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Posttranslational Modifications of Human Thrombin Activatable Fibrinolysis Inhibitor (TAFI): Evidence for a large shift in isoelectric point and reduced solubility upon activation.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningfagfællebedømt

Standard

Posttranslational Modifications of Human Thrombin Activatable Fibrinolysis Inhibitor (TAFI): Evidence for a large shift in isoelectric point and reduced solubility upon activation. / Valnickova, Zuzana; Christensen, Trine; Skottrup, Peter Durand; Thøgersen, Ida; Højrup, Peter; Enghild, Jan Johannes.

I: Biochemistry, Bind 45, Nr. 5, 2006, s. 1525-1535.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningfagfællebedømt

Harvard

Valnickova, Z, Christensen, T, Skottrup, PD, Thøgersen, I, Højrup, P & Enghild, JJ 2006, 'Posttranslational Modifications of Human Thrombin Activatable Fibrinolysis Inhibitor (TAFI): Evidence for a large shift in isoelectric point and reduced solubility upon activation.', Biochemistry, bind 45, nr. 5, s. 1525-1535.

APA

Valnickova, Z., Christensen, T., Skottrup, P. D., Thøgersen, I., Højrup, P., & Enghild, J. J. (2006). Posttranslational Modifications of Human Thrombin Activatable Fibrinolysis Inhibitor (TAFI): Evidence for a large shift in isoelectric point and reduced solubility upon activation. Biochemistry, 45(5), 1525-1535.

Vancouver

Valnickova Z, Christensen T, Skottrup PD, Thøgersen I, Højrup P, Enghild JJ. Posttranslational Modifications of Human Thrombin Activatable Fibrinolysis Inhibitor (TAFI): Evidence for a large shift in isoelectric point and reduced solubility upon activation. Biochemistry. 2006;45(5):1525-1535.

Author

Valnickova, Zuzana ; Christensen, Trine ; Skottrup, Peter Durand ; Thøgersen, Ida ; Højrup, Peter ; Enghild, Jan Johannes. / Posttranslational Modifications of Human Thrombin Activatable Fibrinolysis Inhibitor (TAFI): Evidence for a large shift in isoelectric point and reduced solubility upon activation. I: Biochemistry. 2006 ; Bind 45, Nr. 5. s. 1525-1535.

Bibtex

@article{292622fb499b48ad986d659deadf1c4c,
title = "Posttranslational Modifications of Human Thrombin Activatable Fibrinolysis Inhibitor (TAFI): Evidence for a large shift in isoelectric point and reduced solubility upon activation.",
abstract = "Thrombin-activable fibrinolysis inhibitor (TAFI) is distinct from pancreatic procarboxypeptidase B in several ways. The enzymatic activity of TAFIa is unstable and decays with a half-life of a few minutes. During this study, we observed that (i) the isoelectric point (pI) of TAFI shifts dramatically from pH 5 toward pH 8 upon activation and (ii) TAFIa is significantly less soluble than TAFI. The structural bases for these observations were investigated by characterizing all post-translational modifications, including attached glycans and disulfide connectivity. The analyses revealed that all five potential N-glycosylation sites were utilized including Asn22, Asn51, Asn63, Asn86 (located in the activation peptide), and Asn219 (located in the catalytic domain). Asn219 was also found in an unglycosylated variant. Four of the glycans, Asn51, Asn63, Asn86, and Asn219 displayed microheterogeneity, while the glycan attached to Asn22 appeared to be homogeneous. In addition, bisecting GlcNAc attached to the trimannose core was detected, suggesting an origin other than the liver. Monosaccharide composition and LC-MS/MS analyses did not produce evidence for O glycosylation. TAFI contains eight cysteine residues, of which two, Cys69 and Cys383, are not involved in disulfides and contain free sulfhydryl groups. The remaining six cystines form disulfides, including Cys156-Cys169, Cys228-Cys252, and Cys243-Cys257. This pattern is homologous to pancreatic procarboxypeptidase B, and it is therefore unlikely that permutations in the cysteine connectivity are responsible for the enzymatic instability. LC-MS/MS analyses covering more than 90{\%} of the TAFI amino acid sequence revealed no additional modifications. When these results are taken together, they suggest that the inherent instability of TAFIa is not caused by post-translational modifications. However, after activation, TAFIa loses 80{\%} of the attached glycans, generating a large shift in pI and a propensity to precipitate. These changes are likely to significantly affect the properties of TAFIa as compared to TAFI.",
author = "Zuzana Valnickova and Trine Christensen and Skottrup, {Peter Durand} and Ida Th{\o}gersen and Peter H{\o}jrup and Enghild, {Jan Johannes}",
year = "2006",
language = "English",
volume = "45",
pages = "1525--1535",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "5",

}

RIS

TY - JOUR

T1 - Posttranslational Modifications of Human Thrombin Activatable Fibrinolysis Inhibitor (TAFI): Evidence for a large shift in isoelectric point and reduced solubility upon activation.

AU - Valnickova, Zuzana

AU - Christensen, Trine

AU - Skottrup, Peter Durand

AU - Thøgersen, Ida

AU - Højrup, Peter

AU - Enghild, Jan Johannes

PY - 2006

Y1 - 2006

N2 - Thrombin-activable fibrinolysis inhibitor (TAFI) is distinct from pancreatic procarboxypeptidase B in several ways. The enzymatic activity of TAFIa is unstable and decays with a half-life of a few minutes. During this study, we observed that (i) the isoelectric point (pI) of TAFI shifts dramatically from pH 5 toward pH 8 upon activation and (ii) TAFIa is significantly less soluble than TAFI. The structural bases for these observations were investigated by characterizing all post-translational modifications, including attached glycans and disulfide connectivity. The analyses revealed that all five potential N-glycosylation sites were utilized including Asn22, Asn51, Asn63, Asn86 (located in the activation peptide), and Asn219 (located in the catalytic domain). Asn219 was also found in an unglycosylated variant. Four of the glycans, Asn51, Asn63, Asn86, and Asn219 displayed microheterogeneity, while the glycan attached to Asn22 appeared to be homogeneous. In addition, bisecting GlcNAc attached to the trimannose core was detected, suggesting an origin other than the liver. Monosaccharide composition and LC-MS/MS analyses did not produce evidence for O glycosylation. TAFI contains eight cysteine residues, of which two, Cys69 and Cys383, are not involved in disulfides and contain free sulfhydryl groups. The remaining six cystines form disulfides, including Cys156-Cys169, Cys228-Cys252, and Cys243-Cys257. This pattern is homologous to pancreatic procarboxypeptidase B, and it is therefore unlikely that permutations in the cysteine connectivity are responsible for the enzymatic instability. LC-MS/MS analyses covering more than 90% of the TAFI amino acid sequence revealed no additional modifications. When these results are taken together, they suggest that the inherent instability of TAFIa is not caused by post-translational modifications. However, after activation, TAFIa loses 80% of the attached glycans, generating a large shift in pI and a propensity to precipitate. These changes are likely to significantly affect the properties of TAFIa as compared to TAFI.

AB - Thrombin-activable fibrinolysis inhibitor (TAFI) is distinct from pancreatic procarboxypeptidase B in several ways. The enzymatic activity of TAFIa is unstable and decays with a half-life of a few minutes. During this study, we observed that (i) the isoelectric point (pI) of TAFI shifts dramatically from pH 5 toward pH 8 upon activation and (ii) TAFIa is significantly less soluble than TAFI. The structural bases for these observations were investigated by characterizing all post-translational modifications, including attached glycans and disulfide connectivity. The analyses revealed that all five potential N-glycosylation sites were utilized including Asn22, Asn51, Asn63, Asn86 (located in the activation peptide), and Asn219 (located in the catalytic domain). Asn219 was also found in an unglycosylated variant. Four of the glycans, Asn51, Asn63, Asn86, and Asn219 displayed microheterogeneity, while the glycan attached to Asn22 appeared to be homogeneous. In addition, bisecting GlcNAc attached to the trimannose core was detected, suggesting an origin other than the liver. Monosaccharide composition and LC-MS/MS analyses did not produce evidence for O glycosylation. TAFI contains eight cysteine residues, of which two, Cys69 and Cys383, are not involved in disulfides and contain free sulfhydryl groups. The remaining six cystines form disulfides, including Cys156-Cys169, Cys228-Cys252, and Cys243-Cys257. This pattern is homologous to pancreatic procarboxypeptidase B, and it is therefore unlikely that permutations in the cysteine connectivity are responsible for the enzymatic instability. LC-MS/MS analyses covering more than 90% of the TAFI amino acid sequence revealed no additional modifications. When these results are taken together, they suggest that the inherent instability of TAFIa is not caused by post-translational modifications. However, after activation, TAFIa loses 80% of the attached glycans, generating a large shift in pI and a propensity to precipitate. These changes are likely to significantly affect the properties of TAFIa as compared to TAFI.

M3 - Journal article

VL - 45

SP - 1525

EP - 1535

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 5

ER -

ID: 33226503