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 present study, we describe a method to rescue the intracellular transport and secretion of glycoproteins mutagenized to delete N-linked glycosylation sites. Site-directed mutagenesis was used to delete N-linked glycosylation sites from a chimeric protein, TNFR-IgG1. Deletion of any of the three glycosylation 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 glycosylation 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 activator (tPA) overcame the blockade to intracellular transport, and restored secretion to levels comparable to those achieved with the fully glycosylated molecule. Ligand binding studies suggested that the secreted glycosylation variants possessed binding characteristics similar to the fully glycosylated protein. This study demonstrates that N-terminal sequences of tPA are unexpectedly efficient in facilitating transport from the ER to the GA and suggests that these sequences contain a previously unrecognized structural element that promotes intracellular transport.
Köhne
C
C
Department of Cell Culture and Fermentation Research and Development, Genentech, Inc., South San Francisco, California 94080, USA.
Johnson
A
A
Tom
S
S
Peers
D H
DH
Gehant
R L
RL
Hotaling
T A
TA
Brousseau
D
D
Ryll
T
T
Fox
J A
JA
Chamow
S M
SM
Berman
P W
PW
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D006031:Glycosylation; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007142:Immunoglobulin gamma-Chains; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D007143:Immunoglobulin Heavy Chains; D007142:Immunoglobulin gamma-Chains; D021382:Protein Sorting Signals; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; C099374:Ro 45-2081; D010959:Tissue Plasminogen Activator