A novel keratan sulphate domain preferentially expressed on the large aggregating proteoglycan from human articular cartilage is recognized by the monoclonal antibody 3D12/H7.
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.
Fischer
D C
DC
Institut für Klinische Chemie und Pathobiochemie, Aachen, Germany.
Haubeck
H D
HD
Eich
K
K
Kolbe-Busch
S
S
Stöcker
G
G
Stuhlsatz
H W
HW
Greiling
H
H
D053674:Aggrecans; D000911:Antibodies, Monoclonal; D002240:Carbohydrate Sequence; D002358:Cartilage, Articular / Q000737:chemistry*; D016326:Extracellular Matrix Proteins; D006026:Glycoside Hydrolases; D006801:Humans; D007120:Immunochemistry; D007632:Keratan Sulfate / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D037181:Lectins, C-Type; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011509:Proteoglycans / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D001616:beta-Galactosidase
C508319:ACAN protein, human; D053674:Aggrecans; D000911:Antibodies, Monoclonal; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D011509:Proteoglycans; D007632:Keratan Sulfate; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D001616:beta-Galactosidase