Molecular Cloning of the Heparin/Heparan Sulfate Δ4,5 Unsaturated Glycuronidase from Flavobacterium heparinum, Its Recombinant Expression in Escherichia coli, and Biochemical Determination of Its Unique Substrate Specificity

Abstract

The soil bacterium Flavobacterium heparinum produces several enzymes that degrade heparan sulfate glycosaminoglycans (HSGAGs) in a sequence-specific manner. Among others, these enzymes include the heparinases and an unusual glycuronidase that hydrolyzes the unsaturated Delta4,5 uronic acid at the nonreducing end of oligosaccharides resulting from prior heparinase eliminative cleavage. We report here the molecular cloning of the Delta4,5 glycuronidase gene from the flavobacterial genome and its recombinant expression in Escherichia coli as a highly active enzyme. We also report the biochemical and kinetic characterization of this enzyme, including an analysis of its substrate specificity. We find that the Delta4,5 glycuronidase discriminates on the basis of both the glycosidic linkage and the sulfation pattern within its saccharide substrate. In particular, we find that the glycuronidase displays a strong preference for 1-->4 linkages, making this enzyme specific to heparin/heparan sulfate rather than 1-->3 linked glycosaminoglycans such as chondroitin/dermatan sulfate or hyaluronan. Finally, we demonstrate the utility of this enzyme in the sequencing of heparinase-derived HSGAG oligosaccharides.

Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events

“…Digestion with the heparinases reduces heparin to its component di, tri-and tetrasaccharides and imparts a Δ4,5 bond monitorable at 232 nm; completed in conjunction with treatment with glycuronidase and sulfatase this digest permits the identification of minor species, including those disaccharides containing a modified galacturonic acid (6,7). Thus, concomitant use of a matrix of enzymes, especially in conjunction with LC-MS/MS analysis allows for the complete separation, identification, and quantification of heparin components in the mixture (8).…”

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

“…Both the total and soluble protein expression levels achieved were unsatisfactory, however, especially given our previous successes with recombinantly expressing other HSGAG-degrading enzymes cloned from F. heparinum (1,29,30). As has been the case for most of these enzymes, removal of their putative N-terminal signal sequences greatly facilitated the recombinant expression of soluble protein without compromising their respective specific activities.…”

“…Sulfated glycosaminoglycans such as heparin and the related heparan sulfate (HSGAGs) 1 are complex, linear carbohydrates possessing considerable chemical heterogeneity (2,3). Their structural diversity is largely a consequence of the variable number and position of sulfates present within a single HS-GAG chain.…”

“…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. 2 Whereas Flavobacterium is the commonly used nomenclature, the genus Pedobacter is the currently accepted taxon for this heparinolytic bacteria.…”

“…The molecular cloning of each of these enzymes would be an important first step toward their systematic use in vitro. In addition to heparinase I (20), we have recently cloned one of these enzymes, the ⌬4,5-unsaturated glycuronidase (1). This enzyme has also been recombinantly expressed in Escherichia coli as a highly active enzyme.…”