Characterization of Hydrolysis and Transglycosylation by Testicular Hyaluronidase Using Ion-Spray Mass Spectrometry

Abstract

Various oligosaccharides from hyaluronic acid, which were fluorescence-labeled and blocked by pyridylamination at the reducing terminal, were incubated as substrates or acceptors with bovine testicular hyaluronidase. Fluorescence-labeled reaction products in the reaction mixture were monitored selectively and directly by ion-spray mass spectrometry without chemical derivatization. As a result, several features of the relationship between oligosaccharides, substrates, and testicular hyaluronidase were clarified. When hexasaccharides or larger oligosaccharides having D-glucuronic acid at the nonreducing terminal were used as substrates, they were hydrolyzed sequentially to disaccharides from the nonreducing terminal, and these disaccharides were then transferred to other hexasaccharides. On the other hand, when heptasaccharides or larger oligosaccharides having N-acetyl-D-glucosamine at the nonreducing terminal were used as substrates, trisaccharides were released from the nonreducing terminal, and then also transferred to other hexasaccharides, thus forming nonasaccharides. Thus, the relationship between hydrolysis and transglycosylation reactions with testicular hyaluronidase was characterized using ion-spray mass spectrometry.

Tandem mass spectrometric strategies for determination of sulfation positions and uronic acid epimerization in chondroitin sulfate oligosaccharides

“…For oligosaccharides generated by chondroitinase enzymes, which cleave GalNAcHexA glycosidic bonds by an eliminative mechanism to produce a ⌬-unsaturated HexA residue at the new non-reducing terminus, the monoisotopic peak at m/z 458 is isobaric for oligomers of different lengths, resulting in overlapping isotopic patterns, as shown in Figure 1 nϪ , where X ϭ the number of HexA residues (including the ⌬-unsaturated residue), Y ϭ the number of GalNAc residues, and Z ϭ the number of sulfate groups. Although saturated CS oligosaccharides produced by testicular hyaluronidase, an enzyme acting by a hydrolytic mechanism, form unique m/z values for the most abundant ions from different oligomer lengths, transglycosylase reactions [32,33] limit the usefulness of this enzyme for sequence analy- Reductive amination has been widely used for attachment of chromophores to the reducing end of carbohydrates to facilitate chromatographic separation and quantitation prior to MS [34 -38]. Reaction of the oligosaccharide with an amine has most often been followed by reduction, producing an open ring structure at the former reducing end.…”

Cleavage of the Xylosyl Serine Linkage between a Core Peptide and a Glycosaminoglycan Chain by Cellulases

“…Using this enzyme, it was possible to analyze GAG chains from 50 mg wet weight of animal tissues with the combined use of PA labeling at the reducing terminal of the released sugar chains and HPLC (45). Furthermore, this enzyme is useful in the molar quantification of GAG chains (45), in giving directivity to GAG chains (46), and in preparing artificial substrates for novel endo-type enzymes (35). Therefore, we believe that cellulases with endo-␤-xylosidase activity will become useful tools for structural and functional analysis.…”

Domain Structure of Chondroitin Sulfate E Octasaccharides Binding to Type V Collagen

“…However, not all of the expected oligosaccharides were obtained. Recently, the transglycosylation mechanism of testicular hyaluronidase, which is an endo-␤-N-acetylhexosaminidase, has been investigated with the aim of performing enzymatic synthesis of GAG oligosaccharides (12,13). Using this enzymatic reconstruction system, it was possible to custom synthesize chimeric GAG oligosaccharides with sequences of nonsulfated and monosulfated disaccharide units, GlcA␤1-3GalNAc, GlcA␤1-3GlcNAc, IdoA␣1-3GalNAc, GlcA␤1-3GalNAc4S, and GlcA␤1-3GalNAc6S.…”