Glycosylated and Phosphorylated Proteins—Expression in Yeast and Oocytes of Xenopus: Prospects and Challenges—Relevance to Expression of Thermostable Proteins

Li, Pingzuo; Gao, Xiugong; Arellano, Rogelio O.; Renugopalakrishnan, V.

doi:10.1006/prep.2001.1431

Cited by 26 publications

(22 citation statements)

References 137 publications

(13 reference statements)

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“…So, the D1 Cys heterogeneity is a special feature connected to the expression in yeast cells. The heterogeneity might be caused by a combination of several common modifications of proteins expressed in yeast such as phosphorylation, N-linked glycosylation, O-linked glycosylation and fatty acid acylation (Tanner & Lehle 1987, Simon & Aderem 1992, Li et al 2001. By site-directed mutagenesis of consensus N-linked glycosylation sites and treatment with N-glycosidases, we found no evidence for N-linked glycosylation and it appears to be excluded.…”

Section: Discussionmentioning

confidence: 73%

“…Although the D1 Cys protein expressed in yeast is posttranslationally modified in a different manner to the D1 Cys protein expressed in COS cells the kinetic characteristics are similar. Future experiments will be aimed at purification of the D1 Cys protein and overexpression in other yeast strains such as Pichia pastoris or Hansenula polymorpha, which could result in even higher expression levels and more authentic post-translational modification characteristics (Bill 2001, Li et al 2001.…”

Section: Discussionmentioning

confidence: 99%

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Expression of recombinant membrane-bound type I iodothyronine deiodinase in yeast

Kuiper

Klootwijk²,

Visser³

2005

Journal of Molecular Endocrinology

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The bioactivity of thyroid hormone is determined to a large extent by the monodeiodination of the prohormone thyroxine (T4) by the hepatic selenoenzyme type I iodothyronine deiodinase (D1), i.e. by outer ring deiodination (ORD) to the active hormone triiodothyronine (T3) or by inner ring deiodination (IRD) to the inactive metabolite reverse T3 (rT3). Since D1 is a membrane-bound protein with an N-terminal membrane-spanning domain, the enzyme is very difficult to purify in an active state. This study was undertaken in order to develop a heterologous (over)-expression system that would eventually allow the production of large amounts of purified active D1 protein. We have expressed a mutant rat D1 protein, in which the selenocysteine residue in the core catalytic center was replaced by cysteine (D1 Cys) in yeast cells (Saccharomyces cerevisiae). After yeast cell fractionation, kinetic analysis was performed with dithiothreitol as reducing cofactor. ORD activity was associated with membrane fractions, while no activity could be detected in the cytosolic fraction. The D1 Cys protein displayed a tenfold increase in K m (2 µM) for rT3 as compared with native D1 protein in rat liver microsomes. The D1 protein content is about 65 pmol/mg microsomal protein, as compared with about 3 pmol/mg in rat liver microsomal fraction. SDS-PAGE analysis of N-bromoacetyl-[125 I]T3 affinity-labeled D1 protein showed several labeled protein isoforms with apparent molecular masses between 27 and 32 kDa. Immunoblot analysis with a specific D1 antiserum confirmed the observed D1 protein heterogeneity. Site-directed mutagenesis of several potential N-linked glycosylation sites, phosphorylation sites and a unique myristoylation site established that D1 heterogeneity is not caused by N-linked glycosylation, but probably by a combination of O-linked glycosylation and phosphorylation. Deletion of the endoplasmic reticulum (ER)-signal sequence and the membrane-spanning domain (amino acid residue 2-35), did not result in the production of a soluble D1 enzyme. Although this mutated D1 protein is inactive, the fact that it is still membrane bound indicates the existence of additional membrane attachment site(s) or membrane-spanning domains. Overall, our studies indicate that yeast cells provide a useful system for the expression of relatively high levels of D1 protein which could be used for further structure-function analysis.

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Section: Discussionmentioning

confidence: 73%

Section: Discussionmentioning

confidence: 99%

Expression of recombinant membrane-bound type I iodothyronine deiodinase in yeast

Kuiper

Klootwijk²,

Visser³

2005

Journal of Molecular Endocrinology

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“…A role for glycosylation (40) or hydroxylation of prolines (17) in protein folding, oligomer assembly, structural stability, specific signal transduction, recognition and secretion processes, or clearance of glycoproteins has been described previously. Insect cells are unable to produce several modifications, including proline hydroxylation and complex Nlinked side chains with terminal sialic acid (28,43).…”

Section: Vol 75 2007 Phagocytosis By Cho Cell-produced Sp-a Variantmentioning

confidence: 93%

“…We speculated that incomplete posttranslational modification of the insect cell-expressed proteins accounts for the lower level of cell association. Insect cell-derived proteins lack or are defective in certain mammalian posttranslational modifications (16,40), such as proline hydroxylation and complex N-linked glycosylation, that may be important for enhancement of cell association. In the present study, we expanded on our previous findings.…”

mentioning

confidence: 99%

Surfactant Protein A2 (SP-A2) Variants Expressed in CHO Cells Stimulate Phagocytosis of Pseudomonas aeruginosa More than Do SP-A1 Variants

Mikerov¹,

Wang²,

Umstead

et al. 2007

Infect Immun

112

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Surfactant protein A (SP-A) enhances phagocytosis of Pseudomonas aeruginosa. Two functional genes, SP-A1and SP-A2, encode human SP-A. As we showed before, baculovirus-mediated insect cell-expressed SP-A2 enhances the association of P. aeruginosa with rat alveolar macrophages (rAMs) more than does SP-A1. However, true phagocytosis (internalization) was not shown, and insect cell derived proteins lack or are defective in certain mammalian posttranslational modifications that may be important for SP-A1 and SP-A2 activity and specificity. Here we used SP-A1 (6A 2 , 6A 4 ) and SP-A2 (1A 0 , 1A 1 ) allele variants expressed by CHO (Chinese hamster ovary) mammalian cells to study their effect on association and/or internalization of P. aeruginosa by rAMs and/or human AMs (hAMs) and to study if phagocytosis can be modulated differentially and/or more effectively by CHO cell-expressed SP-A variants than by insect-cell expressed SP-A variants. For cell association and internalization assessments, light microscopy and fluorescence-activated cell sorter analyses were used, respectively. We found the following for the first time.

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“…Many cellular processes, including signal transduction, transcription, protein synthesis, and cell growth and differentiation, are regulated by phosphorylation-dependent mechanisms (17,19). Therefore, it is not surprising that the protein kinases, a family of enzymes that catalyze the phosphorylation of proteins, comprise one of the largest families of genes in eukaryotes.…”

mentioning

confidence: 99%

High-Throughput Screening of the Yeast Kinome: Identification of Human Serine/Threonine Protein Kinases That Phosphorylate the Hepatitis C Virus NS5A Protein

Coito¹,

Diamond²,

Neddermann

et al. 2004

J Virol

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The hepatitis C virus NS5A protein plays a critical role in virus replication, conferring interferon resistance to the virus through perturbation of multiple intracellular signaling pathways. Since NS5A is a phosphoprotein, it is of considerable interest to understand the role of phosphorylation in NS5A function. In this report, we investigated the phosphorylation of NS5A by taking advantage of 119 glutathione S-transferase-tagged protein kinases purified from Saccharomyces cerevisiae to perform a global screening of yeast kinases capable of phosphorylating NS5A in vitro. A database BLAST search was subsequently performed by using the sequences of the yeast kinases that phosphorylated NS5A in order to identify human kinases with the highest sequence homologies. Subsequent in vitro kinase assays and phosphopeptide mapping studies confirmed that several of the homologous human protein kinases were capable of phosphorylating NS5A. In vivo phosphopeptide mapping revealed phosphopeptides common to those generated in vitro by AKT, p70S6K, MEK1, and MKK6, suggesting that these kinases may phosphorylate NS5A in mammalian cells. Significantly, rapamycin, an inhibitor commonly used to investigate the mTOR/p70S6K pathway, reduced the in vivo phosphorylation of specific NS5A phosphopeptides, strongly suggesting that p70S6 kinase and potentially related members of this group phosphorylate NS5A inside the cell. Curiously, certain of these kinases also play a major role in mRNA translation and antiapoptotic pathways, some of which are already known to be regulated by NS5A. The findings presented here demonstrate the use of high-throughput screening of the yeast kinome to facilitate the major task of identifying human NS5A protein kinases for further characterization of phosphorylation events in vivo. Our results suggest that this novel approach may be generally applicable to the screening of other protein biochemical activities by mechanistic class.

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Glycosylated and Phosphorylated Proteins—Expression in Yeast and Oocytes of Xenopus: Prospects and Challenges—Relevance to Expression of Thermostable Proteins

Cited by 26 publications

References 137 publications

Expression of recombinant membrane-bound type I iodothyronine deiodinase in yeast

Expression of recombinant membrane-bound type I iodothyronine deiodinase in yeast

Surfactant Protein A2 (SP-A2) Variants Expressed in CHO Cells Stimulate Phagocytosis of Pseudomonas aeruginosa More than Do SP-A1 Variants

High-Throughput Screening of the Yeast Kinome: Identification of Human Serine/Threonine Protein Kinases That Phosphorylate the Hepatitis C Virus NS5A Protein

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