Protein glycosylation as an adaptive response in Archaea: growth at different salt concentrations leads to alterations in Haloferax volcanii S-layer glycoprotein N-glycosylation.

To cope with life in hypersaline environments, halophilic archaeal proteins are enriched in acidic amino acids. This strategy does not, however, offer a response to transient changes in salinity, as would post-translational modifications. To test this hypothesis, N-glycosylation of the Haloferax volcanii S-layer glycoprotein was compared in cells grown in high (3.4 M NaCl) and low (1.75 M NaCl) salt, as was the glycan bound to dolichol phosphate, the lipid upon which the N-linked glycan is assembled. In high salt, S-layer glycoprotein Asn-13 and Asn-83 are modified by a pentasaccharide, while dolichol phosphate is modified by a tetrasaccharide comprising the first four pentasaccharide residues. When the same targets were considered from cells grown in low salt, substantially less pentasaccharide was detected. At the same time, cells grown at low salinity contain dolichol phosphate modified by a distinct tetrasaccharide absent in cells grown at high salinity. The same tetrasaccharide modified S-layer glycoprotein Asn-498 in cells grown in low salt, whereas no glycan decorated this residue in cells grown in the high-salt medium. Thus, in response to changes in environmental salinity, Hfx. volcanii not only modulates the N-linked glycans decorating the S-layer glycoprotein but also the sites of such post-translational modification.

Different routes to the same ending: comparing the N-glycosylation processes of Haloferax volcanii and Haloarcula marismortui, two halophilic archaea from the Dead Sea.

Recent insight into the N-glycosylation pathway of the haloarchaeon, Haloferax volcanii, is helping to bridge the gap between our limited understanding of the archaeal version of this universal post-translational modification and the better-described eukaryal and bacterial processes. To delineate as yet undefined steps of the Hfx. volcanii N-glycosylation pathway, a comparative approach was taken with the initial characterization of N-glycosylation in Haloarcula marismortui, a second haloarchaeon also originating from the Dead Sea. While both species decorate the reporter glycoprotein, the S-layer glycoprotein, with the same N-linked pentasaccharide and employ dolichol phosphate as lipid glycan carrier, species-specific differences in the two N-glycosylation pathways exist. Specifically, Har. marismortui first assembles the complete pentasaccharide on dolichol phosphate and only then transfers the glycan to the target protein, as in the bacterial N-glycosylation pathway. In contrast, Hfx. volcanii initially transfers the first four pentasaccharide subunits from a common dolichol phosphate carrier to the target protein and only then delivers the final pentasaccharide subunit from a distinct dolichol phosphate to the N-linked tetrasaccharide, reminiscent of what occurs in eukaryal N-glycosylation. This study further indicates the extraordinary diversity of N-glycosylation pathways in Archaea, as compared with the relatively conserved parallel processes in Eukarya and Bacteria.

Glyco-engineering in Archaea: differential N-glycosylation of the S-layer glycoprotein in a transformed Haloferax volcanii strain.

Archaeal glycoproteins present a variety of N-linked glycans not seen elsewhere. The ability to harness the agents responsible for this unparalleled diversity offers the possibility of generating glycoproteins bearing tailored glycans, optimized for specific functions. With a well-defined N-glycosylation pathway and available genetic tools, the haloarchaeon Haloferax volcanii represents a suitable platform for such glyco-engineering efforts. In Hfx. volcanii, the S-layer glycoprotein is modified by an N-linked pentasaccharide. In the following, S-layer glycoprotein N-glycosylation was considered in cells in which AglD, the dolichol phosphate mannose synthase involved in addition of the final residue of the pentasaccharide, was replaced by a haloarchaeal homologue of AglJ, the enzyme involved in addition of the first residue of the N-linked pentasaccharide. In the engineering strain, the S-layer glycoprotein is modified by a novel N-linked glycan not found on this reporter from the parent strain. Moreover, deletion of AglD alone and introduction of the AglJ homologue from Halobacterium salinarum, OE2528R, into the deletion strain resulted in increased biosynthesis of the novel 894 Da glycan concomitant with reduced biogenesis of the pentasaccharide normally N-linked to the S-layer glycoprotein. These findings justify efforts designed to transform Hfx. volcanii into a glyco-engineering 'workshop'.

Crossing the membrane in Archaea, the third domain of life.

Many of the recent advancements in the field of protein translocation, particularly from the structural perspective, have relied on Archaea. For instance, the solved structures of the translocon from the methanoarchaeon Methanocaldococcus jannaschii of the ribosomal large subunit from the haloarchaeon Haloarcula marismortui and of components of the SRP pathway from several archaeal species have provided novel insight into various aspects of the translocation event. Given the major contribution that Archaea have made to our understanding of how proteins enter and traverse membranes, it is surprising that relatively little is known of protein translocation in Archaea in comparison to the well-defined translocation pathways of Eukarya and Bacteria. What is known, however, points to archaeal translocation as comprising a mosaic of eukaryal and bacterial traits together with aspects of the process seemingly unique to this, the third domain of life. Here, current understanding of archaeal protein translocation is considered. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.

Towards glycoengineering in archaea: replacement of Haloferax volcanii AglD with homologous glycosyltransferases from other halophilic archaea.

Like eukarya and bacteria, archaea also perform N-glycosylation. However, the N-linked glycans of archaeal glycoproteins present a variety not seen elsewhere. Archaea accordingly rely on N-glycosylation pathways likely involving a broad range of species-specific enzymes. To harness the enormous applied potential of such diversity for the generation of glycoproteins bearing tailored N-linked glycans, the development of an appropriate archaeal glycoengineering platform is required. With a sequenced genome, a relatively well-defined N-glycosylation pathway, and molecular tools for gene manipulation, the haloarchaeon Haloferax volcanii (Hfx. volcanii) represents a promising candidate. Accordingly, cells lacking AglD, a glycosyltransferase involved in adding the final hexose of a pentasaccharide N-linked to the surface (S)-layer glycoprotein, were transformed to express AglD homologues from other haloarchaea. The introduction of nonnative versions of AglD led to the appearance of an S-layer glycoprotein similar to the protein from the native strain. Indeed, mass spectrometry confirmed that AglD and its homologues introduce the final hexose to the N-linked S-layer glycoprotein pentasaccharide. Heterologously expressed haloarchaeal AglD homologues contributed to N-glycosylation in Hfx. volcanii despite an apparent lack of AglD function in those haloarchaea from where the introduced homologues came. For example, although functional in Hfx. volcanii, no transcription of the Halobacterium salinarum aglD homologue, OE1482, was detected in cells of the native host grown under various conditions. Thus, at least one AglD homologue works more readily in Hfx. volcanii than in the native host. These results warrant the continued assessment of Hfx. volcanii as a glycosylation "workshop."

Protein glycosylation in Archaea: sweet and extreme.

While each of the three domains of life on Earth possesses unique traits and relies on characteristic biological strategies, some processes are common to Eukarya, Bacteria and Archaea. Once believed to be restricted to Eukarya, it is now clear that Bacteria and Archaea are also capable of performing N-glycosylation. However, in contrast to Bacteria, where this posttranslational modification is still considered a rare event, numerous species of Archaea, isolated from a wide range of environments, have been reported to contain proteins bearing Asn-linked glycan moieties. Analysis of the chemical composition of the Asn-linked polysaccharides decorating archaeal proteins has, moreover, revealed the use of a wider variety of sugar subunits than seen in either eukaryal or bacterial glycoproteins. Still, although first reported some 30 years ago, little had been known of the steps or components involved in the archaeal version of this universal posttranslational modification. Now, with the availability of sufficient numbers of genome sequences and the development of appropriate experimental tools, molecular analysis of archaeal N-glycosylation pathways has become possible. Accordingly using halophilic, methanogenic and thermophilic model species, insight into the biosynthesis and attachment of N-linked glycans decorating archaeal glycoproteins is starting to amass. In this review, current understanding of N-glycosylation in Archaea is described.

The VDAC1 N-terminus is essential both for apoptosis and the protective effect of anti-apoptotic proteins.

The release of mitochondrial-intermembrane-space pro-apoptotic proteins, such as cytochrome c, is a key step in initiating apoptosis. Our study addresses two major questions in apoptosis: how are mitochondrial pro-apoptotic proteins released and how is this process regulated? Accumulating evidence indicates that the voltage-dependent anion channel (VDAC) plays a central role in mitochondria-mediated apoptosis. Here, we demonstrate that the N-terminal domain of VDAC1 controls the release of cytochrome c, apoptosis and the regulation of apoptosis by anti-apoptotic proteins such as hexokinase and Bcl2. Cells expressing N-terminal truncated VDAC1 do not release cytochrome c and are resistant to apoptosis, induced by various stimuli. Employing a variety of experimental approaches, we show that hexokinase and Bcl2 confer protection against apoptosis through interaction with the VDAC1 N-terminal region. We also demonstrate that apoptosis induction is associated with VDAC oligomerization. These results show VDAC1 to be a component of the apoptosis machinery and offer new insight into the mechanism of cytochrome c release and how anti-apoptotic proteins regulate apoptosis and promote tumor cell survival.