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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

Distribution of uPA between the supernatant (S) and pellet (P) fractions of cell lysates. Western blot analysis of uPA. The lysates of the following transformants were fractionated by centrifugation: DL/pAM226 (uPA), DL/pAM410 (uPA-C), DLpNR4 (uPA-KDEL) and DL/pNR5 (uPA-C-KDEL). Pellets were dissolved in a 5-fold volume of starting lysate.
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Figure 4: Distribution of uPA between the supernatant (S) and pellet (P) fractions of cell lysates. Western blot analysis of uPA. The lysates of the following transformants were fractionated by centrifugation: DL/pAM226 (uPA), DL/pAM410 (uPA-C), DLpNR4 (uPA-KDEL) and DL/pNR5 (uPA-C-KDEL). Pellets were dissolved in a 5-fold volume of starting lysate.

Mentions: The amount of uPA in the soluble fraction should depend on the rates of uPA synthesis, aggregation and exit from the ER. To prevent the latter process, the uPA and uPA-C variants were modified by the substitution of 12 C-terminal amino acid residues for the KDEL sequence, representing the ER retention signal. This should retain in the ER correctly folded uPA, which is normally secreted, and thus increase proportion of soluble to aggregated uPA in cell lysates. Indeed, such modification abolished the exit of both uPA variants into culture medium (data not shown) and led to the increase of their amounts in cell lysates, though this effect was much more pronounced for uPA-C (Figure 4). Surprisingly, this did not increase the amount of soluble form of both uPA variants in cell lysates. Since full-length uPA showed a high degree of aggregation on its own, the addition of the ER retention signal only slightly increased its aggregation degree. In contrast, the retention in the ER of uPA-C, which normally is less prone to aggregate, led to a drastic increase of amount of aggregated form. At the same time amount of the soluble form of uPA dropped (Figure 4). This indicates that accumulation of uPA in the ER causes its aggregation.


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)

Distribution of uPA between the supernatant (S) and pellet (P) fractions of cell lysates. Western blot analysis of uPA. The lysates of the following transformants were fractionated by centrifugation: DL/pAM226 (uPA), DL/pAM410 (uPA-C), DLpNR4 (uPA-KDEL) and DL/pNR5 (uPA-C-KDEL). Pellets were dissolved in a 5-fold volume of starting lysate.
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Related In: Results  -  Collection

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Figure 4: Distribution of uPA between the supernatant (S) and pellet (P) fractions of cell lysates. Western blot analysis of uPA. The lysates of the following transformants were fractionated by centrifugation: DL/pAM226 (uPA), DL/pAM410 (uPA-C), DLpNR4 (uPA-KDEL) and DL/pNR5 (uPA-C-KDEL). Pellets were dissolved in a 5-fold volume of starting lysate.
Mentions: The amount of uPA in the soluble fraction should depend on the rates of uPA synthesis, aggregation and exit from the ER. To prevent the latter process, the uPA and uPA-C variants were modified by the substitution of 12 C-terminal amino acid residues for the KDEL sequence, representing the ER retention signal. This should retain in the ER correctly folded uPA, which is normally secreted, and thus increase proportion of soluble to aggregated uPA in cell lysates. Indeed, such modification abolished the exit of both uPA variants into culture medium (data not shown) and led to the increase of their amounts in cell lysates, though this effect was much more pronounced for uPA-C (Figure 4). Surprisingly, this did not increase the amount of soluble form of both uPA variants in cell lysates. Since full-length uPA showed a high degree of aggregation on its own, the addition of the ER retention signal only slightly increased its aggregation degree. In contrast, the retention in the ER of uPA-C, which normally is less prone to aggregate, led to a drastic increase of amount of aggregated form. At the same time amount of the soluble form of uPA dropped (Figure 4). This indicates that accumulation of uPA in the ER causes its aggregation.

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