Function of the Carbohydrate Moieties of Glycoproteins

Olden, Kenneth; Bernard, Bruno; White, Sandra L.; Parent, J B

doi:10.1002/jcb.1982.240180306

Cited by 92 publications

(41 citation statements)

References 102 publications

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“…A second possibility is that the oligosaccharides protect the polypeptide from proteolytic degradation, thereby influencing secretion in an indirect way. Protease inhibitors can restore secretion or cell surface expression of several mammalian glycoproteins in TM-blocked cells, supporting a protective role for N-linked glycans (22,23 (2,11,20). If so, one or more of the proteins coprecipitating with nonglycosylated t-PA could be an insect cell version of the immunoglobulin-heavy-chain-binding protein, BiP (2,11,20).…”

Section: Discussionmentioning

confidence: 94%

“…This model requires that the product synthesized in the presence of TM also lacks the prosequence to explain its comigration with the deglycosylated extracellular product and the fact that it is smaller than the deglycosylated intracellular product. Proteolytic removal of the prosequence of intracellular nonglycosylated t-PA might be anticipated on the basis of increased sensitivity of the polypeptide to proteolysis in the absence of N glycosylation (22,23).…”

Section: Resultsmentioning

confidence: 99%

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Glycosylation and secretion of human tissue plasminogen activator in recombinant baculovirus-infected insect cells.

Jarvis¹,

Summers²

1989

Mol. Cell. Biol.

234

134

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Cell lines established from the lepidopteran insect Spodoptera frugiperda (fall armyworm; Sf9) are used routinely as hosts for the expression of foreign proteins by recombinant baculovirus vectors. We have examined the pathway of protein glycosylation and secretion in these cells, using human tissue plasminogen activator (t-PA) as a model. t-PA expressed in Sf9 cells was both N glycosylated and secreted. At least a subset of the N-linked oligosaccharides in extracellular t-PA was resistant to endo-o-N-acetyl-D-glucosaminidase H, which removes immature, high-mannose-type oligosaccharides. This refutes the general conclusion from previous studies that Sf9 cells cannot process immature N-linked oligosaccharides to an endo-4-N-acetyl-D-glucosaminidase H-resistant form. A nonglycosylated t-PA precursor was not detected in Sf9 cells, even with very short pulse-labeling times. This suggests that the mammalian signal sequence of t-PA is efficiently recognized in Sf9 cells and that it can mediate rapid translocation across the membrane of the rough endoplasmic reticulum, where cotranslational N glycosylation takes place. However, t-PA was secreted rather slowly, with a half-time of about 1.6 h. Thus, a rate-limiting step(s) in secretion occurs subsequent to translocation and N glycosylation of the t-PA polypeptide. Treatment of Sf9 cells with tunicamycin, but not with inhibitors of oligosaccharide processing, prevented the appearance of t-PA in the extracellular medium. This suggests that N glycosylation per se, but not processing of the N-linked oligosaccharides, is required directly or indirectly in baculovirusinfected Sf9 cells for the secretion of t-PA. Finally, the relative efficiency of secretion decreased dramatically with time of infection, suggesting that the Sf9 host cell secretory pathway is compromised during the later stages of baculovirus infection.

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Section: Discussionmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

Glycosylation and secretion of human tissue plasminogen activator in recombinant baculovirus-infected insect cells.

Jarvis¹,

Summers²

1989

Mol. Cell. Biol.

234

134

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“…4, lanes d and e). This may be due to the instability of proteins synthesized in the presence of tunicamycin (44) and the low amount of p185 in these cells. However, B104-1-1 cells and DHFR/G-8 cells contained the 170-kDa protein described above, which migrated more slowly than the EGFr core polypeptide (compare Fig.…”

Section: Resultsmentioning

confidence: 99%

p185, a product of the neu proto-oncogene, is a receptorlike protein associated with tyrosine kinase activity.

Stern¹,

Heffernan²,

Weinberg³

1986

Mol. Cell. Biol.

305

165

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The neu oncogene was originally identified in cell lines derived from rat neuroectodermal tumors. neu is related to but distinct from the c-erbB gene, which encodes the epidermal growth factor (EGF) receptor. neu encodes a protein, designated p185, that is serologically related to the EGF receptor. Identification of the normal homolog of p185 encoded by the neu proto-oncogene enabled us to compare the product of the neu proto-oncogene with the mutated version specified by the neu oncogene and with the EGF receptor. The normal form of p185 was structurally similar to its transforming counterpart, indicating that activation of the neu oncogene did not cause major structural alterations in the gene product. Both normal and transforming forms of p185 were associated with tyrosine kinase activity, supporting the idea that normal p185 functions as a growth factor receptor. p185 differed both structurally and functionally from the EGF receptor. p185 and the EGF receptor had distinct electrophoretic mobilities when synthesized under normal culture conditions or in the presence of tunicamycin. EGF did not stimulate increased turnover of p185 and did not bind quantitatively to p185. A number of other growth factors failed to stimulate degradation of p185 or tyrosine phosphorylation of p185 and are therefore unlikely to be ligands for p185.More than 20 different oncogenes have been implicated in virus-mediated tumorigenesis, and a number of these are likely to play a direct role in human cancer (reviewed in references 4 and 35). It is now evident that some of these oncogenes operate by disrupting pathways regulated by polypeptide growth factors (reviewed in references 23, 25, and 59). Thus the v-sis oncogene has been found to encode a protein which is structurally and functionally homologous to platelet-derived growth factor (PDGF) (13,15,30,48,64). v-sis apparently transforms cells by causing them to constitutively secrete a mitogenic factor to which these same cells respond. Two viral oncogenes, v-erbB and v-fms, are derived from cellular genes encoding growth factor receptors. v-erbB specifies a truncated version of the cellular epidermal growth factor receptor (EGFr) (16, 63), while v-fms specifies a protein related to the macrophage colony-stimulating factor 1 receptor (56). The two defective receptor proteins may transform cells by constitutively transmitting growth excitatory signals that are normally produced only in the presence of their respective ligands.We have been studying another oncogene, designated neu, which apparently specifies a mutated growth factor receptor. In contrast to v-erbB and v-fms, which were discovered as transforming genes of retroviruses, the neu oncogene was identified by transfection of DNA from chemically induced tumors (57). DNA from four ethyl-nitrosourea-induced rat neuroectodermal tumor cell lines induced foci when introduced by transfection into NIH 3T3 cell monolayers (57). Subsequent studies revealed that these four neuroectodermal tumor cell lines all contained activated version...

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“…By contrast, non-glycosylated DPP IV synthesized in the presence of tunicamycin failed to reach the cell surface and was rapidly degraded after biosynthesis intracellularly. As has been shown for several glycoproteins, carbohydrate side chains prevent degradation by masking domains which are sensitive to a proteolytic attack (for review see [49]). Moreover, N-glycans may also influence the proper folding of newly synthesized proteins [50, 511.…”

mentioning

confidence: 91%

Biosynthesis and metabolism of dipeptidylpeptidase IV in primary cultured rat hepatocytes and Morris hepatoma 7777 cells

Loch

Tauber

Becker

et al. 1992

European Journal of Biochemistry

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N‐Glycosylation, biosynthesis and degradation of dipeptidylpeptidase IV (EC 3.4.14.5) (DPP IV) were comparatively studied in primary cultured rat hepatocytes and Morris hepatoma 7777 cells (MH 7777 cells). DPP IV had a molecular mass of 105 kDa in rat hepatocytes and of 103 kDa in MH 7777 cells as assessed by SDS/PAGE under reducing conditions. This difference in molecular mass was caused by differences in covalently attached N‐glycans. DPP IV from hepatoma cells contained a higher proportion of N‐glycans of the oligomannosidic or hybrid type and therefore migrated at a slightly lower molecular mass. In both cell types DPP IV was initially synthesized as a 97‐kDa precursor which was completely susceptible to digestion with endo‐β‐N‐acetylglucosaminidase H converting the molecular mass to 84 kDa. The precursor was processed to the mature forms of DPP IV, glycosylated with N‐glycans mainly of the complex type with a half‐life of 20–25 min. The transit of newly synthesized DPP IV to the cell surface displayed identical or very similar kinetics in both cell types with the major portion of DPP IV appearing at the cell surface after 60 min. DPP IV molecules were very slowly degraded in hepatocytes as well as in hepatoma cells with half‐lives of approximately 45 h. Inhibition of oligosaccharide processing with 1‐deoxymannojirimycin led to the formation of DPP IV molecules containing N‐glycans of the oligomannosidic type. This glycosylation variant was degraded with the same half‐life as complex‐type glycosylated DPP IV. By contrast, inhibition of N‐glycosylation with tunicamycin resulted into rapid degradation of non‐N‐glycosylated DPP IV molecules in both cell types. Non‐N‐glycosylated DPP IV could not be detected at the cell surface indicating an intracellular proteolytic process soon after biosynthesis.

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Function of the Carbohydrate Moieties of Glycoproteins

Cited by 92 publications

References 102 publications

Glycosylation and secretion of human tissue plasminogen activator in recombinant baculovirus-infected insect cells.

Glycosylation and secretion of human tissue plasminogen activator in recombinant baculovirus-infected insect cells.

p185, a product of the neu proto-oncogene, is a receptorlike protein associated with tyrosine kinase activity.

Biosynthesis and metabolism of dipeptidylpeptidase IV in primary cultured rat hepatocytes and Morris hepatoma 7777 cells

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