Formation of Isoaspartate at Two Distinct Sites during in vitro Aging of Human Growth Hormone

Johnson, Brett A.; Shirokawa, Jill M.; Hancock, William S.; Spellman, Michael W.; Basa, Louisette J.; Aswad, Dana W.

doi:10.1016/s0021-9258(18)71672-4

Cited by 145 publications

(74 citation statements)

References 54 publications

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“…Methylation Assay. Methylation of rCD4 by protein carboxyl methyltransferase (PCMT) using radiolabeled methyl donor S-adenosyl-L-methionine (SAM) was carried out according to the method described by Johnson et al (1989). rCD4 Direct-Binding ELISA. Microtiter plates (Nunc) were coated for 12 h at 4-8 °C with 100 ML/well of a 2 Mg/mL rgpl20 solution in 50 mM carbonate/bicarbonate, pH 9.5.…”

Section: Experimental Procedures (Materials and Methods)mentioning

confidence: 99%

Deamidation of soluble CD4 at asparagine-52 results in reduced binding capacity for the HIV-1 envelope glycoprotein gp120

Teshima¹,

Porter²,

Yim³

et al. 1991

Biochemistry

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High-performance cation-exchange chromatography of recombinant soluble CD4 (rCD4) allowed the resolution of four charge variants. This charge heterogeneity could be eliminated by neuraminidase treatment of rCD4 and therefore can be attributed to different degrees of sialylation of the carbohydrate portion of this glycoprotein. A single acidic variant was observed upon cation-exchange chromatography of neuraminidase-treated rCD4 that had been stored in liquid solution, pH 7.2, at 25 degrees C for 6 months. This acidic variant was isolated by semipreparative cation-exchange chromatography and subjected to tryptic mapping analysis. Tryptic peptides were characterized by fast atom bombardment mass spectrometry (FABMS). The results of this analysis demonstrated that the acidic variant of neuraminidase-treated rCD4 is generated from deamidation at Asn-52. Digestion of the deamidated rCD4 with endoproteinase Asp-N confirmed Asn-52 as the primary site of deamidation. The ability of the deamidated rCD4 variant to bind gp120 was assessed by use of an ELISA-based binding assay. The binding capacity of the deamidated variant was 24% of the binding capacity of unmodified rCD4. The overall structure of the V1 domain in the deamidated variant was not markedly different from that of the native protein as probed with eight conformationally dependent anti-V1 monoclonal antibodies. Therefore, it appears that Asn-52 is directly involved in binding to gp120.

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Section: Experimental Procedures (Materials and Methods)mentioning

confidence: 99%

Deamidation of soluble CD4 at asparagine-52 results in reduced binding capacity for the HIV-1 envelope glycoprotein gp120

Teshima¹,

Porter²,

Yim³

et al. 1991

Biochemistry

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“…Digestion by Pronase was carried out at pH 7 (the pH optima of the various peptidase activities in Pronase range from 6.5 to 8), whereas digestion by CPY was carried out at pH 5.5. The rate of deamidation of asparagine to isoaspartate is known to increase with increasing pH (Johnson et al, 1989b). The generation of isoaspartate from asparagine during digestion by Pronase and the inability of Pronase to release isoaspartyl dipeptides from two of the three isoaspartatecontaining peptides tested indicate that CPY is a superior protease for detecting isoaspartate in proteins.…”

Section: Resultsmentioning

confidence: 99%

“…A method for detecting isoaspartyl linkages independently of enzymatic methylation would be useful in testing hypotheses concerning the physiological function of the methyltransferase. In recent studies, isoaspartate has been detected by tritium labeling at internal -carboxyl groups (Di Donato et al, 1986) and by a failure to sequence past sites originally occupied by asparagine or aspartate during automated Edman degradation (Gráf et al, 1971;Henderson et al, 1976;McDonald et al, 1983;Ekman et al, 1984;Kanaya & Uchida, 1986;Di Augustine et al, 1987;Johnson et al, 1989b). The tritium labeling method requires the use of high levels of 3H20, and it would be subject to high background signals, especially if applied to complex mixtures in which some proteins might contain aspartate as the carboxyl-terminal amino acid.…”

mentioning

confidence: 99%

Fragmentation of isoaspartyl peptides and proteins by carboxypeptidase Y: release of isoaspartyl dipeptides as a result of internal and external cleavage

Johnson¹,

Aswad²

1990

Biochemistry

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Isoaspartate-containing versions of sea urchin sperm-activating peptide, delta sleep-inducing peptide, and lactate dehydrogenase (231-242) were cleaved at internal sites by carboxypeptidase Y. Cleavage occurred between the isoaspartate and the preceding amino acid, and it was accompanied by sequential digestion of amino acids from the two resulting carboxyl termini. Because the isoaspartyl bonds were not cleaved, isoaspartyl dipeptides were among the final products. The rate of release of isoaspartyl dipeptides was different for the three peptides, a 24-h digestion yielding 0.32 mol of isoaspartylglycine/mol of isoaspartyl sperm-activating peptide, 0.50 mol of isoaspartylalanine/mol of isoaspartyl delta sleep-inducing peptide, and 1.15 mol of isoaspartylserine/mol of isoaspartyl lactate dehydrogenase (231-242). The different rates could be explained by the slow cleavage of amino acids preceded by glycine. Isoaspartyl dipeptides were not detected in digests of the corresponding aspartate- or asparagine-containing forms of the peptides. Release of isoaspartyl dipeptides by carboxypeptidase Y was used to demonstrate the presence of isoaspartylglycine sequences in deamidated adrenocorticotropin (0.54 mol/mol), in a mixture of trypic fragments of base-treated calmodulin (0.20 mol/mol), and in a mixture of tryptic fragments of base-treated triosephosphate isomerase (0.08 mol/mol). These results confirm earlier work suggesting that isoaspartylglycine formation is prevalent in proteins exposed to alkaline conditions. They also provide a methodology that should prove useful in the characterization of natural substrates for protein L-isoaspartyl methyltransferase.

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“…In the latter case, the β-carbon is part of the polypeptide backbone, and the R-carboxyl group is present as an atypical one carbon carboxylic acid side chain available for methylation by isoaspartate O-methyltransferase. Under in vitro conditions, selective deamidation occurs in a number of purified proteins such as growth hormone (30), calmodulin (31), ribonuclease (32), and phosphocarrier protein (33). Robinson and Rudd proposed that deamidation can also occur in vivo, likely related to the aging of the protein (34).…”

mentioning

confidence: 99%

“…An isoaspartate derivative of calbindin generated by deamidation of Asn 56 loses calcium binding by about 10-fold, and the aspartyl derivative actually binds calcium 50% more tightly than the native asparaginyl form (46). Deamidation of Asn residues or isomerization of Asp residues has been shown to occur in a variety of proteinbased pharmaceuticals including human growth hormone (30), tissue plasminogen activator (47), hirudin (48), monoclonal antibodies (43), acidic fibroblast growth factor (49), and interleukin 1 (50), with varying effect on the activity or stability of the drug. In this report, we demonstrate that the deamidation of rhSCF can be induced by prolonged storage at elevated temperature.…”

mentioning

confidence: 99%

Selective Deamidation of Recombinant Human Stem Cell Factor during in Vitro Aging: Isolation and Characterization of the Aspartyl and Isoaspartyl Homodimers and Heterodimers

et al. 1998

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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 Asn10 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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Formation of Isoaspartate at Two Distinct Sites during in vitro Aging of Human Growth Hormone

Cited by 145 publications

References 54 publications

Deamidation of soluble CD4 at asparagine-52 results in reduced binding capacity for the HIV-1 envelope glycoprotein gp120

Deamidation of soluble CD4 at asparagine-52 results in reduced binding capacity for the HIV-1 envelope glycoprotein gp120

Fragmentation of isoaspartyl peptides and proteins by carboxypeptidase Y: release of isoaspartyl dipeptides as a result of internal and external cleavage

Selective Deamidation of Recombinant Human Stem Cell Factor during in Vitro Aging: Isolation and Characterization of the Aspartyl and Isoaspartyl Homodimers and Heterodimers

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