title: Localization of the binding site for transforming growth factor-beta in human alpha2-macroglobulin to a 20-kDa peptide that also contains the bait region.
abstract: alpha2-Macroglobulin (alpha2M) functions as a major carrier of transforming growth factor-beta (TGF-beta) in vivo. The goal of this investigation was to characterize the TGF-beta-binding site in alpha2M. Human alpha2M, which was reduced and denatured to generate 180-kDa subunits, bound TGF-beta1, TGF-beta2, and NGF-beta in ligand blotting experiments. Cytokine binding was not detected with bovine serum albumin that had been reduced and alkylated, and only minimal binding was detected with purified murinoglobulin. To localize the TGF-beta-binding site in alpha2M, five cDNA fragments, collectively encoding amino acids 122-1302, were expressed as glutathione S-transferase (GST) fusion proteins. In ligand blotting experiments, TGF-beta2 bound only to the fusion protein (FP3) that includes amino acids 614-797. FP3 bound 125I-TGF-beta1 and 125I-TGF-beta2 in solution, preventing the binding of these growth factors to immobilized alpha2M-methylamine (alpha2M-MA). The IC50 values were 33 +/- 5 and 26 +/- 6 nM for TGF-beta1 and TGF-beta2, respectively; these values were comparable with or lower than those determined with native alpha2M or alpha2M-MA. A GST fusion protein that includes amino acids 798-1082 of alpha2M (FP4) and purified GST did not inhibit the binding of TGF-beta to immobilized alpha2M-MA. FP3 (0.2 microM) neutralized the activity of TGF-beta1 and TGF-beta2 in fetal bovine heart endothelial (FBHE) cell proliferation assays; FP4 was inactive in this assay. FP3 also increased NO synthesis by RAW 264.7 cells, mimicking an alpha2M activity that has been attributed to the neutralization of endogenously synthesized TGF-beta. Thus, we have isolated a peptide corresponding to 13% of the alpha2M sequence that binds TGF-beta and neutralizes the activity of TGF-beta in two separate biological assays.
authors.affiliation: Department of Pathology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.
authors.forename: D J
authors.initials: DJ
authors.lastname: Webb
authors.forename: J
authors.initials: J
authors.lastname: Wen
authors.forename: L R
authors.initials: LR
authors.lastname: Karns
authors.forename: M G
authors.initials: MG
authors.lastname: Kurilla
authors.forename: S L
authors.initials: SL
authors.lastname: Gonias
mesh_terms: D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002417:Cattle; D002455:Cell Division; D002460:Cell Line; D006801:Humans; D007457:Iodine Radioisotopes; D009569:Nitric Oxide / Q000096:biosynthesis; D011485:Protein Binding; D011487:Protein Conformation; D011869:Radioligand Assay; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D000511:alpha-Macroglobulins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D007457:Iodine Radioisotopes; D011993:Recombinant Fusion Proteins; D016212:Transforming Growth Factor beta; D000511:alpha-Macroglobulins; D009569:Nitric Oxide