βig-h3 Interacts Directly with Biglycan and Decorin, Promotes Collagen VI Aggregation, and Participates in Ternary Complexing with These Macromolecules

Betty Reinboth,John Thomas,Eric Hanssen,Mark A Gibson

doi:10.1074/jbc.m511316200

Betty Reinboth, John Thomas + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m511316200

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Abstract

Recombinant human beta ig-h3 was found to bind 125I-labeled small leucine-rich proteoglycans (SLRPs), biglycan, and decorin, in co-immunoprecipitation experiments. In each instance the binding could be blocked by an excess of the unlabeled proteoglycan, confirming the specificity of the interaction. Scatchard analysis showed that biglycan bound beta ig-h3 more avidly than decorin with Kd values estimated as 5.88 x 10(-8) and 1.02 x 10(-7) M, respectively. In reciprocal blocking experiments both proteoglycans inhibited the others binding to beta ig-h3 indicating that they may share the same binding site or that the two binding sites are in close proximity on the beta ig-h3 molecule. Since beta ig-h3 and the SLRPs are known to be associated with the amino-terminal region of collagen VI in tissue microfibrils, the effects of including collagen VI in the incubations were investigated. Co-immunoprecipitation of 125I-labeled biglycan incubated with equimolar mixtures of beta ig-h3 and pepsin-collagen VI was increased 6-fold over beta ig-h3 alone and 3-fold over collagen VI alone. Similar increases were also observed for decorin. The findings indicate that beta ig-h3 participates in a ternary complex with collagen VI and SLRPs. Static light scattering techniques were used to show that beta ig-h3 rapidly forms very high molecular weight complexes with both native and pepsin-collagen VI, either alone or with the SLRPs. Indeed beta ig-h3 was shown to form a complex with collagen VI and biglycan, which appeared to be much more extensive than that formed by beta ig-h3 with collagen VI and decorin or those formed between the collagen and beta ig-h3, biglycan, or decorin alone. Biglycan core protein was shown to inhibit the extent of complexing of beta ig-h3 with native and pepsin-collagen VI suggesting that the glycosaminoglycan side chains of the proteoglycan were important for the formation of the large ternary complexes. Further studies showed that the direct interaction between beta ig-h3 and biglycan and between biglycan and collagen VI were also important for the formation of these complexes. The globular domains of collagen VI also appeared to have an influence on the interaction of the three components. Overall the results indicate that beta ig-h3 can differentially modulate the aggregation of collagen VI with biglycan and decorin. Thus this interplay is likely to be important in tissues such as cornea where such complexes are considered to occur.

Highlights

Transforming growth factor-␤ (TGF-␤)2-inducible gene-h3 (␤ig-h3), is an extracellular matrix protein expressed in a wide variety of tissues including developing nuchal ligament, aorta, lung, kidney, and cartilage; and mature cornea, skin, bladder, and bone [5,6,7,8,9,10,11]
We have shown that r␤ig-h3 directly binds to biglycan and decorin and in turn forms ternary complexes with collagen VI and these small leucinerich proteoglycans (SLRP)
Recent evidence indicates that SLRPs, decorin and biglycan may be complexed with collagen VI in tissues and that these proteoglycans can influence the rate, size, and shape of collagen VI aggregation in vitro [34, 35]

Summary

EXPERIMENTAL PROCEDURES

Materials—Biglycan and decorin were purified from the nuchal ligaments of 230-day-old fetal calves as described previously [36]. Core proteins of biglycan and decorin were prepared by digestion of the 125I-labeled proteoglycans with chondroitinase ABC as described previously [36]. The test protein (1 ␮g of r␤ig-h3 or 2 ␮g of collagen VI) was incubated with 7 ϫ 104 dpm of 125I-labeled biglycan or decorin for 4 h at 37 °C in 50 ␮l of TBS (10 mM Tris buffer, pH 7.4, containing 0.15 M NaCl and protease inhibitors, EDTA (2 mM), benzamidine (1 mM), ⑀-amino-n-caproic acid (1 mM), and phenylmethylsulfonyl fluoride (0.5 mM)) containing 2 ␮g of BSA. Each experiment was commenced by mixing native or pepsin-collagen VI tetramers (120 nM) with r␤ig-h3 (120 nM) and/or a proteoglycan (biglycan or decorin (480 nM)) in TBS, directly in the cell. The precipitated and nonprecipitated protein fractions were analyzed by SDS-PAGE on 8% gels and Coomassie Blue staining

RESULTS

DISCUSSION