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.
Highlights
That was originally characterized as a broad spectrum proteinase inhibitor [1]
Ligand Blot Analysis of 125I-TGF- Binding to ␣2M—Native ␣2M, ␣2M-MA, and bovine serum albumin (BSA) were denatured in SDS, subjected to SDS-PAGE, and electrotransferred to Polyvinylidene fluoride (PVDF) membranes
125I-transforming growth factor- (TGF-)2 bound to native ␣2M and ␣2M-MA, which were immobilized on PVDF membranes (Fig. 2). 125I-TGF-2 binding was unchanged when the ␣2M was treated with DTT or with IAM prior to electrophoresis. 125I-TGF-2 bound to BSA; this interaction was observed only after DTT
Summary
That was originally characterized as a broad spectrum proteinase inhibitor [1]. The structure of ␣2M consists of four identical subunits, each with 1451 amino acids [2]. Proteinases react with ␣2M by cleaving any of a number of susceptible peptide bonds in the “bait region,” which includes amino acids 666 –706 [1, 3, 5]. In addition to its activity as a proteinase inhibitor, ␣2M functions as a major carrier and regulator of certain cytokines, including isoforms of the transforming growth factor- (TGF-) family. O’Connor-McCourt and Wakefield [19] first identified ␣2M as a physiologically significant carrier of TGF- in human serum [19] Their studies demonstrated that most of the TGF-1 in serum is associated with ␣2M and that the bound TGF-1 is inactive. Huang et al [20] confirmed the role of ␣2M as a TGF--carrier and demonstrated that the TGF- binding activity of ␣2M depends on its conformational state. The function of ␣2M as a significant modulator of TGF- activity in vivo and in vitro has prompted us to elucidate the ␣2M-TGF- interaction on a molecular level
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