The membrane-spanning proteoglycan NG2 binds to collagens V and VI through the central nonglobular domain of its core protein.
NG2 is a membrane-spanning proteoglycan with a primary structure unique among cell surface or extracellular matrix proteins. To characterize the interaction between NG2 and extracellular matrix proteins, we have used a eukaryotic expression system to produce and purify several recombinant fragments covering not only the entire ectodomain of NG2 but also distinct subdomains of the molecule. Using a solid phase binding assay with various extracellular matrix proteins, we have identified two main ligands for NG2, namely, collagens V and VI. Consistent with previous models of glycosaminoglycan attachment, roughly 50% of the recombinant NG2 fragments containing the central domain have chondroitin sulfate chains attached to the protein core. These glycosaminoglycan chains are not directly involved in collagen binding, since chondroitinase-treated fragments exhibit an unimpaired ability to bind to both collagens. Using more restricted recombinant fragments of NG2, we mapped the binding site for both collagens to the central domain of NG2. Electron microscopy after rotary shadowing of native NG2 molecules indicates that this extended nonglobular domain provides a flexible connection joining the two N- and C-terminal globular regions of NG2. Rotary shadowing of mixtures of NG2 and collagen V or VI confirms a direct interaction between the molecules and indicates that the collagens align with the central region of NG2, giving the appearance of a rod between the N- and C-terminal globules.
Tillet
E
E
Burnham Institute, La Jolla Cancer Research Center, La Jolla, California 92037, USA.
Ruggiero
F
F
Nishiyama
A
A
Stallcup
W B
WB
D000941:Antigens / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D001665:Binding Sites; D002460:Cell Line; D002810:Chondroitinases and Chondroitin Lyases; D003094:Collagen / Q000737:chemistry* / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000737:chemistry* / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008854:Microscopy, Electron; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011509:Proteoglycans / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D000099216:Chondroitin Sulfate Proteoglycan 4
D000941:Antigens; D002810:Chondroitinases and Chondroitin Lyases; D003094:Collagen; D016326:Extracellular Matrix Proteins; D006025:Glycosaminoglycans; D010446:Peptide Fragments; D011509:Proteoglycans; D011994:Recombinant Proteins; D000099216:Chondroitin Sulfate Proteoglycan 4