Abstract
Versican/PG-M is a large chondroitin sulfate proteoglycan of the extracellular matrix with a common domain structure to aggrecan and is present in cartilage at low levels. Here, we characterized cartilage versican during development and growth. Immunostaining showed that versican was mainly localized in the interterritorial zone of the articular surface at 2 weeks in mice, whereas aggrecan was in the pericellular zone of prehypertrophic and hypertrophic cells of the growth plate. Although its transcription level rapidly diminished during growth, versican remained in the articular cartilage. Biochemical analysis of normal articular cartilage and aggrecan-null cartilage from cmd (cartilage matrix deficiency)/cmd mice revealed that versican was present as a proteoglycan aggregate with both link protein and hyaluronan. Chondroitin sulfate chains of versican digested with chondroitinase ABC contained 71% nonsulfated and 28% 4-sulfated unsaturated disaccharides, whereas those of aggrecan contained 25% nonsulfated and 70% 4-sulfated. Link protein overexpression in chondrocytic N1511 cells at the early stage of differentiation, in which versican is expressed, enhanced versican deposition in the matrix and prevented subsequent aggrecan deposition. These results suggest that versican is present as an aggregate distinct from the aggrecan aggregate and may play specific roles in the articular surface.
Highlights
Versican/PG-M is a large chondroitin sulfate proteoglycan of the extracellular matrix with a common domain structure to aggrecan and is present in cartilage at low levels
Distribution of Versican, HA, and link protein (LP) in Cartilage—Initially, we investigated the presence and localization of versican, aggrecan, LP, and HA in the growth plate and articular cartilage of 2-week-old mice by immunostaining and with biotinylated hyaluronan-binding protein
Versican is localized in the interterritorial zone of the articular surface, whereas aggrecan is mainly in the territorial zone of hypertrophic cells
Summary
Immunostaining and Detection of HA by HABP in Tissues—Aggrecan-null cartilage matrix deficiency (cmd) lines [22] were maintained in our animal facility. The solution in the tube was fractionated into six tubes, A1-A6, from the bottom To identify these molecules individually, the tissue was extracted using 4 M GdnHCl, Tris-HCl, pH 8.0, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, and 0.36 mM pepstatin A, and the extract was applied to dissociative ultracentrifugation. These molecules were monitored by dot blot and immunoblot analyses. For quantification of versican and aggrecan in the articular surface area, the surface area at day 14 was carefully excised and extracted using ten volumes of 4 M GdnHCl, 50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, and 0.36 mM pepstatin A for 24 h. We treated the above eluate with hyaluronidase from Streptomyces hyalurolyticus (Seikagaku), filtered and analyzed, and confirmed that hyaluronan disaccharide was negligible in the samples
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