Cell-free synthesis of enzymically active tissue-type plasminogen activator

Protein folding determines the extent of N-linked glycosylation

N. J. Bulleid, R. S. Bassel-Duby, R. B. Freedman, J. F. Sambrook, M. J H Gething

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)275-280
Number of pages6
JournalBiochemical Journal
Volume286
Issue number1
StatePublished - 1992

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Glycosylation
Protein folding
Protein Folding
Tissue Plasminogen Activator
Plasminogen Activators
Molecules
Protein Conformation
Proteins
Cell-Free System
Reticulocytes
Oligosaccharides
Disulfides
Conformations
Pancreas
Cells
Dogs
Rabbits
Membranes
Peptides
In Vitro Techniques

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cell-free synthesis of enzymically active tissue-type plasminogen activator : Protein folding determines the extent of N-linked glycosylation. / Bulleid, N. J.; Bassel-Duby, R. S.; Freedman, R. B.; Sambrook, J. F.; Gething, M. J H.

In: Biochemical Journal, Vol. 286, No. 1, 1992, p. 275-280.

Research output: Contribution to journalArticle

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