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Synthesis and protein curing abilities of membrane glycolipids
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

There are many types of membrane lipids throughout Nature. Still little is known about synthesizing pathways and how different lipids affect the embedded membrane proteins. The most common lipids are glycolipids since they dominate plant green tissue. Glycolipids also exist in mammal cells as well as in most Gram-positive bacteria. Glycosyltransferases (GTs) catalyze the final enzymatic steps for these glycolipids. In the bacteria Acholeplasma laidlawii and Streptococcus pneumonie and in the plant Arabidopsis thaliana, GTs for mono-/di-glycosyl-diacylglycerol (-DAG) are suggested to be regulated to keep a certain membrane curvature close to a bilayer/nonbilayer phase transition. The monoglycosylDAGs are nonbilayer-prone with small headgroups, hence by themselves they will not form bilayer structures.

Here we have determined the genes encoding the main glycolipids of A. laidlawii and S. pneumonie. We have also shown that these GTs belong to a large enzyme group widely spread in Nature, and that all four enzymes are differently regulated by membrane lipids. The importance of different lipid properties were traced in a lipid mutant of Escherichia coli lacking the major (75 %), nonbilayer-prone/zwitterionic, lipid phosphatidylethanolamine. Introducing the genes for the GTs of A. laidlawii and two analogous genes from A. thaliana yielded new strains containing 50 percent of glyco-DAG lipids. The monoglyco-DAG strains contain significant amounts of nonbilayer-prone lipids while the diglyco-DAG strains contain no such lipids. Comparing these new strains for viability and the state of membrane-associated functions made it possible to connect different functions to certain lipid properties. In summary, a low surface charge density of anionic lipids is important in E.coli membranes, but this fails to be supportive if the diluting species have a too large headgroup. This indicates that a certain magnitude of the curvature stress is crucial for the membrane bilayer in vivo.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik , 2006. , 48 p.
Keyword [en]
membrane, lipids, glycolipids, nonbilayer-prone
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-1361ISBN: 91-7155-351-7 (print)OAI: oai:DiVA.org:su-1361DiVA: diva2:189959
Public defence
2006-12-15, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00
Opponent
Supervisors
Available from: 2006-11-16 Created: 2006-11-16Bibliographically approved
List of papers
1. Sequence properties of the 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii membranes: Recognition of a large group of lipid glycosyltransferases in eubacteria and archaea
Open this publication in new window or tab >>Sequence properties of the 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii membranes: Recognition of a large group of lipid glycosyltransferases in eubacteria and archaea
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2001 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 25, 22056-22063 p.Article in journal (Refereed) Published
Abstract [en]

Synthesis of the nonbilayer-prone α-monoglucosyldiacylglycerol (MGlcDAG) is crucial for bilayer packing properties and the                     lipid surface charge density in the membrane ofAcholeplasma laidlawii. The gene for the responsible, membrane-bound glucosyltransferase (alMGS) (EC 2.4.1.157) was sequenced and functionally cloned in Escherichia coli, yielding MGlcDAG in the recombinants. Similar amino acid sequences were encoded in the genomes of several Gram-positive                     bacteria (especially pathogens), thermophiles, archaea, and a few eukaryotes. All of these contained the typical EX7E catalytic motif of the CAZy family 4 of α-glycosyltransferases. The synthesis of MGlcDAG by a close sequence analog from                      Streptococcus pneumoniae (spMGS) was verified by polymerase chain reaction cloning, corroborating a connection between sequence and functional similarity                     for these proteins. However, alMGS and  spMGS varied in dependence on anionic phospholipid activators phosphatidylglycerol                     and cardiolipin, suggesting certain regulatory differences. Fold predictions strongly indicated a similarity for alMGS (and                     spMGS) with the two-domain structure of the E. coli MurG cell envelope glycosyltransferase and several amphipathic membrane-binding segments in various proteins. On the basis                     of this structure, the alMGS sequence charge distribution, and anionic phospholipid dependence, a model for the bilayer surface                     binding and activity is proposed for this regulatory enzyme.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-23039 (URN)10.1074/jbc.M102576200 (DOI)000169412700011 ()
Available from: 2006-11-16 Created: 2006-11-16 Last updated: 2017-12-13Bibliographically approved
2. Structural features of glycosyltransferases synthesizing major bilayer and nonbilayer-prone membrane lipids in Acholeplasma laidlawii and Streptococcus pneumoniae
Open this publication in new window or tab >>Structural features of glycosyltransferases synthesizing major bilayer and nonbilayer-prone membrane lipids in Acholeplasma laidlawii and Streptococcus pneumoniae
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2003 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 10, 8420-8428 p.Article in journal (Refereed) Published
Abstract [en]

In membranes of Acholeplasma laidlawii two consecutively acting glucosyltransferases, the (i) alpha-monoglucosyl-diacylglycerol. (MGlcDAG) synthase (aIMGS) (EC 2.4.1.157) and the (ii) alpha-diglucosyl-DAG (DGlcDAG) synthase (alDGS) (EC 2.4.1.208), are involved in maintaining (i) a certain anionic lipid surface charge density and (ii) constant nonbilayer/bilayer conditions (curvature packing stress), respectively. Cloning of the aIDGS gene revealed related uncharacterized sequence analogs especially in several Gram-positive pathogens, thermophiles and archaea, where the encoded enzyme function of a potential Streptococcus pneumoniae DGS gene (cpoA) was verified. A strong stimulation of aIDGS by phosphatidylglycerol (PG), cardiolipin, or nonbilayer-prone 1,3-DAG was observed, while only PG stimulated CpoA. Several secondary structure prediction and fold recognition methods were used together with SWISS-MODEL to build three-dimensional model structures for three MGS and two DGS lipid glycosyltransferases. Two Escherichia coli proteins with known structures were identified as the best templates, the membrane surface-associated two-domain glycosyltransferase MurG and the soluble GlcNAc epimerase. Differences in electrostatic surface potential between the different models and their individual domains suggest that electrostatic interactions play a role for the association to membranes. Further support for this was obtained when hybrids of the N- and C-domain, and full size alMGS with green fluorescent protein were localized to different regions of the E. coli inner membrane and cytoplasm in vivo. In conclusion, it is proposed that the varying abilities to bind, and sense lipid charge and curvature stress, are governed by typical differences in charge (pI values), amphiphilicity, and hydrophobicity for the N- and (catalytic) C-domains of these structurally similar membrane-associated enzymes.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-23040 (URN)10.1074/jbc.M211492200 (DOI)000181466800088 ()
Available from: 2006-11-16 Created: 2006-11-16 Last updated: 2017-12-13Bibliographically approved
3. Monoglucosyldiacylglycerol, a foreign lipid, can substitute for phosphatidylethanolamine in essential membrane-associated functions in Escherichia coli
Open this publication in new window or tab >>Monoglucosyldiacylglycerol, a foreign lipid, can substitute for phosphatidylethanolamine in essential membrane-associated functions in Escherichia coli
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2004 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 11, 10484-10493 p.Article in journal (Refereed) Published
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-23041 (URN)10.1074/jbc.M310183200 (DOI)000220050400100 ()
Note

The mechanisms by which lipid bilayer properties govern or influence membrane protein functions are little understood, but a liquid-crystalline state and the presence of anionic and nonbilayer (NB)-prone lipids seem important. An Escherichia coli mutant lacking the major membrane lipid phosphatidylethanolamine (NB-prone) requires divalent cations for viability and cell integrity and is impaired in several membrane functions that are corrected by introduction of the "foreign" NB-prone neutral glycolipid alpha-monoglucosyldiacylglycerol (MGlcDAG) synthesized by the MGlcDAG synthase from Acholeplasma laidlawii. Dependence on Mg2+ was reduced, and cellular yields and division malfunction were greatly improved. The increased passive membrane permeability of the mutant was not abolished, but protein-mediated osmotic stress adaptation to salts and sucrose was recovered by the presence of MGlcDAG. MGlcDAG also restored tryptophan prototrophy and active transport function of lactose permease, both critically dependent on phosphatidylethanolamine. Three mechanisms can explain the observed effects: NB-prone MGlcDAG improves the quenched lateral pressure profile across the bilayer; neutral MGlcDAG dilutes the high anionic lipid surface charge; MGlcDAG provides a neutral lipid that can hydrogen bond and/or partially ionize. The reduced dependence on Mg2+ and lack of correction by high monovalent salts strongly support the essential nature of the NB properties of MGlcDAG.

Available from: 2006-11-16 Created: 2006-11-16 Last updated: 2017-12-13Bibliographically approved
4. Bilayer forming Diglucosyl- and Digalactosyl-diacylglycerol are not exchangeable supporting membrane-associated processes in Escherichia coli
Open this publication in new window or tab >>Bilayer forming Diglucosyl- and Digalactosyl-diacylglycerol are not exchangeable supporting membrane-associated processes in Escherichia coli
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Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-23042 (URN)
Note
Part of urn:nbn:se:su:diva-1361Available from: 2006-11-16 Created: 2006-11-16 Last updated: 2010-01-13Bibliographically approved

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