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

Abstract

Recombinant human ␤ig-h3 was found to bind 125 I-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 ␤ig-h3 more avidly than decorin with K d values estimated as 5.88 ؋ 10 ؊8 and 1.02 ؋ 10 ؊7 M, respectively. In reciprocal blocking experiments both proteoglycans inhibited the others binding to ␤ig-h3 indicating that they may share the same binding site or that the two binding sites are in close proximity on the ␤ig-h3 molecule. Since ␤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 125 I-labeled biglycan incubated with equimolar mixtures of ␤ig-h3 and pepsin-collagen VI was increased 6-fold over ␤ig-h3 alone and 3-fold over collagen VI alone. Similar increases were also observed for decorin. The findings indicate that ␤ig-h3 participates in a ternary complex with collagen VI and SLRPs. Static light scattering techniques were used to show that ␤ig-h3 rapidly forms very high molecular weight complexes with both native and pepsin-collagen VI, either alone or with the SLRPs. Indeed ␤ig-h3 was shown to form a complex with collagen VI and biglycan, which appeared to be much more extensive than that formed by ␤ig-h3 with collagen VI and decorin or those formed between the collagen and ␤ig-h3, biglycan, or decorin alone. Biglycan core protein was shown to inhibit the extent of complexing of ␤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 ␤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 ␤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.Transforming growth factor-␤ (TGF-␤) 2 -inducible gene-h3 (␤ig-h3) (also known variously as MP78/70 (1, 2), RGD-CAP (3), and keratoepithelin (4)), 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 (5-11). The name ␤ig-h3 stems from its identification and cloning as a major TGF-␤-responsive gene in A549 lung adenocarcinoma cells (12, 13). ␤ig-h3 protein is 76 -78 kDa in size and contains four repeat domains, with homology to the insect protein fasciclin, and 11 cysteine residues most of which ar...

Comparative Proteomic Analysis of Normal and Collagen IX Null Mouse Cartilage Reveals Altered Extracellular Matrix Composition and Novel Components of the Collagen IX Interactome

“…Although the function of Tgfbi is not fully understood, the inverse relationship between Tgfbi expression and chondrocyte maturation suggests an inhibitory effect on hypertrophy and matrix mineralization (40,53). Tgfbi is attached at periodic intervals with collagen VI microfibrils extracted under native conditions (18) and Tgfbi⅐biglycan complexes enhance the formation of collagen VI networks in vitro (54), supporting a role for Tgfbi in matrix organization. In addition, Tgfbi interacts with fibronectin and is thought to facilitate cell-matrix interactions via an integrin-binding RGD motif (55).…”

Transforming growth factor-β–induced protein (TGFBIp/β ig-h3) activates platelets and promotes thrombogenesis

“…TGFBIp is also known to bind proteoglycans such as small leucine-rich biglycan and decorins. 12,32 The presence of proteoglycans on human platelets 33 suggests that proteoglycans on platelets could be the receptor of TGFBIp.…”

BMP2-induced gene profiling in dental epithelial cell line

“…It is expressed in several tissues, including lung, kidney, skin, bone, and cartilage (25,50,51). βig -h3 has been shown to bind not only to ECM proteins such as FN, laminin, and collagens, but also to cell surface molecules such as αvβ5 integrin, decorin, and biglycan (52,53). To confirm βig -h3 expression, we performed immunohistochemical analysis using a commercially available antibody.…”