Structure of Recombinant Human Interleukin 5 Produced by Chinese Hamster Ovary Cells

Minamitake, Yoshiharu; Kodama, Shiho; Katayama, Toyoko; Adachi, Hideki; Tanaka, Shoji; Tsujimoto, Masafumi

doi:10.1093/oxfordjournals.jbchem.a123041

Cited by 58 publications

(22 citation statements)

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“…The mechanism of unusual polymannosylation may be further elucidated by the construction of mutants glycosylated at a site other than position 49. Our previous result, that mutant lysozyme was glycosylated at a position in the most N-terminal site of the mutant proteins attempted in the experiment, supports the proposal that the efficiency of the glycosylation decreases with the distance of the N-terminus in proteins [4][5][6]. Therefore, we attempted to construct a mutant lysozyme having an N-glycosylation signal sequence at a more N-terminal site and on the molecular surface.…”

Section: Polymannosylationsupporting

confidence: 61%

Polymannosylation to asparagine‐19 in hen egg white lysozyme in yeast

1994

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Complementary DNA encoding hen egg white lysozyme (HEWL) was subjected to site-directed mutagenesis to have the N-glycosylation signal sequence (Asn"-Ty? '-Th?) by substituting Arg with Thr at position 21. The mutant lysozyme (R2lT) was expressed in Succharomyces cerevisiae carrying the yeast expression plasmid inserting the mutant HEWL cDNA. The mutant lysozyme was expressed in the glycosylated forms which are mainly a polymannosyl form with a small amount of oligomannosyl form. The polymannosyl lysozyme was susceptible to Endo H cleavage of the carbohydrate chain. The length of the polymannose chain was predicted to be approximately 340 residues/mol of lysozyme from carbohydrate analysis. According to the estimation with low-angle laser light scattering combined with HPLC, the average molecular weight of polymannosyl lysozyme was 75 kDa, which is consistent with the value obtained from the carbohydrate analysis. The size of polymannosyl lysozyme R2lT is similar or somewhat larger than that of G49N reported previously. Thus, it was confirmed that the unusually large polymannose chain was attached to heterologous mutant lysozyme, regardless of the N-linked position, in yeast.

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Section: Polymannosylationsupporting

confidence: 61%

Polymannosylation to asparagine‐19 in hen egg white lysozyme in yeast

1994

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“…29 Using size exclusion filtration, we demonstrated that immunoreactive IL-5 was completely detectable in a large-molecularweight fraction (>100 kd) after mepolizumab infusion. IL-5 is normally produced as a homodimer (estimated molecular weight is 52 kd), 30,31 and indeed, in control experiments both recombinant IL-5 and naturally produced IL-5 were detectable in the low-molecularweight fraction. To further elucidate the nature of high-molecular-weight immunoreactive IL-5 after mepolizumab therapy, we demonstrated that IL-5 was completely precipitated by means of incubation with protein A/G, thereby providing evidence that its high molecular weight was due to an immunoglobulin/IL-5 complex.…”

Section: Discussionmentioning

confidence: 87%

Anti–IL-5 (mepolizumab) therapy reduces eosinophil activation ex vivo and increases IL-5 and IL-5 receptor levels

Stein

Villanueva

Buckmeier

et al. 2008

Journal of Allergy and Clinical Immunology

130

115

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“…There are precedents for the antiparallel orientation of the monomeric chains of a dimer although there are none among the glycosyltransferases. Cys 42 and Cys 84 of one subunit of interleukin 5 are joined in an antiparallel manner to Cys 84 and Cys 42 of the other subunit (44,45). Similarly, the monomeric subunits of plateletderived growth factor are joined by two intermolecular disulfides between Cys 43 and Cys 52 to form an antiparallel arrangement (46,47).…”

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confidence: 99%

Disulfide Bonds of GM2 Synthase Homodimers

Li¹,

Yen²,

Allende³

et al. 2000

Journal of Biological Chemistry

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GM2 synthase is a homodimer in which the subunits are joined by lumenal domain disulfide bond(s). To define the disulfide bond pattern of this enzyme, we analyzed a soluble form by chemical fragmentation, enzymatic digestion, and mass spectrometry and a fulllength form by site-directed mutagenesis. Ganglioside synthesis is regulated during differentiation, development, and malignant transformation (1-3) and occurs in the Golgi apparatus by the stepwise addition of monosaccharides to glycolipid acceptors by membrane bound glycosyltransferases. The simple gangliosides GM3, 1 GD3, and GT3 are the precursors of the a-, b-, and c-series of gangliosides, respectively, and are synthesized by the addition of 1, 2, or 3 molecules of sialic acid to lactosylceramide (LacCer). Complex gangliosides are formed by the addition of GalNAc to simple gangliosides by the action of UDP-GalNAc:lactosylceramide/ GM3/GD3 ␤-1,4-N-acetylgalactosaminyltransferase (GM2 synthase) followed by the attachment of Gal and additional sialic acid residues (1). Thus, GM2 synthase is a key enzyme in ganglioside biosynthesis, controlling the balance between the expression of simple and complex gangliosides (4). Genetic ablation of GM2 synthase in mice resulted in male sterility (5) as well as decreased myelination and axonal degeneration of the central and peripheral nervous system (6).Previously we showed that GM2 synthase is a homodimer formed by disulfide bond(s) in the lumenal domain (7). The importance of Cys residues of glycosyltransferases has been shown in several functional and structural studies (8, 9) and also by the fact that the Cys residues of each glycosyltransferase family are conserved in spacing (10, 11). In addition a few glycosyltransferases have been shown to be disulfide bonded dimers although the majority are monomeric (see "Discussion").In this report we have utilized protein chemistry experiments, coupled with mass spectrometric analyses, to determine: 1) that all Cys of the soluble form of GM2 synthase are involved in disulfide bonds; and 2) which disulfides are responsible for dimer formation. These results demonstrate that in the dimer the NH 2 terminus of one subunit is close to the COOH terminus of the other subunit in space. EXPERIMENTAL PROCEDURESGM2 Synthase-CHO cell clone GTm1 which stably expresses a soluble form of myc-tagged GM2 synthase was described previously (12). Large scale cell culture was achieved in roller bottles using the serum-free medium CHO-S-SFM II (Life Technologies) according to Kolhekar et al. (13) and in bioreactors at the National Cell Culture Center, Minneapolis, MN. The soluble form of GM2 synthase was partially purified from culture supernatants by SP-Sepharose chromatography using a 0 -0.25 M NaCl gradient in 50 mM Hepes, pH 7.6, 5 mM MnCl 2 .

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Structure of Recombinant Human Interleukin 5 Produced by Chinese Hamster Ovary Cells

Cited by 58 publications

References 0 publications

Polymannosylation to asparagine‐19 in hen egg white lysozyme in yeast

Polymannosylation to asparagine‐19 in hen egg white lysozyme in yeast

Anti–IL-5 (mepolizumab) therapy reduces eosinophil activation ex vivo and increases IL-5 and IL-5 receptor levels

Disulfide Bonds of GM2 Synthase Homodimers

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