Biochimica et Biophysica Acta, 1091 (1990) 197-204 © 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0167-4889/91/$03.50 ADONIS 016748899100081V

197

BBAMCR 12854

Reaction of a -macroglobulin with plasmin increases binding of transforming growth factors-ill and t 2 Jonathan LaMarre ~, Gordon K. Wollenberg 1, Steven L. Gonias 2 and M. Anthony Hayes 1 t Department of Pathology, University of Guelph, Guelph (Canada) and 2 the Departments of Pathology and Biochemistry, University of Virginia Health Sciences Center, Charlottesville, VA (U.S.A.)

(Received 27 February 1990) (Revised manuscript received 26 September 1990)

Key words: a-Macroglobulin; Plasmin; Transforming growth factor ill; Transforming growth factor t2

The binding of IZSl-transforming growth factors-j01 and if2 (TGF-Ill and TGF-Il2) to az-macroglobuUn (a:M) was studied before and after reaction with plasmin, thrombin, trypsin, or methylamine. Complex formation between TGF-Il and native or reacted forms of a2M was demonstrated by non-denaturing polyacrylamide gel electrophoresis a~d autoradiography. Reaction of native azM with plasmin or methylamine markedly increased the binding of 2Sl-TGF-ill and IZSl-TGF-il2 to azM. The azM-plasmin/TGF-fl complexes were mitn~mally dissociated by heparin. Reaction of azM with thrombin or trypsin reduced the binding of I-TGF-Ill and I-TGF-Il2; the resulting complexes were readily dissociated by heparin. Complexes between TGF-Il2 and native or reacted forms of azM were less dissociable by heparin than the equivalent complexes with TGF-fll. These studies demonstrate that the TGF-il-binding activity of a2M is significantly affected by plasmin, thrombin, trypsin and methylamine. Observations that a2M-plasmin preferentially binds TGFs-fl suggest a mechanism by which azM may regulate availability of TGFs-il to target cells in vivo. Introduction a2-Macroglobulin (a2M) is a high molecular weight (Mr = 718000) glycoprotein from plasma which reacts with a wide range of proteinases [1]. Recently, a2M has been shown to bind several growth factors and cytokines, specifically, transforming growth factor-ill (TGF-Ill) [2,3], basic fibroblast growth factor (bFGF) [4], platelet-derived growth factor (PDGF) [5,6], nerve growth factor (NGF) [7,8], intedeukin-lil (!L-lit) [9] and interleukin-6 (IL-6) [10]. Native a2M is a tetramer of four identical subunits [11,12] which undergoes a specific conformational change following reaction with proteinases [12] or methylamine [13] from a "slow" to a "fast" migrating form during non-denaturing polyacryl-

Abbreviations: a2M, az-macroglobulin; azM-MA, a2M-methylamine; TGF-fl, transforming growth factor-t; bFGF, basic fibroblast growth factor; PDGF, platelet derived growth factor; NGF, nerve growth factor; IL-lfl, interleukin-lfl; IL-6, interleukin-6; NPGB, pnitrophenyl p'-guanadino benzoate. Correspondence: M.A. Hayes, Department of Pathology, University of Guelph, Guelph, Canada N1G 2Wl.

amide gel electrophoresis. The growth factor binding activity of a2M may be increased or decreased coincident with this conformational change, depending on the growth factor/cytokine involved and whether the conformational change is induced by methylamine or trypsin. For example, the binding of 125I-TGF-Ill and 1251bFGF to a2M is increased relative to native a2M by reaction with methylamine [3, 4] but is either reduced (TGF-Ill)[3] or unchanged (bFGF)[4] by reaction with trypsin. In contrast, reaction of aEM with methylamine reduces its tendency to form a complex with PDGF [5]. Apperently,125I-ILolil does not bind to the native form of a2M but does bind to a2M-trypsin or a2M-methylamine [9]. When bound to a2M, different cytokines behave non-uniformly with respect to specific cellular receptorinteractions and biologic activity. PDGF [5], NGF [8] and IL-6 [10] remain biologically active when complexed with UEM in various cell culture assays. Conversely, IL-lfl and bFGF demonstrate markedly decreased activity in the presence of excess a2M [4,9]. The TGF-Ill/aEM complex from serum is a biologically latent form of the growth factor [2,3]. O'Connor-McCourt and Wakefield [2] were unable to demonstrate a

198 significant decrease in TGF-fll activity in the presence of high concentrations of native a2 M using two different cell lines. However, in primary cultures of rat hepatocytes the mitoinhibitory activity of transforming growth factor-fl2 (TGF-fl2) was counteracted by a2M or serum to a greater extent than the activity of TGF-~I [14]. ~ e s e findings suggest that a2M might modify the biologic activity of the TGFs-fl differently under specific circumstances. Recent studies have also shown that mitoinhibitory activity toward aortic smooth muscle cells can be dissociated from the TGF-fll/a2M complex by heparin [15]. The transforming growth factors-/~ are a family of potent, polypeptide growth factors (see Ref. 16 for review) secreted as inactive precursors by stromal cells and platelets [17-20]. Several forms of TGF-B have been characterized. TGF-/~I and TGF-fl2 [22,23] are 70% homologous by amino acid sequence [23] and have similar biological effects on most cells [22], including, normal and neoplastic hepatocytes [24-26]. The precursor forms are activated by pH extremes [19] and some proteinases, such as plasmin and cathepsin D [21]. By electron microscopy, a2M-plasmin is similar to t~2M-trypsin [27]. Both complexes undergo rapid receptor-mediated endocytosis in vitro and in vivo [28]. Native t~2M is not recognized by the a2M receptor. ~x2Mplasmin is likely present both in serum, where latent TGF-fl activity is associated with a2M [2,3] and is vivo where pro-TGF-fl is activated by plasmin [21]. These unique features suggest that cx2M-plasmin might function as the major form of a2M responsible for binding and clearance of TGFs-fl in vivo. The studies presented here support this hypothesis, demonstrating that a2Mplasmin binds more r2SI-TGF-#I and 1251-TGF.,f12than native a2M, a2M-thrombin or ~x2M-trypsin and that the ot2M-plasmin/TGF.,8 complex is virtually not dissociated by heparin,

Experimental procedures Materials Methylamine HCI and p-nitrophenyl-p'-guanadinobenzoate-HCL (NPGB), were purchased from Sigma Chemical (St. Louis, MO), Coomassie blue stain was purchased from Bio-Rad Laboratories (Richmond, CA).

Proteins Native a,M was purified from fresh human plasma as described previously [29]. Trypsin (from bovine panereas) and heparin (from porcine intestine) were purchased from Sigma. Purified human or bovine plasmin (2 U/rag) and thrombin (30 U/mg) were from Boehringer-Mannheim (Dorval, Canada). In some experiments, highly purified human plasminogen was prepared as described by Deutsch and Mertz [30] and reacted with a2M as described previously [31]. Highly

urified (> 97%) TGF-fll, TGF-fl2, 12SI-TGF-f11 and 5I-TGF-fl2 were purchased from R&D Systems (Minneapolis, MN). Preparation of reacted forms of a2M. Purified, human, native a2M (1 #M) was incubated with plasmin (2 /~M), thrombin (2 #M) or trypsin (2 #M) in 50 mM Tris-HCl (pH 7.60) at 37°C. Residual proteinase activity was inhibited after 2 h by the addition of NPGB to a final concentration of 100 #M [32]. Methylamine reaction of ot2M was performed by dialysis of 1.0 mg of purified, human, native a2M against 250 ml of 300 mM methylamine for 8 h at 25°C followed by dialysis against 4 1 150 mM NaCI buffered with 10 mM NaHPO4 (pH 7.4) for 24 h at 4°C. All proteinase and methylamine-reacted forms of a2M migrated primarily as "fast" forms on non-denaturing gel electrophoresis except the thrombin-reacted species which migrated at a position intermediate between the native and "fast" forms as reported previously [33].

Non-denaturing polyacrylamide gel electrophoresis. Native, proteinase-reacted or methylamine-reacted forms of a2M (0.15 #M) or 5 #1 of normal human serum were incubated for 1 h at 37°C with 0.8 nM 12SI-TGF-fll or 12SI-TGF-fl2 in 100 #1 50 mM Tris-HCl (pH 7.6). In some experiments, heparin (100 #g/ml) was added to the solutions. Samples for electrophoresis were mixed with an equal volume of reservoir buffer (0.041 M Tris, 0.04 M sodium borate, pH 8.6) containing 10% glycerol. Samples (25 ml) were separated by electrophoresis for 3 h at constant 150 V on non-denaturing 5% acrylamide electrophoresis gels with a 4% acrylamide stacking gel, according to the method of Van Leuven et al. [13]. Gels were stained with Coomasfie blue, destained, dried and exposed to preflashed (10 -3 s, to background ,4540=0.15) Kodak X-Omat AR-5 film at -70°C using Lanex regular intensifying screens. To determine the incubation period required for maximal binding, 12SI-TGF-fll (0.4 nM) was incubated with 10 nM ot2M or a2M-methylamine , as above, for periods of 0.25, 0.5, 1, 1.5, 2, 3 or 4 h. Each preparation was electrophoresed as above and radioactivity was measured in the slow or fast t~2M bands cut from the gels. In additional experiments to compare binding of TGF-fll versus TGF-fl2, to aaMs, 0.4 nM 12SI-TGF-fll was incubated with 10 nM a2M-methylamine in 50 mM Tris-HCl (pH 7.6), for 1 h, alone or in the presence of a 10-, 20-, 50- or 100-times excess of unlabeled TGF-fll or TGF-fl2, followed by non-denaturing gel electrophoresis and autoradiography as described above. Identical experiments with 12SI-TGF-,82 were also performed. To estimate the binding capacity of native ,t M and a2M-methylamine, various concentrations of I~sITGF-fll (0.02-200 nM) were incubated with either native a2M or ,t2M-methylamine (20 nM) for 2 h and then electrophoresed, as above. The proportions of ad-

199 ded radioactivity recovered in the slow or fast ot2M bands cut from the gels were determined for TGF-fll at 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1 and 10-times the concentration of a2M. To compare preferential binding of TGF-fls to azM-plasmin versus native a 2 M , 0.8 nM 125I-TGF-fll or 1251-TGF-f12 was incubated with 150 nM native et2M and the following concentrations of a2M-plasmin: 0, 1.5, 3, 7.5, 15, 75, or 150 nM in 100/tl Tris-HCl (pH 7.6), for I h (note: all residual plasmin activity was first inhibited by 100/tM NPGB). Further experiments were performed as follows: native a2M (150 nM) was incubated with 125I-TGF-fll or 12SI-TGF-f12 for 1 h followed by the addition of 150 nM ct2M-plasmin and incubation for an additional 1 h period (final concentrations: 75 nM native a2M and 75 nM a2M-plasmin). In each experiment, 25-/tl aliquots were subjected to nondenaturing polyacrylamide gel electrophoresis and autoradiography as previously explained. Results

Binding of t251-TGF-fll and 1251-TGF-f12 to serum proteins and purified ~t2M. 125I-TGF-f11 formed complexes with native and methylamine-reacted human a 2M (data not shown), in agreement with reports of others [2,3]. 1251-TGF-f12 also formed complexes with purified native a 2 M (Fig. 1) and with a high molecular weight protein migrating at a position identical to ~21~4 in human serum (data not shown), aEM-methylamine bound more I25I-TGF-fll and t251-TGF-f12 than native a 2 M (Table I and Fig. 1). The amounts of 125I-TGF-fll and tESI-TGF-f12 bound to aEM-methylamine were sim-

TABLE I

Binding of TGF-fll to native a,M and a,M-methylamine after various periods of incubation Incubation Time (hours)

125I-TGF-fl! bound to native a2M 1

]2SI-TGF-fll bound to a2M.methylamine 1

0.25 0.5 1.0 1.5 2.0 3.0 4.0

14 16 17 17 17 17 18

43 46 48 49 49 49 54

t 1251.TGF.fll bound to a2M is the amount of radioactivity recovered in the a2M bands expressed as a percentage of the total radioactivity loaded onto the gel (20000 cpm/lane).

ilar; in cross-competition studies, unlabeled TGF-fll displaced 1251-TGF-B2 and, unlabeled TGF-fl2 displaced I:51-TGF-fll from a2M-methylamine (Fig. 2A). In direct competition studies, unlabeled TGF-fl2 also displaced 125I-TGF-f12 from a2M-methylamine (Fig. 2B). The minimum incubation period required for maximal binding of 125I-TGF-fll to a2M or a2M-methylamine was 0.5 h (Table I). The percentage of t2Sl-TGFbound to a 2M-methylamine was consistently 5-10-times greater than that recovered in association with native a2M over a wide concentration range of TGF-fll (Fig. 3~5" With 10-fold molar excess of TGF-fll to a 2 M , I-TGF-fll bound at approximately a 1 : 1 molar ratio with aEM-methylamine, but 0.1:1 with native ~tEM. Differences in amounts of lzSI-TGF-fl bound to OtEMor

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Fig. 1. Non-denaturing gel electrophoresis and autoradiography of native a2M, a2M-trypsin and a2M-methylamine/~251-TGF-f12 complexes. Reaction mixtures containing 12sI-TGF-fl2 only (lane 1), or 125I-TGF-f12 with native a2M (lane 2), a2M-trypsin (lane 3), or a2M-methylamine (lane 4) were subjected to non-denaturing gel electrophoresis, Coomassie blue staining (A) and autoradiography (B) as described under Experimental Procedures. Native a2M migrates in the"slow" (s) positions, whereas modified ot2Ms migrate in "fast" (f) positions.

200 a2M-methylamine were not due to differences in the rate of binding (Table I). Binding of proteinase-reacted forms of a2M to J251TGF-fll and 1251.TGF..f12. a2M-plasmin bound greater amounts of 1251-TGF-fll and 125I-TGF-f12 than native a 2 M (Fig. 4). When t251-TGF-f11 or 1251-TGF-f12 were incubated with premixed solutions of native a2M and a:,M-plasmin, the radiolabelled growth factors bound primarily to a2M-plasmin, even in the presence of a 10-20-fold excess of native t~2M (Fig. 5), Furthermore, a fraction of 1251-TGF-f11 or 1251-TGF-f12 which was allowed to form a complex with native a2M (1 h incubation) subsequently became bound to a:M-plasrain when a:M-plasmin was added to the reaction mixture prior to electrophoresis (Fig. 6). tqM-plasmin also incorporated additional radiolabeled growth factor from the incubation mixture as indicated by the greater density of the a2M-plasmin band on autoradiography (Fig. 6). In contrast to the increase in ~2~I-TGF-/~I or 12SlTGF-~82 binding observed with a~M-plasmin or a2Mmethylamine, we observed a decrease in the binding of ~2SI-TGF-~81 or ~2SI-TGF-fl2 to a2M-thrombin (Fig. 4). We also demonstrated reduced binding of ~2~I-TGF-fll to a2M-trypsin, confirming the work of others [3] . Similar results were obtained with 'eSI-TGF-fl2 (Fig. 1). In these experiments, residual trypsin activity was inhibited with NPGB. , Effects of heparin on the binding of_ ~"~I-TGF-~BI and ~'~!.TGF.~2 to different proteinase-reacted forms of a : M.

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Fig. 3. Saturation binding profiles for 12SI-TGF-fll with native a2M compared with a2M-methylamine. Curves represent the amounts of t2Sl-TGF-fll recovered bound to the native or fast a2M bands cut from gels, expressed as a molar ratio of TGF-,81 recovered to the amount of a2M loaded (1 pmol). Various concentrations of t2"~I-TGFfll (0.02-200 nM) were incubated with either native a2M or a2Mmethylamine (20 nM) for 2 h and then electrophoresed, as above.

Others ha' c reported that the a2M-TGF-fll complex is dissociated by heparin [15]. In our studies, heparin dissociated complexes of TGF-fll with native a2M, a2M-thrombin and azM-methylamine (Fig. 7A). The complexes formed between ~251-TGF-f12 and ot2M-MA proteinase-reacted forms of a2M were less susceptible to dissociation by heparin (Fig. 7B). 1251-TGF-fll or 1251-TGF-f12 complexes with a2M-plasmin were only

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Fig. 2.(A) Cross competition between 1251-TGF.~81 and unlabeled TGF-/32 or 12Si-TGF-fl2 and unlabeled TGF-/~I for binding to a2M-methylamine. Gel autoradiographs of a,M-methylamine (10 riM) incubated with 1251-TGF-fll (0.4 nM) and unlabeled TGF-p2 added to final concentrations of 0 (lane 1), 2 nM (lane 2), 4 nM (lane 3), or 20 nM (lane 4), respectively, a2M-methylamine was similarly incubated with t~I-TGF-~2 (0.4 nM) and unlabeled TGF-/]I added at concentrations of 0 (lane 5), 2 nM (lane 6), 4 nM (lane 7), or 20 nM (lane 8). Non-denaturing polyac~lamide gels were subjected to autoradiography under the conditions described in the Ex erimental Pr corn tition betwee ' ~ • p ocedures. (B) Direct pe n I-TGF-.82 and unlabeled TGF-fl2 for binding to a2M-methylamine, a2M-methylamine (10 nM) was incubated with I~I-TGF-~2 (0.4 nM) and unlabeled TGF-,82 to final concentrations of 0 nM (lane 1), 4 nM (lane 2), 20 nM (lane 3), or 40 nM (lane 4) followed by non-denaturing gel electrophoresis and autoradiography as described under Experimental Procedures.

201

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Reaction of alpha 2-macroglobulin with plasmin increases binding of transforming growth factors-beta 1 and beta 2.

The binding of 125I-transforming growth factors-beta 1 and beta 2 (TGF-beta 1 and TGF-beta 2) to alpha 2-macroglobulin (alpha 2M) was studied before a...
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