Localization of the Binding Site for Transforming Growth Factor-β in Human α2-Macroglobulin to a 20-kDa Peptide That Also Contains the Bait Region

Abstract

and TGF-␤2 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 ␣ 2 M activity that has been attributed to the neutralization of endogenously synthesized TGF-␤. Thus, we have isolated a peptide corresponding to 13% of the ␣ 2 M sequence that binds TGF-␤ and neutralizes the activity of TGF-␤ in two separate biological assays.1 is a 718-kDa glycoprotein that was originally characterized as a broad spectrum proteinase inhibitor (1). The structure of ␣ 2 M consists of four identical subunits, each with 1451 amino acids (2). The subunits are linked into dimers by disulfide bonds and into intact homotetramers by noncovalent interactions (3, 4). Proteinases react with ␣ 2 M by cleaving any of a number of susceptible peptide bonds in the "bait region," which includes amino acids 666 -706 (1, 3, 5). Bait region cleavage causes ␣ 2 M to undergo a major conformational change, which effectively "traps" the attacking proteinase in a complex that is nondissociable, even when the proteinase and the inhibitor are not covalently linked (1, 6 -8).Conformational change also reveals binding sites for the ␣ 2 M receptor/low density lipoprotein receptor-related protein (LRP) (9). These binding sites have been localized to 18-kDa peptides at the C terminus of each ␣ 2 M subunit; Lys-1370 and Lys-1374 play particularly important roles (10 -13).Like the complement components, C3 and C4, each ␣ 2 M subunit contains a novel thiol ester bond, which is formed from the side chains of . The thiol esters may be instrumental in determining the conformational state of ␣ 2 M (17, 18). When ␣ 2 M reacts with a proteinase, the thiol esters emerge from within hydrophobic, solvent-restricted clefts and are cleaved by nucleophiles or H 2 O (14, 18). Small primary amines, such as methylamine, penetrate the hydrophobic clefts and react with ␣ 2 M thiol esters independently of proteinases, inducing an equivalent or nearly equivalent conformational change (6, 7).In addition to its activity as a proteinase inhibitor, ␣ 2 M 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 ␣ 2 M as a physiologically significant carrier of TGF-␤ in human serum (19). Their studies demonstrated that nearly all of the TGF-␤1 in serum is associated with ␣ 2 M and that the bound TGF-␤1 is inactive. Huang et al. (20) More recent studies have demonstrated the function of ␣ 2 M as a TGF-␤-carrier in animal model systems. When radioiodinated TGF-␤1 is injected intravascularly in mice, the cytokine is cleared rapidly at first; however, this is followed by a slow clearance phase, during which time the TGF-␤ is almost entirely ␣ 2 M-associated (21-23). In cell culture systems, ␣ 2 M neutralizes both exogenously added and endogenously synthesized TGF-␤ (24 -28). Neutralization of endogenously synthesized TGF-␤ results in altered gene expr...

The Conformation-Dependent Interaction of alpha sub2 -Macroglobulin with Vascular Endothelial Growth Factor: A Novel Mechanism of alpha sub2 -Macroglobulin/Growth Factor Binding

“…In certain cases, such as TGF-β1 and PDGF, both the biochemical interaction of these molecules with α 2 M and the ability of α 2 M to regulate their function have been extensively studied (12,14,16,17,43,44). With regards to VEGF, an earlier study demonstrated that radiolabeled VEGF added to human plasma coprecipitates with α 2 M (22).…”

“…Numerous cytokines and growth factors are known to bind to α 2 M and α 2 M-MA, including transforming growth factor beta (TGF-β), types 1 (11,12) and 2 (13,14), platelet derived growth factor (PDGF) (15)(16)(17), nerve growth factor (NGF) (17,18), tumor necrosis factor alpha (TNF-α) (19), basic fibroblast growth factor (bFGF) (20), interleukin six (IL-6) (21), and vascular endothelial growth factor (VEGF) (22). In some cases, such as TGF-β1, the nature of the interaction is well investigated whereas in others, such as VEGF, the nature of the interaction is not well understood.…”

The NMDA receptor functions independently and as an LRP1 co-receptor to promote Schwann cell survival and migration

“…The hemopexin domain of MMP9, which includes the LRP1-binding site in MMP9 (MMP9-PEX; Mantuano et al, 2008a,b), was expressed as a GST fusion protein in bacteria. MMP9-PEX was partially purified by selective detergent extraction (Webb et al, 1998) and then purified to homogeneity by chromatography on a glutathione-Sepharose column (Mantuano et al, 2008a,b). All GST fusion proteins were subjected to urea treatment, dialysis and chromatography on Detoxi-Gel endotoxin-removing columns (Pierce).…”

Distinct Binding Sites in the Structure of α2-Macroglobulin Mediate the Interaction with β-Amyloid Peptide and Growth Factors

“…␣ 2 M-peptide-GST Fusion Proteins-Six previously described fusion proteins, which collectively encode amino acids 99 -1451 of the human ␣ 2 M sequence, were expressed in BL-21 cells (17,18). These fusion proteins include: FP1 (aa 99 -392), FP2 (aa 341-590), FP3 (aa 591-774), FP4 (aa 775-1059), FP5 (aa 1030 -1279), and FP6 (aa 1242-1451).…”

“…The first involves a region within intron 17, at the 5Ј splice acceptor site for exon 18 (14). This exon is important because it encodes part of the bait region, where proteinases initiate reaction with ␣ 2 M by cleaving susceptible peptide bonds (15,16), and a segment of the growth factor binding sequence (17)(18)(19). In the second A2M gene polymorphism, Val-1000 is replaced by Ile (20).…”