Increased biological activity of deglycosylated recombinant human granulocyte/macrophage colony-stimulating factor produced by yeast or animal cells.

Moonen, Peter; Mermod, Jean‐Jacques; Ernst, Joachim F.; Hirschi, Marlis; DeLamarter, J F

doi:10.1073/pnas.84.13.4428

Cited by 115 publications

(41 citation statements)

References 31 publications

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“…The suppression of biological activities by N-linked glycosylation has been reported for other proteins, including LCAT (22), the human immunodeficiency virus-1 envelope protein gp120 (23), influenza virus hemagglutinin (24), human protein C (25), and human granulocytemacrophage colony-stimulating factor (26). For example, the abolishment of N-linked glycosylation at Asn-384 of LCAT enhanced its catalytic activity by 2-fold, and it was proposed that glycosylation at this site sterically hindered the access of substrate to the catalytic site (22).…”

Section: Discussionmentioning

confidence: 99%

Glycosylation of endothelial lipase at asparagine-116 reduces activity and the hydrolysis of native lipoproteins in vitro and in vivo

Brown

Miller

Griffon

et al. 2007

Journal of Lipid Research

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We previously identified that four of five putative N-linked glycosylation sites of human endothelial lipase (EL) are utilized and suggested that the substitution of asparagine-116 (Asn-116) with alanine (Ala) (N116A) increased the hydrolytic activity of EL. The current study demonstrates that mutagenesis of either Asn-116 to threonine (Thr) or Thr-118 to Ala also disrupted the glycosylation of EL and enhanced catalytic activity toward synthetic substrates by 3-fold versus wild-type EL. Furthermore, we assessed the hydrolysis of native lipoprotein lipids by EL-N116A. EL-N116A exhibited a 5-fold increase in LDL hydrolysis and a 1.8-fold increase in HDL 2 hydrolysis. Consistent with these observations, adenovirus-mediated expression of EL-N116A in mice significantly reduced the levels of both LDL and HDL cholesterol beyond the reductions observed by the expression of wild-type EL alone. Finally, we introduced Asn-116 of EL into the analogous positions within LPL and HL, resulting in N-linked glycosylation at this site. Glycosylation at this site suppressed the LPL hydrolysis of synthetic substrates, LDL, HDL 2 , and HDL 3 but had little effect on HL activity. These data suggest that N-linked glycosylation at Asn-116 reduces the ability of EL to hydrolyze lipids in LDL and HDL 2 .-Brown, R. J., G. C. Miller, N. Griffon, C. J. Long, and D. J. Rader. Glycosylation of endothelial lipase at asparagine-116 reduces activity and the hydrolysis of native lipoproteins in vitro and in vivo. J. Lipid Res. 2007Res. . 48: 1132Res. -1139. Supplementary key words lipaseEndothelial lipase (EL) belongs to a superfamily of lipases (EC 3.1.1.3) that includes LPL and HL (1-6). These three lipases have both triglyceride (TG) lipase and phospholipase activity, but EL has relatively more phospholipase activity compared with LPL, which has predominantly TG lipase activity (7). Overexpression of EL in mice was shown to significantly reduce high density lipoprotein cholesterol (HDL-C) (1,8,9), whereas loss-of-function studies in mice result in significantly elevated plasma 10,11).In vitro and in vivo studies using chimeric proteins of LPL and HL have shown that the differences in substrate specificity between these two lipases are governed by a 22 amino acid loop, or "lid domain," within the N-terminal domain of the respective lipases that covers the catalytic site (12)(13)(14). The shorter 19 amino acid lid domain within EL partially contributes to its substrate specificity (15); other elements affecting substrate specificity remain to be elucidated.Human EL is translated as a 500 amino acid 57 kDa peptide that is processed into a mature 480 amino acid protein with an apparent molecular mass of 68 kDa after the loss of its signal peptide and the addition of N-linked glycosylation (1, 2). EL has five putative N-linked glycosylation sites [identified by the presence of asparagine-X-serine/threonine (Asn-Xaa-Ser/Thr) motifs]. We previously reported that four of the five sites, specifically are utilized (16). Abolishment of N-linke...

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Section: Discussionmentioning

confidence: 99%

Glycosylation of endothelial lipase at asparagine-116 reduces activity and the hydrolysis of native lipoproteins in vitro and in vivo

Brown

Miller

Griffon

et al. 2007

Journal of Lipid Research

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“…The cDNA-predicted sequence of mature recombinant human GM-CSF (rhGM-CSF) contains 127 amino acid residues with four cysteine residues, two potential attachment sites for N-linked carbohydrate(s), and a 21-residue signal peptide. The presence of N-linked carbohydrate inhibits in vitro biological activity significantly (Kaushansky et al, 1987;Moonen et al, 1987), even though naturally occurring hGM-CSF is a glycoprotein.…”

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confidence: 99%

Isolation and characterization of a resistant core peptide of recombinant human granulocyte‐macrophage colony‐stimulating factor (gm‐csf); confirmation of the gm‐csf amino acid sequence by mass spectrometry

Tsarbopoulos¹,

Pramanik²,

Labdon³

et al. 1993

Protein Science

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A trypsin-resistant core peptide of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) was isolated and analyzed by high-energy Cs' liquid secondary-ion (LSI) mass spectrometric analysis. This analysis provided successful detection of the high-mass disulfide-linked core peptide as well as information confirming the existence of disulfide pairing. Similarly, LSI mass spectrometric analysis of the peptide fragments isolated chromatographically from a Staphylococcus aureus V8 protease digest of rhGM-CSF provided rapid confirmation of the cDNA-derived sequence and determination of the existing disulfide bonds between cysteine residues 54-96 and 88-121. Electrospray ionization mass spectrometry was employed to measure the molecular weight of the intact protein and to determine the number of the disulfide bonds in the protein molecule by comparative analysis of the protein before and after reduction with P-mercaptoethanol.

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“…DeLamarter (Biogen, Geneva, Switzerland), and had a specific activity of 1.1 • l0 s U/Bg in the CML assay [11]. The material had an endotoxin concentration of 0.5 EU/10 ~tg in the limulus amebocyte lysate assay.…”

Section: Cytokinesmentioning

confidence: 99%

Granulocyte-activating mediators (GRAM)

et al. 1987

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Increased biological activity of deglycosylated recombinant human granulocyte/macrophage colony-stimulating factor produced by yeast or animal cells.

Cited by 115 publications

References 31 publications

Glycosylation of endothelial lipase at asparagine-116 reduces activity and the hydrolysis of native lipoproteins in vitro and in vivo

Glycosylation of endothelial lipase at asparagine-116 reduces activity and the hydrolysis of native lipoproteins in vitro and in vivo

Isolation and characterization of a resistant core peptide of recombinant human granulocyte‐macrophage colony‐stimulating factor (gm‐csf); confirmation of the gm‐csf amino acid sequence by mass spectrometry

Granulocyte-activating mediators (GRAM)

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