Characterization of Recombinant Human Interleukin 4 Receptor from CHO Cells: Role of N-Linked Oligosaccharides

Rajan, Nithya; Tsarbopoulos, Anthony; Kumarasamy, Ramasamy; O’Donnell, Robert; Taremi, S. Shane; Baldwin, Samuel; Seelig, G F; Fan, Xuedon; Pramanik, Birendra; Le, Hung V.

doi:10.1006/bbrc.1995.1098

Cited by 23 publications

(12 citation statements)

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“…The patients with non-ON attacks of MS also had significantly increased plasma concentrations of sIL4r. which may inhibit the biological effects of IL-4 (Garrone et af., 199 1 ; Rajan et al, 1995). Thus.…”

Section: Discussionmentioning

confidence: 94%

Cytokines and soluble IL‐4 in patients with acute optic neuritis and multiple sclerosis

Sellebjerg

Bendtzen

Christiansen

et al. 1997

Euro J of Neurology

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We measured the cerebrospinal fluid (CSF) and plasma concentrations of interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)-α, TNF-β, interferon (IFN)-γ, the IL-1 receptor antagonist, and soluble IL-4 receptor (sIL-4r) by ELISA in 12 patients each with acute, monosymptomatic, idiopathic optic neuritis (ON), ON as part of MS, other attack forms of MS, and in neurological control subjects. CSF concentrations of IL-1β, IL-2 and IFN-γ differed significantly between the different patient groups and were detected most commonly at the highest concentrations in patients with non-ON attacks of MS. TNF-β was detected exclusively in CSF from neurological control patients. The patients with non-ON attacks of MS also had significantly elevated concentrations of sIL-4r in plasma. Increased CSF concentrations of IL-1β, IL-2 and IFN-γ together with increased plasma concentrations of sIL-4r support the concept of MS as an autoimmune disease with preferential activation of proinflammatory or T-helper type 1-like cells. Patients with idiopathic ON or ON as part of MS may, however, differ immunologically from patients with other attack forms of MS.

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Section: Discussionmentioning

confidence: 94%

Cytokines and soluble IL‐4 in patients with acute optic neuritis and multiple sclerosis

Sellebjerg

Bendtzen

Christiansen

et al. 1997

Euro J of Neurology

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“…The original IL4-BP sequence was expressed in CHO cells, and the purified protein migrated as a broad band with an approximate molecular mass of 44-50 kDa ( The increase in apparent size of the ILCBP expressed in the insect and animal cells results from extensive glycosylation. IL4-BP contains six potential N-glycosylation sites, which are probably all modified (Rajan et al, 1995 ;Conradt, H., personal communication). Complex-type oligosaccarides with bi-, tri-, and tetraantennary structures that are sialylated to different extents have been identified in the CHO cell-derived protein.…”

Section: Il4-bp Variantsmentioning

confidence: 99%

Global and Local Determinants for the Kinetics of Interleukin‐4/Interleukin‐4 Receptor α Chain Interaction

Shen

Hage

Sebald

1996

European Journal of Biochemistry

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An engineered interleukin-4-binding protein (IL4-BP) representing the extracellular domain of the human interleukin-4 (IL-4) receptor a chain was expressed in Sf9 cells. The purified IL4-BP was immobilized via a single biotinylated SH group near the carboxyl end to a biosensor matrix and analysed in real time for interaction with IL-4 and IL-4 variants. IL-4 was bound to IL4-BP at a molar ratio of approximately 1 : l . The association and dissociation at pH 7.4 and 150 mM NaCl had rate constants of 1.9 2 0.3 X 10' M-' s-I and 2 ? 1 X lo-' s-', respectively. Glycosylation and engineered amino acid substitutions of IL4-BP did not alter the kinetic constants as shown by a parallel analysis of IL4-BP variants produced in Eschevichia coli or Chinese hamster ovary cells. The rate of association was only slighly affected in binding-deficient variants [E9Q]IL-4 and [R88Q]IL-4 and by acidic pH down to values of 4.5, but it was reduced up to fivefold at higher ionic strength. The rate of dissociation was increased 70-fold and 150-fold with the 1L-4 variants and fivefold at an acidic pH of 4.5, but it was not affected by high ionic strength. Temperatures between 6°C and 37°C yielded similar rates of IL-4 dissociation and only a marginally reduced rate of IL-4 association at 6°C.These results indicate that the high-affinity binding of IL-4 to its receptor (Kd=lOO pM) is mainly the result of an unusually high association rate. The IL-4/IL4-BP interaction appears to be dominated by charge effects. The exceedingly high rate of IL-4IIU-BP association is augmented by the overall electrostatic potentials of both proteins (electrostatic steering). Localized charges and the formation of ion pairs may control the rate of complex dissociation.

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“…The cytokine trap fusion technology described by Stahl et al (US patent 6,472,179) avoids significant alteration of the glycosylation sites (Stahl et al, 1999;Stahl and Yancopoulos, 2002). A study that describes the expression of the IL-4 receptor protein in CHO cells reported the protein to be highly glycosylated (Rajan et al, 1995). Additionally, other IL, cytokine trap, and IL receptor molecules expressed in CHO cells have been reported to have significant glycosylation (Gawlitzek et al, 2000;Kaszubska et al, 2000;Lee et al, 1996;Tsarbopoulos et al, 1995).…”

Section: Introductionmentioning

confidence: 99%

Gene‐expression profiles for five key glycosylation genes for galactose‐fed CHO cells expressing recombinant IL‐4/13 cytokine trap

Clark

Griffiths

Bailey

et al. 2005

Biotech & Bioengineering

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Recombinant protein glycosylation profiles have been shown to affect the in-vivo half-life, and therefore the efficacy and economics, for many therapeutics. While much research has been conducted correlating the effects of various stimuli on recombinant protein glycosylation characteristics, relatively little work has examined glycosylation-related gene-expression profiles. In this study, the effects of galactose feeding on the gene-expression profiles for five key glycosylation-related genes were determined for Chinese hamster ovary cells producing a recombinant IL-4/13 cytokine trap fusion. The genes investigated were sialidase, a putative alpha2,3-sialyltransferase, CMP-sialic acid transporter, beta1,4-galactosyltransferase, and UDP-galactosyltransferase. Additionally, the sialic acid content (sialylation) of the recombinant protein was examined. The peak sialic acid content of the IL-4/13 cytokine trap fusion protein was observed to be similar for the control and galactose-fed cultures. The gene-expression profiles for four of the glycosylation genes were observed to be sensitive to the glucose concentration and not significantly different for the control and galactose-fed cultures prior to glucose depletion. However, the sialidase gene-expression profiles were different for the control and galactose-fed cultures. The sialidase gene-expression profile increased significantly for the galactose-fed cultures prior to glucose depletion, whereas for the control cultures, the sialidase gene-expression profiles did not increase until the late stationary phase. The intracellular sialidase enzyme activity decreased exponentially with time for the control cultures; however, for the galactose-fed cultures, the intracellular sialidase enzyme activity decreased initially and then remained relatively high compared to the control cultures. These results indicate that the galactose feeding may increase the potential for desialylation, which offsets any improvements in the sialylation rate due to increased substrate levels. Thus, galactose feeding is an unnecessary expense for the production of the IL-4/13 cytokine trap fusion protein in a batch process.

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Characterization of Recombinant Human Interleukin 4 Receptor from CHO Cells: Role of N-Linked Oligosaccharides

Cited by 23 publications

References 0 publications

Cytokines and soluble IL‐4 in patients with acute optic neuritis and multiple sclerosis

Cytokines and soluble IL‐4 in patients with acute optic neuritis and multiple sclerosis

Global and Local Determinants for the Kinetics of Interleukin‐4/Interleukin‐4 Receptor α Chain Interaction

Gene‐expression profiles for five key glycosylation genes for galactose‐fed CHO cells expressing recombinant IL‐4/13 cytokine trap

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