Abstract
Type IX collagen is covalently bound to the surface of type II collagen fibrils within the cartilage extracellular matrix. The N-terminal, globular noncollagenous domain (NC4) of the α1(IX) chain protrudes away from the surface of the fibrils into the surrounding matrix and is available for molecular interactions. To define these interactions, we used the NC4 domain in a yeast two-hybrid screen of a human chondrocyte cDNA library. 73% of the interacting clones encoded fibronectin. The interaction was confirmed using in vitro immunoprecipitation and was further characterized by surface plasmon resonance. Using whole and pepsin-derived preparations of type IX collagen, the interaction was shown to be specific for the NC4 domain with no interaction with the triple helical collagenous domains. The interaction was shown to be of high affinity with nanomolar K(d) values. Analysis of the fibronectin-interacting clones indicates that the constant domain is the likely site of interaction. Type IX collagen and fibronectin were shown to co-localize in cartilage. This novel interaction between the NC4 domain of type IX collagen and fibronectin may represent an in vivo interaction in cartilage that could contribute to the matrix integrity of the tissue.
Highlights
A member of the fibril-associated collagens with an interrupted triple helix (FACIT) family of collagens, type IX is a heterotrimer of three genetically distinct polypeptide chains, ␣1(IX), ␣2(IX), and ␣3(IX), stabilized by interchain disulfide bonds
Yeast Two-hybrid Screening of a Human Chondrocyte cDNA Library—Screening 6.35 ϫ 105 yeast chondrocyte library transformants identified 26 clones encoding proteins that potentially interacted with NC4 bait causing expression of all three reporter genes
The reconstruction of the interaction in yeast with all stated controls demonstrated that only the presence of both the NC4 domain and FN resulted in the expression of all three reporter genes (Fig. 3)
Summary
A member of the fibril-associated collagens with an interrupted triple helix (FACIT) family of collagens, type IX is a heterotrimer of three genetically distinct polypeptide chains, ␣1(IX), ␣2(IX), and ␣3(IX), stabilized by interchain disulfide bonds. Type IX Collagen Interacts with Fibronectin domain are lost from the territorial and interterritorial matrices after maturation but are maintained in the pericellular matrix in articular cartilage throughout life [1, 18].
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