Cell-free synthesis of enzymically active tissue-type plasminogen activator. Protein folding determines the extent of N-linked glycosylation.
Tissue-type plasminogen activator (t-PA) is synthesized in mammalian cells as a mixture of two forms that differ in their extent of N-linked glycosylation. We have investigated the mechanism underlying this variation in glycosylation, using a cell-free system that consists of a rabbit reticulocyte lysate optimized for the formation of disulphide bonds and supplemented with dog pancreas microsomal membranes. Molecules of human t-PA synthesized in vitro are enzymically active and responsive to natural activators and inhibitors, and are glycosylated in a pattern identical with that of the protein produced in vivo. This demonstrates that t-PA synthesized in vitro folds into the same conformation as the protein synthesized in vivo. We show that the extent of glycosylation of individual t-PA molecules is dependent on the state of folding of the polypeptide chain, since the probability of addition of an oligosaccharide side chain at Asn-184 is decreased under conditions that promote the formation of enzymically active molecules. This variation in glycosylation is independent of the rate of protein synthesis.
Bulleid
N J
NJ
Department of Biochemistry and Molecular Biology, University of Manchester, U.K.
Bassel-Duby
R S
RS
Freedman
R B
RB
Sambrook
J F
JF
Gething
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D002474:Cell-Free System; D004285:Dogs; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010179:Pancreas / Q000378:metabolism; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D014158:Transcription, Genetic
D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator