Synthesis of a Cys949Tyr alpha 2-macroglobulin thiol ester mutant: co-transfection with wild-type alpha 2-macroglobulin in an episomal expression system.
A full-length alpha 2-macroglobulin (alpha 2M) cDNA was cloned into the episomal expression vectors pREP7 and pMEP4. Electroporation of the cell lines WI-L2-729HF2, U-937, K-562 and an Epstein-Barr virus-transformed cell line resulted in stable transfectants only with K-562 cells. Stable expression was obtained exclusively with pMEP4-alpha 2M and was driven from the inducible human metallothionein IIA promoter. Expression of the wild-type alpha 2M cDNA resulted in a recombinant protein (r alpha 2M) that could not be distinguished from plasma alpha 2M (p-alpha 2M): the transfected K-562 cells secreted tetrameric alpha 2M with intact internal thiol esters, a functional bait domain and a latent receptor-binding domain. r alpha 2M inhibited trypsin and elastase from cleaving a high-molecular-mass substrate. When the Cys-949 involved in the formation of the internal thiol ester was mutated to tyrosine (C949Y-r alpha 2M), a tetrameric alpha 2M was secreted, with the electrophoretic mobility of methylamine-treated p-alpha 2M (p-alpha 2M/MA) and with a functional receptor-binding domain. The C949Y-r alpha 2M did not possess proteinase-inhibiting capacity. Heterozygosity was mimicked by co-transfecting the K-562 cells with wild-type and mutant expression vectors. In this case, r alpha 2M was secreted with zero, one, two, three or four internal thiol esters. A comparison of the interaction of interleukin 1 beta and basic fibroblast growth factor with native p-alpha 2M, p-alpha 2M/MA and the mutant C949Y-r alpha 2M revealed that when assayed under nondenaturing conditions, no binding occurred to 'slow' p-alpha 2M whereas quantitatively similar binding was observed to 'fast' p-alpha 2M/MA and C949Y-r alpha 2M. Covalent binding, however, was essentially limited to p-alpha 2M/MA, suggesting the involvement of Cys-949 in the process. Covalent binding of insulin, on the contrary, was only observed when it was present during hydrolysis of the internal thiol esters of p-alpha 2M by trypsin treatment, and thus involves the activated Glx residue.
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D004952:Esters; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism; D008744:Methylamines / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors; D010957:Plasmids / Q000235:genetics*; D017354:Point Mutation / Q000235:genetics; D011487:Protein Conformation; D011973:Receptors, LDL / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D013438:Sulfhydryl Compounds; D014162:Transfection; D014357:Trypsin / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000494:pharmacology; D000511:alpha-Macroglobulins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D016207:Cytokines; D004952:Esters; D007328:Insulin; D008744:Methylamines; D011973:Receptors, LDL; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D014361:Trypsin Inhibitors; D000511:alpha-Macroglobulins; D016222:Fibroblast Growth Factor 2; C027451:methylamine; D010196:Pancreatic Elastase; D014357:Trypsin