Heparinase I from Flavobacterium heparinum

Godavarti, Ranga; Sasisekharan, Ram

doi:10.1074/jbc.273.1.248

Cited by 32 publications

(22 citation statements)

References 28 publications

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“…The pattern for this enzyme is predominantly exolytic, processive (23), and not endolytic as suggested previously (27). It might be possible to generate an exclusively exolytic or a nonprocessive heparinase I by protein engineering (28). Such an enzyme would be suited more ideally for sequencing.…”

Section: Discussionmentioning

confidence: 99%

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

Rhomberg

Ernst

Sasisekharan

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Discussionmentioning

confidence: 99%

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

Rhomberg

Ernst

Sasisekharan

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

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“…Recombinant heparin lyase I (EC 4.2.2.7), II (no EC number), and III (E.C. 4.2.2.8) were prepared as described previously (23). ⌬ 4,5 -Glycuronidase was isolated from Flavobacterium heparinum (24).…”

Section: Methodsmentioning

confidence: 99%

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

Liu

Shriver

Pope

et al. 2002

Journal of Biological Chemistry

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153

116

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Herpes simplex virus type 1 utilizes cell surface heparan sulfate as receptors to infect target cells. The unique heparan sulfate saccharide sequence offers the binding site for viral envelope proteins and plays critical roles in assisting viral infections. A specific 3-O-sulfated heparan sulfate is known to facilitate the entry of herpes simplex virus 1 into cells. The 3-O-sulfated heparan sulfate is generated by the heparan sulfate D-glucosaminyl-3-O-sulfotransferase isoform 3 (3-OST-3), and it provides binding sites for viral glycoprotein D (gD). Here, we report the purification and structural characterization of an oligosaccharide that binds to gD. The isolated gDbinding site is an octasaccharide, and has a binding affinity to gD around 18 M, as determined by affinity coelectrophoresis. The octasaccharide was prepared and purified from a heparan sulfate oligosaccharide library that was modified by purified 3-OST-3 enzyme. The molecular mass of the isolated octasaccharide was determined using both nanoelectrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry. The results from the sequence analysis suggest that the structure of the octasaccharide is a heptasulfated octasaccharide. The proposed structure of the octasaccharide is ⌬UA-GlcNSIdoUA2S-GlcNAc-UA2S-GlcNS-IdoUA2S-GlcNH 2 3S6S. Given that the binding of 3-O-sulfated heparan sulfate to gD can mediate viral entry, our results provide structural information about heparan sulfate-assisted viral entry.Heparan sulfates (HS), 1 highly sulfated polysaccharides, are present on the surface of mammalian cells and in the extracellular matrix in large quantities. HS play critical roles in a variety of biological interactions, including assisting viral infection, regulating blood coagulation and embryonic development, suppressing tumor growth, and controlling the eating behavior of mice by interacting with specific regulatory proteins (1-5). HS is initially synthesized as a copolymer of glucuronic acid and N-acetylated glucosamine by D-glucuronyl and N-acetyl-D-glucosaminyl transferase, followed by various modifications (6). These modifications include C 5 -epimerization of glucuronic acid to form iduronic acid residues, 2-O-sulfation of iduronic and glucuronic acid, N-deacetylation and N-sulfation of glucosamine, as well as 6-O-sulfation and 3-O-sulfation of glucosamine. Numerous HS biosynthetic enzymes have been cloned and characterized (for review, see Esko and Lindahl (7)).The specific sulfated saccharide sequences play critical roles in determining the functions of HS. A recent report suggests that the expression levels of various isoforms of each class of HS biosynthetic enzyme contribute to the synthesis of specific saccharide sequences in specific tissues (8). HS N-deacetylase/ N-sulfotransferase, 3-O-sulfotransferase, and 6-O-sulfotransferase are present in multiple isoforms, and each isoform is believed to recognize the saccharide sequence around the modification site to generate a specific sulfated saccharid...

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“…We have shown that Cys 135 , His 203 , and Lys 199 are important in the catalytic mechanism of heparinase I (9,11,12). Through extensive site-directed mutagenesis studies, we identified other positively charged residues (Lys 132 and Lys…”

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confidence: 99%

“…) that provide the necessary microenvironment for heparinase I catalysis (12). Recent studies of heparin degradation by heparinase I revealed a processive catalytic mechanism in which heparin is predominantly degraded exolytically from the nonreducing end (13).…”

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confidence: 99%

“…Heparin-POROS Chromatography-The heparin-POROS chromatography study of heparinases I was essentially carried out following the procedure described previously (12). Briefly, ϳ30 -40 g of ϪL recombinant heparinase I and the various mutant enzymes were injected into a heparin-POROS column (4.6 ϫ 100 mm; PerSeptive BioSystems, Framingham, MA) connected to a BioCAD system (PerSeptive BioSystems).…”

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confidence: 99%

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The Calcium-binding Sites of Heparinase I fromFlavobacterium heparinum Are Essential for Enzymatic Activity

Liu

Shriver

Godavarti³

et al. 1999

Journal of Biological Chemistry

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, and Glu 381 in CB-2) that are important for calcium binding and heparinase I enzymatic activity. Mutations in CB-1 resulted in a lower k cat , but did not change the product profile of heparinase I action on heparin; conversely, mutations in CB-2 not only altered the k cat for heparinase I, but also resulted in incomplete degradation, leading to longer saccharides. Fluorescence competition experiments along with heparin affinity chromatography suggested that mutations in CB-1 alter heparinase I activity primarily through decreasing the enzyme's affinity for its calcium cofactor without altering heparin binding to heparinase I. Compared with CB-1 mutations, mutations in CB-2 affected calcium binding to a lesser extent, but they had a more pronounced effect on heparinase I activity, suggesting a different role for CB-2 in the enzymatic action of heparinase I. These results, taken together with our accompanying study, led us to propose a model for calcium binding to heparinase I that includes both CB-1 and CB-2 providing critical interactions, albeit via a different mechanism. Through binding to CB-1 and/or CB-2, we propose that calcium may play a role in the catalytic mechanism and/or in the exolytic processive mechanism of heparin-like glycosaminoglycan depolymerization by heparinase I.

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Heparinase I from Flavobacterium heparinum

Cited by 32 publications

References 28 publications

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

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

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

The Calcium-binding Sites of Heparinase I fromFlavobacterium heparinum Are Essential for Enzymatic Activity

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