Substrate Promiscuity: AglB, the Archaeal Oligosaccharyltransferase, Can Process a Variety of Lipid-Linked Glycans

Abstract

bAcross evolution, N-glycosylation involves oligosaccharyltransferases that transfer lipid-linked glycans to selected Asn residues of target proteins. While these enzymes catalyze similar reactions in each domain, differences exist in terms of the chemical composition, length and degree of phosphorylation of the lipid glycan carrier, the sugar linking the glycan to the lipid carrier, and the composition and structure of the transferred glycan. To gain insight into how oligosaccharyltransferases cope with such substrate diversity, the present study analyzed the archaeal oligosaccharyltransferase AglB from four haloarchaeal species. Accordingly, it was shown that despite processing distinct lipid-linked glycans in their native hosts, AglB from Haloarcula marismortui, Halobacterium salinarum, and Haloferax mediterranei could readily replace their counterpart from Haloferax volcanii when introduced into Hfx. volcanii cells deleted of aglB. As the four enzymes show significant sequence and apparently structural homology, it appears that the functional similarity of the four AglB proteins reflects the relaxed substrate specificity of these enzymes. Such demonstration of AglB substrate promiscuity is important not only for better understanding of N-glycosylation in Archaea and elsewhere but also for efforts aimed at transforming Hfx. volcanii into a glycoengineering platform. N-glycosylation is a posttranslational modification that occurs in all three domains of life. In each case, a core or fully assembled glycan is assembled on a phosphorylated polyprenol lipid carrier, namely, dolichol in Eukarya and Archaea and undecaprenol in Bacteria, and transferred to select Asn residues of a target protein by the actions of an oligosaccharyltransferase (OST) (1-4). In higher Eukarya, the OST is a multisubunit complex, with the Stt3 (staurosporine-and temperature-sensitive) protein serving as the catalytic subunit (5, 6). In Bacteria and Archaea, the OST comprises a single subunit, namely, the Stt3 homologues PglB and AglB, respectively (7,8).While members of all three domains perform N-glycosylation, the diversity presented by the N-linked glycans added to target proteins varies greatly across evolution. In particular, the Nlinked glycans that decorate archaeal glycoproteins offer a degree of architectural and compositional variability not seen in either their bacterial or eukaryal counterparts (4, 9). Archaeal N-glycosylation also presents diversity at the level of the glycan-charged lipid carrier, with both dolichol phosphate (DolP) and dolichol pyrophosphate (DolPP) reportedly serving this role (10-15). Moreover, archaeal N-glycosylation relies on several different sugars to provide the link between the oligosaccharide and either the lipid carrier or target protein Asn residues (4). As such, the archaeal OST must cope with a degree of substrate variety not encountered by either its eukaryal or bacterial homologues. Finally, while the presence of a sequon (the motif Asn-X-Ser/Thr, where X is any residue but proline) is requi...

Comparative Analysis of Archaeal Lipid-linked Oligosaccharides That Serve as Oligosaccharide Donors for Asn Glycosylation

“…Exceptionally, in the total lipid extract from H. salinarum, a tetrasaccharide-charged Dol-PP was detected in addition to the Dol-P type LLO (17). The tetrasaccharide chain was similar but not identical to the N-glycan attached to the Asn-2 position of the H. salinarum S-layer glycoprotein reported previously (23).…”

“…In parallel, they performed the normal phase liquid chromatography-electrospray ionization tandem mass spectrometry (NPLC-ESI-MS/MS) analysis of total lipid extracts from cultured cells, to elucidate the chemical structures of the LLOs. In addition to H. volcanii, they analyzed the LLOs from other haloarchaea, including Halobacterium salinarum, Haloferax mediterranei, and Haloarcula marismortui (17). They also extended their MS analysis to the LLOs from non-haloarchaea, including the thermoacidophilic archaeon, S. acidocaldarius (18), and the hyperthermophilic archaeon, Pyrococcus furiosus (19).…”

Complementation of an aglB Mutant of Methanococcus maripaludis with Heterologous Oligosaccharyltransferases

“…acidocaldarius , on the other hand, is a thermoacidophile, growing optimally at 70–75°C and pH 2–3. Since the catalytic site in AglB is predicted to be orientated extracellularly [34], the enzymes from Hfx . volcanii and S .…”

“…In in vitro experiments, neither enzyme could process the lipid-linked glycan of the other organism [33], indicating specificity of the enzyme but it is unclear whether this is due to the structure of the different glycans or other factors. More recently, Eichler’s group showed substrate promiscuity of AglB from various extreme halophiles [34]. Specifically, AglB from Haloarcula marismortui , Halobacterium salinarum and Haloferax mediterranei could all functionally replace the oligosaccharyltransferase activity in a Hfx .…”

Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes

“…But, the catalytic promiscuity of enzymes is not a new concept (1,2) and has been reported frequently. (3)(4)(5)(6) The mechanism of promiscuity has been discussed recently in several reviews. (7)(8)(9)(10)(11) Promiscuous enzymes generally show different catalytic efficiencies for different substrates.…”