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...

The interactions of the dermatan sulfate proteoglycans biglycan and decorin have been investigated with the elastic fiber components, tropoelastin, fibrillin-containing microfibrils, and microfibril-associated glycoproteins (MAGP) 1 and 2. Both proteoglycans were found to bind tropoelastin and fibrillin-containing microfibrils but not MAGPs 1 and 2 in solid phase binding assays. The specificity of the binding of biglycan and decorin to tropoelastin was confirmed by co-immunoprecipitation experiments and by the blocking of the interactions with elastin-derived peptides. Isolated core proteins from biglycan and decorin bound to tropoelastin more strongly than the intact proteoglycans, and there were no differences in the tropoelastin binding characteristics of distinct glucuronate-rich and iduronate-rich glycoforms of biglycan. These findings indicated that the binding sites were contained in the protein cores of the proteoglycans rather than the glycosaminoglycan side chains. Scatchard analysis showed that biglycan bound more avidly than decorin to tropoelastin with K d values estimated as 1.95 ؋ 10 ؊7 M and 5.3 ؋ 10 ؊7 M, respectively. In blocking experiments each proteoglycan showed extensive inhibition of binding of the other to tropoelastin but was most effective at blocking its own binding. This result suggested that biglycan and decorin had closely spaced but distinct binding sites on tropoelastin. Addition of the elastin-binding protein MAGP-1 to the assays enhanced the binding of biglycan to tropoelastin but had no effect on the decorin-tropoelastin interaction. Co-immunoprecipitation experiments showed that MAGP-1 interacted with biglycan but not decorin in the solution phase. The results indicated that biglycan specifically formed a ternary complex with tropoelastin and MAGP-1. Overall the study supports the concept that biglycan may have a specific role in the elastinogenic phase of elastic fiber formation.

Transforming growth factor-␤ induced gene-h3 (␤ig-h3) was found to co-purify with collagen VI microfibrils, extracted from developing fetal ligament, after equilibrium density gradient centrifugation under both nondenaturing and denaturing conditions. Analysis of the collagen VI fraction from the non-denaturing gradient by gel electrophoresis under non-reducing conditions revealed the present of a single high molecular weight band that immunostained for both collagen VI and ␤ig-h3. When the fraction was analyzed under reducing conditions, collagen VI ␣ chains and ␤ig-h3 were the only species evident. The results indicated that ␤ig-h3 is associated with collagen VI in tissues by reducible covalent bonding, presumably disulfide bridges. Rotary shadowing and immunogold staining of the collagen VI microfibrils and isolated tetramers indicated that ␤ig-h3 was specifically and periodically associated with the double-beaded region of many of the microfibrils and that this covalent binding site was located in or near the amino-terminal globular domain of the collagen VI molecule. Using solid phase and co-immunoprecipitation assays, recombinant ␤ig-h3 was found to bind both native and pepsin-treated collagen VI but not individual pepsin-collagen VI ␣ chains. Blocking experiments indicated that the major in vitro ␤ig-h3 binding site was located in the pepsin-resistant region of collagen VI. In contrast to the tissue situation, the in vitro interaction had the characteristics of a reversible non-covalent interaction, and the K d was measured as 1.63 ؋ 10Rotary shadowing of immunogold-labeled complexes of recombinant ␤ig-h3 and pepsin-collagen VI indicated that the in vitro ␤ig-h3 binding site was located close to the amino-terminal end of the collagen VI triple helix. The evidence indicates that collagen VI may contain distinct covalent and non-covalent binding sites for ␤ig-h3, although the possibility that both interactions use the same binding region is discussed. Overall the study supports the concept that ␤ig-h3 is extensively associated with collagen VI in some tissues and that it plays an important modulating role in collagen VI microfibril function.Transforming growth factor-␤-inducible gene h3 (␤ig-h3) 1 was first cloned from A549 lung adenocarcinoma cells that had been stimulated with transforming growth factor-␤1 (1, 2). ␤ig-h3 is also known variously as MP78/70 (3, 4), RGD-CAP (5), and keratoepithelin (6). ␤ig-h3 has been established as a extracellular matrix protein in a wide variety of tissues including developing nuchal ligament, aorta, lung, and kidney and mature cornea, skin, bladder, and bone (7-12). ␤ig-h3 is a 76 -78-kDa protein containing 4 repeat regions, with homology to the insect protein fasciclin, and 11 cysteine residues, most of which are clustered in a distinct amino-terminal region. The ␤ig-h3 molecule appears to undergo partial processing at the carboxylterminal end to yield a 68 -70 kDa isoform (2). ␤ig-h3 has been shown to bind in vitro to a number of other matrix components including fibro...

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The glycosaminoglycan chondroitin sulfate is significantly increased in the peritumoral stroma of prostate tumors compared with normal stroma and is an independent predictor of prostate-specific antigen (PSA) relapse following radical prostatectomy. In this study, we determined whether specific alterations in the sulfation pattern of glycosaminoglycan chains in clinically organ-confined prostate cancer are associated with PSA relapse. Immunoreactivity to distinct glycosaminoglycan disaccharide epitopes was assessed by manually scoring the staining intensity in prostate tissues from patients with benign prostatic hyperplasia (n = 19), early-stage cancer (cohort 1, n = 55 and cohort 2, n = 275), and advanced-stage cancer (n = 20). Alterations to glycosaminoglycans in benign and malignant prostate tissues were determined by cellulose acetate chromatography and high-pressure liquid chromatography. Glycosaminoglycan disaccharide epitopes were localized to the peritumoral stroma of clinically localized prostate cancer. The level of immunostaining for unsulfated disaccharides (C0S) in the peritumoral stroma, but not for 4-sulfated (C4S) or 6-sulfated disaccharides (C6S), was significantly associated with the rate of PSA relapse following radical prostatectomy. High levels of C0S immunostaining were determined to be an independent predictor of PSA relapse (1.6-fold, P = 0.020). Advanced-stage prostate cancer tissues exhibited reduced electrophoretic mobility for chondroitin sulfate and increased unsulfated disaccharides when compared with benign prostatic hyperplasia tissues, whereas the sulfated disaccharide levels were unaffected. The level of C0S immunostaining in the peritumoral stroma is an independent determinant of PSA failure in clinically localized prostate cancer. Specific alterations to chondroitin sulfate side chains occurring during tumor development may be a crucial step for disease progression in prostate cancer. (Cancer Epidemiol Biomarkers Prev 2008;17(9):2488 -97)