Glycosylation of rat NGF receptor ectodomain in the yeast <i>Saccharomyces cerevisiae</i>

Holkeri, Heidi; Simonen, Mika; Pummi, Tiina; Vihinen, Helena; Makarow, Marja

doi:10.1016/0014-5793(96)00264-5

Cited by 5 publications

(5 citation statements)

References 25 publications

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“…In addition to these expected forms, we observed that part of the yeast‐expressed penaeidins were produced as O‐glycosylated peptides, the nonsubstituted molecules representing about 50% and 25% of Pen‐2 and ‐3a, respectively. Such an unexpected glycosylation has been already reported for other proteins expressed in yeast [25–27], and as in the case of penaeidins, these recombinant glycopeptides were bearing a dimannosyl O‐substitution with some of them being extensively mannosylated. The dimannosyl substitution induced, in half of the tested strains, a two‐ to four‐fold decrease in penaeidin activity.…”

Section: Discussionsupporting

confidence: 69%

Recombinant expression and range of activity of penaeidins, antimicrobial peptides from penaeid shrimp

Destoumieux

Bulet

Strub

et al. 1999

European Journal of Biochemistry

160

134

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Penaeidins are 5.5-to 6.6-kDa antimicrobial peptides recently isolated from the plasma and haemocytes of the tropical shrimp Penaeus vannamei. These molecules differ from the other classes of antimicrobial peptides in that they are composed of a proline-rich N-terminus and of a C-terminus containing six cysteine residues engaged in three disulfide bridges. In order to gain information on their antimicrobial activity, two penaeidins (Pen-2 and Pen-3a) were expressed in Saccharomyces cerevisiae. The recombinant Pen-2 and -3a were characterized in terms of primary structure by Edman degradation, mass spectrometry and gas chromatography. A protocol was then established to purify the amount of penaeidins required for the determination of their activity spectrum. We demonstrate in this study that expression in yeast is appropriate for the large-scale production of functional penaeidins, whose activities are almost indistinguishable from those of the native molecules. Data on Pen-2 and -3a activity demonstrate that penaeidins have a broad spectrum of antifungal properties associated with a fungicidal activity, and that their antibacterial activities are essentially directed against Gram-positive bacteria, with a strain-specific inhibition mechanism. Despite a better efficiency of Pen-3a on most of the tested strains, similar activity spectra and inhibition mechanisms were observed for both Pen-2 and -3a. Finally, no synergistic effect could be observed between the two molecules.

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Section: Discussionsupporting

confidence: 69%

Recombinant expression and range of activity of penaeidins, antimicrobial peptides from penaeid shrimp

Destoumieux

Bulet

Strub

et al. 1999

European Journal of Biochemistry

160

134

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“…SDS‐PAGE analysis of the precipitates revealed NGFR e , migrating like a 48 kDa protein (Fig. 1 A, lane 1), known to carry one N ‐glycan of about 3 kDa [16]. However, during the chase NGFR e disappeared with a half‐life of about 7 min (Fig.…”

Section: Resultsmentioning

confidence: 98%

“…Hsp150Δ is an N‐terminal fragment (signal peptide plus 303 amino acids) of the yeast secretory glycoproteins Hsp150 [15]. To keep Hsp150Δ‐NGFR e in the pre‐Golgi compartment, we used sec18‐1 cells, where ER‐derived vesicles do not fuse with the Golgi at 37°C [16]. DTT treatment resulted in retardation of electrophoretic migration of Hsp150Δ‐NGFR e (lane 2) as compared to native molecules (lane 1).…”

Section: Resultsmentioning

confidence: 99%

Different degradation pathways for heterologous glycoproteins in yeast

Holkeri¹,

Makarow²

1998

FEBS Letters

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Rat nerve growth factor receptor ectodomain (NGFR e ) and Escherichia coli L L-lactamase were translocated into the yeast endoplasmic reticulum (ER), glycosylated, misfolded and rapidly degraded. NGFR e underwent ATPdependent thermosensitive degradation independently of vesicular transport. Since no evidence for degradation by the cytoplasmic 26S proteosome complex could be obtained, NGFR e appeared to be degraded in the ER. L L-Lactamase exited the ER by vesicular traffic and was transported from the Golgi via the Vps10 receptor pathway to the vacuole for degradation. Machineries in the ER and the Golgi appear to recognize distinct structural features on misfolded heterologous proteins and guide them to different degradation pathways.z 1998 Federation of European Biochemical Societies.

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“…Thus, we fused FucTe to the Hsp150D carrier derived form the secretory S. cerevisiae glycoprotein Hsp150. We have shown that the Hsp150D carrier promotes proper folding of several heterologous proteins in the yeast ER, and confers them secretion competence both in S. cerevisiae and P. pastoris [18,19,[23][24][25][26][27]29,50]. The newly synthesized fusion protein Hsp150D-FucTe was translocated into the yeast ER, and the FucTe portion acquired a catalytically active conformation.…”

Section: Discussionmentioning

confidence: 99%

“…However, mammalian proteins usually cannot exit the yeast endoplasmic reticulum (ER) due to misfolding [17–22]. This secretion block can be overcome by fusing the heterologous protein to the Hsp150Δ carrier, which helps the heterologous protein to acquire its proper conformation [23–26]. The Hsp150Δ carrier consists of an N‐terminal fragment of Hsp150, a glycoprotein of S. cerevisiae , which is secreted rapidly and efficiently to the yeast culture medium [27,28].…”

Section: Introductionmentioning

confidence: 99%

Co-expression of two mammalian glycosyltransferases in the yeast cell wall allows synthesis of sLex

Salo¹,

Sievi²,

Suntio³

et al. 2005

FEMS Yeast Research

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Interactions between selectins and their oligosaccharide-decorated counter-receptors play an important role in the initiation of leukocyte extravasation in inflammation. L-selectin ligands are O-glycosylated with sulphated sialyl Lewis X epitopes (sulpho-sLex). Synthetic sLex oligosaccharides have been shown to inhibit adhesion of lymphocytes to endothelium at sites of inflammation. Thus, they could be used to prevent undesirable inflammatory reactions such as rejection of organ transplants. In vitro synthesis of sLex glycans is dependent on the availability of recombinant glycosyltransferases. Here we expressed the catalytic domain of human alpha-1,3-fucosyltransferase VII in the yeasts Saccharomyces cerevisiae and Pichia pastoris. To promote proper folding and secretion competence of this catalytic domain in yeast, it was fused to the Hsp150 delta carrier, which is an N-terminal fragment of a secretory glycoprotein of S. cerevisiae. In both yeasts, the catalytic domain acquired an active conformation and the fusion protein was externalised, but remained mostly attached to the cell wall in a non-covalent fashion. Incubation of intact S. cerevisiae or P. pastoris cells with GDP-[14C]fucose and sialyl-alpha-2,3-N-acetyllactosamine resulted in synthesis of radioactive sLex, which diffused to the medium. Finally, we constructed an S. cerevisiae strain co-expressing the catalytic domains of alpha-2,3-sialyltransferase and alpha-1,3-fucosyltransferase VII, which were targeted to the cell wall. When these cells were provided with N-acetyllactosamine, CMP-sialic acid and GDP-[14C]fucose, radioactive sLex was produced to the medium. These data imply that yeast cells can provide a self-perpetuating source of fucosyltransferase activity immobilized in the cell wall, useful for the in vitro synthesis of sLex.

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Glycosylation of rat NGF receptor ectodomain in the yeast Saccharomyces cerevisiae

Cited by 5 publications

References 25 publications

Recombinant expression and range of activity of penaeidins, antimicrobial peptides from penaeid shrimp

Recombinant expression and range of activity of penaeidins, antimicrobial peptides from penaeid shrimp

Different degradation pathways for heterologous glycoproteins in yeast

Co-expression of two mammalian glycosyltransferases in the yeast cell wall allows synthesis of sLex

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