Comparison of the activity of two chondroitin AC lyases on dermatan sulfate

Gu, Kenan; Liu, Jian; Pervin, Azra; Linhardt, Robert J.

doi:10.1016/0008-6215(83)85014-9

Cited by 36 publications

(25 citation statements)

References 13 publications

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“…51 In contrast, chondroitinase B only cleaves β-1,4-galactosaminid-ic bonds between 4-O-sulfoGalNAc and IdoA residues, acting exclusively on CS-B. 50,51 The sequential use of these chondroitinases followed by PAGE analysis confirmed that the bikunin was a CS GAG, containing no IdoA residues. The disaccharide products of exhaustive depolymerization of bikunin GAG with chondroitinase ABC and endolytic chondroitinase AC were analyzed by RP-IP-HPLC using postcolumn fluorescence detection.…”

Section: Bikunin Gag Mixturementioning

confidence: 99%

Structural Analysis of Bikunin Glycosaminoglycan

et al. 2008

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The structure of an intact glycosaminoglycan (GAG) chain of the bikunin proteoglycan (PG) was analyzed using a combined top-down and bottom-up sequencing strategy. PGs are proteins with one or more linear, high-molecular weight, sulfated GAG polysaccharides O-linked to serine or threonine residues. GAGs are often responsible for the biological functions of PGs, and subtle variations in the GAG structure have pronounced physiological effects. Bikunin is a serine protease inhibitor found in human amniotic fluid, plasma, and urine. Bikunin is posttranslationally modified with a chondroitin sulfate (CS) chain, O-linked to a serine residue of the core protein.Recent studies have shown that the CS chain of bikunin plays an important role in the physiological and pathological functions of this PG. While no PG or GAG has yet been sequenced, bikunin, the least complex PG, offers a compelling target. Electrospray ionization Fourier transform-ion cyclotron resonance mass spectrometry (ESI FTICR-MS) permitted the identification of several major components in the GAG mixture having molecular masses in a range of 5505-7102 Da. This is the first report of a mass spectrum of an intact GAG component of a PG. FTICR-MS analysis of a size-uniform fraction of bikunin GAG mixture obtained by preparative polyacrylamide gel electrophoresis, allowed the determination of chain length and number of sulfo groups in the intact GAGs.

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Section: Bikunin Gag Mixturementioning

confidence: 99%

Structural Analysis of Bikunin Glycosaminoglycan

et al. 2008

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“…1B, lanes 2 and 3), whereas endogenous polysaccharide was sensitive only to chondroitin ABC lyase (Seikagaku), affording unsulfated and monosulfated disaccharide products that could not be visualized with Alcian blue staining (Fig. 1B, lanes 4-6) [14].…”

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Microscale isolation and analysis of heparin from plasma using an anion-exchange spin column

Zhang¹,

Sun²,

Muñoz³

et al. 2006

Analytical Biochemistry

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“…3 -5 show the presence of residual products larger than disaccharides. This may be the result of resistant sequences [37] or the reduced ability of lyases to act on low molecular weight substrates [38]. It should also be noted that, while the amount of these larger products looks substantial, quantitative interpretation of these data is complicated by the inability to visualize the disaccharide product by staining [37].…”

Section: Product Profiles Of Enzymatic Depolymerizationmentioning

confidence: 99%

“…The mode of action of this enzyme on chondroitin substrates has been established as random endolytic [11]. Dermatan sulfate has been demonstrated to inhibit F. heparinum AC lyase (K i f 0.2 AM) with respect to chondroitin-6-sulfate as substrate [24].…”

Section: Introductionmentioning

confidence: 99%

Role of arginine 292 in the catalytic activity of chondroitin AC lyase from Flavobacterium heparinum

Capila

Rethwisch

et al. 2002

Biochimica et Biophysica Acta (BBA) - Protein Structure and Mol

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Chondroitin AC lyase (chondroitinase EC 4.2.2.5), an eliminase from Flavobacterium heparinum, cleaves chondroitin sulfate glycosaminoglycans (GAGs) at 1,4 glycosidic linkages between N-acetylgalactosamine and glucuronic acid residues. Cleavage occurs through h-elimination in a random endolytic action pattern. Crystal structures of chondroitin AC lyase (wild type) complexed with oligosaccharides reveal a binding site within a narrow and shallow protein channel, suggesting several amino acids as candidates for the active site residues. Site-specific mutagenesis studies on residues within the active-site tunnel revealed that only the Arg to Ala 292 mutation (R292A) retained activity. Furthermore, structural data suggested that R292 was primarily involved in recognition of N-acetyl or O-sulfo moieties of galactosamine residues and did not directly participate in catalysis. The current study demonstrates that the R292A mutation affords f 10-fold higher K m values but no significant change in V max , consistent with hypothesis that R292 is involved in binding the O-sulfo moiety of the saccharide residues. Change in chondroitin sulfate viscosity, as a function of its enzymatic cleavage, affords a shallower concave curve for the R292A mutant, suggesting its action pattern is neither purely random endolytic nor purely random exolytic. Product studies using gel electrophoresis confirm the altered action pattern of this mutant. Thus, these data suggest that the R292A mutation effectively reduces binding affinity, making it possible for the oligosaccharide chain, still bound after initial endolytic cleavage, to slide through the tunnel to the catalytic site for subsequent, processive, step-wise, exolytic cleavage. D

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Comparison of the activity of two chondroitin AC lyases on dermatan sulfate

Cited by 36 publications

References 13 publications

Structural Analysis of Bikunin Glycosaminoglycan

Structural Analysis of Bikunin Glycosaminoglycan

Microscale isolation and analysis of heparin from plasma using an anion-exchange spin column

Role of arginine 292 in the catalytic activity of chondroitin AC lyase from Flavobacterium heparinum

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