Interaction of cartilage oligomeric matrix protein/thrombospondin 5 with aggrecan

Abstract

Cartilage oligomeric matrix protein/thrombospondin 5 (COMP/TSP5) is a major component of the extracellular matrix (ECM) of the musculoskeletal system. Its importance is underscored by its association with several growth disorders. In this report, we investigated its interaction with aggrecan, a major component of cartilage ECM. We also tested a COMP/TSP5 mutant, designated MUT3 that accounts for 30% of human pseudoachondroplasia cases, to determine if the mutation affects function. Using a solid-phase binding assay, we have shown that COMP/TSP5 can bind aggrecan. This binding was decreased with MUT3, or when COMP/TSP5 was treated with EDTA, indicating the presence of a conformation-dependent aggrecan binding site. Soluble glycosaminoglycans (GAGs) partially inhibited binding, suggesting that the interaction was mediated in part through aggrecan GAG side chains. Using affinity co-electrophoresis, we showed that COMP/TSP5, in its calcium-replete conformation, bound to heparin, chondroitin sulfates, and heparan sulfate; this binding was reduced with EDTA treatment of COMP/TSP5. MUT3 showed weaker binding than calcium-repleted COMP/TSP5. Using recombinant COMP/TSP5 fragments, we found that the "signature domain" could bind to aggrecan, suggesting that this domain can mediate the interaction of COMP/TSP5 and aggrecan. In summary, our data indicate that COMP/TSP5 is an aggrecan-binding protein, and this interaction is regulated by the calcium-sensitive conformation of COMP/TSP5; interaction of COMP with aggrecan can be mediated through the GAG side chains on aggrecan and the "signature domain" of COMP/TSP5. Our results suggest that COMP/TSP5 may function to support matrix interactions in cartilage ECM.

FIGURE 1. Schematic presentation of recombinant COMP/TSP5 fragments

Recombinant fragments of COMP/TSP5 include E4T3C5 that spans the last type 2 repeat (oval modules), the type 3 repeats (rectangles), and the C-terminal domain (globe), and T3C5 that includes the type 3 repeats and the C-terminal domain, and C5, which is composed of just the C-terminal domain. The site of mutation that gives rise to MUT3 (D469del) is also indicated.

FIGURE 2. Binding of COMP/TSP5 to heparin-Sepharose

Serum-free COMP/TSP5-conditioned medium was loaded on to a heparin-Sepharose column. Unbound material was collected as flow-through. After binding, the column was washed with TBS/C containing 0.15 M NaCl. The bound material was eluted with TBS/C containing 0.55 M NaCl.

FIGURE 3. Binding of COMP/TSP5, and MUT3 to aggrecan

The solid-phase binding assay was carried out as described under “Materials and Methods” using antibody against CS. COMP/TSP5 or MUT3 was coated in the presence of calcium. Half of the samples were kept in the calcium-replete conformation while the other half was treated with EDTA before adding aggrecan. Maximal binding of COMP/TSP5 to aggrecan in the presence of calcium ions was set as 100%. Data are expressed as % maximal binding. Shown are the average data of three experiments.

FIGURE 4. Affinity co-electrophoresis of COMP/TSP5 with heparin and chondroitin 6-sulfate

ACE was carried out as described under supplemental data. Proteins were in solutions containing 2 mM CaCl₂, or 5 mM EDTA as indicated in the figure. Concentrations of COMP/TSP5 or MUT3 embedded in each lane are indicated between the gels in nM. Radiolabeled heparin or C6S was loaded into the slot lane at the top of the gel. Where there is an interaction of the protein with the labeled GAGs, the migration of the GAGs is retarded during electrophoresis. The K_d values are calculated as described in the text as a function of the protein concentration and the electrophoretic mobility shift of GAGs in each lane.

FIGURE 5. Competition of GAGs for aggrecan binding to COMP/TSP5

The solid-phase binding assay was carried out as described in the legend to Fig. 3 with COMP/TSP5 coated in the presence of calcium. GAGs at a final concentration of 0.5 mg/ml were incubated with coated COMP/TSP5 before aggrecan was added to the plate. The binding was expressed as % binding of COMP/TSP5 to aggrecan without any GAG competitors.

FIGURE 6. Binding of COMP/TSP5 fragments to aggrecan molecule

The solid-phase binding assay was carried out as described in the legend to Fig. 3 except that antibody against KS was used in this experiment. Proteins were coated in the presence of 2 mM CaCl₂. Shown are the average ±S.D.

FIGURE 7. Competition of GAGs for aggrecan binding to C5 of COMP/ TSP5

The solid-phase binding assay was carried out as described in the legend to Fig. 3 with C5 coated in the presence of calcium at 2.5 μg/ml, which is equivalent in molar concentration to COMP/TSP5 at 7.5 μg/ml. GAGs at a final concentration of 0.5 mg/ml were incubated with coated C5 before aggrecan was added to the plate. The binding was expressed as % binding of aggrecan to C5 without any GAG competitors.