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

Institute of Experimental Cardiology, Cardiology Research Center, 3rd Cherepkovskaya Str, 15A, Moscow, 121552, Russia.
BMC Molecular Biology (Impact Factor: 2.19). 11/2002; 3(1):15. DOI: 10.1186/1471-2199-3-15
Source: PubMed


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.
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.
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.

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Available from: Michael Agaphonov, Mar 31, 2015
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    • "The S. cerevisiae strain BY4741 (MATa his3-D1 leu2-D0 met15-D0 ura3-D0) [25] and its derivatives disrupted for the VPS8, VPS10, and VPS17 genes were obtained from the Euroscarf collection. To study uPA secretion, these strains were transformed with the EcoRV-digested pNR9 plasmid [23] "
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    ABSTRACT: Human urokinase-type plasminogen activator (uPA) is poorly secreted by yeast cells. Here, we have selected Hansenula polymorpha mutants with increased productivity of active extracellular uPA. Several of the obtained mutants also demonstrated a defect of sorting of carboxypeptidase Y to the vacuole and the mutant loci have been identified in six of them. All these mutations damaged genes involved in protein traffic between the Golgi apparatus and the vacuole, namely PEP3, VPS8, VPS10, VPS17, and VPS35. We have shown that inactivation of the VPS10 gene encoding the vacuolar protein sorting receptor does not increase uPA secretion but stimulates its proteolytic processing.
    FEMS Yeast Research 12/2005; 5(11):1029-35. DOI:10.1016/j.femsyr.2005.07.003 · 2.82 Impact Factor
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    • "To distinguish the S. cerevisiae and H. polymorpha genes or proteins, they are designated when necessary by Sc and Hp, respectively. Culture conditions used for cultivation of H. polymorpha strains are described elsewhere (Agaphonov et al., 2002). "
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    ABSTRACT: Human urokinase-type plasminogen activator (uPA) is poorly secreted and aggregates in the endoplasmic reticulum of yeast cells due to inefficient folding. A screen for Hansenula polymorpha mutants with improved uPA secretion revealed a gene encoding a homologue of the Saccharomyces cerevisiae protein-O-mannosyltransferase Pmt1p. Expression of the H. polymorpha PMT1 gene (HpPMT1) abolished temperature sensitivity of the S. cerevisiae pmt1 pmt2 double mutant. As in S. cerevisiae, inactivation of the HpPMT1 gene affected electrophoretic mobility of the O-glycosylated protein, extracellular chitinase. In contrast to S. cerevisiae, disruption of HpPMT1 alone caused temperature sensitivity. Inactivation of the HpPMT1 gene decreased intracellular aggregation of uPA, suggesting that enhanced secretion of uPA was due to improvement of its folding in the endoplasmic reticulum. Unlike most of the endoplasmic reticulum membrane proteins, HpPmt1p possesses the C-terminal KDEL retention signal.
    Yeast 10/2005; 22(13):1037-47. DOI:10.1002/yea.1297 · 1.63 Impact Factor
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    • "Yeast expression systems for uPA have also been attempted because yeast are capable of glycosylating proteins during exocytic transport. However, secretion of uPA by yeast was hampered due to the formation of large aggregates in the endoplasmic reticulum (Agaphonov et al. 2002 "
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    ABSTRACT: The development of specific catalytic inhibitors for the serine protease urokinase-type plasminogen activator (uPA) has been hindered due to difficulties in producing sufficient amounts of active recombinant uPA that is catalytically equivalent to native uPA. The purpose of this study was to develop an efficient system for the expression of recombinant human uPA that exhibits comparable proteolytic activity to that of the native protein. Since post-translational modifications (e.g. glycosylations) of uPA are necessary for efficient proteolytic activity, we have used a mammalian cell line [Chinese hamster ovary (CHO)-S] to express recombinant human uPA. CHO-S cells were selected to stably express full-length recombinant human uPA containing a hexahistidine tag at its C-terminus to permit purification by nickel-based affinity chromatography. Secretion of recombinant uPA into the culture media was confirmed by immunoblotting and the presence of an N-linked glycosylation was confirmed by PNGase sensitivity. Enzymatic activity of purified recombinant uPA was demonstrated using zymography and quantitatively compared to native uPA by kinetic analysis using an uPA-specific substrate. Native uPA and the recombinant uPA demonstrated comparable K(m) values (55.7 and 39 muM, respectively). Furthermore, inhibition studies using benzamidine resulted in a K(i) of 195 muM for native uPA, while recombinant uPA had a K(i) of 112 muM. These data indicate that recombinant human uPA expressed by CHO-S cells is functionally comparable to native uPA.
    Cytotechnology 10/2005; 49(1):25-37. DOI:10.1007/s10616-005-4637-7 · 1.75 Impact Factor
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