Abstract
The reducing end of heparan sulfate has been known for a long time, but information on the non-reducing end has been lacking. Recent studies indicate that the non-reducing end of heparan sulfate might be the place where fibroblast growth factor signaling complex forms. The non-reducing end also changes with heparanase digestion and, thus, might serve as a marker for tumor pathology. Using high performance liquid chromatography-coupled mass spectrometry, we have identified and characterized the non-reducing end of bovine kidney heparan sulfate. We find that the non-reducing end region is highly sulfated and starts with a glucuronic acid (GlcA) residue. The likely sequence of the non-reducing end hexasaccharides is GlcA-GlcNS6S-UA+/-2S-GlcNS+/-6S-Ido2S-GlcNS+/-6S (where GlcNS is N-sulfate-D-glucosamine, S is sulfate, UA is uronic acid, and Ido is iduronic acid). Our data suggests that the non-reducing end of bovine kidney heparan sulfate is not trimmed by heparanase and is capable of supporting fibroblast growth factor signaling complex formation.
Highlights
Despite these known structural information and biological functions of Heparan sulfate (HS), the non-reducing end (NRE) of HS has not been studied
Theoretical Calculation of the Monoisotopic m/z Value of a Terminal Oligosaccharide Released from the Non-reducing End of an HS chain by Bacterial Lyases—An oligosaccharide released from internal sections of an HS chain with bacterial lyases begins with a ⌬4,5-uronic acid (⌬UA); the terminal oligosaccharide released from the NRE of an HS chain should begin with a saturated residue (Fig. 1)
M/z 514.05 had a solution with Equation 2, suggesting that it belongs to a terminal oligosaccharide with a UA at the NRE
Summary
Materials—Bovine kidney heparan sulfate, heparinase, and heparitinase I and II were obtained from Seikagaku America (Falmouth, MA). These enzymes were reconstituted at 0.3 milliunits/l according to the manufacturer’s direction. Stable Isotope Labeling of Heparan Sulfate—34S incorporation by 6-OST-1 was done as described previously [26]. Heparan Sulfate Oligosaccharide -Glucuronidase Digestion—Briefly, 10 g of HS lyase digest was treated with 1 l of -glucuronidase (50 units) in 15 l of digestion buffer (0.05 mM sodium acetate, pH 5.0, and 0.1 mg/ml bovine serum albumin). A gradient elution was performed using a binary solvent system composed of water (eluent A) and 70% aqueous methanol (eluent B), both containing 8 mM acetic acid and 5 mM ion-pairing agent. Total ion chromatograms and mass spectra were processed with the Data Explorer software version 3.0
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