Molecular Phylogenetics and Evolution 68 (2013) 327–339 Contents lists available at SciVerse ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Phylogenetic- and genome-derived insight into the evolution of N-glycosylation in Archaea ⇑ Lina Kaminski a, Mor N. Lurie-Weinberger b, Thorsten Allers c, Uri Gophna b, Jerry Eichler a, a Department of Life Sciences, Ben Gurion University, Beersheva 84105, Israel b Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Tel Aviv 69978, Israel c School of Biology, University of Nottingham, Nottingham NG7 2UH, UK article info abstract Article history: N-glycosylation, the covalent attachment of oligosaccharides to target protein Asn residues, is a post- Received 25 January 2013 translational modification that occurs in all three domains of life. In Archaea, the N-linked glycans that Revised 23 March 2013 decorate experimentally characterized glycoproteins reveal a diversity in composition and content Accepted 26 March 2013 unequaled by their bacterial or eukaryal counterparts. At the same time, relatively little is known of Available online 6 April 2013 archaeal N-glycosylation pathways outside of a handful of model strains. To gain insight into the distri- bution and evolutionary history of the archaeal version of this universal protein-processing event, 168 Keywords: archaeal genome sequences were scanned for the presence of aglB, encoding the known archaeal oli- Archaea gosaccharyltransferase, an enzyme key to N-glycosylation. Such analysis predicts the presence of AglB N-glycosylation Oligosaccharyltransferase in 166 species, with some species seemingly containing multiple versions of the protein. Phylogenetic analysis reveals that the events leading to aglB duplication occurred at various points during archaeal evolution. In many cases, aglB is found as part of a cluster of putative N-glycosylation genes. The pres- ence, arrangement and nucleotide composition of genes in aglB-based clusters in five species of the hal- ophilic archaeon Haloferax points to lateral gene transfer as contributing to the evolution of archaeal N- glycosylation. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction Igura et al., 2008; Kelly et al., 2009; Peyfoon et al., 2010; Ng et al., 2011; Matsumoto et al., 2012; Vinogradov et al., 2012). Apart Originally thought to be a process restricted to Eukarya, it is from the two similar Methanococcus N-linked glycans, distinct pro- now clear that Bacteria and Archaea can also modify proteins via tein-bound oligosaccharides are seen in each species. Moreover, in the addition of oligosaccharides to selected Asn residues, i.e. per- both Halobacterium salinarum and Haloferax volcanii, the S-layer form N-glycosylation (Calo et al., 2010; Nothaft and Szymanski, glycoprotein is simultaneously modified by two distinct N-linked 2010; Larkin and Imperiali, 2011; Eichler, 2013). At present, under- glycans (Wieland et al., 1983; Lechner et al., 1985; Guan et al., standing of archaeal N-glycosylation lags behind that of the paral- 2012). lel process in Eukarya and Bacteria. Nonetheless, analysis of even a Given the species-specific profile of N-linked glycans that deco- limited number of archaeal glycoproteins has made it clear that rate archaeal glycoproteins, pathways of oligosaccharide assembly archaeal N-linked glycans show a diversity of content and struc- unique to a given archaeon likely exist. Indeed, in the four Agl ture that is not seen elsewhere (Schwarz and Aebi, 2011; Eichler, (archaeal glycosylation) pathways responsible for N-glycosylation 2013). To date, N-linked glycans decorating glycoproteins or repor- studied to date, namely those of the halophile Hfx. volcanii, the ter peptides from Archaeoglobus fulgidus, Halobacterium salinarum, methanogens M. voltae and M. maripaludis, and the thermoacido- Haloferax volcanii, Methanococcus maripaludis, Methanococcus vol- phile S. acidocaldarius (Calo et al., 2010; Jarrell et al., 2010; Albers tae, Methanothermus fervidus, Pyrococcus furiosus, Sulfolobus acido- and Meyer, 2011; Eichler, 2013), few common components are caldarius and Thermoplasma acidophilum have been characterized seen. The oligosaccharyltransferase (OST) AglB, responsible for (Wieland et al., 1983; Lechner et al., 1985; Kärcher et al., 1993; delivery of the assembled glycan and its precursors from a phos- Zähringer et al., 2000; Voisin et al., 2005; Abu-Qarn et al., 2007; phorylated dolichol lipid carrier to target protein Asn residues, is, however, present in each pathway. With this is mind, Magidovich and Eichler (2009) relied on the presence of aglB, encoding the only OST currently identified in Archaea, to predict the existence of a N- ⇑ Corresponding author. Address: Department of Life Sciences, Ben Gurion University, P.O. Box 653, Beersheva 84105, Israel. Fax: +972 8647 9175. glycosylation pathway in 54 of the 56 species for which complete E-mail address: [email protected] (J. Eichler). genome sequence information was available at the time. 1055-7903/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ympev.2013.03.024 328 L. Kaminski et al. / Molecular Phylogenetics and Evolution 68 (2013) 327–339 Today, the number of publicly available archaeal genome into the prevalence, and by extension, the importance of N- sequences, including those of several phylogenetically proximal glycosylation in Archaea, may be obtained. Of the 168 genomes species, is approaching 200. This wealth of genomic data lends considered, 166 contained AglB-encoding sequences (Table 1), itself to a detailed examination of archaeal N-glycosylation from including A. fulgidus AF0329, Hfx. volcanii HVO_1530, M. voltae an evolutionary perspective. Accordingly, the examination of MVO1749, M. maripaludis MMP1424 and P. furiosus PF0156, AglB putative N-glycosylation pathway components across genome proteins all experimentally demonstrated to possess OST activity lines reported here offers novel insight into the evolution of (Chaban et al., 2006; Abu-Qarn et al., 2007; Igura et al., 2008; the archaeal version of this universal post-translational protein VanDyke et al., 2009; Matsumoto et al., 2012). In fact, AglB is modification. predicted to exist in members of all five archaeal phyla, i.e. Crenarchaeota, Euryarchaeota, Korarchaeota, Nanoarchaeota and 2. Materials and methods Thaumarchaeota, further pointing to N-glycosylation as being a common trait in Archaea. It should, however, be noted that in 2.1. Databases the vast majority of cases, neither N-glycosylation nor transcrip- tion of the predicted AglB-encoding gene has been confirmed. The list of AglB proteins, identified as containing a multi-mem- Finally, and as previously reported (Magidovich and Eichler, brane-spanning N-terminal domain and a soluble C-terminal do- 2009), no aglB sequence was detected in either Aeropyrum pernix main that includes the WWDYG consensus motif implicated in or Methanopyrus kandleri, suggesting that these species do not OST function across evolution (Yan and Lennarz, 2002; Maita perform N-glycosylation. Alternatively, given that Aeropyrum et al., 2010; Lizak et al., 2011), was obtained by scanning the fol- pernix and Methanopyrus kandleri are characterized by an atypical lowing: GT family 66 at the Carbohydrate-Active Enzymes data- gene content (Brochier et al., 2004), it is possible that a different, base (http://www.cazy.org), the Integrated Microbial Genomes – currently unrecognized OST mediates N-glycosylation in these Genome Encyclopedia of Bacteria and Archaea Genomes (IMG/ species. GEBA) (http://img.jgi.doe.gov/cgi-bin/w/main.cgi), using the term ‘EC 2.4.1.119’ as query, and the NCBI Protein Database (http:// 3.2. Multiple versions of AglB appeared throughout evolution www.ncbi.nlm.nih.gov/protein) sites, using the terms ‘Stt3’ or ‘AglB’ as query. These searches were complemented by manual In 31 of the 113 euryarchaeal species considered, and in only 2 searches of non-annotated proteins for the presence of WWDXG, of the 55 non-euryarchaeal species addressed, two or more aglB se- a relaxed form of the WWDYG motif. quences were identified. Of the 31 euryarchaeal species, 14 were methanogens (out of a total of 49 methanogens considered). In examining those methanoarchaeal species containing two or more 2.2. Phylogenetic analysis copies of aglB, no common phenotypic trait, such as an ability to grow under a given condition, is apparent. On the other hand, in The sequences of Haloferax AglB proteins were retrieved from addressing thermo- and hyperthermophilic euryarchaea, two or the IMG/GEBA website utilizing the ‘‘Gene Neighborhood’’ func- more aglB sequences were identified in all nine Thermococcus spe- tion. Homologs were aligned using MUSCLE (Edgar, 2004). The cies, in six of the seven Pyrococcus species considered, and in two of Halorubrum lacusprofundi AglB sequence served as an out-group. the three Archaeoglobus species examined. Yet, the possibility that The alignment was manually edited and ambiguously aligned posi- multiplicity of AglB in a given species is related to an elevated opti- tions were removed. The tree was then constructed utilizing the mal growth temperature is unlikely, since of the 45 crenarcheal PhyML server (http://www.atgc-montpellier.fr/phyml/)(Guindon species, all of which are thermo- or hyperthermophiles, only two, et al., 2010), using the JTT model + 4 gamma categories to approx- belonging to different genera, contain a pair of predicted AglB- imate the different