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Title: Synthesis of a Cys949Tyr alpha 2-macroglobulin thiol ester mutant: co-transfection with wild-type alpha 2-macroglobulin in an episomal expression system
Authors: Van Rompaey, L ×
Van den Berghe, Herman
Marynen, Peter #
Issue Date: Nov-1995
Publisher: Portland press
Series Title: The Biochemical journal. vol:312 ( Pt 1) pages:183-90
Abstract: A full-length alpha(2)-macroglobulin (alpha(2)M) 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(2)M and was driven from the inducible human metallothionein II, promoter. Expression of the wild-type alpha(2)M cDNA resulted in a recombinant protein (r alpha(2) M) that could not be distinguished from plasma alpha(2)M (p-alpha(2)M): the transfected K-562 cells secreted tetrameric alpha(2)M with intact internal thiol esters, a functional bait domain and a latent receptor-binding domain. r alpha(2)M 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(2)M), a tetrameric alpha(2)M was secreted, with the electrophoretic mobility of methylamine-treated p-alpha(2)M (p-alpha(2)M/MA) and with a functional receptor-binding domain. The C949Y-r alpha(2)M 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(2)M was secreted with zero, one, two, three or four internal thiol esters. A comparison of the interaction of interleukin IB and basic fibroblast growth factor with native p-alpha(2)M, p-alpha(2)M/MA and the mutant C949Y-r alpha(2)M revealed that when assayed under nondenaturing conditions, no binding occurred to 'slow' p-alpha(2)M whereas quantitatively similar binding was observed to 'fast' p-alpha(2)M/MA and C949Y-r alpha(2)M. Covalent binding, however, was essentially limited to p-alpha(2)M/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(2)M by trypsin treatment, and thus involves the activated Glx residue.
ISSN: 0264-6021
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Molecular Genetics Section (-)
Clinical Genetics Section (-)
Department of Human Genetics - miscellaneous
× corresponding author
# (joint) last author

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