1 2 Defining the topology of the N-glycosylation pathway in the halophilic archaea 3 Haloferax

34 Pages·2008·0.53 MB·English

JB Accepts, published online ahead of print on 17 October 2008
J. Bacteriol. doi:10.1128/JB.01200-08
Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
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3 Defining the topology of the N-glycosylation pathway in the halophilic archaea
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4 Haloferax volcanii
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8 Noa Plavner and Jerry Eichler* a
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10 Department of Life Sciences, Ben Gurion University, Beersheva 84105 Israel h
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14 *author for correspondence: Prof. Jerry Eichler, Dept. of Life Sciences, Ben Gurion J
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15 University, P.O. Box 653, Beersheva 84105, Israel. Tel: [phone]; Fax [phone] a
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16 9175; email: [email protected] ,
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19 Keywords: Archaea, Haloferax volcanii, N-glycosylation, post-translational modification,
20 topology
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22 Running title: The topology of H. volcanii N-glycosylation
1 ABSTRACT
2 In Eukarya, N-glycosylation involves the actions of enzymes working on both faces of
3 the endoplasmic reticulum membrane. The steps of bacterial N-glycosylation, by contrast,
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4 essentially transpire on the cytoplasmic side of the plasma membrane, with only transfer
5 of the assembled glycan to the target protein occurring on the external surface oEf the cell.
6 In Archaea, virturally nothing is known of the topology of enzymes involved in
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7 assembling those glycans subsequently N-linked to target proteins on the external surface w
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8 of the cell. To remedy this situation, sub-cellular localization and topology predictive a
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9 algorithms, protease accessibility and immunoblotting, together with cysteine f
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10 modification following site-directed mutagenesis, were enlisted to define the topology of h
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11 Haloferax volcanii proteins experimentally proven to participate in the N-glycosylation /
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12 process. As such, AglJ and AglD, respectively involved in the earliest and latest stages of m
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13 assembly of the pentasaccharide decorating the H. volcanii S-layer glycoprotein, were g
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14 shown to present their soluble N-terminal domain, likely containing the putative catalytic J
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15 site of each enzyme, to the cytosol. The same holds true for Alg5-B, Dpm1-A and Mpg1- a
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16 D, proteins putatively involved in this post-translational event. The results thus point to ,
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17 the assembly of the pentasaccharide linked to select Asn residues of the H. volcanii S-

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18 layer glycoprotein as occurring within the cell. u
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1 INTRODUCTION
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3 It has been long recognized that Archaea originating from a variety of diverse
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4 environments are able to N-glycosylate numerous proteins (7 and references within).
5 Indeed, the first non-eukaryal N-glycosylated protein described was derived froEm an
6 archaeal source, i.e. the S-layer glycoprotein of the halophilic archaeaon, Halobacterium
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7 salinarum (15). Still, little is known of the pathway responsible for the covalent w
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8 attachment of glycan moieties to select Asn residues in archaeal glycoproteins. Of late, a
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9 however, steps aimed at remedying this situation have been taken. In both the halophilic f
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10 archaeon Haloferax volcanii and the methanogen Methanococcus voltae, genes involved h
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11 in the N-glycosylation process have been defined (1-3,5,21,28). Additional insight into /
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12 the archaeal version of this post-translational modification has come with the m
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13 development of an in vitro assay to test Pyrococcus furiosus Stt3/AglB activity (11) and g
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14 the recent solution of the three-dimensional structure of the C-terminal soluble domain of J
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15 the protein (9), thought to comprise the sole unit of the archaeal oligosaccharide a
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16 transferase (1,5). ,
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18 Thus, while progress is being made in identifying the different enzymes responsible for u
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19 the various steps leading to archaeal N-glycosylation, little is known of the topology of
20 such reactions. Archaeal N-glycosylation is thought to share steps with the parallel
21 processes in Bacteria and Eukarya (1,4,9,13,27). In Campylobacter jejuni, the sole
22 bacterial species for which the N-glycosylation pathway has been delineated, a
23 heptasaccharide is assembled from soluble nucleotide-activated sugars onto a
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1 cytoplasmically-oriented lipid carrier present in the plasma membrane. The
2 oligosaccharide-charged lipid is then ‘flipped’ across the membrane to face the cell
3 exterior, where the glycan moiety is transferred to select Asn residues in target proteins
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4 by the actions of PglB, the oligosaccharide transferase in this species (for review, see
5 23,25). In Eukarya, such as Saccharomyces cerevisiae, N-glycosylation also beEgins with
6 the assembly of cytoplasmically-located, soluble nucleotide-activated sugars into a
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7 heptasaccharide chain of defined composition on a lipid carrier associated with the w
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8 cytoplasmic face of the endoplasmic reticulum (ER) membrane. Once assembled, the a
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9 lipid-charged heptasaccharide is reoriented, such that the oligosaccharide now faces the f
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10 ER lumen. Next, an additional seven sugar subunits, each derived from their own h
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11 individual lipid carriers, charged on the cytoplasmic face of the ER membrane and /
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12 flipped to face the ER lumen, are added to yield a 14-member oligosaccharide. This m
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13 oligosaccharide is now transferred, en bloc, to select Asn residues of a nascent g
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14 polypeptide translocating into the ER, via the actions of the multimeric oligosaccharide J
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15 transferase complex (for review, see 4,8). a
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16 ,
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17 In the case of archaeal N-glycosylation, comparatively less is known of the topology of

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18 the process. Based on studies following the modification of cell-impermeant peptide u
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19 reporters of N-glycosylation (14) or through the use of bacitracin, an antibiotic that
20 interferes with the regeneration of the dolichol pyrophosphate oligosaccharide carrier
21 presumably used in archaeal N-glycosylation (26), the transfer of lipid-linked
22 oligosaccharides to target proteins was assigned to the external surface of the archaeal
23 plasma membrane. By contrast, the biosynthesis of nucleotide-activated sugars in
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1 Archaea, likely recruited for N-glycosylation, has been shown to occur in the cytoplasm
2 (17,18). Thus, apart from the first and last phases of the process, virtually nothing is
3 known of the topology of archaeal N-glycosylation. Accordingly, the topologies of both
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4 proven (i.e. AglD and AglJ) and putative components (i.e. Alg5-B, Dpm1-A and Mpg1-
5 D) of the H. volcanii N-glycosylation pathway were considered experimentallyE in this
6 study.
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1 MATERIALS AND METHODS
2 Materials
3 Cellulose, novobiocin and phenylmethylsulfonyl fluoride (PMSF) were obtained from
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4 Sigma (St. Louis MO). Proteinase K came from Boehringer (Mannheim, Germany).
5 Yeast extract came from Pronadisa (Madrid, Spain), while tryptone came from EUSB
6 (Cleveland, OH). 4-acetoamido-4-maleimidylstilbene-2,2-disulfonic, disodium salt
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7 (AMS) came from Invitrogen (Carlsbad, CA), while [14C] N-ethylmaleimide (NEM; 20- ow
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8 40 mCi/mmol) came from Perkin-Elmer (Boston, MA). A 3.3 mM working solution of a
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9 [14C]-NEM was prepared by dilution into NEM prepared in ethanol. f
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11 Culture conditioCns /jb
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12 H. volcanii cells were grown in rich medium containing 3.4 M NaCl, 0.15 M m
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13 MgSO •7H 0, 1 mM MnCl , 4 mM KCl, 3 mM CaCl , 0.3% (w/v) yeast extract, 0.5% g
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14 (w/v) tryptone, 50 mM Tris-HCl, pH 7.2, at 40°C (16). J
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16 In silico topology analysis ,
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17 To define protein topology, the HMMTOP (http://www.enzim.hu/hmmtop/), SOSUI

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18 (http://bp.nuap.nagoya-u.ac.jp/sosui/), TMHMM u
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19 (http://www.cbs.dtu.dk/services/TMHMM-2.0/) TMpred
20 (http://www.ch.embnet.org/software/TMPRED_form.html) and TopPred
21 (http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html) topology prediction programs,
22 as found at www.expasy.ch, were consulted. The H. volcanii proteins considered by these
23 algorithms included those previously identified as homologues of eukaryal or bacterial N-
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1 glycosylation proteins (1). For reasons described below, only those proteins predicted by
2 the algorithms to possess an N-terminus sequestered within the cytosol were considered
3 for further analysis in the study.
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5 Plasmid construction E
6 The aglD gene was amplified from H. volcanii strain WR536 (H53) genomic DNA using
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7 the primers listed in Table 1, designed to respectively introduce NdeI and KpnI restriction w
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8 sites on the 5’ and 3’ ends of the aglD coding region, and ligated into the pGemT-Easy a
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9 vector (Promega). The aglD gene was then excised upon digestion with NdeI and KpnI f
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10 and inserted into the pWL-CBD vector (10), also pre-digested with the same restriction h
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11 enzymes, resulting in a plasmid encoding the Clostridium thermocellum cellulose-binding /
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12 domain (CBD) (GenBank accession number 2554722) fused to the 5’-end of the AglD- m
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13 encoding gene. CBD-AglJ- (aglJ GenBank accession number FM210664), CBD-Alg5-B- g
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14 , CBD-Dpm1-A- and CBD-Mpg1-D-encoding plasmids were similarly generated, using J
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15 the appropriate primers. a
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17 Immunoblotting
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18 Immunoblotting was performed as described previously (12), using polyclonal antibodies u
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19 raised against H. volcanii SRP54 (1:1,000)(24) or the C. thermocellum cellulose-binding
20 domain (obtained from Ed Bayer, Weizmann Institute of Science; 1:10,000). Antibody
21 binding was detected using goat anti-rabbit horseradish peroxidase (HRP)-conjugated
22 antibodies (1:4000, BioRad, Hercules, CA) and an ECL enhanced chemiluminescence kit
23 (Amersham, Buckingham, UK).
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2 Determination of protease accessibility
3 To assess the protease accessibility of the CBD-tagged proteins, 1 ml aliquots of the
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4 transformed cells were challenged with proteinase K (1 mg/ml, 55°C). Aliquots were
5 removed at time zero and at subsequent 30-60 min intervals and transferred to iEce. The
6 samples were then centrifuged (3,000 x g, 3 min, 4°C) and resuspended in 1 ml of lysis
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7 buffer (1% Triton X-100 (v/v), 1.8 M NaCl, 50 mM Tris-HCl, pH 7.2) containing 1 mM w
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8 PMSF. The mixtures were rocked (10 min, RT), after which time 50 µl of a 10% (w/v) a
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9 solution of cellulose beads were added. After a 20 min rocking at RT, the suspension was f
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10 centrifuged (3,000 x g, 3 min, RT), the supernatant was discarded and the cellulose pellet h
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11 was washed with 2 M NaCl, 50 mM Tris-HCl, pH 7.2. This washing procedure was /
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12 repeated twice. After the final wash, the cellulose beads were centrifuged (5,000 x g, 3 m
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13 min, RT), the supernatant was removed and the cellulose pellet was resuspended in 40 µl g
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14 SDS-PAGE sample buffer. The samples were then boiled for 5 min and centrifuged J
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15 (5,000 x g, 5 min) to release any cellulose-bound proteins, which were then examined by a
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16 10% SDS-PAGE and immunoblotting using anti-CBD antibodies. ,
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18 Site-directed mutagenesis u
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19 To generate single or reduced cysteine-containing versions of AglD and Dpm1-A, site-
20 directed mutagenesis was performed using the Quickchange (Stratagene) protocol,
21 according to the manufacturer’s instructions, together with those plasmids encoding
22 CBD-tagged versions of AglD or Dpm1-A, respectively, as template. Oligonucleotide
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1 primers used to introduce the various mutations are listed in Table 1. The introduction of
2 mutations was confirmed by sequencing.
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4 Cysteine modification
5 Cysteine modification was achieved using two different cysteine-reactive reageEnts,
6 namely membrane-permeant [14C]-NEM and membrane-impermeant AMS. H. volcanii
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7 cells transformed to express the various single or reduced cysteine-containing versions of w
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8 proven or putative N-glycosylation pathway proteins fused to CBD were challenged with a
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9 [14C]-NEM (15 µl of the working solution, 20 min, 30ºC, with rocking), in some cases f
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10 followed by AMS (5 mM, final concentration, 30 min, 37ºC). Alternatively, cells were h
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11 first challenged with AMS and then incubated with [14C] NEM. In other cases, the cells //
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12 were incubated with 1% Triton X-100 (5 min, RT) prior to incubation with the cysteine- m
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13 reactive reagents. In all cases, cysteine modification was terminated by addition of g
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14 dithiothreitol (DTT) to a final concentration of 50 mM (10 min, 30ºC). The CBD-based J
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15 fusion proteins were then cellulose-purified, separated by 10% SDS-PAGE and a
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1 RESULTS AND DISCUSSION
2 Predicted topology of proteins putatively or proven to participate in H. volcanii N-
3 glycosylation
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4 As a first step in delineating the topology of components of the H. volcanii N-
5 glycosylation machinery, both sequences of proteins putatively involved in the Eprocess
6 (1) as well as sequences of those proteins experimentally verified as participating in this
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7 post-translational modification (2) were analyzed by predictive topology software. Only w
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8 those proteins predicted by the algorithms to possess an N-terminus sequestered within a
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9 the cytosol were considered for further study (the reasons for this selection are expanded f
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10 upon below). This list included Alg5-B (HVO_0704; http://archaea.ucsc.edu), Dpm1-A h
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11 (HVO_2061), and Mpg1-D (HVO_A0586), all H. volcanii homologues of eukaryal /
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12 proteins known to participate in N-glycosylation (1), as well as AglD (HVO_0798), m
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13 previously verified as contributing to the N-glycosylation of the H. volcanii S-layer g
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14 glycoprotein (2). AglJ (HVO_1517), recently observed to participate in this post- J
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15 translational modification (M. Abu-Qarn, P.G. Hitchen, F. Battaglia, A. Dell and J. a
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16 Eichler, in preparation), was also included. In each case, at least three of the topology ,
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17 prediction programs assigned the N-terminus as being intracellular.

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19 The various topology prediction programs were next employed to define the sub-cellular
20 localization of the five proteins considered. Accordingly, Alg5-B was predicted to be a
21 cytoplasmic protein by all of the programs consulted, while Mpg1-D was predicted to lie
22 within the cytoplasm by three of the five topology programs. The other two programs
23 predicted Mpg1-D to be a single-spanning membrane protein, with its N-terminus found
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