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. Author manuscript; available in PMC: 2016 Oct 15.
Published in final edited form as: J Immunol. 2015 Sep 4;195(8):3654–3664. doi: 10.4049/jimmunol.1500283

Figure 3.

Figure 3

Ctr2−/− BMMCs contain more heparin and less chondroitin sulfate proteoglycans than WT cells. GAGs were isolated from WT and Ctr2−/− BMMCs, and depolymerized to disaccharides by treatment with chondroitinase ABC and heparinase I, II, and III, respectively. Disaccharides were separated by RPIP-HPLC. (A) The disaccharide composition of chondroitin sulfate (CS) recovered from WT and Ctr2−/− BMMCs. (B) The disaccharide composition of heparin/heparan sulfate recovered from WT and Ctr2−/− BMMCs. (C, D) Percentage of the respective disaccharide units in chondroitin sulfate (C) and heparin/heparan sulfate (D) recovered from WT and Ctr2−/− cells, respectively. Data are presented as mean ± S.D. from three biological replicates. The disaccharide composition is given as picomoles per 106 cells (BMMCs). (E) Schematic figure depicting critical enzymatic steps in heparin and chondroitin sulfate synthesis; analyzed enzymes are indicated in bold. Xyl1/2 catalyze transfer of Xylose residues to Ser residues of the protein core (both in heparin/heparan sulfate and chondroitin sulfate); EXT1/2 catalyze chain elongation in heparin/heparan sulfate; NDSTs catalyze the N-deacetylation and subsequent N-sulfation of selected N-acetylglucosamine (GlcNAc) residues in heparin/heparan sulfate; HS2ST and HS6STs catalyze the 2-O- and 6-O-sufation of heparin/heparan sulfate; C4STs catalyze the 4-O-sulfation of selected N-acetylgalactosamine (GalNAc) residues of chondroitin sulfate. (F) Total RNA was prepared from WT and Ctr2−/− BMMCs and expression of critical enzymes involved in chondroitin sulfate and heparin synthesis were analyzed by qPCR. Data presented as mean ± S.E.M. from three biological replicates. Chondroitin sulfate disaccharides (A, C): OS CS/Hya, nonsulfated disaccharides of chondroitin sulfate or hyaluronan origin; 4S, Hexuronic acid (HexA)-GalNAc(4-O-SO3−); 6S, HexA-GalNAc(6-O-SO3−); 2S, HexA(2-O-SO3−)-GalNAc; 4S6S, HexAGalNAc(4,6-di-O-SO3−); 2S4S, HexA(2-O-SO3−)-GalNAc(4-O-SO3−); 2S6S, HexA(2-O-SO3−)-GalNAc(6-O-SO3−); 2S4S6S, HexA(2-O-SO3−)-GalNAc(4,6-di-O-SO3−). Heparin/HS disaccharides (B, D): NAc, HexA-GlcNAc; NS, HexA-GlcNSO3−; 6S, HexA-GlcNAc(6- O-SO3−); 2S, HexA(2-O-SO3−)-GlcNAc; NS6S, HexA-GlcNSO3−(6- O-SO3−); NS2S, HexA(2-O-SO3−)-GlcNSO3−; 2S6S, HexA(2-O-SO3−)-GlcNAc(6-O-SO3−); NS6S2S, HexA(2-O-SO3−)-GlcNSO3−(6-O-SO3−). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.