The sulphation pattern in chondroitin sulphate chains investigated by chondroitinase ABC and ACII digestion and reactivity with monoclonal antibodies

Hardingham, Timothy E.; Fosang, Amanda J.; Hey, Nina J.; Hazell, Paula K.; Kee, Wai Jing; Ewins, R.J.F.

doi:10.1016/s0008-6215(00)90982-0

Cited by 26 publications

(13 citation statements)

References 16 publications

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“…Sandwich ELISAs confirmed that some of the lubricin present in synovial fluid was decorated with CS or KS that was reactive with the MAbs 3-B-3 and 5-D-4, respectively. Aggrecan has also been reported to be decorated with the MAb 3-B-3 CS structure [24] and KS [25] and was found to co-purify with lubricin in many of the DEAE-enriched fractions analyzed in this study. Lubricin has been shown to bind to various extracellular matrix molecules including aggrecan in solid-phase assays [26].…”

Section: Discussionsupporting

confidence: 53%

Not All Lubricin Isoforms Are Substituted with a Glycosaminoglycan Chain

Lord

Estrella

Chuang

et al. 2011

Connective Tissue Research

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Lubricin, also referred to as superficial zone protein, has been reported to be a proteoglycan. However, the structure of its glycosaminoglycan chain has not been well characterized, and this study was undertaken to investigate the structure of the glycosaminoglycan chain that decorated lubricin in human synovial fluid to provide insight into its biological role. Lubricin was detected as a major band at approximately 360 kDa which co-migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a chondroitin sulfate (CS)-containing proteoglycan that was detected by both monoclonal antibodies (MAb) 2-B-6 and MAb 3-B-3 after chondroitinase ABC treatment and keratan sulfate (KS) that was detected by MAb 5-D-4. Further analysis of lubricin-containing fractions that eluted from an anion exchange column indicated that the major population of lubricin could be separated from the CS and KS stubs which indicated that this fraction of lubricin was not decorated with glycosaminoglycan chain and was the glycoprotein form of lubricin. Lubricin present in fractions that also contained CS was found to be decorated with CS structures which were reactive with MAb 3-B-3 after chondroitinase ABC digestion using a sandwich enzyme-linked immunosorbent assay approach. Aggrecan was not found to form complexes with lubricin in synovial fluid which confirmed that the MAb 3-B-3 CS and MAb 5-D-4 KS structures decorated lubricin. These data demonstrate that lubricin present in human synovial fluid was a heterogeneous population with both glycoprotein and proteoglycan forms.

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Section: Discussionsupporting

confidence: 53%

Not All Lubricin Isoforms Are Substituted with a Glycosaminoglycan Chain

Lord

Estrella

Chuang

et al. 2011

Connective Tissue Research

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“…On the other hand, the conventional preparation of chondroitinase ABC has been claimed to have a non-random endolytic action (27). In contrast, the same preparation has also been reported to release di-, tetra-, and hexasaccharides from the non-reducing ends of CS polysaccharides in an exolytic fashion (32). Thus, it had become necessary to define its endolytic or exolytic actions to apply it as an analytical tool.…”

Section: -Mhz 1 H Nmr Spectroscopy-fractionsmentioning

confidence: 99%

Characteristic Hexasaccharide Sequences in Octasaccharides Derived from Shark Cartilage Chondroitin Sulfate D with a Neurite Outgrowth Promoting Activity

Nadanaka¹,

Clement²,

Masayama³

et al. 1998

Journal of Biological Chemistry

154

132

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Chondroitin sulfate (CS)1 chains occur in animal tissues usually as proteoglycans (PGs), in which these polysaccharide chains are covalently attached to a core protein. CS-PGs are present on cell surfaces and in the extracellular matrix of most tissues and are involved in many biological activities. However, the structure of CS chains is only poorly understood. The backbone structure of CS chains consists of repeating disaccharide units, each containing a glucuronic acid (GlcUA) residue and an N-acetylgalactosamine (GalNAc) residue. Diversity in estersulfation of both saccharide residues results in structural variability of CS chains, which is assumed to be the basis for its functional diversity. Immunological studies using monoclonal antibodies (mAbs) have revealed that the sulfation profile of CS chains changes with concomitant specific spatio-temporal patterns in various tissues, suggesting that CS isoforms differing in sulfation position and degree perform distinct functions in development (1). Disaccharide units with various sulfation profiles are non-randomly distributed in the CS chains as detected by mAbs (2).It had been shown previously that the CS-PG named DSD-1-PG from developing mouse brain is located on the surface of immature glial cells in the central nervous system and promotes neurite outgrowth (3). The neurite outgrowth stimulating capacity of DSD-1-PG was strongly reduced by mAb 473HD, which recognizes a glycosaminoglycan epitope present in DSD-1-PG. Surprisingly, shark cartilage CS-C inhibited the interactions between the mAb 473HD and the DSD-1 epitope *

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“…There is a commercially available chondroitin sulfate ABC lyase ("chondroitinase ABC" from P. vulgaris, the product of Seikagaku Corp.) which has found wide applications including the quantification of chondroitin sulfate and dermatan sulfate (12), the structural analysis of the carbohydrate moiety of proteoglycans (13)(14)(15)(16), the preparation of core proteins from proteoglycans (13,17), the formation of antigenic epitopes to prepare anti-proteoglycan monoclonal antibodies (18), and the medical use for intervertebral disc protrusion (19,20). Because of the increased use of the enzyme, more attention has been directed in our laboratories toward understanding its purity, specificity, and mode of action.…”

mentioning

confidence: 99%

Two Distinct Chondroitin Sulfate ABC Lyases

Hamai

Hashimoto

Mochizuki

et al. 1997

Journal of Biological Chemistry

178

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Crude enzyme obtained from chondroitin sulfate-induced Proteus vulgaris NCTC 4636 has been fractionated into 1) an endoeliminase capable of depolymerizing chondroitin sulfate and related polysaccharides to produce, as end products, a mixture of ⌬ 4 -unsaturated tetra-and disaccharides and 2) an exoeliminase preferentially acting on chondroitin sulfate tetra-and hexasaccharides to yield the respective disaccharides. Isolation of the two enzymes was achieved by a simple two-step procedure: extracting the enzymes from intact P. vulgaris cells with a buffer solution of nonionic surfactant and then treating the extract by cation-exchange chromatography. Each of the enzymes thus prepared was apparently homogeneous as assessed by SDS-polyacrylamide gel electrophoresis and readily crystallized from polyethylene glycol solutions. Both enzymes acted on various substrates such as chondroitin sulfate, chondroitin sulfate proteoglycan, and dermatan sulfate at high, but significantly different, initial rates. They also attacked hyaluronan but at far lower rates and were inactive to keratan sulfate, heparan sulfate, and heparin. Our results show that the known ability of the conventional enzyme called "chondroitinase ABC" to catalyze the complete depolymerization of chondroitin sulfates to unsaturated disaccharides may actually result from the combination reactions by endoeliminase (chondroitin sulfate ABC endolyase) and exoeliminase (chondroitin sulfate ABC exolyase).Chondroitin sulfate ABC lyase (EC 4.2.2.4) was first purified from extracts of Proteus vulgaris NCTC 4636 adapted to chondroitin 6-sulfate (1). It is believed to be an endoeliminase that splits ␤1,4-galactosaminidic bonds between N-acetylgalactosamine and either D-glucuronic acid or L-iduronic acid and degrades, therefore, a variety of glycosaminoglycans of the chondroitin sulfate and dermatan sulfate type to the respective unsaturated disaccharides. Using this enzyme, both chondroitin sulfates and dermatan sulfates were shown to contain, in addition to predominant 4-or 6-sulfated disaccharide residues, a smaller proportion of nonsulfated or disulfated disaccharide residues, or both (2). A variety of studies have since shown that the proportion of these disaccharide residues varies greatly with species and anatomical sites, during development and aging, and in pathology (3-11 among others). Many of these data suggest that variation in the carbohydrate sequence and sulfation pattern may be used to specify the functional properties of chondroitin sulfate and dermatan sulfate proteoglycans.There is a commercially available chondroitin sulfate ABC lyase ("chondroitinase ABC" from P. vulgaris, the product of Seikagaku Corp.) which has found wide applications including the quantification of chondroitin sulfate and dermatan sulfate (12), the structural analysis of the carbohydrate moiety of proteoglycans (13-16), the preparation of core proteins from proteoglycans (13, 17), the formation of antigenic epitopes to prepare anti-proteoglycan monoclonal antibodies (18), and ...

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The sulphation pattern in chondroitin sulphate chains investigated by chondroitinase ABC and ACII digestion and reactivity with monoclonal antibodies

Cited by 26 publications

References 16 publications

Not All Lubricin Isoforms Are Substituted with a Glycosaminoglycan Chain

Not All Lubricin Isoforms Are Substituted with a Glycosaminoglycan Chain

Characteristic Hexasaccharide Sequences in Octasaccharides Derived from Shark Cartilage Chondroitin Sulfate D with a Neurite Outgrowth Promoting Activity

Two Distinct Chondroitin Sulfate ABC Lyases

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