Fig. 6

Composition of Sdc2-linked HS chains is N-terminal domain-dependent. a, b HUVEC were transduced with adenovirus expressing the indicated syndecan extracellular domain (ED). Secreted EDs were purified by ionic-exchange chromatography (DEAE) followed by affinity chromatography (anti-HA resin). Sdc-linked HS chains were digested with Heparinase I–III and analyzed by SAX-HPLC (a, n = 4–8, each dot is an independent HS chain isolation) and LC–MS (b, 3 independent isolations). a Figure inset, Sdc2 HS chains (Sdc2) showed higher frequency of 6-O sulfation compared to Sdc4 HS chains. A Sdc4 chimera ED expressing Sdc2 D1 region in place of Sdc4 D1 (Sdc2^D1/Sdc4^D2) showed increased 6-O sulfation that was comparable to that seen in Sdc2 (top right inset, ^##P < 0.01 vs. Sdc2, ⁺⁺P < 0.01 vs. Sdc4, ±precedes the standard deviation value). b Results of LC–MS analysis are presented after normalization to Sdc2 (shown as dotted line in red at y = 1). The analysis was repeated three times with similar results (a representative image is shown). Downward arrows highlight a reduction in 6-O sulfated disaccharides. c Secreted syndecan EDs were collected, digested with 24 h with pronase and GAG chains were concentrated/enriched with DEAE column chromatography. GAG-binding plate were coated with same amount of purified chains (1 µg) and tested for binding of biotinylated-VEGFA₁₆₅ (left panel) or FGF2 (right panel). Errors bars represent SEM. Statistical analysis was performed by unpaired t test (N.S. not significant, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001)