Fluorescent-tagged heparan sulfate precursor oligosaccharides to probe the enzymatic action of heparitinase I.
Heparitinase I, a key lyase enzyme essential for structural analysis of heparan sulfate (HS), degrades HS domains that are undersulfated at glucuronyl residues through an elimination mechanism. Earlier studies employed viscosimetric measurements and electrophoresis to deduce the mechanism of action of heparitinase I and two other related lyases, heparitinase II and heparitinase III. However, these findings lack molecular evidence for the intermediates formed and could not distinguish whether the cleavage occurred from the reducing end or the nonreducing end. In the current study, 2-aminoacridone (2-AMAC)-labeled HS precursor oligosaccharides of various sizes were prepared to investigate the mechanism of heparitinase I-mediated depolymerization using sensitive and quantitative methodologies. Furthermore, fluorescent (2-AMAC) tagging of HS precursor oligosaccharides allowed us to distinguish fragments that result from cleavage of the substrates at various time intervals and sites farther away from the reducing and nonreducing ends of oligosaccharide substrates. This study provides the first direct molecular evidence for a predominantly random endolytic mechanism of cleavage of HS precursor oligosaccharides by heparitinase I. This robust strategy can be adapted to deduce the mechanism of action of other heparitinases and also to deduce structural information of complex HS oligosaccharides of biological importance.
Babu
Ponnusamy
P
Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112, USA.
Kuberan
Balagurunathan
B
D000609:Aminoacridines / Q000378:metabolism*; D055162:Biocatalysis; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D057075:Enzyme Assays / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism*; D019763:Heparin Lyase / Q000378:metabolism*; D006497:Heparan Sulfate / Q000737:chemistry / Q000378:metabolism*; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013194:Staining and Labeling / Q000379:methods*; D013997:Time Factors
D000609:Aminoacridines; D005456:Fluorescent Dyes; D019763:Heparin Lyase; D006497:Heparan Sulfate; D009844:Oligosaccharides; C072515:2-aminoacridone