Mutation of the protein-O-mannosyltransferase enhances secretion of the human urokinase-type plasminogen activator inHansenula polymorpha

Agaphonov, Michael O.; Sokolov, Svyatoslav S.; Romanova, Nina V.; Sohn, Jung‐Hoon; Kim, Soyoung; Калебина, Т.С.; Choi, Eui‐Sung; Ter‐Avanesyan, Michael D.

doi:10.1002/yea.1297

Cited by 16 publications

(21 citation statements)

References 44 publications

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“…Changes in the O-glycosylation of chitinase are often observed as a consequence of limited activity of the O-glycosylation pathway (Strahl-Bolsinger et al, 1999;Agaphonov et al, 2001;Janik et al, 2003;Zakrzewska et al, 2003;Agaphonov et al, 2005;Kim et al, 2006). Shortage of GDPmannose production (Agaphonov et al, 2001) and limited activity of protein O-mannosyltransferases (Strahl-Bolsinger et al, 1999;Agaphonov et al, 2005) in addition to blocked activity of DPM synthase (Janik et al, 2003) resulted in the production of under O-glycosylated chitinase or for an S. cerevisiae dpm1-6 mutant a totally non O-glycosylated form.…”

Section: Discussionmentioning

confidence: 96%

Cloning and functional analysis of the dpm2 and dpm3 genes from Trichoderma reesei expressed in a Saccharomyces cerevisiae dpm1Δ mutant strain

Zembek

Perlińska-Lenart²,

Rawa³

et al. 2011

Biological Chemistry

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In Trichoderma reesei, dolichyl phosphate mannose (dpm) synthase, a key enzyme in the O-glycosylation process, requires three proteins for full activity. In this study, the dpm2 and dpm3 genes coding for the DPMII and DPMIII subunits of T. reesei DPM synthase were cloned and functionally analyzed after expression in the Saccharomyces cerevisiae dpm1Δ [genotype (BY4743; his3Δ1; /leu2Δ0; lys2Δ0; /ura3Δ0; YPR183w::kanMX4] mutant. It was found that apart from the catalytic subunit DPMI, the DPMIII subunit is also essential to form an active DPM synthase in yeast. Additional expression of the DPMII protein, considered to be a regulatory subunit of DPM synthase, decreased the enzymatic activity. We also characterized S. cerevisiae strains expressing the dpm1, 2, 3 or dpm1, 3 genes and analyzed the consequences of dpm expression on protein O-glycosylation in vivo and on the cell wall composition.

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Section: Discussionmentioning

confidence: 96%

Cloning and functional analysis of the dpm2 and dpm3 genes from Trichoderma reesei expressed in a Saccharomyces cerevisiae dpm1Δ mutant strain

Zembek

Perlińska-Lenart²,

Rawa³

et al. 2011

Biological Chemistry

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“…O-Glycosylation Analysis of Chitinase-Native chitinases from supernatants of saturated cultures were isolated, and their degrees of O-glycosylation were analyzed by SDS-PAGE as described previously (21). Thirty ml of culture supernatants of H. polymorpha cells grown in YPD for 24 h were collected and transferred into centrifuge tubes.…”

Section: Methodsmentioning

confidence: 99%

Functional Characterization of the Hansenula polymorpha HOC1, OCH1, and OCR1 Genes as Members of the Yeast OCH1 Mannosyltransferase Family Involved in Protein Glycosylation

Kim

et al. 2006

Journal of Biological Chemistry

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The ␣-1,6-mannosyltransferase encoded by Saccharomyces cerevisiae OCH1 (ScOCH1) is responsible for the outer chain initiation of N-linked oligosaccharides. To identify the genes involved in the first step of outer chain biosynthesis in the methylotrophic yeast Hansenula polymorpha, we undertook the functional analysis of three H. polymorpha genes, HpHOC1, HpOCH1, and HpOCR1, that belong to the OCH1 family containing seven members with significant sequence identities to ScOCH1. The deletions of these H. polymorpha genes individually resulted in several phenotypes suggestive of cell wall defects. Whereas the deletion of HpHOC1 (Hphoc1⌬) did not generate any detectable changes in N-glycosylation, the null mutant strains of HpOCH1 (Hpoch1⌬) and HpOCR1 (Hpocr1⌬) displayed a remarkable reduction in hypermannosylation. Although the apparent phenotypes of Hpocr1⌬ were most similar to those of S. cerevisiae och1 mutants, the detailed structural analysis of N-glycans revealed that the major defect of Hpocr1⌬ is not in the initiation step but rather in the subsequent step of outer chain elongation by ␣-1,2-mannose addition. Most interestingly, Hpocr1⌬ showed a severe defect in the O-linked glycosylation of extracellular chitinase, representing HpOCR1 as a novel member of the OCH1 family implicated in both N-and O-linked glycosylation. In contrast, addition of the first ␣-1,6-mannose residue onto the core oligosaccharide Man 8 GlcNAc 2 was completely blocked in Hpoch1⌬ despite the comparable growth of its wild type under normal growth conditions. The complementation of the S. cerevisiae och1 null mutation by the expression of HpOCH1 and the lack of in vitro ␣-1,6-mannosyltransferase activity in Hpoch1⌬ provided supportive evidence that HpOCH1 is the functional orthologue of ScOCH1. The engineered Hpoch1⌬ strain with the targeted expression of Aspergillus saitoi ␣-1,2-mannosidase in the endoplasmic reticulum was shown to produce human-compatible high mannosetype Man 5 GlcNAc 2 oligosaccharide as a major N-glycan.

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“…We found that all our mutants secreted similar amounts of proteins compared to the control. A different effect was observed for chitinase secreted by the pmt mutant of Hansenula where an under-O-mannosylated enzyme was secreted in a decreased amount (Agaphonov et al 2005). On the other hand, glucoamylase I from Aspergillus awamori expressed in the Saccharomyces cerevisiae pmt1 mutant, although O-glycosylated to a lesser extent, was secreted in the same amount as in a wild type strain (Goto et al 1999).…”

Section: Discussionmentioning

confidence: 93%

“…Changes in both types of glycosylation were observed for a Hansenula polymorpha pmt mutant, where O-mannosylation of chitinase was partially inhibited together with total inhibition of N-glycosylation of invertase or heterologously expressed human urinary type plasminogen activator (Agaphonov et al 2005). The nature of the influence of a limited protein O-mannosyltransferase activity on the N-glycosylation process is not clear, although many authors have reported a close interdependence of the two glycosylation processes (Harty et al 2001;Ecker et al 2003;Nakatsukasa et al 2004).…”

Section: Discussionmentioning

confidence: 94%

“…In this strain, N-glycosylated proteins such as heterologously expressed human urinary type plasminogen activator were produced and secreted in higher amounts although they were unglycosylated (Agaphonov et al 2005). It is important that the stimulation of protein production and secretion concerned only N-glycoproteins whereas production and secretion of the O-glycosylated chitinase were significantly decreased.…”

Section: Introductionmentioning

confidence: 98%

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Integration of additional copies of Trichoderma reesei gene encoding protein O-mannosyltransferase I results in a decrease of the enzyme activity and alteration of cell wall composition

et al. 2011

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In fungi, transfer of the first mannosyl residue to proteins during their O-glycosylation is catalyzed by protein O-mannosyltransferases. Integration of additional copies of the pmt1 gene into Trichoderma reesei genome unexpectedly resulted in the silencing of pmt1 expression. Strains carrying the additional copies of pmt1 gene exhibited lower total activity of protein O-mannosyltransferases, lower O- and N-glycosylation of secreted proteins and showed defects in their cell wall composition. Moreover, the strains grew slowly on solid medium and were hypersensitive to an antifungal reagent, Calcofluor white. These results indicate that protein O-mannosyltransferases are required for proper cell wall formation, and their decreased activity influences not only O- but also N-glycosylation.

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Mutation of the protein-O-mannosyltransferase enhances secretion of the human urokinase-type plasminogen activator inHansenula polymorpha

Abstract: Abstract

Cited by 16 publications

References 44 publications

Cloning and functional analysis of the dpm2 and dpm3 genes from Trichoderma reesei expressed in a Saccharomyces cerevisiae dpm1Δ mutant strain

Cloning and functional analysis of the dpm2 and dpm3 genes from Trichoderma reesei expressed in a Saccharomyces cerevisiae dpm1Δ mutant strain

Functional Characterization of the Hansenula polymorpha HOC1, OCH1, and OCR1 Genes as Members of the Yeast OCH1 Mannosyltransferase Family Involved in Protein Glycosylation

Integration of additional copies of Trichoderma reesei gene encoding protein O-mannosyltransferase I results in a decrease of the enzyme activity and alteration of cell wall composition

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