Analysis of 3-O-Sulfated Heparan Sulfate Using Isotopically Labeled Oligosaccharide Calibrants

Abstract

The 3-O-sulfated glucosamine in heparan sulfate (HS) is a low-abundance structural component, but it is a key saccharide unit for the biological activities of HS. A method to determine the level of 3-O-sulfated HS is lacking. Here, we describe a LC–MS/MS based method to analyze the structural motifs. We determined the levels of 3-O-sulfated structural motifs from pharmaceutical heparin manufactured from bovine, porcine, and ovine. We discovered that saccharide chains carrying 3-O-sulfation from enoxaparin, an FDA-approved low-molecular weight heparin, displayed a slower clearance rate than non-3-O-sulfated sugar chains in a mouse model. Lastly, we detected the 3-O-sulfated HS from human brain. Furthermore, we found that a specific 3-O-sulfated structural motif, tetra-1, is elevated in the brain HS from Alzheimer’s disease patients (n = 5, p = 0.0020). Our method offers a practical solution to measure 3-O-sulfated HS from biological sources with the sensitivity and quantitative capability.

Heparan Sulfate Proteoglycans in Tauopathy

“…Compared to the control brains, GAGs isolated from the AD brains showed decreased binding to growth factors, such as fibroblast growth factor 2 (FGF-2), brain-derived neurotrophic factor and vascular endothelial growth factor (VEGF), and increased binding to tau, heparin-binding EGF-like growth factor and pleiotrophin [ 92 ], reflecting GAG structural alteration in AD brain. This has been supported by a recent study showing multiple sulfated disaccharides (ΔUA2S-GlcNS, ΔUA2S-GlcNAc, ΔUA-GlcNAc6S, ΔUA2S-GlcNAc6S) and a tetrasaccharide with rare 3S (ΔUA-GlcNAc6S-GlcA-GlcNS3S6S) were increased in AD [ 137 ]. These increased di- and tetrasaccharides are rich in N -, 6- O -, and 3- O -sulfation that bind tau [ 137 ] and may increase tau’s propensity for aggregation.…”

“…This has been supported by a recent study showing multiple sulfated disaccharides (ΔUA2S-GlcNS, ΔUA2S-GlcNAc, ΔUA-GlcNAc6S, ΔUA2S-GlcNAc6S) and a tetrasaccharide with rare 3S (ΔUA-GlcNAc6S-GlcA-GlcNS3S6S) were increased in AD [ 137 ]. These increased di- and tetrasaccharides are rich in N -, 6- O -, and 3- O -sulfation that bind tau [ 137 ] and may increase tau’s propensity for aggregation.…”

“…They also observed that HS is most abundantly expressed in the basement membrane of capillary endothelial cells [ 136 ]. Other groups’ studies confirmed the abnormal HS expression in the AD brain [ 92 , 137 ].…”

The 3-O sulfation of heparan sulfate proteoglycans contributes to the cellular internalization of tau aggregates

“…Around 4 × 10 6 cells were collected for HS extraction. The HS extraction procedures were published previously with minor modification [ 52 , 53 ]. Briefly, the cell pellet was suspended in water and proteolyzed with pronase E (10 mg:1 g (w/w), pronase E/protein) at 55 °C for 24 h to digest the protein.…”

“…The YM-3KDa spin column was applied to desalt the elute, and the retentate was subjected to heparin lyases digest. Before the digestion, a known amount 13 C-labeled 3-O-sulfation oligosaccharide calibrants was added to the retentate [53]. The 100 μL of digestion solution containing 7.5 μL of enzymatic buffer (100 mM sodium acetate/2 mM calcium acetate buffer (pH 7.0) containing 0.1 g/L BSA), and 1.25 μL heparinase I (1.2 U/ml) and 2.5 μL heparinase II (1.2 U/m).…”