Structural Features of Glycosyltransferases Synthesizing Major Bilayer and Nonbilayer-prone Membrane Lipids in Acholeplasma laidlawii and Streptococcus pneumoniae*,

Maria EdmanDagger §, Stefan BergDagger §, Patrik Storm||, Malin Wikström||, Susanne Vikström**, Anders ÖhmanDagger , and Åke Wieslander||DaggerDagger

From the Dagger  Department of Biochemistry and the ** Department of Odontology, Umeå University, 90187 Umeå, Sweden and the || Department of Biochemistry and Biophysics, Stockholm University, 10691 Stockholm, Sweden

Received for publication, November 11, 2002, and in revised form, November 29, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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In membranes of Acholeplasma laidlawii two consecutively acting glucosyltransferases, the (i) alpha -monoglucosyldiacylglycerol (MGlcDAG) synthase (alMGS) (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 alDGS 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 alDGS 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.

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Lipid bilayer properties, important for membrane barrier and protein function, are regulated at several levels in cells. Acholeplasma laidlawii and Escherichia coli membrane lipids are metabolically designed to yield a bilayer "window" with certain features between the gel and nonbilayer phases (1, 2). In A. laidlawii this aims at maintaining (i) a certain lipid bilayer surface charge density and (ii) a constant radius of spontaneous curvature (elastic packing stress), including similar bilayer/nonbilayer transition temperatures (1, 3). Regulation of (i) and (ii) resides mainly at the polar headgroup level (4), whereas in E. coli curvature is regulated at the acyl chain level (2), with more or less constant headgroup composition.

The bilayer-forming glucolipid alpha -diglucosyldiacylglycerol (DGlcDAG)1 is one of the major lipids in the small cell wall-less A. laidlawii, the other is the nonbilayer-prone alpha -monoglucosyldiacylglycerol (MGlcDAG), cf. pathway below. Another pathway leads from phosphatidic acid to phosphatidylglycerol (PG). Under certain circumstances more acylated, and more nonbilayer-prone, variants of MGlcDAG and DGlcDAG, i.e. MAMGlcDAG and MADGlcDAG, respectively, are synthesized (5). A. laidlawii can only synthesize saturated acyl chains, and to still be able to adapt to membrane stress and new environments, the amounts of the two major glucolipids are strongly adjusted to maintain a functional membrane bilayer. The MGlcDAG-synthesizing enzyme alMGS (EC 2.4.1.157) ((i) above) was recently cloned (6) and found to be a surface-associating protein with no transmembrane segments, where bilayer binding was dependent on phosphatidylglycerol and stimulated by small amounts of nonbilayer lipids.2 This seemed logical given the strong involvement of this enzyme in the lipid surface charge regulation, i.e. (i) above (8).


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Scheme 1.   Glucolipid pathway.

The 1,2-diacylglycerol-3-alpha -glucose (1 right-arrow 2)-alpha -glucosyl transferase (DGS) (EC 2.4.1.208) catalyzes the consecutive transfer of glucose from UDP-Glc to MGlcDAG to yield DGlcDAG (cf. (ii) above). This reaction is activated in an essential manner by certain anionic lipids and stimulated by other additives promoting nonbilayer tendencies both in vivo (1, 9) and with crude or pure enzymes in vitro (10, 11), keeping or restoring bilayer packing conditions. Furthermore, an additional modulation of the activity is achieved by certain phosphorylated metabolites, double-stranded DNA (12), and by a low redox potential (7). Due to their small polar headgroups, nonbilayer-prone lipids of the reversed type, like MGlcDAG, make the two monolayers in a bilayer each want to curl concavely toward the water phase. This induces a curvature elastic stress (increased spontaneous curvature), with an increased chain order and a closer approach to a bilayer-nonbilayer phase transition. Similar packing features can be inferred for the major galactolipids in the membranes of chloroplasts and photosynthetic bacteria (13). A number of peripheral and integral membrane proteins are functionally affected by this lateral stress, but for none the exact mechanisms are known (14), although correlation with the lipid properties is very evident (e.g. Refs. 15-17).

Hence, a question of large importance is how the A. laidlawii DGS is able to sense the curvature stress and respond by increased or decreased synthesis of DGlcDAG. The cloned gene (this work) yielded an active enzyme from E. coli, and the deduced amino acid sequence and the predicted secondary and three-dimensional structure models strongly indicate this enzyme to be, unexpectedly, very similar in structure and surface association as the preceding MGlcDAG synthase (glucosyltransferase) and several other glycosyltransferases. Hence, the bilayer/nonbilayer lipid balance, and the connected bilayer physical forces, are sensed at the bilayer surface by the DGlcDAG synthase. Data base searches revealed a new group of related lipid glycosyltransferases in Gram-positive bacteria and archaea. Functional cloning of CpoA from Streptococcus pneumoniae suggests this group to be responsible for synthesis of alpha -diglycosyldiacylglycerols in a number of species. However, CpoA responds differently to the properties of the surrounding bilayer, and the latter is substantiated by certain differences in the enzyme structure models.

    MATERIALS AND METHODS
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Genomic Preparation-- A. laidlawii strain A-EF22 was grown in 28 °C in tryptose/bovine serum albumin medium (11), supplemented with 0.12 mM oleic acid (18:1c). The cells were harvested, and the DNA was prepared with the GenomicPrepTM kit (Amersham Biosciences). The S. pneumoniae strain 19FCCUG 3030 was grown in Todd-Hewitt medium, pH 7.8 at 37 °C overnight. The DNA was prepared by heating the resuspended cell pellet at 95 °C for 10 min. After the subsequent centrifugation, the DNA was present in the supernatant.

Cloning of DGS Genes-- Degenerated DNA primers were made based on the N-terminal amino acid sequence of the purified A. laidlawii DGlcDAG synthase (alDGS) determined through Edman degradation (10). An internal sequence was also determined after proteolytic cleavage and fragment purification by reverse phase-high performance liquid chromatography. The primers 5'-GGT CGT GCT TTT TAT CA(C/T) CA(A/G) AAA-3' and 5'-AAT AAC AGC ACC (A/G)TC IAC (A/G)TG-3' were used in a PCR amplification procedure that resulted in a ~600-bp DNA fragment. The PCR products were purified and ligated into the pCR-Script SK(+) cloning vector (Stratagene) linearized with SrfI and then sequenced. The purified PCR product was also radioactively labeled by alpha -35S-dATP and used as a probe in a Southern blot procedure with completely HindIII/EcoRI digested DNA from A. laidlawii. The hybridization results were visualized by electronic autoradiography (Packard Instant ImagerTM). The DNA fragments with corresponding size to the hybridization band were purified and ligated into the pCR-Script SK(+) cloning vector and transformed into E. coli TOP 10F heat-shock competent cells. Positive clones were found by colony replicating to nitrocellulose filter and hybridization (18) with the radioactively labeled probe. The nucleotide sequence of the inserted DNA fragment was determined using both gene-specific and vector-specific primers with ABI PRISM® Big Dye terminator cycle sequencing ready reaction kit (PE Applied Biosystems). Using these results, a construct was made where the gene ALdgs from A. laidlawii A-EF22 strain was cloned into a pET15b-vector and transformed into E. coli BL21(DE3). This recombinant was used for the enzymatic assays.

The gene coding for the spDGS in S. pneumoniae (cpoA), identified with the alDGS aa sequence in a computer search, was isolated by PCR with the primers 5'-TAG TTA TGG AGA AAA AGA AAT TAC G-3' and 5'-TAC CTC ACT TTT TAC TTT CTC CC-3', which bind to the Shine-Dalgarno and stop codon sequence regions, respectively. The PCR product was purified, ligated into a SmaI-digested pCR-script vector, and transformed into TOP10F.

Lipids and Amphiphiles Used-- Synthetic rac-1,3-dioleoylglycerol (1,3-DOG) was purchased from Larodan (Malmö, Sweden). The MGlcDAG and DGlcDAG were prepared as described (8). Synthetic alpha -MGalDAG was obtained from Dr. D. Mannock (cf. Dahlqvist et al. (11)). Synthetic DOPA and DOPG were purchased from Avanti Polar Lipids (Alabaster, AL) and CHAPS detergent from Roche Molecular Biochemicals.

Growth of Recombinants-- Protein expression in the recombinant strains was performed in 1× LB medium supplemented with 100 µg of carbenicillin/ml. The strains were grown at 37 °C, and 0.3 mM isopropyl-1-thio-beta -D-galactopyranoside was added at OD600 = 0.75. Cells were harvested by centrifugation after another 16 h at 23 °C. Harvested recombinant E. coli cells were solubilized in assay buffer (110 mM Tris-HCl, pH 8.0, 22 mM MgCl2, and 22 mM CHAPS) to a total protein concentration of ~1.3 mg/ml, by 4 × 30 s in a sonication bath and three times extensive vortexing during incubation on ice for 3 h, before enzymatic assays.

Enzymatic Assays-- In the standard assay for glycolipid synthesis 25 µl of protein solution (cf. above) was added to 20 µl of lipid micellar solution and incubated on ice for 30 min. The enzymatic reaction was started by addition of 5 µl of UDP-[14C]galactose or UDP-[14C]glucose to a concentration of 1 mM (30 GBq mol-1) in a total volume of 50 µl. Standard lipid concentration was 10 mM (1 mM MGlcDAG substrate in addition to the activator DOPG (or CL) and the nonbilayer-prone 1,3-DOG, with DGlcDAG as balance). After 30 min of incubation at 28 °C, the reaction was stopped with 375 µl of methanol/chloroform 2:1 (v/v), and the lipids were extracted and separated by TLC (10, 19). The lipid products on the TLC plates were visualized and quantified by electronic autoradiography (Packard Instant ImagerTM). All assays were done in duplicate. A variety of glycolipids was used as TLC references (20).

Localization of alMGS-GFP Fusion Variants-- Full-size (398 aa), N- (aa 1-226), and C-domain (aa 227-398) alMGS, respectively, were cloned upstream and in-frame with the gene for green fluorescent protein (GFP) using a PCR primer approach based on the alMGS sequence (6) and the vector pGFPuv (Clontech), in E. coli JM109. Ligation, transformation, and selection followed standard procedures, and the obtained constructs were verified by DNA sequencing. Enzymatic activities of the hybrid proteins were analyzed after induction with isopropyl-1-thio-beta -D-galactopyranoside by labeling of E. coli lipids in vivo with radioactive acetate (6) and by assay for MGlcDAG synthesis after detergent solubilization in vitro (6). Localization of the MGS-GFP variants in E. coli was analyzed by fluorescence microscopy at various growth and induction conditions, using a Zeiss Axioplan2 microscope, fluorescein isothiocyanate filters, a CCD camera (Sony), and Image Access 3.0 software (Imagic Bildverarbeitung AG).

Sequence Analysis and Structure Prediction-- To find homologs to the alDGS and to predict a function for the genes in flanking regions, PSI-BLAST (21) at NCBI in the nonredundant data base and the data base for finished and unfinished microbial genomes, was used. Preliminary genomic sequence data were obtained from The Institute for Genomic Research. Features of the primary structure of the alDGS sequence were further analyzed with tools available at the ExPASy Molecular Biology Server (www.expasy.ch) (Swiss Institute of Bioinformatics) and with the Wisconsin Package Version 9.1 (Genetics Computer Group, Madison, WI).

Potential secondary and three-dimensional structure models were predicted from aa sequences by several local structure (PsiPred, Target99, and Jpred2) and fold recognition methods (FFAS, 3D-PSSM, GenTHREADER, mGenTHREADER, INBGU, Sam-T99, and FUGUE) and jointly evaluated using the Pcons consensus method (22) at the MetaServer (www.bioinfo.pl) (23). The scores stating the similarity between the obtained models are derived from Levitt and Gerstein (cf. Ref. 22). The aa sequences from several known glycosyltransferase structures were analyzed ("benchmarked") in a similar manner (with PDB Test, www.bioinfo.pl) and the suggested PDB structures compared with the DALI method (24).

Structural models of the glycosyltransferases were built, based on the sequence alignments and recognized folds given by the MetaServer (above), using the Swiss-PdbViewer program for alignment (Version 3.7; www.expasy.ch/spdbv/) (25) and the optimize-mode of the SWISS-MODEL program server (Version 3.5; www.expasy.ch/swissmod/SWISS-MODEL.html) for automatic model building. For the MGS sequences the three protein structures of ecMurG (PDB-ID 1F0K), ecEpim (PDB-ID 1F6D), and aoGtfB (PDB-ID 1IIR) were used as templates, while the DGS sequences were modeled with the first two (ecMurG and ecEpim) as templates. All five proteins were successfully modeled. Although the models obtained from the SWISS-MODEL server are already energy minimized, they were subjected to an additional global energy minimization. Prior to the final minimization a few side chains were adjusted as they were trapped within the ring of aromatic side chains, a situation an energy minimization would not be able to correct. For the global energy minimization the Swiss-PdbViewer implementation of the GROMOS96 43B1 force field was used, together with the SWISS-MODEL server minimization protocol, namely 200 steps of steepest descent, followed by 300 steps of conjugate gradient minimization. The quality of the models was accessed using the WhatCheck program (26). Identification of secondary structure elements, calculation of the electrostatic surface potential, and visualization were made using the program MOLMOL (27). The electrostatic potentials were calculated assuming point charges on heavy atoms, dielectric constants of 2 and 80 for protein and solvent, respectively, an ionic strength of 0.3 M, a 2-Å salt radius, and a boundary condition of zero potential at 15 Å. A solvent radius of 1.4 Å was used when the accessible van der Waals surfaces of the heavy atoms of each model were identified. The relative solvent accessibility of residues was calculated from their coordinates and divided into three groups; buried (0-5%), intermediate (5-25%) and exposed (25-100%) (28).

Nucleotide Sequence Accession Number-- Nucleotide sequence data for A. laidlawii DGlcDAG synthase have been deposited at GenBankTM with accession number AY078412.

    RESULTS AND DISCUSSION
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Gene Cloning and Organization-- The N-terminal and an internal amino acid sequence of the purified DGlcDAG synthase from A. laidlawii (alDGS) were determined by Edman degradation (10). Designed degenerated PCR primers yielded a gene-specific probe, which was used in a Southern blot procedure identifying a 2.2-kbp DNA fragment that was cloned. The sequence of this contig (Table I) revealed an open reading frame (ORF) of 999 bp coding for the alDGS sequence named ALdgs (Fig. 1) with a potential Shine-Dalgarno sequence starting at position -16. The G+C content was 33%, which is typical for A. laidlawii DNA (31.7-35.7% (29)). The translated ORF found just downstream the ALdgs gene was related to proteins from the aldo/keto-reductase family. The sequence further downstream show similarity to trigger factor, a conserved chaperone found in all prokaryotes. No ORFs in the opposite direction were found. The A. laidlawii contigs containing the preceding glucosyltransferase gene ALmgs cloned earlier (6) and the ALdgs determined here (Table I) have no nucleotide sequence in common and were not localized to the same DNA fragment according to hybridization. Hence, it was concluded that these two glycosyltransferase (GT) genes are not adjacent to each other on the chromosome and do not belong to the same operon.

                              
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Table I
Analysis of the content of flanking regions to DGS in A. laidlawii


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Fig. 1.   Alignment of alDGS and close homologs/analogs. aa alignment (CLUSTALW) of alDGS to three hits (Table II) found by BLAST in the data base of finished and unfinished microbial genomes at NCBI. Shaded residues are conserved in all the four proteins. Residues marked in bold are conserved in all best hits (Table II). Boxed residues belong to the EX7E motif. Amino acid sequences obtained by Edman degradation of the N terminus and a proteolytic fragment of the purified protein are underlined. Ala, A. laidlawii; Spy, S. pyogenes (48% identity); Hal, Halobacterium sp. NRC-1 (30% identity); Spn, S. pneumoniae CpoA (30% identity). The latter is shown here (Fig. 2) to be functionally analogous to alDGS.

Homologs and Analogs to alDGS-- BLAST searches in the finished/unfinished and the nonredundant data bases at NCBI with the alDGS amino acid sequence gave several significant hits in eubacteria and archaea (Table II). All the top hits were Gram-positive sequences, and most were from pathogenic species. The best score showed a gene in Enterococcus faecium with 49% aa identity to the alDGS, and sequences from streptococcal or other closely related species (Lactococcus) had an identity above 45%. These species do all contain an alpha -diglycosyldiacylglycerol identical to the A. laidlawii one (30, 31), and the sugar moieties are solely glucose. In the corresponding glycolipid from S. pneumoniae (cf. Table II) the outer glucose is replaced by a galactose. For the alMGS glucosyltransferase aa identities of 31 and 29% were recorded to similar enzymes in S. pneumoniae and Borrelia burgdorferi, and these were shown to encode homologous functions to the A. laidlawii enzyme (6). Most important, alDGS and all the sequences of Table II contain the conserved EX7E motif characteristic for the retaining (alpha ) GTs of CAZy family 4 of glycosyltransferases (32); the preceding enzyme alMGS (cf. Introduction) also belongs to this family (6). They also contain a typical motif for glycosyltransferases belonging to Pfam family 00534 (conserved domain search). Proteins in this family transfer nucleoside diphosphate-linked sugars, like glucose, galactose, mannose, and X-glucose, to a variety of acceptor substrates such as glycogen and lipopolysaccharides.

                              
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Table II
DGS homologs/analogs
The best hits found by BLAST in finished and unfinished microbial genomes data base at NCBI. Homologs/analogs are found in Gram-positive bacteria and archaea. Homologs to the alMGS are also found by similarity in almost all organisms in the table. All are members of CAZy family 4 of GTs.

The BLAST searches revealed no proteins with an established function, since many of them are from recently published genome projects and so far lack annotation. However, the S. pneumoniae gene in Table II was first sequenced by Grebe et al. (33) and named cpoA. This protein was proposed to be involved in resistance to beta -lactams and was isolated from first-step piperacillin-resistant mutants. The cpoA and the consecutive ORF5 gene, both part of the same operon, were suggested (but not shown) to be involved in formation of the linkage unit in lipoteichoic acid or polymerization of teichoic acid precursors prior to translocation (33). We showed recently that this ORF5 encoded an enzyme homologous in function to alMGS (cf. step (i) in Introduction) and that was named spMGS. This enzyme was of similarly high sequence identity to alMGS as the CpoA protein is to alDGS (cf. Table II and Fig. 1). Most intriguing, the majority of organisms with a potential alDGS homolog (Table II) also encode a homolog to alMGS, according to BLAST sequence analyses (data not shown) (6). The fact that the homologs of DGS and MGS genes in S. pneumoniae were adjacent to each other (above), potentially in an operon organization, was true also for most of the species in Table II. However, not for A. laidlawii and the related species Clostridium acetobutylicum and not for Pyrococcus furiosus (data not shown). Since the function of the S. pneumoniae MGS enzyme (i.e. ORF5, see above) has been verified (6), the adjacent gene cpoA was PCR-cloned (see "Materials and Methods") to establish its function (cf. below).

The BLAST searches also revealed analogs in the three archaeal species Halobacterium sp. NRC-1, Pyrococcus horikoshii and P. furiosus (Table II); all sequences are of unknown function. The membrane of Halobacterium spp. contains the major glycolipid 3-HSO3-beta -Galp-(1-6)-alpha -Manp-(1-2)-alpha -Glcp-(1-1)-diether and several minor glycolipids. Since alDGS, and all CAZy family 4 members (32), are retaining enzymes the Halobacterium analog (Vng0600c, Table II) can be proposed to catalyze the glycosylation with the second mannose, and the adjacent Vng0598c (6), the first glucose. In another thermophile Thermotoga maritima, a DGlcDAG was found with the rare alpha (1 right-arrow 4) diglucosyl structure (34), and an analog (pir C72340) with low score to alDGS was found by BLAST (Table II). Hence, the A. laidlawii alDGS glucosyltransferase gene has homologs in a number of especially Gram-positive pathogens, and analogs in archaea and thermophiles (cf. Table II). Most of these homologs are encoded next to an alMGS homolog, most likely in an operon organization.

Gene Function-- Enzymatic synthesis of A. laidlawii DGlcDAG from MGlcDAG, with crude native cell proteins or purified alDGS, only yielded the alpha -DGlcDAG product; no other glucose acceptor than alpha -MGlcDAG was active (like beta  or Gal variants) (10, 11). Here, alDGS and S. pneumoniae CpoA were cloned and overexpressed in E. coli and enzymatic activity assays verified the function (see "Materials and Methods"). As expected a new lipid product was synthesized by the cloned alDGS when the assay mixture was supplemented with alpha -MGlcDAG purified from A. laidlawii and radiolabeled UDP-Glc (Fig. 2, lane 2). This lipid, tentatively identified as the 1,2-diacyl-3-O-[alpha -D-glucopyranosyl-(1 right-arrow 2)-O-alpha -D-glucopyranosyl]-sn-glycerol (DGlcDAG), showed the same TLC migration as the lipid product obtained from corresponding syntheses using native, crude or purified, DGS enzyme from A. laidlawii (Fig. 2, lane 1) (10), and several diglycosyl-DAG reference lipids (20). Assays with alpha -MGalDAG or UDP-Gal yielded no products.


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Fig. 2.   Synthesis of diglycosyldiacylglycerol by clones. Glycolipid synthesis from solubilized recombinant E. coli cells from autoradiograms of silica gel TLC plates. All assays contained cell protein, CHAPS, DOPG, and substrate alpha -MGalDAG or alpha -MGlcDAG, and UDP-Glc or UDP-Gal. Substrates and enzymes (top of figure): lane 1, MGlcDAG plus radiolabeled UDP-Glc and purified native DGlcDAG synthase from A. laidlawii membranes (10). Lane 2, as in lane 1 but with overexpressed alDGS (ec). Lanes 3-6, overexpressed spDGS and MGalDAG plus radiolabeled UDP-Glc (lane 3), radiolabeled UDP-Gal (lane 4), MGlcDAG plus radiolabeled UDP-Glc (lane 5), or radiolabeled UDP-Gal (lane 6). Enzyme assays were performed, and TLC plates were developed and analyzed as described under "Material and Methods." A, application spot; PG, phosphatidylglycerol; DG, diglycosyl diacylglycerol; MG, monoglucosyl diacylglycerol. Lane 7, reference lipids from A. laidlawii. The migration of DGlcDAG and GalGlcDAG was similar to several other disugar-DAGs (20). Both alDGS and spDGS use alpha -MGlcDAG as lipid acceptor, but discriminate between sugars where spDGS uses UDP-Gal and alDGS uses UDP-Glc.

The sequence similarity between alDGS and CpoA (Fig. 1) suggested the latter to possess a similar function as alDGS. All combinations of the lipid substrates alpha -MGalDAG and alpha -MGlcDAG, and the radiolabeled nucleotide sugars UDP-Gal and UDP-Glc, were tried. All assays were supplemented with the anionic DOPG. Fig. 2 (lane 3-6) shows that the overexpressed CpoA (spDGS) was able to catalyze the glycosylation of only alpha -MGlcDAG by using the sugar nucleotide UDP-Gal. This is in agreement with earlier characterization of the lipid composition in S. pneumoniae, where the two major glycolipids were characterized to be monoglucosyl diacylglycerol (MGlcDAG) and galactosyl-glucosyldiacylglycerol (GalGlcDAG) (36, 37), and where all sugars were in alpha -configuration and identically linked as in A. laidlawii (38, 39). We have recently characterized the ORF5 gene adjacent downstream to cpoA as encoding the S. pneumoniae alpha -MGlcDAG glucosyltransferase (spMGS) (6). Hence, the CpoA protein is the consecutively acting spDGS, analogous to the alDGS above. Furthermore, synthesis of DAG, MGlcDAG, and GalGlcDAG could be achieved in a joint CHAPS extract of three E. coli clones separately expressing each of a phosphatidic acid phosphatase identified by us, spMGS (6) and spDGS (this work), respectively (S. pneumoniae genes sp0489, sp1076, and sp1075 (40)), data not shown. Consequently, an efficient synthesis of this lipid pathway can be achieved without the genes in an operon, cf. biosynthetic route in introduction.

Modulation by Anionic and Nonbilayer Lipids-- The native alDGS enzyme is very responsive toward the presence of certain anionic and nonbilayer lipids, as well as some phosphorylated metabolites and the redox state (7, 10, 12). These features are likely to constitute the basis for regulation of the nonbilayer/bilayer packing conditions in the A. laidlawii membrane. Packing properties brought by nonbilayer-prone molecules (curvature elastic stress) can be qualitatively analyzed also in mixed lipid-detergent bilayer-like aggregates (41-43). Assays (see "Materials and Methods") for the DGlcDAG synthase from A. laidlawii and the GalGlcDAG synthase from S. pneumoniae (CpoA/spDGS) showed that both enzymes respond to PG in a similar manner, but that only the A. laidlawii variant was stimulated by CL, and strongly by the nonbilayer-prone 1,3-DOG (Fig. 3). Activation of alDGS correlated with the amount of negatively charged lipid phosphates in the mixed micelles; CL has two phosphates per mole lipid compared with one in PG. 1,3-DOG behaves similarly to 1,2-DOG, which has a well established ability to shift the phase equilibria of membrane lipids toward nonbilayer aggregates (reversed type) (44). The combined effect of CL and 1,3-DOG was a very strong stimulation of alDGS (Fig. 3, top), perhaps also promoted by the nonbilayer-prone character of the unsaturated CL used. It is very evident that spDGS responds differently to the immediate lipid environment (Fig. 3, bottom). In S. pneumoniae CL is a major membrane component and larger than PG (45), as is GalGlcDAG (37). Reasons why CL did not activate spDGS may be either that the enzyme does not bind (unlikely) alternatively that binding gives an unfavorable orientation at the membrane or that the larger negative potential from the lipid surface repels the acidic C-domain (cf. pI in Table III), abolishing enzyme activity. The lack of response for spDGS toward the nonbilayer-prone 1,3-DOG may indicate that this S. pneumoniae enzyme is not designed to sense such packing properties. In A. laidlawii strains there is a correlation between low MGlcDAG amounts, and hence more DGlcDAG, and high CL amounts (46, 47). Yet, in A. laidlawii A-EF22 CL is lacking. Most important, the response here for the cloned alDGS (Fig. 3) is in agreement with the purified native enzyme (10, 12). The differences in response toward CL between alDGS and spDGS are analogous to what was recorded for the preceding alMGS and spMGS enzymes versus CL amounts (6). Hence, these A. laidlawii and S. pneumoniae lipid GTs must have certain differentiating features in their sequence properties.


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Fig. 3.   Modulation of activity by lipids. Effect of anionic and nonbilayer lipids on the activity of the cloned DGSs as seen with assays in mixed CHAPS/lipid micelles. alDGS refers to DGS from A. laidlawii and spDGS to DGS from S. pneumoniae. A and B, lipid composition was changed from 0 to 30 mol % activator DOPG (open symbols) or CL (filled symbols), with 3 mol % substrate alpha -MGlcDAG; A. laidlawii DGlcDAG was used as balance. C and D, at 18 mol % activator, the nonbilayer-prone 1,3-DOG was added stepwise. Note that the scales for activity on the y axes are different, with substantially higher rates for alDGS with CL plus the nonbilayer 1,3-DOG (C) and lower for spDGS (B and D).

                              
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Table III
Theoretical pI for selected glycosyltransferases

Sequence Features and Properties-- Fig. 1 shows the sequences of some selected homologs/analogs to alDGS (Table II), including the cloned CpoA/spDGS with an established function (above). Conserved residues are spread along the whole sequences but concentrated to two regions: one contains the triplet Arg170, Lys171, Gly172 in the aa stretch 161-192 in alDGS, and the other includes ~30 aa around the characteristic EX7E-motif of CAZy GT family 4 (aa 242-250 in alDGS). The most variable region is the end of the C-terminal part. However, a Conserved domain search (cf. "Materials and Methods") proposed the second half of the alDGS sequence to contain a nucleotide-binding domain. Mutant studies of the alpha -mannosyltransferase AceA (also CAZy family 4) suggested three aa positions (Lys211, Glu287, and Glu295) to be involved in the catalytical mechanism (48). We propose Lys171, Glu242, and Glu250 to be the corresponding residues in alDGS, all conserved in the two regions with higher identity. A pair-wise alignment with BLAST2 (not shown) revealed a similar region between the two A. laidlawii glucosyltransferases at position 181-346 (alMGS) and 129-294 (alDGS), with 28% identical residues (cf. Figs. 1 and 5). The alDGS, and its homologs/analogs (in Fig. 1), have not any predicted transmembrane segments (HMMTOP (49)), yet DGS is associated with the membrane (10, 50). Likewise, the main portion of the CpoA was found in the membrane fraction, suggesting that it associates with membrane components in vivo (33).

A high pI value is typical for membrane-associated proteins (51), and essentially all membrane lipid surfaces are negatively charged due to their anionic lipids (low pKa values), e.g. the alMGS enzyme binds especially to phospholipids like PG.2 alDGS has a calculated pI around 9.4 (Table III), hence is positively charged at physiological pH, and charged residues do frequently appear as pairs. The N-terminal half is predominantly positively charged, with a calculated pI of 9.7. These charge features were not conserved among the closest analogs and the pI (full size) varies from 4.7 (Halobacterium) to 9.1 (Streptococcus pyogenes). The characteristic highly positive N-terminal half was also the case for the proteins from S. pyogenes and Streptococcus mutans. A common pattern for other homologs/analogs was an N-terminal half with higher pI and a C-terminal half with lower pI (Table III). Note the substantially lower pI values for the two S. pneumoniae enzymes compared with the A. laidlawii ones, especially for the C-terminal domains (Table III). This may constitute the basis for the different response toward CL of the A. laidlawii and S. pneumoniae GTs (Fig. 3 and 4).


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Fig. 4.   Structural modeling of the glycosyltransferases. Ribbon representations, showing the side-views, and electrostatic surface potentials, visualizing the bottom views, of the modeled proteins alMGS, spMGS, csMGS, alDGS, and spDGS. Models were made in SWISS-MODEL and figures were prepared in MOLMOL (27), see "Materials and Methods." The template structures, ecMurG, ecEpim, and aoGtfB, are also shown. N-terminal and/or C-terminal sequences, which could not be modeled, are indicated as yellow patches on the electrostatic surface (electrostatic surface potentials: blue, positive; red, negative; gray, mainly hydrophobic). The bottom faces are the probable lipid bilayer-binding areas, and the large positively charged area at the N-terminal bottom face of e.g. alMGS extends into the N-terminal side-end face (data not shown). The middle clefts are the active site regions, where the two conserved E residues are indicated in blue on the C-terminal side to the right (ribbon version), involved in binding nucleoside diphosphate-sugar. Note the similarity of the cucumber beta -MGalDAG synthase GT to the four acholeplasma and streptococcal alpha -GTs.

Fold Recognition and Structure Modeling-- A number of established local structure prediction and fold recognition methods (23), with the Pcons evaluation method (22), were used to analyze the potential structural relationships for alDGS, spDGS, and alMGS (Table IV in the Supplemental Material) and spMGS. A substantial similarity was proposed to the three-dimensional structures of the glycosyltransferases MurG, GtfB, and the soluble UDP-GlcNAc 2-epimerase, which are all strongly related to phage T4 beta -glucosyltransferase. The predicted sequence alignments were the basis for an alignment in Swiss-PdbViewer and model building in SWISS-MODEL. The proteins alMGS, spMGS, and csMGS were modeled onto the template protein structures of ecMurG, GlcNAc-epimerase, and aoGtfB, while modeling of alDGS and spDGS were performed with only the first two templates (see Supplement Material and Refs. 53-57). A presentation of the structural models and their surface charge potentials are shown in Fig. 4, and some important quality indicators of the models are given in Table V (see the Supplemental Material). In general the model structures have normal (fairly good) packing quality and Ramachandran plot behavior, together with distinct energy minima. Note the general similarity of the cucumber beta -MGalDAG synthase GT to the four acholeplasma and streptococcal alpha -GTs. Hence, these glycosyltransferases, most of which are membrane-associated, seem to conform to a similar structural design despite low sequence similarities.

Membrane Association-- The established binding properties of alMGS to anionic phospholipid bilayers (6), the look of the potential active-site clefts, and the bilayer localization of the DAG and MGlcDAG acceptor lipid substrates, strongly support the positively charged bottom faces indicated (Fig. 4) as the lipid bilayer-binding surfaces of the enzymes. These model proposals were further supported by extensive sequence segment comparisons (data not shown) with 11 proteins of known structures of which most bind to lipid membranes3 and to the sequences of another 10 proteins established to bind to lipids and/or membranes.4 For both A. laidlawii enzymes sequence similarities were confined to six regions, with multiple hits to each region. In most instances, the proposed structure of a certain alDGS or alMGS segment (as in Fig. 4) corresponded to the structure in the target protein, provided the sequence similarities were sufficiently high. This was the case for certain amphiphilic helices, some beta -strands, and especially several, more hydrophobic loops at the bottom surface and active site clefts. Such loops, flanked by or containing positively charged residues, are important lipid-binding segments in several proteins like the C2 domains in human Factor V and phosphatidylinositol-phospholipase Cdelta (58, 59). Furthermore, no sequence segment similarities were recorded for the structures of several interface-anchored integral proteins (prostaglandin synthase and squalene cyclase).

Potential lipid surface- or hydrophobic interface-associating segments were predicted by the Membrane Protein Explorer.5 Several obvious differences were noted (Fig. 5), e.g. alDGS has more exposed hydrophobic segments than spDGS and notably one stronger in the C-domain. These two also differ by two sequence gaps in their in the N-domain. The soluble epimerase reveals no hydrophobic stretch, and MurG has one in the N-domain (Fig. 4), also suggested to be the membrane-binding (amphiphilic) segment (52). The latter seems to have counterparts at similar locations in the three MGS enzymes. These proposals indicate important differences in charge and hydrophobicity between alDGS and spDGS, which may be related to their different behavior toward the anionic CL activator and nonbilayer DOG additive. Likewise, similar differences between alDGS and alMGS may be the basis for the strong stimulation of alDGS by nonbilayer-prone lipids.


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Fig. 5.   Alignment of the lipid glycosyltransferases with model template sequences. The aa sequences of the five lipid glycosyltransferases were aligned to their corresponding template sequences according to the MetaServer predictions. The predicted secondary structures are from the SWISS-MODEL (Fig. 4), and the template MurG and epimerase ones from PDB. Residues shaded by light and dark gray indicates alpha -helical and beta -strand structure, respectively. Residues conserved among the eight most similar alMGS analogs (6) and/or the eight best alDGS analogs (Table II) are in bold. Underlined sequences are identified by the MPEx prediction algorithms5 as lipid bilayer surface-associated (thin lines) or penetrating into the hydrophobic interface (thick lines), based on a thermodynamic hydrophobicity scale (7) and an aa structure exposure of 25%. In addition, the characteristic EX7E-motif, and corresponding one in csMGS and MurG, is boxed. Note the presence of some gaps.

Domain Charge and Membrane Binding in Vivo-- The N- and C-domains of alMGS have a high and low calculated pI, respectively (Table III), which should yield different membrane binding. DNA encoding these domains, and full-size alMGS, were ligated in front (in frame) of the gene for GFP in the pGFPuv vector and transformed into E. coli JM109 wt (see "Materials and Methods"). Full-size, but not the N- and C-domain alMGS hybrids, displayed enzymatic activity as revealed from synthesis of radioactively labeled MGlcDAG by E. coli in vivo, as well as after detergent solubilization in vitro (data not shown), similar to the cloned enzyme (6). Localization of the GFP fusion proteins was assayed by fluorescence microscopy at various growth and gene induction conditions (Fig. 6). A membrane association in E. coli of the full size alMGS-GFP hybrid was evident (Fig. 6A), very similar to a MinD-GFP hybrid (cell division protein) of established phospholipid binding properties (60). The low pI of the native GFP protein should prevent membrane binding, as is indicated in Fig. 6D. Likewise, the C-domain-GFP hybrid had a cytoplasmic distribution, similar to the native GFP, with an occasional localization to an intracellular structure (Fig. 6C). A membrane distribution of the N-domain-GFP with a strong enrichment to the cell poles (Fig. 6B) is similar to the GFP fusion of the E. coli integral membrane, secretion protein YidC (61), and MinD-GFP under control by MinE (62). The alMGS N-domain-GFP was released from the cell poles to the cytoplasm by a slight increase in NaCl concentration (data not shown). Furthermore, the GelK N-domain (high pI, cf. Table III), but not the C-domain (low pI), binds to the acidic E. coli membrane (63). Similar pI values are also valid for mannosyltransferase AceA (Table III), which is found associated with the membrane (48). Clearly, the three GFP variants of the MGS domains have different membrane binding properties in vivo.


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Fig. 6.   Localization of alMGS-GFP hybrids. Full-size and N- and C-domains of alMGS were fused to the N terminus of GFP. Cellular localization of full-size alMGS-GFP (A), N-domain-GFP of alMGS (B), C-domain-GFP of alMGS (C), and native GFP (D) was analyzed by fluorescence microscopy (Zeiss Axioplan 2) after induction of the GFP-hybrid genes in E. coli JM109. Black and white presentations of the colored original images are shown. The fused enzyme in A displayed enzymatic activity.

E. coli membrane lipids contain ~25 mol % anionic lipids (PG plus CL), sufficient to yield binding of purified alMGS to defined liposomes in vitro.2 CL promotes binding and activity of alMGS, and activity of alDGS (Fig. 3), better than PG (6).2 It has been shown by different techniques that the cell poles of E. coli are enriched in the anionic CL compared with the average membrane (64, 65). The different in vivo binding properties of the alMGS domains, correlating to protein/domain charge (Table III), is analogous to the differences recorded for C1 and C2 membrane lipid targeting domains in many peripheral proteins (35). The latter yields a synergistic and/or regulatory interplay between targeting and activation (35). Substantial differences between the alDGS and alMGS N- and C-domains were revealed here, and the homologous spMGS and spDGS are both more similar to alMGS in this respect (Table III). Most likely this will affect the binding, activities, and responsiveness of the various enzymes, as is indicated in Fig. 3 and Ref. 6.

Conclusions-- From experimental data, the predicted structural models, and the comparisons with many lipid-associating proteins, it is evident that these lipid glucosyltransferases are associated with the membrane lipid interface. Hence, the sensing of the bilayer packing properties and the regulatory activities must be confined to the exact mechanisms by how the N- and C-domains of these enzymes are anchored and activated, as indicated from alDGS, spDGS, alMGS, and spMGS here. This is analogous to the C1 and C2 membrane targeting domains in many eukaryotic peripheral proteins. The different amounts of charged and hydrophobic residues exposed on the enzyme bilayer-binding surfaces (see Figs. 4 and 5) support that lipid surface charge and curvature elastic packing stress might affect the orientation and potentially the conformation between the N- and C-terminal domains. This can be coupled to the interactions of the soluble and lipid substrates and to other stimulating agents like metabolic phosphates and DNA.

    ACKNOWLEDGEMENTS

We thank Viola Tegman for excellent technical assistance, Dr. Mari Norgren for the S. pneumoniae strain, Hanna Eriksson for constructing the GFP hybrids, Petra Björk and Dr. Lars Wieslander for microscope access, and Dr. Anders Berglund and Dr. Arne Elofsson for valuable discussions.

    FOOTNOTES

* This work was supported by the Swedish Science Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY078412.

The on-line version of this article (availabe at http://www.jbc.org) contains supplemental text, supplemental Tables IV and V, and supplemental Refs. 1-8.

§ Both authors have contributed equally to this work.

Present address: Dept. of Microbiology, Colorado State University, Fort Collins, CO 80523-0015.

Dagger Dagger To whom correspondence should be addressed. Tel.: 46-8-16-24-63; Fax: 46-8-15-36-79; E-mail: ake@dbb.su.se.

Published, JBC Papers in Press, December 2, 2002, DOI 10.1074/jbc.M211492200

2 L. Li, submitted for publication.

3 E. coli SRP receptor FtsY and D-lactate dehydrogenase, Neisseria meningitidis glycosyltransferase LgtC, B. subtilis glycosyltransferase SpsA, cytochrome c (several species), human Factor V C2 domain, galactose oxidase A, phosphatidylinositol-phospholipase Cdelta , synaptotagmin C2 domain, human pleckstrin, and Epsin 1.

4 E. coli proteins DnaA, SecA, phosphatidylserine synthase, RecA and PutA, rat phosphocholine cytidylyltransferase, A. xylium glycosyltransferase AceA, mouse beta -spectrin, and human dynamin.

5 S. Jaysinghe, K. Hristova, and S. H. White (blanco.biomol.uci. edu/mpex).

    ABBREVIATIONS

The abbreviations used are: DGlcDAG, 1,2-diacyl-3-O-[alpha -D-glucopyranosyl-(1right-arrow2)-O-alpha -D-glucopyranosyl]-sn-glycerol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; MGlcDAG, 1,2-diacyl-3-O-(alpha -D-glucopyranosyl)-sn-glycerol; GalGlcDAG, 1,2-diacyl-3-O-[alpha -D-glucopyranosyl-(1right-arrow2)-O-alpha -D-galactopyranosyl]-sn-glycerol; 1, 3-DOG, 1,3-dioleoylglycerol; MGalDAG, 1,2-diacyl-3-O-(alpha -D-galactopyranosyl)-sn-glycerol; MAMGlcDAG, 1,2-diacyl-3-O-[6-O-acyl(alpha -D-glucopyranosyl)]-sn-glycerol; MADGlcDAG, 1,2-diacyl-3-O-[alpha -D-glucopyranosyl-(1right-arrow2)-O-(6-O-acyl-alpha -D-glucopyranosyl)]-sn-glycerol; DGS, disugar-glycolipid synthase; MGS, monosugar-glycolipid synthase; PG, phosphatidylglycerol; DOPG, dioleoylphosphatidylglycerol; DOPA, dioleoylphosphatidic acid; CL, cardiolipin; GT, glycosyltransferase; GFP, green fluorescent protein; aa, amino acids; ORF, open reading frame.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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