Document Detail


Incorporation of ammonium into intracellular UDP-activated N-acetylhexosamines and into carbohydrate structures in glycoproteins.
MedLine Citation:
PMID:  10397879     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The negative effects of ammonia on animal cells, especially in vitro cultures, are well known, but the mechanism of how ammonia inhibits cell growth and influences the glycosylation of proteins is not completely understood. We investigated the ammonium action on the synthesis of the intracellular UDP-N-acetylhexos- amines (UDPGNAc), which are precursors of glycosylation as well as on N-linked oligosaccharides of a recombinant human IL-2 mutant variant model glycoprotein expressed in BHK-21 cells under defined and controlled culture conditions in a continuously perfused bioreactor. The examinations were based on our previous observations that increased ammonia concentrations in the medium lead to the intracellular formation and accumulation of UDPGNAc (Ryll et al., 1994). The kinetics of formation of the UDPGNAc pool after adding ammonia and its reconstitution to normal conditions are shown. To study the pathway leading to the intracellular increase of UDPGNAc, the uptake and incorporation of 15NH4+ was confirmed by the detection of 15N in UDP-N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc was purified using high pH anion-exchange chromatography with pulsed amperometric detection and analyzed by GC/MS. The proportion of UDP-GlcNAc containing 15N was approximately 60% and corresponds quantitatively to the increased intracellular concentration of UDP-GlcNAc. In order to confirm the direct influence of ammonia on protein glycosylation, the human IL-2 mutant glycoprotein variant IL-Mu6, bearing a novel N-glycosylation site, has been produced under defined protein-free medium conditions in the presence of 15NH4Cl. IL-Mu6 glycoprotein was purified and N-glycans released were analyzed by matrix-assisted laser desorption ionization time of flight mass spectroscopy. Maximally 60-80% of N-acetylated sugars in N-glycan structures contained 15N indicating that ammonium is used as a building block during synthesis of the carbohydrate structures expressed from in vitro cultivated mammalian cells.
Authors:
U Valley; M Nimtz; H S Conradt; R Wagner
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Biotechnology and bioengineering     Volume:  64     ISSN:  0006-3592     ISO Abbreviation:  Biotechnol. Bioeng.     Publication Date:  1999 Aug 
Date Detail:
Created Date:  1999-08-24     Completed Date:  1999-08-24     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  7502021     Medline TA:  Biotechnol Bioeng     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  401-17     Citation Subset:  IM    
Copyright Information:
Copyright 1999 John Wiley & Sons, Inc.
Affiliation:
University of Leipzig, Institute of Clinical Immunology and Transfusion Medicine, Department of Medical Biotechnology, Delitzscher Strasse 141, D-04129 Leipzig, Germany.
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MeSH Terms
Descriptor/Qualifier:
Animals
Bioreactors
Carbohydrate Conformation
Cell Line
Cricetinae
Glycoproteins / chemistry*
Hexosamines / chemistry*
Humans
Kinetics
Methylation
Quaternary Ammonium Compounds / chemistry*
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Uridine Diphosphate / chemistry*
Chemical
Reg. No./Substance:
0/Glycoproteins; 0/Hexosamines; 0/Quaternary Ammonium Compounds; 58-98-0/Uridine Diphosphate

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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