Reinboth Betty, Thomas John, Hanssen Eric, Gibson Mark A
Department of Pathology, University of Adelaide, Adelaide, South Australia, SA 5005, Australia.
J Biol Chem. 2006 Mar 24;281(12):7816-24. doi: 10.1074/jbc.M511316200. Epub 2006 Jan 24.
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.
在共免疫沉淀实验中发现重组人βig-h3能结合125I标记的富含亮氨酸的小分子蛋白聚糖（SLRPs）、双糖链蛋白聚糖和核心蛋白聚糖。在每种情况下，未标记的蛋白聚糖过量时可阻断这种结合，证实了相互作用的特异性。Scatchard分析表明，双糖链蛋白聚糖比核心蛋白聚糖更紧密地结合βig-h3，其解离常数（Kd）值分别估计为5.88×10^(-8)和1.02×10^(-7)M。在相互阻断实验中，两种蛋白聚糖均抑制对方与βig-h3的结合，这表明它们可能共享相同的结合位点，或者两个结合位点在βig-h3分子上位置相近。由于已知βig-h3和SLRPs与组织微原纤维中胶原蛋白VI的氨基末端区域相关联，因此研究了在孵育体系中加入胶原蛋白VI的影响。与单独的βig-h3相比，125I标记的双糖链蛋白聚糖与βig-h3和胃蛋白酶处理的胶原蛋白VI等摩尔混合物共同孵育后的共免疫沉淀增加了6倍，与单独的胶原蛋白VI相比增加了3倍。核心蛋白聚糖也观察到类似的增加。这些发现表明βig-h3参与了与胶原蛋白VI和SLRPs的三元复合物的形成。静态光散射技术用于显示βig-h3能与天然和胃蛋白酶处理的胶原蛋白VI迅速形成非常高分子量的复合物，无论是单独存在还是与SLRPs一起。事实上，已证明βig-h3与胶原蛋白VI和双糖链蛋白聚糖形成复合物，其复合物似乎比βig-h3与胶原蛋白VI和核心蛋白聚糖形成的复合物或胶原蛋白与βig-h3、双糖链蛋白聚糖或核心蛋白聚糖单独形成的复合物更为广泛。双糖链蛋白聚糖核心蛋白可抑制βig-h3与天然和胃蛋白酶处理的胶原蛋白VI的复合程度，这表明蛋白聚糖的糖胺聚糖侧链对形成大型三元复合物很重要。进一步研究表明，βig-h3与双糖链蛋白聚糖之间以及双糖链蛋白聚糖与胶原蛋白VI之间的直接相互作用对这些复合物的形成也很重要。胶原蛋白VI的球状结构域似乎也对这三种成分之间的相互作用有影响。总体而言，结果表明βig-h3可以差异性地调节胶原蛋白VI与双糖链蛋白聚糖和核心蛋白聚糖的聚集。因此，这种相互作用在诸如角膜等被认为会发生此类复合物的组织中可能很重要。
