Figure 2: GlcNAc6ST-1 exclusively catalyzes sulfation of N-glycans in mouse PNS myelin.
From: GlcNAc6ST-1 regulates sulfation of N-glycans and myelination in the peripheral nervous system
(A) PA-N-glycans from young adult mouse PNS myelin were analyzed by DEAE HPLC. Peaks 5–9 were numbered in order of elution time from the anionic fractions. The elution positions of peaks 5 and 6 were mono- and di-sialyl N-glycans, respectively. The peak 5–9 fractions were collected and further analyzed individually. The asterisk indicates the peak derived from contaminants. Arrowheads indicate the elution positions of mono-, di-, tri-, and tetra-sialyl standard PA-oligosaccharides. (B–E) The anionic fractions of peaks 5 (B), 6 (C), 7 + 8 (D) and 9 (E) from mouse PNS myelin were individually analyzed by NP-HPLC after desialylation (peaks 5 and 6) and desulfation (peaks 7 + 8 and 9). The major peaks 5a, 5b, 5c (B), 6′ (C), (7 + 8)i, (7 + 8)ii (D) and 9′ (E) were numbered. (F) The N-glycans of peaks 5a, 5b, 5c (B), 6′ (C), (7 + 8)i, (7 + 8)ii (D) and 9′ (E) in Fig. 2B–E were further analyzed (Supplementary Fig. S3), and the N-glycan structures from peaks 5–6 were identified. (G) The percentages of sulfated N-glycans from mouse CNS and PNS myelin were measured. Error bars indicate the mean ± SD (CNS myelin, n = 4; PNS myelin, n = 7). (H) The mRNA expression levels of four GlcNAc6STs in sciatic nerves were analyzed by RT-PCR. (I) PA-N-glycans from PNS myelin of young adult GlcNAc6ST-1-KO mice (red) were separated using a DEAE column. The elution positions of peaks 5–8 coincided with those from PNS myelin of WT mice (black; Fig. 2A). (J) The percentages of N-glycans from PNS myelin of young adult WT and GlcNAc6ST-1-KO mice were measured as the peak areas on DEAE HPLC. Error bars indicate the mean ± SD (WT mice, n = 7; GlcNAc6ST-1-KO mice, n = 3).
