Secretion of glycosylation site mutants can be rescued by the signal/pro sequence of tissue plasminogen activator

Strategies that prevent the attachment of N-linked carbohydrates to nascent glycoproteins often impair intracellular transport and secretion. In the p resent study, we describe a method to rescue the intracellular transport an d secretion of glycoproteins mutagenized to delete N-linked glycosylation s ites. Site-directed mutagenesis was used to delete N-linked glycosylation s ites from a chimeric protein, TNFR-IgG1. Deletion of any of the three glyco sylation sites in the TNFR portion of the molecule, alone or in combination , resulted in a moderate or near total blockade of TNFR-IgG1 intracellular transport and secretion. Pulse chase experiments suggested that the glycosy lation site mutants accumulated in the endoplasmic reticulum (ER) and were inefficiently exported to the Golgi apparatus (GA). Replacement of the TNFR signal sequence with the signal/pro sequence of human tissue plasminogen a ctivator (tPA) overcame the blockade to intracellular transport, and restor ed secretion to levels comparable to those achieved with the fully glycosyl ated molecule. Ligand binding studies suggested that the secreted glycosyla tion variants possessed binding characteristics similar to the fully glycos ylated protein. This study demonstrates that N-terminal sequences of tPA ar e unexpectedly efficient in facilitating transport from the ER to the GA an d suggests that these sequences contain a previously unrecognized structura l element that promotes intracellular transport. J. Cell. Biochem. 75:446-4 61, 1999. (C) 1999 Wiley-Liss. Inc.