAc(6S)β1 - 3Galβ1 - 4GlcNAc(6S)β1 (type 2- type 2 keratan sulfate). Removal of sulfate from GlcNAc(6S) or addition of sulfate to Gal abolished the binding activity almost completely. We also examined the binding specificity of TRA-1-60/81 in the same assay system. The minimum epitope structure was shown to be Galβ1 - 3GlcNAcβ1 - 3Galβ1 - 4GlcNAcβ1 in agreement with the previous study involving glycan arrays (Natunen et al., Glycobiology, 21, 1125-1130 (2011)). Interestingly, however, TRA-1-60/81 was shown to bind to Galβ1 - 3GlcNAc(6S)β1 - 3Galβ1 - 4GlcNAc(6S)β1 (type 1- type 2 keratan sulfate) dose-dependently, being more than one-third the binding activity toward Galβ1 - 3GlcNAcβ1 - 3Galβ1 - 4GlcNAcβ1 than in the case of TRA-1-60. In addition, a substrate specificity study on keratanase II revealed that keratanase II degraded not only "type 2-type 2 keratan sulfate" but also "type 1-type 2 keratan sulfate", significantly.

Keratan sulfate (KS)‐proteoglycans and neuronal regulation in health and disease: the importance ofKS‐glycodynamics and interactive capability with neuroregulatory ligands

“…MAb 5-D-4 was the first KS antibody developed to high-charge density KS epitopes similar to those detected by MAb MZ-15 ). Antibodies to regions of intermediate to low-charge density such as MAb, 1-B-4 identify KS tetrasaccharides Tang et al 1986) while MAbs R-10G and BCD-4 recognize low-sulfation KS motifs (Kawabe et al 2013;Nakao et al 2017). MAb BKS-1(+) was raised to a keratanase-generated [N-acetylglucosamine-6-sulfate (GlcNAc-6-S)] stub neoepitope at the non-reducing terminus of corneal and skeletal KS (Akhtar et al 2008).…”

Keratan sulfate, a complex glycosaminoglycan with unique functional capability

“…Low sulfation KS would not have been detected. Since MAb 1-B-4 ( Mehmet et al 1986 ) and MAb R-10G ( Kawabe et al 2013 ; Nakao et al 2017 ) which detect these were not used in this study.…”

“…KS is synthesized by the chick cornea between E5 and E7, but only becomes highly sulfated by E14 ( Hart 1976 ) with a switch in KS biosynthesis from an unsulfated form to sulfated KS between E12 and E15 ( Cornuet et al 1994 ). The low sulfation form of KS in early corneal development displays antigenic determinants in the poly- N -acetyllactosamine chain identified by EMCA-2, EMCA-3, TRA-1-60/TRA-1-81 and R-10G ( Kawabe et al 2013 ; Nakao et al 2017 ). Human auto-antibodies to the blood group substances i- and I-antigen also identify determinants in embryonic KS chains ( Liles et al 2010 ).…”

“…GCTM-2 is an epitope on the core protein of a mucin-like KS/CS pericellular matrix PG, podocalyxcin expressed by human pluripotent stem cells ( Pebay et al 2005 ). MAb TRA-1-60 and TRA-1-81 also recognize epitopes on carbohydrate side chains on the same molecule ( Nakao et al 2017 ). A related diagnostic MAb to EMCA detects serum epitheliamucin epitopes arising from breast cancer tumors ( Dixon et al 1993 ).…”

“…These epitopes are sensitive to endo-β- d -galactosidase, keratanase-I, keratanase II and N -glycanase digestion and are completely lost upon differentiation of the stem cells. Podocalyxcin, a cell surface mucin-like stem cell KSPG bears the TRA-1-60, TRA-1-81 KS epitopes ( Andrews et al 1984 ; Nakao et al 2017 ). Antibody R-10G also identifies low sulfation KS chains on podocalyxcin ( Cooper et al 2002 ) in induced pluripotent stem cells and embryonic stem cells distinguishing these from embryonal carcinoma cells expressing podocalyxcin containing oversulfated KS chains detected using MAb 5-D-4.…”