The relationship of N-linked glycosylation and heavy chain-binding protein association with the secretion of glycoproteins.

Abstract

Abstract. The relationship of N-linked glycosylation and association with heavy chain binding protein (BiP) to the secretion of Factor VIII (FVIIO, von Willebrand Factor (vWF), and tissue plasminogen activator (tPA) was studied in Chinese hamster ovary (CHO) cells. FVHI has a heavily glycosylated region containing 20 clustered potential N-linked glycosylation sites. A significant proportion of FVIII was detected in a stable complex with BiP and not secreted. Deletion of the heavily glycosylated region resulted in reduced association with BiP and more efficient secretion. Tunicamycin treatment of cells producing this deleted form of FVIII resulted in stable association of unglycosylated FVIII with BiP and inhibition of efficient secretion, vWF contains 17 potential N-linked glycosylation sites scattered throughout the molecule, vWF was transiently associated with BiP and efficiently secreted demonstrating that CHO cells are competent to secrete a highly glycosylated protein, tPA, which has three utilized N-linked glycosylation sites, exhibited low level association with BiP and was efficiently secreted. Disruption of N-linked glycosylation of tPA by either site-directed mutagenesis or tunicamycin treatment resulted in reduced levels of secretion and increased association with BiP. This effect was enhanced by high levels of tPA expression. The glycosylation state and extent of association with BiP could be correlated with secretion efficiency.

Heat Shock Proteins hsp60 and hsp70: Their Roles in Folding, Assembly and Membrane Translocation of Proteins

“…The role of BiP could be to retain the misfolded proteins. The inhibition of N-glycosylation increased the association with BiP, as shown for immunoglobulin heavy chains (BOLE et al 1986) and several human serum glycoproteins (DORNER et al 1987). The extent and stability of BiP association were inversely correlated with secretion efficiency.…”

Analysis in vivo of GRP78-BiP/substrate interactions and their role in induction of the GRP78-BiP gene.

“…As GRP78-BiP has often been found associated in a semistable manner with incompletely assembled or malfolded proteins, it has been proposed that it may play a role in retention of proteins in the ER (Bole et al, 1986;Dorner et al, 1987;. Therefore, it was of interest to investigate the fate of the HN truncation and deletion mutants in the exocytic pathways given their differing association with GRP78-BiP.…”

“…Although the precise function of GRP78-BiP has not yet been defined, more recently it has been suggested to have a general role in the folding and assembly of proteins in the ER (Gething et al, 1986;Munro and Pelham, 1986;Pelham, 1986), to mark aberrantly folded proteins destined for degradation (Gething et al, 1986;Dorner et al, 1987;Lodish, 1988;, or to aid in solubilizing aggregated proteins during times of stress (Munro and Pelham, 1986). Studies using viral membrane glycoproteins as models have implicated a role for GRP78-BiP in the general folding and assembly of proteins in the exocytic pathway.…”

Expression of a defective mouse mammary tumor virus envelope glycoprotein precursor which binds stably to GRP78 within the lumen of the endoplasmic reticulum is associated with decreased glucocorticoid-induced apoptosis in mouse lymphoma cells

“…GRP78 has a high degree of amino acid sequence homology to the 70 kDa heat shock protein (HSP70) and is, therefore, considered a member of the HSP70 family of stress proteins (Hunt and Morimoto, 1985). GRP78 functions as a molecular chaperone, associating transiently with nascent proteins, facilitating their translocation into the ER (Sanders et al, 1992) and aiding in their folding and assembly into oligomeric structures (Bole et al, 1986;Dorner et al, 1987). For example, in B lymphocytes, nascent immunoglobulin heavy chains and light chains associate with GRP78 until they assemble to form complete immunoglobulin molecules (Haas and Wabl, 1983).…”