Mass spectrometric and capillary electrophoretic investigation of the enzymatic degradation of heparin-like glycosaminoglycans

Abstract

Difficulties in determining composition and sequence of glycosaminoglycans, such as those related to heparin, have limited the investigation of these biologically important molecules. Here, we report methodology, based on matrix-assisted laser desorption ionization MS and capillary electrophoresis, to follow the time course of the enzymatic degradation of heparin-like glycosaminoglycans through the intermediate stages to the end products. MS allows the determination of the molecular weights of the sulfated carbohydrate intermediates and their approximate relative abundances at different time points of the experiment. Capillary electrophoresis subsequently is used to follow more accurately the abundance of the components and also to measure sulfated disaccharides for which MS is not well applicable. For those substrates that produce identical or isomeric intermediates, the reducing end of the carbohydrate chain was converted to the semicarbazone. This conversion increases the molecular weight of all products retaining the reducing terminus by the ''mass tag'' (in this case 56 Da) and thus distinguishes them from other products. A few picomoles of heparin-derived, sulfated hexa-to decasaccharides of known structure were subjected to heparinase I digestion and analyzed. The results indicate that the enzyme acts primarily exolytically and in a processive mode. The methodology described should be equally useful for other enzymes, including those modified by site-directed mutagenesis, and may lead to the development of an approach to the sequencing of complex glycosaminoglycans.The growing interest in glycoprotein, glycolipid, and proteoglycan function in biology increases the demand for methods to characterize the structure of oligosaccharides (1). Heparan sulfate proteoglycans are probably the most complex glycoconjugates (2). One or more heparin-like glycosaminoglycan chains (HLGAGs)-heterogenous in length, composition, and sequence-are linked covalently to a single core protein and can account for 50-90% of the molecular weight of the proteoglycan (3). HLGAGs are linear chains of 20-100 disaccharide units comprised of a uronic acid (iduronic or glucuronic acid) and a glucosamine. Various disaccharide modifications are introduced by enzymatic deacetylation, epimerization, and sulfation resulting in chemically highly heterogeneous, mature HLGAG chains (3). Many proteins have been shown to bind HLGAGs with high affinity, but only in a few cases (e.g., antithrombin III and basic fibroblast growth factor) has the HLGAG sequence responsible for high affinity binding been elucidated (4,5). This is, at least in part, because of the lack of effective and simple methods to isolate and characterize HLGAG oligosaccharides. Full length HLGAGs cannot be separated into individual components because of the large number of different HLGAG chains of closely related structure. Attempts at sequencing therefore have focused on oligosaccharide fragments obtained by HL-GAG depolymerization. Such fragments are generated either ch...

Competing fragmentation processes in tandem mass spectra of heparin-like glycosaminoglycans

“…The abundances of the charge states observed under each condition are dictated by factors including the molecular conformation of the ion in solution 14 , instrumental ion source conditions [15], pH [16], solvent polarity [17], and solvent volatility [18,19]. In these experiments the pH and instrument operating conditions were held constant.…”

The Heparin/Heparan Sulfate 2-O-Sulfatase from Flavobacterium heparinum

“…Molecular Mass Determinations by MALDI Mass Spectrometry-The molecular weight of the 2-O-sulfatase NH 2 -truncated enzyme (2-O ⌬N 1-24 ) was determined by MALDI mass spectrometry essentially as described (19). The NH 2 -terminal histidine tag of the recombinant protein was cleaved by thrombin prior to mass analysis.…”

“…One example of an important class of GAG-degrading enzyme is the heparin lyases (heparinases) originally isolated from the Gram-negative soil bacterium Flavobacterium heparinum. 2 Each of the three heparinases encoded by this microorganism cleaves both heparin and heparan sulfate with a substrate specificity that is generally based on the differential sulfation pattern that exists within each GAG chain (18,19). In fact, F. heparinum uses several additional enzymes in an apparently sequential man- 1 The abbreviations used are: HSGAG, heparin/heparan sulfate glycosaminoglycan; ⌬U, uronic acid with a ⌬4,5 double bond; 2-O ⌬N , recombinant 2-O-sulfatase lacking NH 2 -terminal signal sequence composed of first 24 amino acids; MALDI, matrix-assisted laser desorption ionization; GAG, glycosaminoglycan; RP-HPLC, reverse phase high pressure liquid chromatography; MES, 4-morpholineethanesulfonic acid; ADA, [(carbamoylmethyl)imino]diacetic acid.…”

Characterization of a Heparan Sulfate Octasaccharide That Binds to Herpes Simplex Virus Type 1 Glycoprotein D

“…The approach for the analysis of oligosaccharides followed a previously described method with modifications (30). Briefly, the analysis was carried out on a Beckman P/ACE MDQ unit using an uncoated fused silica capillary (inner diameter ϭ 75 m; L tot ϭ 106 cm).…”