Soluble forms of YlCrh1p and YlCrh2p, cell wall proteins of <i>Yarrowia lipolytica</i>, have β‐1,3‐glycosidase activity

Hwang, Ji-Sook; Seo, Dong-Hyuck; Kim, Jeong‐Yoon

doi:10.1002/yea.1395

Cited by 10 publications

(7 citation statements)

References 20 publications

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“…The pIMR53-AUX vector (11), containing the XPR2 promoter and terminator and the URA3 gene, was used for the secretory expression of Trichoderma reesei endoglucanase I (EGI) and Y. lipolytica lipase tagged with six histidine residues. To express EGI and lipase, the recombinant Y. lipolytica cells were cultivated on YPDm medium (1% yeast extract, 1% proteose peptone, 1% glucose, and 50 mM phosphate-buffered saline, pH 6.8) at 28°C.…”

Section: Methodsmentioning

confidence: 99%

Engineering of the Yeast Yarrowia lipolytica for the Production of Glycoproteins Lacking the Outer-Chain Mannose Residues of N-Glycans

Song

Choi

Park

et al. 2007

Appl Environ Microbiol

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In an attempt to engineer a Yarrowia lipolytica strain to produce glycoproteins lacking the outer-chain mannose residues of N-linked oligosaccharides, we investigated the functions of the OCH1 gene encoding a putative ␣-1,6-mannosyltransferase in Y. lipolytica. The complementation of the Saccharomyces cerevisiae och1 mutation by the expression of YlOCH1 and the lack of in vitro ␣-1,6-mannosyltransferase activity in the Yloch1 null mutant indicated that YlOCH1 is a functional ortholog of S. cerevisiae OCH1. The oligosaccharides assembled on two secretory glycoproteins, the Trichoderma reesei endoglucanase I and the endogenous Y. lipolytica lipase, from the Yloch1 null mutant contained a single predominant species, the core oligosaccharide Man 8 GlcNAc 2 , whereas those from the wild-type strain consisted of oligosaccharides with heterogeneous sizes, Man 8 GlcNAc 2 to Man 12 GlcNAc 2 . Digestion with ␣-1,2-and ␣-1,6-mannosidase of the oligosaccharides from the wild-type and Yloch1 mutant strains strongly supported the possibility that the Yloch1 mutant strain has a defect in adding the first ␣-1,6-linked mannose to the core oligosaccharide. Taken together, these results indicate that YlOCH1 plays a key role in the outer-chain mannosylation of N-linked oligosaccharides in Y. lipolytica. Therefore, the Yloch1 mutant strain can be used as a host to produce glycoproteins lacking the outer-chain mannoses and further developed for the production of therapeutic glycoproteins containing human-compatible oligosaccharides.Yeast can secrete a variety of proteins in much the same way that mammalian cells do. The presence of yeast-specific outerchain mannosylation, however, has been a primary hindrance to the exploitation of yeast for therapeutic glycoprotein production, because glycoproteins decorated with yeast-specific glycans are immunogenic and show poor pharmacokinetic properties in humans (1, 24). In the budding yeast Saccharomyces cerevisiae, the N-linked oligosaccharides assembled on glycoproteins include hypermannose structures with outer chains that may contain up to 200 mannose units (6). Elongation of the outer chain is initiated by the Och1 protein, which adds the first ␣-1,6-linked mannose to the core N-linked oligosaccharides upon their arrival in the Golgi apparatus in S. cerevisiae (17). Following the addition of the first ␣-1,6-mannose by Och1p, the core oligosaccharide is elongated by additional ␣-1,6-mannosyltransferases, Mnn9p and Van1p, which extend the ␣-1,6-linked polymannose backbone, and the core oligosaccharide is further branched by the addition of ␣-1,2-and ␣-1,3-linked mannoses (5, 8). Other yeast species, including Pichia pastoris, Hansenula polymorpha, and Schizosaccharomyces pombe, also use the Och1 protein to extend the mannose outer chain of N-glycans (14, 24, 26, 28). Therefore, the elimination of the Och1 protein was performed to block the yeast-specific outer-chain mannosylation, followed by further engineering of yeast N-glycosylation pathways for the production of glycoproteins with hum...

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Section: Methodsmentioning

confidence: 99%

Engineering of the Yeast Yarrowia lipolytica for the Production of Glycoproteins Lacking the Outer-Chain Mannose Residues of N-Glycans

Song

Choi

Park

et al. 2007

Appl Environ Microbiol

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“…Fungal Bgl2p also has transglucosidase activity in vitro (Mouyna et al , 1998). Crh proteins in Yarrowia lipolytica possess endo‐1,3‐β‐glucosidase activity in vitro (Hwang et al , 2006; Mrša et al , 1993), but it is yet unknown whether they also have transglucosidase activity. Crh‐family members in both S. cerevisiae and C. albicans are cell cycle‐regulated and their cell‐surface localization largely coincides with spots of chitin incorporation (Pardini et al , 2006; Rodríguez‐Peña et al , 2000).…”

Section: Introductionmentioning

confidence: 99%

Mass spectrometric identification of covalently bound cell wall proteins from the fission yeast Schizosaccharomyces pombe

Groot

Yin

Weig

et al. 2007

Yeast

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The cell wall of Schizosaccharomyces pombe is bilayered, consisting of an inner layer of mainly polysaccharides and an outer layer of galactomannoproteins. We present a detailed analysis of the cell wall proteome. Six covalently-bound cell wall proteins (CWPs) were identified using tandem mass spectrometry, including four predicted GPI-dependent CWPs (Gas1p, Gas5p, Ecm33p and Pwp1p) and two alkali-sensitive CWPs (Psu1p and Asl1p). Gas1p and Gas5p belong to glycoside hydrolase family 72, and are believed to be involved in 1,3-β-glucan elongation. Ecm33p belongs to a ubiquitous fungal protein family with an unknown but crucial function in cell wall integrity. Pwp1p is an abundant protein with an unknown but probably nonenzymatic function. All four CWPs were present in HF-pyridine extracts, indicating that they are linked via a phosphodiester bridge to the glucan network. Psu1p is a homologue of the Saccharomyces cerevisiae Sun family, whereas Asl1p has no homologues in S. cerevisiae but is related to Aspergillus fumigatus and Ustilago maydis proteins. Finally, although the protein content of Sz. pombe cell walls is only slightly less than in S. cerevisiae and Candida albicans, the amount of carbohydrate added to the proteins was found to be two-to three-fold decreased, consistent with earlier reported differences in outer chain N -glycosylation.

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“…Utr2 is found in K. lactis cell wall exclusively in logarithmic growth phase on either glucose or lactose or ethanol. In the cell wall of Y. lipolytica, Hwang et al [58] found two cell wall proteins YlCrh1 and YlCrh2 showing high amino acid sequence similarity to S. cerevisiae Crh1 and Crh2. Deletion of the YlCRH1 and YlCRH2 resulted in increased sensitivity to Congo red and Calcofluor white.…”

Section: Cell Wall Proteins With Enzyme Activitiesmentioning

confidence: 99%

“…The YlCrh1 protein showed to be capable to hydrolyze β-1,4and β-1,6-linkages as well, although with lower specific activities than that on β-1,3-linkage. Both YlCrh1 and YlCrh2 cannot degrade the substrate of exo-β-1,3-glucanases (4-nitrophenyl-β-D-glucopyranoside), indicating that they are endo β-1,3-glucanases [58].…”

Section: Cell Wall Proteins With Enzyme Activitiesmentioning

confidence: 99%

Evolutionary Overview of Molecular Interactions and Enzymatic Activities in the Yeast Cell Walls

Teparić

Lozančić

Mrša

2020

IJMS

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Fungal cell walls are composed of a polysaccharide network that serves as a scaffold in which different glycoproteins are embedded. Investigation of fungal cell walls, besides simple identification and characterization of the main cell wall building blocks, covers the pathways and regulations of synthesis of each individual component of the wall and biochemical reactions by which they are cross-linked and remodeled in response to different growth phase and environmental signals. In this review, a survey of composition and organization of so far identified and characterized cell wall components of different yeast genera including Saccharomyces, Candida, Kluyveromyces, Yarrowia, and Schizosaccharomyces are presented with the focus on their cell wall proteomes.

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Soluble forms of YlCrh1p and YlCrh2p, cell wall proteins of Yarrowia lipolytica, have β‐1,3‐glycosidase activity

Cited by 10 publications

References 20 publications

Engineering of the Yeast Yarrowia lipolytica for the Production of Glycoproteins Lacking the Outer-Chain Mannose Residues of N-Glycans

Engineering of the Yeast Yarrowia lipolytica for the Production of Glycoproteins Lacking the Outer-Chain Mannose Residues of N-Glycans

Mass spectrometric identification of covalently bound cell wall proteins from the fission yeast Schizosaccharomyces pombe

Evolutionary Overview of Molecular Interactions and Enzymatic Activities in the Yeast Cell Walls

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