Abstract
Interleukin-11 (IL-11) has been expected as a drug on severe thrombocytopenia caused by myelo-suppressive chemotherapy. Whereas, development of IL-11 inhibitor is also expected for a treatment against IL-11 related cancer progression. Here, we will demonstrate the creation of various kinds of genetically modified hIL-11s. Modified vectors were constructed by introducing N- or O-glycosylation site on the region of hIL-11 that does not belong to the core α-helical motif based on the predicted secondary structure. N-terminal (N: between 22 to 23 aa), the first loop (M1:70 to 71 aa), the second loop (M2:114–115 aa), the third loop (M3:160–161 aa) and C-terminal (C: 200- aa) were selected for modification. A large scale production system was established and the characteristics of modified hIL-11s were evaluated. The structure was analyzed by amino acid sequence and composition analysis and CD-spectra. Glycan was assessed by monosaccharide composition analysis. Growth promoting activity and biological stability were analyzed by proliferation of T1165 cells. N-terminal modified proteins were well glycosylated and produced. Growth activity of 3NN with NASNASNAS sequence on N-terminal was about tenfold higher than wild type (WT). Structural and biological stabilities of 3NN were also better than WT and residence time in mouse blood was longer than WT. M1 variants lacked growth activity though they are well glycosylated and secondary structure is very stable. Both of 3NN and OM1 with AAATPAPG on M1 associated with hIL-11R strongly. These results indicate N-terminal and M1 variants will be expected for practical use as potent agonists or antagonists of hIL-11.
| Original language | English |
|---|---|
| Pages (from-to) | 205-217 |
| Number of pages | 13 |
| Journal | Biochimica et Biophysica Acta - General Subjects |
| Volume | 1861 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - 2017 Feb 1 |
| Externally published | Yes |
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Keywords
- Agonist
- Antagonist
- Genetically modified hIL-11s
- Glycosylated hIL-11s
- Recombinant CHO cells
- Structural stability
ASJC Scopus subject areas
- Biophysics
- Biochemistry
- Molecular Biology
Cite this
Production and characterization of genetically modified human IL-11 variants. / Sano, Emiko; Takei, Toshiaki; Ueda, Takuya; Tsumoto, Kouhei.
In: Biochimica et Biophysica Acta - General Subjects, Vol. 1861, No. 2, 01.02.2017, p. 205-217.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Production and characterization of genetically modified human IL-11 variants
AU - Sano, Emiko
AU - Takei, Toshiaki
AU - Ueda, Takuya
AU - Tsumoto, Kouhei
PY - 2017/2/1
Y1 - 2017/2/1
N2 - Interleukin-11 (IL-11) has been expected as a drug on severe thrombocytopenia caused by myelo-suppressive chemotherapy. Whereas, development of IL-11 inhibitor is also expected for a treatment against IL-11 related cancer progression. Here, we will demonstrate the creation of various kinds of genetically modified hIL-11s. Modified vectors were constructed by introducing N- or O-glycosylation site on the region of hIL-11 that does not belong to the core α-helical motif based on the predicted secondary structure. N-terminal (N: between 22 to 23 aa), the first loop (M1:70 to 71 aa), the second loop (M2:114–115 aa), the third loop (M3:160–161 aa) and C-terminal (C: 200- aa) were selected for modification. A large scale production system was established and the characteristics of modified hIL-11s were evaluated. The structure was analyzed by amino acid sequence and composition analysis and CD-spectra. Glycan was assessed by monosaccharide composition analysis. Growth promoting activity and biological stability were analyzed by proliferation of T1165 cells. N-terminal modified proteins were well glycosylated and produced. Growth activity of 3NN with NASNASNAS sequence on N-terminal was about tenfold higher than wild type (WT). Structural and biological stabilities of 3NN were also better than WT and residence time in mouse blood was longer than WT. M1 variants lacked growth activity though they are well glycosylated and secondary structure is very stable. Both of 3NN and OM1 with AAATPAPG on M1 associated with hIL-11R strongly. These results indicate N-terminal and M1 variants will be expected for practical use as potent agonists or antagonists of hIL-11.
AB - Interleukin-11 (IL-11) has been expected as a drug on severe thrombocytopenia caused by myelo-suppressive chemotherapy. Whereas, development of IL-11 inhibitor is also expected for a treatment against IL-11 related cancer progression. Here, we will demonstrate the creation of various kinds of genetically modified hIL-11s. Modified vectors were constructed by introducing N- or O-glycosylation site on the region of hIL-11 that does not belong to the core α-helical motif based on the predicted secondary structure. N-terminal (N: between 22 to 23 aa), the first loop (M1:70 to 71 aa), the second loop (M2:114–115 aa), the third loop (M3:160–161 aa) and C-terminal (C: 200- aa) were selected for modification. A large scale production system was established and the characteristics of modified hIL-11s were evaluated. The structure was analyzed by amino acid sequence and composition analysis and CD-spectra. Glycan was assessed by monosaccharide composition analysis. Growth promoting activity and biological stability were analyzed by proliferation of T1165 cells. N-terminal modified proteins were well glycosylated and produced. Growth activity of 3NN with NASNASNAS sequence on N-terminal was about tenfold higher than wild type (WT). Structural and biological stabilities of 3NN were also better than WT and residence time in mouse blood was longer than WT. M1 variants lacked growth activity though they are well glycosylated and secondary structure is very stable. Both of 3NN and OM1 with AAATPAPG on M1 associated with hIL-11R strongly. These results indicate N-terminal and M1 variants will be expected for practical use as potent agonists or antagonists of hIL-11.
KW - Agonist
KW - Antagonist
KW - Genetically modified hIL-11s
KW - Glycosylated hIL-11s
KW - Recombinant CHO cells
KW - Structural stability
UR - http://www.scopus.com/inward/record.url?scp=84997840777&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84997840777&partnerID=8YFLogxK
U2 - 10.1016/j.bbagen.2016.11.028
DO - 10.1016/j.bbagen.2016.11.028
M3 - Article
C2 - 27884519
AN - SCOPUS:84997840777
VL - 1861
SP - 205
EP - 217
JO - Biochimica et Biophysica Acta - General Subjects
JF - Biochimica et Biophysica Acta - General Subjects
SN - 0006-3002
IS - 2
ER -