N-glycosylation in Archaea: Unusual sugars and unique modifications.

Archaea are microorganisms that comprise a distinct branch of the universal tree of life and which are best known as extremophiles, residing in a variety of environments characterized by harsh physical conditions. One seemingly universal trait of Archaea is the ability to perform N-glycosylation. At the same time, archaeal N-linked glycans present variety in terms of both composition and architecture not seen in the parallel eukaryal or bacterial processes. In this mini-review, many of the unique and unusual sugars found in archaeal N-linked glycans as identified by nuclear magnetic resonance spectroscopy are described.

Agl28 and Agl29 are key components of a Halobacterium salinarum N-glycosylation pathway.

Although Halobacterim salinarum provided the first example of N-glycosylation outside the Eukarya, only recently has attention focused on delineating the pathway responsible for the assembly of the N-linked tetrasaccharide decorating selected proteins in this haloarchaeon. In the present report, the roles of VNG1053G and VNG1054G, two proteins encoded by genes clustered together with a set of genes demonstrated to encode N-glycosylation pathway components, were considered. Relying on both bioinformatics and gene deletion and subsequent mass spectrometry analysis of known N-glycosylated proteins, VNG1053G was determined to be the glycosyltransferase responsible for addition of the linking glucose, while VNG1054G was deemed to be the flippase that translocates the lipid-bound tetrasaccharide across the plasma membrane to face the cell exterior, or to contribute to such activity. As observed with Hbt. salinarum lacking other components of the N-glycosylation machinery, both cell growth and motility were compromised in the absence of VNG1053G or VNG1054G. Thus, given their demonstrated roles in Hbt. salinarum N-glycosylation, VNG1053G and VNG1054G were re-annotated as Agl28 and Agl29, according to the nomenclature used to define archaeal N-glycosylation pathway components.

Distinct Regions of the Haloferax volcanii Dolichol Phosphate-Mannose Synthase AglD Mediate the Assembly and Subsequent Processing of the Lipid-Linked Mannose.

Haloferax volcanii AglD is currently the only archaeal dolichol phosphate (DolP)-mannose synthase shown to participate in N-glycosylation. However, the relation between AglD and Pyrococcus furiosus PF0058, the only archaeal DolP-mannose synthase for which structural information is presently available, was unclear. In this report, similarities between the PF0058 and AglD catalytic domains were revealed. At the same time, AglD includes a transmembrane domain far longer than that of PF0058 or other DolP-mannose synthases. To determine whether this extension affords AglD functions in addition to generating mannose-charged DolP, a series of Hfx. volcanii strains expressing truncated versions of AglD was generated. Mass spectrometry revealed that a version of AglD comprising the catalytic domain and only two of the six to nine predicted membrane-spanning domains could mediate mannose addition to DolP. However, in cells expressing this or other truncated versions of AglD, mannose was not transferred from the lipid to the protein-bound tetrasaccharide precursor of the N-linked pentasaccharide normally decorating Hfx. volcanii glycoproteins. These results thus point to AglD as contributing to additional aspects of Hfx. volcanii N-glycosylation beyond charging DolP with mannose. Accordingly, the possibility that AglD, possibly in coordination with AglR, translocates DolP-mannose across the plasma membrane is discussed.

Identifying Components of a Halobacterium salinarum N-Glycosylation Pathway.

Whereas N-glycosylation is a seemingly universal process in Archaea, pathways of N-glycosylation have only been experimentally verified in a mere handful of species. Toward expanding the number of delineated archaeal N-glycosylation pathways, the involvement of the putative Halobacterium salinarum glycosyltransferases VNG1067G, VNG1066C, and VNG1062G in the assembly of an N-linked tetrasaccharide decorating glycoproteins in this species was addressed. Following deletion of each encoding gene, the impact on N-glycosylation of the S-layer glycoprotein and archaellins, major glycoproteins in this organism, was assessed by mass spectrometry. Likewise, the pool of dolichol phosphate, the lipid upon which this glycan is assembled, was also considered in each deletion strain. Finally, the impacts of such deletions were characterized in a series of biochemical, structural and physiological assays. The results revealed that VNG1067G, VNG1066C, and VNG1062G, renamed Agl25, Agl26, and Agl27 according to the nomenclature used for archaeal N-glycosylation pathway components, are responsible for adding the second, third and fourth sugars of the N-linked tetrasaccharide decorating Hbt. salinarum glycoproteins. Moreover, this study demonstrated how compromised N-glycosylation affects various facets of Hbt. salinarum cell behavior, including the transcription of archaellin-encoding genes.

A KLK4 proteinase substrate capture approach to antagonize PAR1.

Proteinase-activated receptor-1 (PAR1), triggered by thrombin and other serine proteinases such as tissue kallikrein-4 (KLK4), is a key driver of inflammation, tumor invasiveness and tumor metastasis. The PAR1 transmembrane G-protein-coupled receptor therefore represents an attractive target for therapeutic inhibitors. We thus used a computational design to develop a new PAR1 antagonist, namely, a catalytically inactive human KLK4 that acts as a proteinase substrate-capture reagent, preventing receptor cleavage (and hence activation) by binding to and occluding the extracellular R41-S42 canonical PAR1 proteolytic activation site. On the basis of in silico site-saturation mutagenesis, we then generated KLK4S207A,L185D, a first-of-a-kind 'decoy' PAR1 inhibitor, by mutating the S207A and L185D residues in wild-type KLK4, which strongly binds to PAR1. KLK4S207A,L185D markedly inhibited PAR1 cleavage, and PAR1-mediated MAPK/ERK activation as well as the migration and invasiveness of melanoma cells. This 'substrate-capturing' KLK4 variant, engineered to bind to PAR1, illustrates proof of principle for the utility of a KLK4 'proteinase substrate capture' approach to regulate proteinase-mediated PAR1 signaling.

Revisiting N-glycosylation in Halobacterium salinarum: Characterizing a dolichol phosphate- and glycoprotein-bound tetrasaccharide.

Although Halobacterium salinarum provided the first example of N-glycosylation outside the Eukarya, much regarding such post-translational modification in this halophilic archaea remains either unclear or unknown. The composition of an N-linked glycan decorating both the S-layer glycoprotein and archaellins offers one such example. Originally described some 40 years ago, reports from that time on have presented conflicted findings regarding the composition of this glycan, as well as differences between the protein-bound glycan and that version of the glycan attached to the lipid upon which it is assembled. To clarify these points, liquid chromatography-electrospray ionization mass spectrometry was employed here to revisit the composition of this glycan both when attached to selected asparagine residues of target proteins and when bound to the lipid dolichol phosphate upon which the glycan is assembled. Such efforts revealed the N-linked glycan as corresponding to a tetrasaccharide comprising a hexose, a sulfated hexuronic acid, a hexuronic acid and a second sulfated hexuronic acid. When attached to dolichol phosphate but not to proteins, the same tetrasaccharide is methylated on the final sugar. Moreover, in the absence of the oligosaccharyltransferase AglB, there is an accumulation of the dolichol phosphate-linked methylated and disulfated tetrasaccharide. Knowing the composition of this glycan at both the lipid- and protein-bound stages, together with the availability of gene deletion approaches for manipulating Hbt. salinarum, will allow delineation of the N-glycosylation pathway in this organism.

N-Glycosylation Is Important for Halobacterium salinarum Archaellin Expression, Archaellum Assembly and Cell Motility.

Halobacterium salinarum are halophilic archaea that display directional swimming in response to various environmental signals, including light, chemicals and oxygen. In Hbt. salinarum, the building blocks (archaellins) of the archaeal swimming apparatus (the archaellum) are N-glycosylated. However, the physiological importance of archaellin N-glycosylation remains unclear. Here, a tetrasaccharide comprising a hexose and three hexuronic acids decorating the five archaellins was characterized by mass spectrometry. Such analysis failed to detect sulfation of the hexuronic acids, in contrast to earlier reports. To better understand the physiological significance of Hbt. salinarum archaellin N-glycosylation, a strain deleted of aglB, encoding the archaeal oligosaccharyltransferase, was generated. In this ΔaglB strain, archaella were not detected and only low levels of archaellins were released into the medium, in contrast to what occurs with the parent strain. Mass spectrometry analysis of the archaellins in ΔaglB cultures did not detect N-glycosylation. ΔaglB cells also showed a slight growth defect and were impaired for motility. Quantitative real-time PCR analysis revealed dramatically reduced transcript levels of archaellin-encoding genes in the mutant strain, suggesting that N-glycosylation is important for archaellin transcription, with downstream effects on archaellum assembly and function. Control of AglB-dependent post-translational modification of archaellins could thus reflect a previously unrecognized route for regulating Hbt. salinarum motility.

Sialic Acid-Like Sugars in Archaea: Legionaminic Acid Biosynthesis in the Halophile Halorubrum sp. PV6.

N-glycosylation is a post-translational modification that occurs in all three domains. In Archaea, however, N-linked glycans present a degree of compositional diversity not observed in either Eukarya or Bacteria. As such, it is surprising that nonulosonic acids (NulOs), nine-carbon sugars that include sialic acids, pseudaminic acids, and legionaminic acids, are routinely detected as components of protein-linked glycans in Eukarya and Bacteria but not in Archaea. In the following, we report that the N-linked glycan attached to the S-layer glycoprotein of the haloarchaea Halorubrum sp. PV6 includes an N-formylated legionaminic acid. Analysis of the Halorubrum sp. PV6 genome led to the identification of sequences predicted to comprise the legionaminic acid biosynthesis pathway. The transcription of pathway genes was confirmed, as was the co-transcription of several of these genes. In addition, the activities of LegI, which catalyzes the condensation of 2,4-di-N-acetyl-6-deoxymannose and phosphoenolpyruvate to generate legionaminic acid, and LegF, which catalyzes the addition of cytidine monophosphate (CMP) to legionaminic acid, both heterologously expressed in Haloferax volcanii, were demonstrated. Further genome analysis predicts that the genes encoding enzymes of the legionaminic acid biosynthetic pathway are clustered together with sequences seemingly encoding components of the N-glycosylation pathway in this organism. In defining the first example of a legionaminic acid biosynthesis pathway in Archaea, the findings reported here expand our insight into archaeal N-glycosylation, an almost universal post-translational modification in this domain of life.

Evolutionary history and biogeography of Mandragora L. (Solanaceae).

Mandragora L. (Solanaceae) is the only genus of the tribe Mandragoreae, one of the two tribes of the cosmopolitan nightshade family, which occur exclusively in Eurasia and northern Africa. The genus occurs discontinuously in the Mediterranean region, Turanian region, and on the Tibetan Plateau, representing a classical disjunction pattern in the biogeography of the Old World flora. In this study, we reconstructed the genus phylogeny using AFLP, eight plastid DNA regions and one nuclear (ITS) gene, and evaluated the taxonomic value of quantitative traits time to flowering, fruit and seed size. We also analyzed the evolutionary history of the genus based on a phylogenetic framework and dating inferred from a combined data set of seven plastid regions with one fossil calibration point. Our data suggest that Mandragora originated in the Eocene, apparently along the Tethyan coast in broadleaf deciduous mesophytic forests that covered most of the Mediterranean region at that time. The Mediterranean-Turanian clade diverged from the Tibetan Plateau clade about 20.5 million years ago (Ma) as a result of the plateau uplift which probably was enhanced by aridification in the interior of Eurasia. A second split within the genus occurred about 11.1 Ma and resulted in Western Mediterranean and Near East-Turanian clades. Mandragora turcomanica was found to have very closely related evolutionary history with plants from the Near East indicating a possible ancient human assisted migration from Israel to Persia in historic times. In the Tibetan Plateau area, the morphologically distinctive M. chinghaiensis is nested within the M. caulescens clade indicating a very recent diversification within this lineage.

A bi-specific inhibitor targeting IL-17A and MMP-9 reduces invasion and motility in MDA-MB-231 cells.

The cytokine IL-17A is associated with the progression of various cancers, but little is known about the molecular cross-talk between IL-17A and other tumor-promoting factors. Previous studies have shown that the IL-17A-mediated invasion of breast cancer cells can be inhibited by selective antagonists of the matrix metalloproteinase 9 (MMP-9), suggesting that the cross-talk between IL-17A and MMP-9 may promote cancer invasiveness and metastasis. Here, we present a novel strategy for developing cancer therapeutics, based on the simultaneous binding and inhibition of both IL-17A and MMP-9. To this end, we use a bi-specific heterodimeric fusion protein, comprising a natural inhibitor of MMPs (N-TIMP2) fused with an engineered extracellular domain (V3) of the IL-17A receptor. We show that, as compared with the mono-specific inhibitors of IL-17A (V3) and MMP-9 (N-TIMP2), the engineered bi-specific fusion protein inhibits both MMP-9 activation and IL-17A-induced cytokine secretion from fibroblasts and exhibits a synergistic inhibition of both the migration and invasion of breast cancer cells. Our findings demonstrate, for the first time, that dual targeting of inflammatory (IL-17A) and extracellular matrix remodeling (MMP) pathways can potentially be used as a novel therapeutic approach against cancer. Moreover, the platform developed here for generating the bi-specific IL-17A/MMP-9 inhibitor can be utilized for generating bi-specific inhibitors for other cytokines and MMPs.

Increased Potency of a Bi-specific TL1A-ADAM17 (TACE) Inhibitor by Cell Surface Targeting.

Inflammatory bowel disease (IBD) is a multifactorial disease characterized by the dysregulated activity of many pro-inflammatory factors. Thus, bi-specific inhibitors for the simultaneous inhibition of two pro-inflammatory factors can exhibit high therapeutic potential. Here, we developed a novel bi-specific inhibitor targeting the TL1A cytokine and ADAM17/TACE metalloprotease. Biochemical analysis of the bi-specific inhibitor revealed high TL1A binding and TACE inhibition that is similar to the two respective mono-specific inhibitors. Interestingly, cell based assays for TL1A inhibition revealed strong synergism between the inhibitory domains showing an up to 80-fold increase in potency of the bi-specific inhibitor. The dramatic increase in potency is associated with binding to cell membranes through the TACE inhibitory domain leading to increased concentration of the inhibitor on the cell surface. Our study highlights the high potential of the simultaneous targeting of cell surface metalloprotease (TACE) and soluble pro-inflammatory cytokine (TL1A) as a potential therapeutic approach in IBD.

Directed evolution of a soluble human DR3 receptor for the inhibition of TL1A induced cytokine secretion.

TNF-like 1A (TL1A) is a cytokine belonging to the TNF superfamily that promotes inflammation in autoimmune diseases. Inhibiting the interaction of TL1A with the endogenous death-domain receptor 3 (DR3) offers a therapeutic approach for treating TL1A-induced autoimmune diseases. Here, we generated improved DR3 variants showing increased TL1A binding affinity and stability using a directed evolution approach. Given the high cysteine content and post-translational modification of DR3, we employed yeast surface display and expression in mammalian cell lines for screening, expression and characterization of improved DR3 variants. A cell-based assay performed with the human TF-1 cell line and CD4+ T cells showed that two improved DR3 mutants efficiently inhibited TL1A-induced cell death and secretion of IFN-γ, respectively. These DR3 mutants can be used as drug candidates for the treatment of inflammatory bowel diseases and for other autoimmune diseases, including rheumatic arthritis and asthma.

Directed evolution of a soluble human IL-17A receptor for the inhibition of psoriasis plaque formation in a mouse model.

Interleukin-17 (IL-17) is a T-cell-derived cytokine that promotes inflammatory pathology in autoimmune diseases. Blocking IL-17A interactions with its endogenous IL-17 receptor (IL-17RA) can constitute an important target for therapeutic intervention. Here, we utilized a directed evolution approach to generate soluble IL-17RA mutants that exhibit increased IL-17A binding affinity and thermostability, relative to the wild-type. Human fibroblast cell-based assay and in vivo analysis in mice indicated that two improved IL-17RA mutants efficiently inhibit the secretion of IL-17A-induced proinflammatory cytokines. Analysis of one of these mutants in a psoriasis mouse model showed its efficacy in promoting the recovery of psoriasis plaques. This mutant can be used as a promising drug candidate for the treatment of psoriasis and may be a therapeutic agent for various other autoimmune diseases.

Tight coevolution of proliferating cell nuclear antigen (PCNA)-partner interaction networks in fungi leads to interspecies network incompatibility.

The structure and connectivity of protein-protein interaction (PPI) networks are maintained throughout evolution by coordinated changes (coevolution) of network proteins. Despite extensive research, relatively little is known regarding the molecular basis and functional implications of the coevolution of PPI networks. Here, we used proliferating cell nuclear antigen, a hub protein that mediates DNA replication and repair in eukaryotes, as a model system to study the coevolution of PPI networks in fungi. Using a combined bioinformatics and experimental approach, we discovered that PCNA-partner interactions tightly coevolved in fungal species, leading to specific modes of recognition. We found that fungal proliferating cell nuclear antigen-partner interaction networks diverged into two distinct groups as a result of such coevolution and that hybrid networks of these groups are functionally noncompatible in Saccharomyces cerevisiae. Our results indicate that the coevolution of PPI networks can form functional barriers between fungal species, and thus can promote and fix speciation.

Differential expression of fungal genes at preinfection and mycorrhiza establishment between Terfezia boudieri isolates and Cistus incanus hairy root clones.

Changes in gene expression by isolates of Terfezia boudieri during mycorrhization with Cistus incanus hairy roots were followed. Four fungus-hairy root clone combinations were cultivated under two sets of conditions, in which the root and the fungus were separated by a cellophane sheet or were allowed physical contact. One of the combinations produced endomycorrhizas, the other three solely ectomycorrhizas. Fragments isolated by cDNA-AFLP analysis from cellophane-separated cultures (preinfection) were used to identify differentially expressed genes by reverse Northern analysis. Genes showing no homology to known sequences constituted the largest group under both growth conditions. Some fungal genes were expressed transiently, while others exhibited altered expression patterns as conditions changed from individually growing through the preinfection stage to mycorrhizas. Genes expressed exclusively under combinations allowing either ectomycorrhiza or endomycorrhiza under a particular condition were detected. Our results point, for the first time, to some of the genes that might be involved in determining the type of association that will be formed: ecto- or endomycorrhiza.