Selective deamidation of recombinant human stem cell factor during in vitro aging

Isolation and characterization of the aspartyl and isoaspartyl homodimers and heterodimers

Yueh Rong Hsu, Wen Chang Chang, Elizabeth A. Mendiaz, Shinichi Hara, David T. Chow, Michael B. Mann, Keith E. Langley, Hsieng S. Lu

Research output: Contribution to journalArticle

49 Citations (Scopus)

Abstract

During in vitro aging, deamidation of recombinant human stem cell factor produced in Escherichia coli was detected by HPLC analysis and by the release of soluble ammonia. The deamidation rate is very slow in buffers at low pH or at low temperatures; however, the rate is significantly accelerated in alkaline buffers such as sodium bicarbonate in combination with elevated temperatures. HPLC isolation of various deamidated forms followed by peptide mapping and mass spectrometric analyses revealed that the deamidation involves Asn10 in the sequence -T9NNV- near the N-terminus of the protein. Following peptide mapping analysis, significant amounts of aspartyl and isoaspartyl peptides were identified, indicating the conversion of asparagine into both aspartate and isoaspartate residues. As a result of spontaneous association-dissociation of stem cell factor dimer, a total of five deamidated forms, including two homodimers and three heterodimers, were detected and isolated. Cell proliferation assays showed that two rhSCF heterodimeric species, derived from dimerization between isoaspartyl and other stem cell factor monomers, retain only approximately half of the biological activity. The homodimer with isoaspartic acid in place of Ash10 is 50-fold less potent, while the aspartyl homodimer, either isolated during deamidation experiments or recombinantly prepared by site-directed mutagenesis (e.g., N10D and N10D/N11D variants), exhibits higher activity than the standard molecule. In comparison, synthetic N10A and N10E variants, though missing the deamidation site, are significantly less active. All these variants lacking the Asn10 deamidation site are relatively more stable than those containing the asparagine residue. The results indicate that the biological function and chemical stability of stem cell factor are influenced by the nature of the residue at position 10.

Original languageEnglish
Pages (from-to)2251-2262
Number of pages12
JournalBiochemistry
Volume37
Issue number8
DOIs
Publication statusPublished - Feb 24 1998
Externally publishedYes

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Stem Cell Factor
Isoaspartic Acid
Aging of materials
Peptide Mapping
Asparagine
Peptides
Buffers
High Pressure Liquid Chromatography
Mutagenesis
Sodium Bicarbonate
Temperature
Dimerization
Chemical stability
Cell proliferation
Site-Directed Mutagenesis
Bioactivity
Ammonia
Aspartic Acid
Dimers
Escherichia coli

ASJC Scopus subject areas

  • Biochemistry

Cite this

Selective deamidation of recombinant human stem cell factor during in vitro aging : Isolation and characterization of the aspartyl and isoaspartyl homodimers and heterodimers. / Hsu, Yueh Rong; Chang, Wen Chang; Mendiaz, Elizabeth A.; Hara, Shinichi; Chow, David T.; Mann, Michael B.; Langley, Keith E.; Lu, Hsieng S.

In: Biochemistry, Vol. 37, No. 8, 24.02.1998, p. 2251-2262.

Research output: Contribution to journalArticle

Hsu, Yueh Rong ; Chang, Wen Chang ; Mendiaz, Elizabeth A. ; Hara, Shinichi ; Chow, David T. ; Mann, Michael B. ; Langley, Keith E. ; Lu, Hsieng S. / Selective deamidation of recombinant human stem cell factor during in vitro aging : Isolation and characterization of the aspartyl and isoaspartyl homodimers and heterodimers. In: Biochemistry. 1998 ; Vol. 37, No. 8. pp. 2251-2262.
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abstract = "During in vitro aging, deamidation of recombinant human stem cell factor produced in Escherichia coli was detected by HPLC analysis and by the release of soluble ammonia. The deamidation rate is very slow in buffers at low pH or at low temperatures; however, the rate is significantly accelerated in alkaline buffers such as sodium bicarbonate in combination with elevated temperatures. HPLC isolation of various deamidated forms followed by peptide mapping and mass spectrometric analyses revealed that the deamidation involves Asn10 in the sequence -T9NNV- near the N-terminus of the protein. Following peptide mapping analysis, significant amounts of aspartyl and isoaspartyl peptides were identified, indicating the conversion of asparagine into both aspartate and isoaspartate residues. As a result of spontaneous association-dissociation of stem cell factor dimer, a total of five deamidated forms, including two homodimers and three heterodimers, were detected and isolated. Cell proliferation assays showed that two rhSCF heterodimeric species, derived from dimerization between isoaspartyl and other stem cell factor monomers, retain only approximately half of the biological activity. The homodimer with isoaspartic acid in place of Ash10 is 50-fold less potent, while the aspartyl homodimer, either isolated during deamidation experiments or recombinantly prepared by site-directed mutagenesis (e.g., N10D and N10D/N11D variants), exhibits higher activity than the standard molecule. In comparison, synthetic N10A and N10E variants, though missing the deamidation site, are significantly less active. All these variants lacking the Asn10 deamidation site are relatively more stable than those containing the asparagine residue. The results indicate that the biological function and chemical stability of stem cell factor are influenced by the nature of the residue at position 10.",
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T1 - Selective deamidation of recombinant human stem cell factor during in vitro aging

T2 - Isolation and characterization of the aspartyl and isoaspartyl homodimers and heterodimers

AU - Hsu, Yueh Rong

AU - Chang, Wen Chang

AU - Mendiaz, Elizabeth A.

AU - Hara, Shinichi

AU - Chow, David T.

AU - Mann, Michael B.

AU - Langley, Keith E.

AU - Lu, Hsieng S.

PY - 1998/2/24

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N2 - During in vitro aging, deamidation of recombinant human stem cell factor produced in Escherichia coli was detected by HPLC analysis and by the release of soluble ammonia. The deamidation rate is very slow in buffers at low pH or at low temperatures; however, the rate is significantly accelerated in alkaline buffers such as sodium bicarbonate in combination with elevated temperatures. HPLC isolation of various deamidated forms followed by peptide mapping and mass spectrometric analyses revealed that the deamidation involves Asn10 in the sequence -T9NNV- near the N-terminus of the protein. Following peptide mapping analysis, significant amounts of aspartyl and isoaspartyl peptides were identified, indicating the conversion of asparagine into both aspartate and isoaspartate residues. As a result of spontaneous association-dissociation of stem cell factor dimer, a total of five deamidated forms, including two homodimers and three heterodimers, were detected and isolated. Cell proliferation assays showed that two rhSCF heterodimeric species, derived from dimerization between isoaspartyl and other stem cell factor monomers, retain only approximately half of the biological activity. The homodimer with isoaspartic acid in place of Ash10 is 50-fold less potent, while the aspartyl homodimer, either isolated during deamidation experiments or recombinantly prepared by site-directed mutagenesis (e.g., N10D and N10D/N11D variants), exhibits higher activity than the standard molecule. In comparison, synthetic N10A and N10E variants, though missing the deamidation site, are significantly less active. All these variants lacking the Asn10 deamidation site are relatively more stable than those containing the asparagine residue. The results indicate that the biological function and chemical stability of stem cell factor are influenced by the nature of the residue at position 10.

AB - During in vitro aging, deamidation of recombinant human stem cell factor produced in Escherichia coli was detected by HPLC analysis and by the release of soluble ammonia. The deamidation rate is very slow in buffers at low pH or at low temperatures; however, the rate is significantly accelerated in alkaline buffers such as sodium bicarbonate in combination with elevated temperatures. HPLC isolation of various deamidated forms followed by peptide mapping and mass spectrometric analyses revealed that the deamidation involves Asn10 in the sequence -T9NNV- near the N-terminus of the protein. Following peptide mapping analysis, significant amounts of aspartyl and isoaspartyl peptides were identified, indicating the conversion of asparagine into both aspartate and isoaspartate residues. As a result of spontaneous association-dissociation of stem cell factor dimer, a total of five deamidated forms, including two homodimers and three heterodimers, were detected and isolated. Cell proliferation assays showed that two rhSCF heterodimeric species, derived from dimerization between isoaspartyl and other stem cell factor monomers, retain only approximately half of the biological activity. The homodimer with isoaspartic acid in place of Ash10 is 50-fold less potent, while the aspartyl homodimer, either isolated during deamidation experiments or recombinantly prepared by site-directed mutagenesis (e.g., N10D and N10D/N11D variants), exhibits higher activity than the standard molecule. In comparison, synthetic N10A and N10E variants, though missing the deamidation site, are significantly less active. All these variants lacking the Asn10 deamidation site are relatively more stable than those containing the asparagine residue. The results indicate that the biological function and chemical stability of stem cell factor are influenced by the nature of the residue at position 10.

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