Minimum substrate requirements of endoglycosidase activities toward dermatan sulfate by electrospray ionization-tandem mass spectrometry.

Abstract:

:The catabolism of dermatan sulfate (DS) commences with endohydrolysis of the polysaccharide to oligosaccharides by proposed endo-beta-N-acetylhexosaminidase and endohexuronidase activities. To investigate the substrate specificities of these activities, we developed an assay to measure specific products of their action upon oligosaccharide substrates. Tetra- to tetradecasaccharides, rich in glucuronic acid (GlcA) or iduronic acid (IdoA), were obtained from chondroitinase ABC digests of chondroitin sulfate (CS)-A and DS, respectively, separated by gel-filtration chromatography and characterized by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Endo-beta-N-acetylhexosaminidase and endohexuronidase cleavage of these oligosaccharides was then assessed by incubating with cell homogenate (source of endoglycosidase activity) and measuring di- to octasaccharide products derived from the nonreducing end of the substrate by ESI-MS/MS. We found that both activities preferentially degraded the GlcA-rich substrate, with minor activity toward the IdoA-rich substrate and that a minimum of four and five monosaccharides were required on the reducing side of the target glycosidic linkage for endo-beta-N-acetylhexosaminidase and endohexuronidase cleavage, respectively. Thus, the minimum-sized substrates were a hexasaccharide for endo-beta-N-acetylhexosaminidase and an octasaccharide for endohexuronidase. We observed that endo-beta-N-acetylhexosaminidase sequentially removed tetrasaccharides from the nonreducing end of oligosaccharides when unrestricted by substrate length, whereas endohexuronidase activity was random and comparatively low. The activities displayed acidic pH optima and were shown by subcellular fractionation to reside in lysosomes and late endosomes. We suggest that these activities represent the known Hyal-1 and endo-beta-glucuronidase enzymes and that these enzymes act in concert to degrade GlcA-rich domains of DS but are less active toward regions containing IdoA.

journal_name

Glycobiology

journal_title

Glycobiology

authors

Nielsen TC,Meikle PJ,Hopwood JJ,Fuller M

doi

10.1093/glycob/cwn097

subject

Has Abstract

pub_date

2008-12-01 00:00:00

pages

1119-28

issue

12

eissn

0959-6658

issn

1460-2423

pii

cwn097

journal_volume

18

pub_type

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