A novel keratan sulphate domain preferentially expressed on the large aggregating proteoglycan from human articular cartilage is recognized by the monoclonal antibody 3D12/H7

Abstract

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.

Immunohistochemical Classification of Primary and Recurrent Macular Corneal Dystrophy in Germany: Subclassification of Immunophenotype I A Using a Novel Keratan Sulfate Antibody

“…Monoclonal antibody 5-D-4 (Medac, Hamburg, Germany) recognizes disulfated domains of keratan sulfate chains both of corneal and skeletal KS (Fischer et al, 1996;Caterson et al, 1983). In contrast, mAb 3D12/H7 recognizes an epitope localized in the binding region of skeletal type KS-chains to the core protein.…”

“…In contrast, mAb 3D12/H7 recognizes an epitope localized in the binding region of skeletal type KS-chains to the core protein. Supernatant from the clone 3D12/H7 was diluted 1 : 2 and mAb 5-D-4 (delivered as ascites) was diluted 1 : 100 (Fischer et al, 1996). Normal human cartilage (knee joint) used as a positive control, showed positive staining of both chondrocytes and cartilage extracellular matrix using both monoclonal antibodies (5-D-4 more intense than 3D12/H7).…”

“…Determination of keratan sulfate concentration in serum was performed using the monoclonal antibody 5-D-4 (Medac) in an antigen-inhibition assay as described previously (Fischer et al, 1994(Fischer et al, , 1996Ku È chle et al, 1999;Cursiefen et al, 2000b).…”

“…The established mAb 5-D-4 was used to evaluate the distribution of the different immunophenotypes in German patients and to evaluate whether the newly discovered immunophenotype I A also occurs outside Saudi Arabia. In addition, a new mAb (3D12/H7; Fischer et al, 1996) was used which in contrast to mAb 5-D-4 recognizes an epitope localized in the binding region of skeletal type KS-chains to the core protein. This may allow further subclassi®cation of MCD immunophenotypes and analysis of the binding region between KS-chains and proteoglycan core protein.…”

Ocular findings in Fryns syndrome

“…In addition, sulfated keratan sulfate was detectable in corneal endothelial cells. This represents a possible marker of normal corneal endothelial cell function (Kü chle et al 1999;Fischer et al 1996;Hyldahl et al 1986). Nevertheless, the ultrastructural abnormalities of the Descemet's membrane detected in this patient (lack of banded collagen fibrils, type VIII collagen; Marshall et al 1993) could point to a dysfunction of corneal endothelial extracellular matrix production.…”