Studies of a monoclonal antibody to skeletal keratan sulphate. Importance of antibody valency.
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ABSTRACT: A mouse monoclonal antibody (AN9P1) to keratan sulphate is described. In a competitive-inhibition solution-phase radioimmunoassay employing 125I-labelled intact proteoglycan, it reacts preferentially with keratan sulphate bound to the core protein of adult human articular-cartilage proteoglycan and to a much lesser degree with keratan sulphate purified from this proteoglycan. Proteolytic cleavage of the proteoglycan by pepsin and trypsin has little effect on antibody binding, but treatment with papain decreases binding considerably and more than does treatment with keratanase. An even greater decrease in binding is observed after treatment with alkaline borohydride. A comparison of binding of antibody AN9P1 with that of another previously described monoclonal antibody, 1/20/5-D-4, to keratan sulphate [Caterson, Christner & Baker (1983) J. Biol. Chem. 258, 8848-8854] revealed similar binding characteristics, both showing much diminished binding after papain digestion of proteoglycan and even less with purified skeletal keratan sulphate. Removal of the Fc piece of antibody AN9P1 had no significant effect on the differential binding of divalent F(ab')2 fragment to proteoglycan, to papain-digested proteoglycan and to keratan sulphate, although there was a small decrease in binding to papain-digested proteoglycan. Conversion of the antibody into univalent Fab fragment with removal of the Fc piece resulted in diminished binding to proteoglycan, compared with that observed with IgG, and in enhanced binding to free keratan sulphate and to papain-digested proteoglycan. These results suggest that close proximity of keratan sulphate chains on the core protein of proteoglycans favours preferential reactivity of bivalent antibody with these species through cross-bridging of chains by antibody. Conversely, much decreased binding to keratan sulphate on proteoglycan core-protein fragments and to free keratan sulphate results from a lack of close proximity of keratan sulphate. By using univalent Fab fragment in these assays these differences in binding are minimized by preventing cross-bridging and thereby enhancing detection of smaller fragments without sacrificing too much sensitivity of detection of larger proteoglycan species. The persistent preferential binding of Fab fragment to proteoglycan is probably in part the result of the increased epitope density in the intact molecule compared with keratan sulphate in a more disperse form.
Project description:Monoclonal antibodies (mAbs) were prepared against aggrecan which has been isolated from human articular cartilage and purified by several chromatographic steps. One of these mAbs, the aggrecan-specific mAb 3D12/H7, was selected for further characterization. The data presented indicate that this mAb recognizes a novel domain of keratan sulphate chains from aggrecan: (1) immunochemical staining of aggrecan is abolished by treatment with keratanase/keratanase II, but not with keratanase or chondroitin sulphate lyase AC/ABC; (2) after chemical deglycosylation of aggrecan no staining of the core-protein was observed; (3) different immunochemical reactivity was observed against keratan sulphates from articular cartilage, intervertebral disc and cornea for the mAbs 3D12/H7 and 5D4. For further characterization of the epitope, reduced and 3H-labelled keratan sulphate chains were prepared. In an IEF-gel-shift assay it was shown that the 3H-labelled oligosaccharides obtained after keratanase digestion of reduced and 3H-labelled keratan sulphate chains were recognized by the mAb 3D12/H7. Thus it can be concluded that the mAb 3D12/H7 recognizes an epitope in the linkage region present in, at least some, keratan sulphate chains of the large aggregating proteoglycan from human articular cartilage. Moreover, this domain seems to be expressed preferentially on those keratan sulphate chains which occur in the chondroitin sulphate-rich region of aggrecan, since the antibody does not recognize the keratan sulphate-rich region obtained after combined chondroitinase AC/ABC and trypsin digestion of aggrecan.
Project description:Peptido-keratan sulphate fragments were isolated from the nucleus pulposus of bovine intervertebral discs (2-year-old animals) after digestion with chondroitin ABC lyase followed by digestion with diphenylcarbamoyl chloride-treated trypsin of A1D1 proteoglycans and gel-permeation chromatography on Sepharose CL-6B. The peptido-keratan sulphate fragments were subjected to alkaline borohydride reduction. The reduced chains were treated with keratanase in the presence of the sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, and the digest was subjected to alkaline borohydride reduction. This produced oligosaccharides with galactitol at their reducing ends. This reduced digest was chromatographed on a Nucleosil 5 SB anion-exchange column and individual oligosaccharides were isolated. One of these was shown by 600 MHz 1H-n.m.r. spectroscopy to have the following structure: NeuAc alpha 2-6Gal beta 1-4GlcNAc(6-SO4)beta 1-3Gal-ol The structure of this oligosaccharide shows that keratan sulphate chains from bovine intervertebral disc have non-reducing termini with N-acetylneuraminic acid linked alpha(2----6) as well as alpha(2----3) to an unsulphated galactose.
Project description:The absence of keratan sulphate synthesis from skeletal tissues of young and mature mice and rats has been confirmed by (1) analysis of specific enzyme degradation products of newly synthesized glycosaminoglycans, and (2) immunohistochemistry and radioimmunoassay using a monoclonal antibody directed against keratan sulphate. Approx. 98% of the [35S]glycosaminoglycans synthesized in vivo by mouse and rat costal cartilage, and all of those of lumbar disc, are chondroitin sulphate. The remainder in costal cartilage were identified as heparan sulphate in mature rats. In contrast, [35S]glycosaminoglycans synthesized by cornea of both species comprised both chondroitin sulphate and keratan sulphate. In mice keratan sulphate accounted for 12-25% and in rats 40-50% of the total [35S]glycosaminoglycans, depending on the age of the animal. Experiments in vitro with organ culture of cartilage and cornea confirm these results. Absence of keratan sulphate from mouse costal cartilage and lumbar disc D1-proteoglycans was corroborated by inhibition radioimmunoassay with the monoclonal antibody MZ15 and by lack of staining for keratan sulphate in indirect immunofluorescence studies using the same antibody.
Project description:Keratan sulphate was extracted from a shark/whale cartilage preparation and examined by 400 MHz 1H- and 100 MHz 13C-n.m.r. spectroscopy. Assignment of the majority of the resonances was facilitated by two-dimensional 13C-1H correlation by using a modified COLOC procedure and a COSY-45 experiment. The spectra are consistent with an N-acetyl-lactosamine repeating unit that is predominantly sulphated at C-6 of both galactose and N-acetylglucosamine. Gel chromatography of a keratanase digest of the shark keratan sulphate confirmed the high degree of galactose sulphation.
Project description:Keratan sulphate and chondroitin sulphate (KS and CS) in the 2-fold helical configurations that are prevalent in solution are of very similar tacticity. The chiral centres, anionic sites and hydrophobic patches are in identical conformations. Only the position of the acetamido group varies from CS to KS, but part of its intramolecular H-bonding potential in CS is retained in KS. The formation of tertiary aggregates, observed in vitro and in tissues, is explicable on these bases. The proposal that KS may be a functional substitute for CS [Scott & Haigh (1988) J. Anat. 158, 95-108] under low-O2 conditions is relevant.
Project description:Peptido-keratan sulphate fragments were isolated from the nucleus pulposus of bovine intervertebral discs (6-year-old animals) after chondroitin ABC lyase digestion followed by digestion of A1D1 proteoglycans by diphenylcarbamoyl chloride-treated trypsin and gel-permeation chromatography on Sepharose CL-6B. Treatment of these peptido-keratan sulphate fragments with alkaline NaB3H4 yielded keratan sulphate chains with [3H]galactosaminitol end-labels, and these chains were further purified by gel-permeation chromatography on Sephadex G-50 and ion-exchange chromatography on a Pharmacia Mono-Q column in order to exclude any contamination with O-linked oligosaccharides. The chains were then treated with keratanase, and the digest was chromatographed on a Bio-Gel P-4 column followed by anion-exchange chromatography on a Nucleosil 5 SB column. Two oligosaccharides, each representing 18% of the recovered radiolabel, were examined by 500 MHz 1H-n.m.r. spectroscopy, and shown to have the following structures: [formula: see text] The structure of oligosaccharide (I) confirms the N-acetylneuraminylgalactose substitution at position 3 of N-acetylgalactosamine in the keratan sulphate-protein linkage region found by Hopwood & Robinson [(1974) Biochem. J. 141, 57-69] but additionally shows the presence of a 6-sulphated N-acetylglucosamine. Electron micro-probe analysis specifically confirmed the presence of sulphur in this sample. This sulphate ester group differentiates the keratan sulphate linkage region from similar structures derived from O-linked oligosaccharides [Lohmander, De Luca, Nilsson, Hascall, Caputo, Kimura & Heinegård (1980) J. Biol. Chem. 255, 6084-6091].
Project description:Keratan sulphate chains were isolated from bovine tracheal ring cartilage (15-18-month-old animals) after papain digestion of the tissue followed by ethanol fractionation, chondroitinase ABC digestion and alkaline borohydride reduction. The keratan sulphate chains were further purified by anion-exchange chromatography on a Pharmacia Mono-Q column in order to remove any contaminating chondroitin sulphate and O-linked oligosaccharides. The chains were then treated with keratanase and the digest was subjected to alkaline borohydride reduction, producing oligosaccharides with galactitol at their reducing ends. The reduced digest was chromatographed on a Nucleosil 5 SB anion-exchange column and individual oligosaccharides were isolated. One of these, oligosaccharide (I), was shown by 500 MHz 1H-n.m.r. spectroscopy to have the following structure: NeuAc alpha 2-3Gal beta 1-4GlcNAc(6SO4) beta 1-3Gal-ol (I) The structure of this oligosaccharide shows that keratan sulphate chains from bovine tracheal ring cartilage may be terminated with N-acetylneuraminic acid linked alpha (2-3) to an unsulphated galactose. Keratan sulphate chains were also isolated from bovine femoral head cartilage (15-18-month-old animals) using an identical protocol, but with keratanase which was subsequently shown to have sialidase activity. This yielded oligosaccharide (II), the unsialyated version of (I): Gal beta 1-4GlcNAc(6SO4) beta 1-3Gal-ol (II).
Project description:A monoclonal antibody of the immunoglobulin M class was produced against mouse kidney ornithine decarboxylase. Screening for the antibody was carried out using alpha-difluoromethyl[5-3H]ornithine-labelled ornithine decarboxylase. The antibody reacted with this antigen and with native ornithine decarboxylase. The antibody attached to Sepharose could be used to form an immunoaffinity column that retained mammalian ornithine decarboxylase. The active enzyme could then be eluted in a highly purified form by 1.0M-sodium thiocyanate. The monoclonal antibody could also be used to precipitate labelled ornithine decarboxylase from homogenates of kidneys from androgen-treated mice given [35S]methionine. Only one band, corresponding to Mr of about 55000, was observed. The extensive labelling of this band is consistent with the rapid turnover of ornithine decarboxylase protein, since this enzyme represents only about 1 part in 10000 of the cytosolic protein.
Project description:Keratan sulphate was isolated from bovine intervertebral disc and bovine nasal septum after hydrolysis with proteinases and treatment with dilute alkali. Each preparation was found to contain, per keratan sulphate chain: (a) 1 residue of mannose; (b) 3 residues of N-acetylneuraminic acid (2 residues after alkali treatment); (c) 1 residue of N-acetylgalactosamine (lost after alkali treatment); (d) 1 residue or less of fucose. N-Acetyl-neuraminic acid residues were at non-reducing termini and were bonded to keratan sulphate through galactose residues. Evidence is presented for two different types of linkage between skeletal keratan sulphate and protein. Consideration of molecular parameters and compositions leads to a proposed structure for keratan sulphate-protein as found in skeletal proteoglycans.
Project description:Cocaine abuse remains prevalent worldwide and continues to be a major health concern; nonetheless, there is no effective therapy. Immunopharmacotherapy has emerged as a promising treatment strategy by which anti-cocaine antibodies bind to the drug blunting its effects. Previous passive immunization studies using our human monoclonal antibody, GNCgzk, resulted in protection against cocaine overdose and acute toxicity. To further realize the clinical potential of this antibody, a recombinant IgG form of the antibody has been produced in mammalian cells. This antibody displayed a high binding affinity for cocaine (low nanomolar) in line with the superior attributes of the GNCgzk antibody and reduced cocaine-induced ataxia in a cocaine overdose model.