Em Ab Research Articles

Interaction of human thrombospondin with types I-V collagen: direct binding and electron microscopy.

Binding of thrombospondin (TSP) to types I-V collagen was examined by direct binding assays using 125I-TSP and by visualization of rotary-shadowed intermolecular complexes in the electron microscope. The binding of TSP was highest to type V collagen in the absence of Ca, while lower but significant levels of binding were observed to all other collagen types in the presence or absence of Ca. Unlike intact TSP, the trimeric collagen-binding domain of TSP composed of 70-kD chains showed no Ca dependence in its binding to type V collagen. Further evidence for binding of TSP to types I and III collagen was obtained by competition studies in which these soluble collagens effectively inhibited binding of 125I-TSP to immobilized type V collagen. The binding of TSP to type V collagen was inhibited by heparin and fucoidin, both high-affinity ligands of TSP's heparin-binding domain. mAb A6.1, which binds to the 70-kD domain of TSP, is also the best of a panel of anti-TSP mAbs at inhibiting the TSP-collagen interaction. Electron microscopy of rotary-shadowed replicas of TSP-collagen complexes revealed that all five types of collagen examined had a binding site for TSP at one end of the pepsinized, triple helical molecule. The specificity of this site was tested by examining the ability of BSA to form a complex with the end of the pepsinized collagens. Rotary-shadowed replicas revealed a low frequency of apparent BSA-collagen complexes, and histograms of these data showed no evidence for the preferential association of BSA with the end of the collagen molecules. In addition to the specific end site, type V collagen had an internal binding site for TSP located about two-thirds of the distance along the length of the collagen molecule from the end site. The internal binding site for TSP on type V collagen is apparently the site responsible for the higher affinity binding of TSP to that protein observed in direct binding assays. The trimeric 70-kD collagen-binding domain of TSP bound to the same sites on the collagens as did intact TSP.

The epithelial cell surface antigen 17-1A, a target for antibody-mediated tumor therapy: its biochemical nature, tissue distribution and recognition by different monoclonal antibodies.

The antigen defined by MAb 17-1A, raised against colorectal cancer cells and currently used in immunotherapy trials, is a 37 kDa protein containing N-linked glycans as demonstrated by inhibition of glycosylation with tunicamycin. The 17-1A antigen (17-1A Ag) is broadly distributed in normal epithelial tissues and is also found in various types of carcinoma. Quantitative differences in expression between normal and malignant tissues were only apparent in gastric carcinomas. After immunizing mice with fresh colon carcinoma tissue, 4 MAbs were obtained that showed the same tissue reaction pattern as MAb 17-1A and recognized the 17-1A Ag as judged from precipitation and immunoblotting experiments. MAbs M72 and M74 reacted with an anti-idiotypic serum directed against the original MAb 17-1A and were able to block 17-1A binding. In contrast, M77 and M79 are directed against a different epitope. This makes them potentially interesting for passive immunotherapy in regimens involving a combination of MAbs.