Binding of transforming growth factor-beta 1 to methylamine-modified alpha 2-macroglobulin and to binary and ternary alpha 2-macroglobulin-proteinase complexes.
The binding of 125I-labelled transforming growth factor-beta 1 (TGF-beta 1) to human alpha 2-macroglobulin (alpha 2M) was studied by native PAGE and autoradiography. TGF-beta 1 bound preferentially to alpha 2M-methylamine and minimally, if at all, to native alpha 2M. Preparations of alpha 2M-proteinase complex were generated by incubating a standard concentration of alpha 2M (0.4 microM) with different concentrations of trypsin, chymotrypsin or neutrophil elastase (0.04-2.0 microM). The 125I-TGF-beta 1-binding activity depended on the initial ratio of active proteinase to alpha 2M, or r value, used to form the alpha 2M-proteinase complex. With all three proteinases, r values of 2 or greater yielded preparations with unchanged or decreased TGF-beta 1-binding activity relative to native alpha 2M. By contrast, r values near 1 yielded preparations with significantly increased TGF-beta 1-binding activity. The results of [3H]thymidine-incorporation studies performed in mouse keratinocytes were consistent with the 125I-TGF-beta-binding experiments. alpha 2M-trypsin and alpha 2M-chymotrypsin prepared at an r value of 1.0 counteracted the activity of TGF-beta 1, whereas the equivalent complexes prepared at an r value of 3.0 had no effect. As determined by SDS/PAGE, 125I-TGF-beta 1 binding to alpha 2M-methylamine was at least 80% non-covalent. Reaction of alpha 2M-methylamine with iodoacetamide or 5,5'-dithiobis-(2-nitrobenzoic acid) decreased the percentage of covalent binding but had no effect on total binding. Neuraminidase treatment had no effect on the binding of 125I-TGF-beta 1 to alpha 2M-methylamine. Cleavage of the 'bait regions' in alpha 2M-methylamine by prolonged treatment with trypsin also had no effect. These studies suggest that TGF-beta 1 binding to alpha 2M is enhanced by conformational change in the proteinase inhibitor resulting from reaction with proteinase or amine. If both proteinase-binding sites in a single alpha 2M molecule are occupied, TGF-beta 1-binding activity is decreased or perhaps eliminated.
Department of Pathology, University of Virginia Health Sciences Center, Charlottesville 22908.
S W
SW
Hall
J
J
LaMarre
L B
LB
Marshall
M A
MA
Hayes
S L
SL
Gonias
D000818:Animals; D002478:Cells, Cultured; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism; D008744:Methylamines / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D009439:Neuraminidase / Q000378:metabolism; D010196:Pancreatic Elastase / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000737:chemistry / Q000378:metabolism*
D008744:Methylamines; D016212:Transforming Growth Factor beta; D000511:alpha-Macroglobulins; D009439:Neuraminidase; D010450:Endopeptidases; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D014357:Trypsin