HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 278, Issue 51, 51068-51074, December 19, 2003

This Article Full Text Full Text (PDF) All Versions of this Article: 278/51/51068    most recent M307470200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mironova, R. Articles by Ivanov, I. Search for Related Content PubMed PubMed Citation Articles by Mironova, R. Articles by Ivanov, I. Social Bookmarking                      What's this?

Glycation and Post-translational Processing of Human Interferon- Expressed in Escherichia coli^*

Roumyana Mironova, Toshimitsu Niwa¶, Rositsa Dimitrova, Maya Boyanova, and Ivan Ivanov

From the Department of Gene Regulations, Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria and the ^¶Department of Clinical Preventive Medicine, Nagoya University School of Medicine, Nagoya 466-8560, Japan

Until recently, nonenzymatic glycosylation (glycation) was thought to affect the proteins of long living eukaryotes only. However, in a recent study (Mironova, R., Niwa, T., Hayashi, H., Dimitrova, R., and Ivanov, I. (2001) Mol. Microbiol. 39, 1061-1068), we have shown that glycation takes place in Escherichia coli as well. In the present study, we demonstrate that the post-translational processing (proteolysis and covalent dimerization) observed with cysteineless recombinant human interferon- (rhIFN-) is tightly associated with its in vivo glycation. Our results show that, at the time of isolation, rhIFN- contained early (but not advanced) glycation products. Using reverse phase high performance liquid chromatography in conjunction with fluorescence measurements, enzyme-linked immunosorbent assay, and mass spectrometry, we found that advanced glycation end products arose in rhIFN- during storage. The latter were identified mainly in the Arg/Lys-rich C terminus of the protein, which was also the main target of proteolysis. Mass spectral analysis and N-terminal sequencing revealed four major (Arg¹⁴⁰Arg¹⁴¹, Phe¹³⁷Arg¹³⁸, Met¹³⁵Leu¹³⁶, and Lys¹³¹Arg¹³²) and two minor (Lys¹⁰⁹Ala¹¹⁰ and Arg⁹⁰Asp⁹¹) cleavage sites in this region. Tryptic peptide mapping indicated that the covalent dimers of rhIFN- originating during storage were formed mainly by lateral cross-linking of the monomer subunits. Antiviral assay showed that proteolysis lowered the antiviral activity of rhIFN-, whereas covalent dimerization completely abolished it.

Received for publication, July 11, 2003 , and in revised form, October 2, 2003.

^* This work was supported by Contract 02/008 from the International Center for Genetic Engineering and Biotechnology (Trieste, Italy). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Gene Regulations, Inst. of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria. Tel.: 359-2-979-26-52; Fax: 359-2-73-62-27; E-mail: rumym{at}obzor.bio21.bas.bg.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				J. C. Aon, R. J. Caimi, A. H. Taylor, Q. Lu, F. Oluboyede, J. Dally, M. D. Kessler, J. J. Kerrigan, T. S. Lewis, L. A. Wysocki, et al. Suppressing Posttranslational Gluconoylation of Heterologous Proteins by Metabolic Engineering of Escherichia coli Appl. Envir. Microbiol., February 15, 2008; 74(4): 950 - 958. [Abstract] [Full Text] [PDF]

				M. B. Johansen, L. Kiemer, and S. Brunak Analysis and prediction of mammalian protein glycation Glycobiology, September 1, 2006; 16(9): 844 - 853. [Abstract] [Full Text] [PDF]

				Y.-G. Choi, J.-I. Kim, Y.-C. Jeon, S.-J. Park, E.-K. Choi, R. Rubenstein, R. J. Kascsak, R. I. Carp, and Y.-S. Kim Nonenzymatic Glycation at the N Terminus of Pathogenic Prion Protein in Transmissible Spongiform Encephalopathies J. Biol. Chem., July 16, 2004; 279(29): 30402 - 30409. [Abstract] [Full Text] [PDF]