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Aggregation and retention of human urokinase type plasminogen activator in the yeast endoplasmic reticulum.

Agaphonov MO, Romanova NV, Trushkina PM, Smirnov VN, Ter-Avanesyan MD - BMC Mol. Biol. (2002)

Bottom Line: Secretion of recombinant proteins in yeast can be affected by their improper folding in the endoplasmic reticulum and subsequent elimination of the misfolded molecules via the endoplasmic reticulum associated protein degradation pathway.Mutation abolishing N-glycosylation decreased the efficiency of uPA secretion and increased its aggregation degree.Accumulation of uPA within the endoplasmic reticulum disturbs its proper folding and leads to formation of high molecular weight aggregates.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Experimental Cardiology, Cardiology Research Center, 3rd Cherepkovskaya Str, 15A, Moscow, 121552, Russia. aga@cardio.ru

ABSTRACT

Background: Secretion of recombinant proteins in yeast can be affected by their improper folding in the endoplasmic reticulum and subsequent elimination of the misfolded molecules via the endoplasmic reticulum associated protein degradation pathway. Recombinant proteins can also be degraded by the vacuolar protease complex. Human urokinase type plasminogen activator (uPA) is poorly secreted by yeast but the mechanisms interfering with its secretion are largely unknown.

Results: We show that in Hansenula polymorpha overexpression worsens uPA secretion and stimulates its intracellular aggregation. The absence of the Golgi modifications in accumulated uPA suggests that aggregation occurs within the endoplasmic reticulum. Deletion analysis has shown that the N-terminal domains were responsible for poor uPA secretion and propensity to aggregate. Mutation abolishing N-glycosylation decreased the efficiency of uPA secretion and increased its aggregation degree. Retention of uPA in the endoplasmic reticulum stimulates its aggregation.

Conclusions: The data obtained demonstrate that defect of uPA secretion in yeast is related to its retention in the endoplasmic reticulum. Accumulation of uPA within the endoplasmic reticulum disturbs its proper folding and leads to formation of high molecular weight aggregates.

No MeSH data available.


Related in: MedlinePlus

Western blot analysis of uPA in intracellular aggregates and culture medium. Lanes 1 and 2, pellet fraction of cell lysate of the DLU strain expressing full-length uPA before and after treatment with EndoH, respectively; lanes 3 and 4, culture medium of the same strain before and after EndoH treatment, respectively; lane 5, pellet fraction of cell lysate of the DLCQ strain expressing uPA-CQ.
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Figure 2: Western blot analysis of uPA in intracellular aggregates and culture medium. Lanes 1 and 2, pellet fraction of cell lysate of the DLU strain expressing full-length uPA before and after treatment with EndoH, respectively; lanes 3 and 4, culture medium of the same strain before and after EndoH treatment, respectively; lane 5, pellet fraction of cell lysate of the DLCQ strain expressing uPA-CQ.

Mentions: Further analysis has shown that intracellular uPA accumulated in a form of high molecular weight aggregates, since it could be precipitated by centrifugation of cell lysates at 10,000 g for 10 min in the presence of a detergent, solubilizing membrane associated proteins (Figure 1B). uPA from culture supernatants of the single copy integrants migrated on the SDS PAGE as a broad smear (Figure 2, lane 3), which could be converted into the distinct ~30 kDa band by treatment with endoglycosidase H (EndoH) (Figure 2, lane 4). In human cells uPA is synthesized as a 48 kDa zymogen consisting of three domains: two N-terminal domains, not essential for the enzymatic activity, and the C-terminal serine protease domain (Figure 1A). uPA zymogen is activated by hydrolysis of the K158-I159 peptide bond. The accuracy of the cleavage is essential for uPA activation, since hydrolysis of the R156-F157 peptide bond by thrombin produces inactive protein [for review, see [12]]. EndoH-treated extracellular uPA migrated faster than its N-terminally truncated variant lacking the N-glycosylation site (Figure 2, lane 5), indicating that it is a product of the uPA cleavage at the activation site, because cleavage at other sites would result in uPA derivatives showing either different molecular weight or no activity.


Aggregation and retention of human urokinase type plasminogen activator in the yeast endoplasmic reticulum.

Agaphonov MO, Romanova NV, Trushkina PM, Smirnov VN, Ter-Avanesyan MD - BMC Mol. Biol. (2002)

Western blot analysis of uPA in intracellular aggregates and culture medium. Lanes 1 and 2, pellet fraction of cell lysate of the DLU strain expressing full-length uPA before and after treatment with EndoH, respectively; lanes 3 and 4, culture medium of the same strain before and after EndoH treatment, respectively; lane 5, pellet fraction of cell lysate of the DLCQ strain expressing uPA-CQ.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC130179&req=5

Figure 2: Western blot analysis of uPA in intracellular aggregates and culture medium. Lanes 1 and 2, pellet fraction of cell lysate of the DLU strain expressing full-length uPA before and after treatment with EndoH, respectively; lanes 3 and 4, culture medium of the same strain before and after EndoH treatment, respectively; lane 5, pellet fraction of cell lysate of the DLCQ strain expressing uPA-CQ.
Mentions: Further analysis has shown that intracellular uPA accumulated in a form of high molecular weight aggregates, since it could be precipitated by centrifugation of cell lysates at 10,000 g for 10 min in the presence of a detergent, solubilizing membrane associated proteins (Figure 1B). uPA from culture supernatants of the single copy integrants migrated on the SDS PAGE as a broad smear (Figure 2, lane 3), which could be converted into the distinct ~30 kDa band by treatment with endoglycosidase H (EndoH) (Figure 2, lane 4). In human cells uPA is synthesized as a 48 kDa zymogen consisting of three domains: two N-terminal domains, not essential for the enzymatic activity, and the C-terminal serine protease domain (Figure 1A). uPA zymogen is activated by hydrolysis of the K158-I159 peptide bond. The accuracy of the cleavage is essential for uPA activation, since hydrolysis of the R156-F157 peptide bond by thrombin produces inactive protein [for review, see [12]]. EndoH-treated extracellular uPA migrated faster than its N-terminally truncated variant lacking the N-glycosylation site (Figure 2, lane 5), indicating that it is a product of the uPA cleavage at the activation site, because cleavage at other sites would result in uPA derivatives showing either different molecular weight or no activity.

Bottom Line: Secretion of recombinant proteins in yeast can be affected by their improper folding in the endoplasmic reticulum and subsequent elimination of the misfolded molecules via the endoplasmic reticulum associated protein degradation pathway.Mutation abolishing N-glycosylation decreased the efficiency of uPA secretion and increased its aggregation degree.Accumulation of uPA within the endoplasmic reticulum disturbs its proper folding and leads to formation of high molecular weight aggregates.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Experimental Cardiology, Cardiology Research Center, 3rd Cherepkovskaya Str, 15A, Moscow, 121552, Russia. aga@cardio.ru

ABSTRACT

Background: Secretion of recombinant proteins in yeast can be affected by their improper folding in the endoplasmic reticulum and subsequent elimination of the misfolded molecules via the endoplasmic reticulum associated protein degradation pathway. Recombinant proteins can also be degraded by the vacuolar protease complex. Human urokinase type plasminogen activator (uPA) is poorly secreted by yeast but the mechanisms interfering with its secretion are largely unknown.

Results: We show that in Hansenula polymorpha overexpression worsens uPA secretion and stimulates its intracellular aggregation. The absence of the Golgi modifications in accumulated uPA suggests that aggregation occurs within the endoplasmic reticulum. Deletion analysis has shown that the N-terminal domains were responsible for poor uPA secretion and propensity to aggregate. Mutation abolishing N-glycosylation decreased the efficiency of uPA secretion and increased its aggregation degree. Retention of uPA in the endoplasmic reticulum stimulates its aggregation.

Conclusions: The data obtained demonstrate that defect of uPA secretion in yeast is related to its retention in the endoplasmic reticulum. Accumulation of uPA within the endoplasmic reticulum disturbs its proper folding and leads to formation of high molecular weight aggregates.

No MeSH data available.


Related in: MedlinePlus