N-linked glycosylation in Archaea: two paths to the same glycan

N‐linked protein glycosylation occurs in all three branches of life, eukaryotes, bacteria and archaea. The simplest system is that of the bacterium, Campylobacter jejuni, in which a heptasaccharide glycan is added to multiple proteins from a single lipid carrier molecule. In the eukaryotic system a conserved tetradecasaccharide modification is first added to target proteins, but is then modified by trimming and addition of other glycans from additional carrier molecules resulting in a diverse array of glycans of distinct functionality. In the halophilic Archaea from the Dead Sea, Haloferax volcanii, the surface array or S‐layer protein is glycosylated with a pentasaccharide. This glycan is synthesized from two separate carrier molecules, one that carries a tetrasaccharide and another that carries the terminal mannose, in a process that is analogous to that of eukaryotes. In this issue of Molecular Microbiology the glycosylation of the S‐layer of another halophilic Archaea from the Dead Sea, Haloarcula marismortui is characterized ( Calo et al., 2011 ). This S‐layer is glycosylated with the same pentasaccharide as that of Hfx. volcanii, but the intact pentasaccharide is synthesized on a single carrier molecule in Har. marismortui in a process that more closely resembles that of the bacterial N‐linked system.     

Pseudomonas aeruginosa 1244 pilin glycosylation: glycan substrate recognition

The pilin of Pseudomonas aeruginosa 1244 is glycosylated with an oligosaccharide that is structurally identical to the O-antigen repeating unit of this organism. Concordantly, the metabolic source of the pilin glycan is the O-antigen biosynthetic pathway. The present study was conducted to investigate glycan substrate recognition in the 1244 pilin glycosylation reaction. Comparative structural analysis of O subunits that had been previously shown to be compatible with the 1244 glycosylation machinery revealed similarities among sugars at the presumed reducing termini of these oligosaccharides. We therefore hypothesized that the glycosylation substrate was within the sugar at the reducing end of the glycan precursor. Since much is known of PA103 O-antigen genetics and because the sugars at the reducing termini of the O7 (strain 1244) and O11 (strain PA103) are identical (beta-N-acetyl fucosamine), we utilized PA103 and strains that express lipopolysaccharide (LPS) with a truncated O-antigen subunit to test our hypothesis. LPS from a strain mutated in the wbjE gene produced an incomplete O subunit, consisting only of the monosaccharide at the reducing end (beta-d-N-acetyl fucosamine), indicating that this moiety contained substrate recognition elements for WaaL. Expression of pilAO(1244) in PA103 wbjE::aacC1, followed by Western blotting of extracts of these cells, indicated that pilin produced has been modified by the addition of material consistent with a single N-acetyl fucosamine. This was confirmed by analyzing endopeptidase-treated pilin by mass spectrometry. These data suggest that the pilin glycosylation substrate recognition features lie within the reducing-end moiety of the O repeat and that structures of the remaining sugars are irrelevant.     

Core glycan in the yeast multicopper ferroxidase, Fet3p: A case study of N-linked glycosylation, protein maturation, and stability

Glycosylation is essential to the maintenance of protein quality in the vesicular protein trafficking pathway in eukaryotic cells. Using the yeast multicopper oxidase, Fet3p, the hypothesis is tested that core glycosylation suppresses Fet3p nascent chain aggregation during synthesis into the endoplasmic reticulum (ER). Fet3p has 11 crystallographically mapped N‐linked core glycan units. Assembly of four of these units is specifically required for localization of Fet3p to the plasma membrane (PM). Fet3 protein lacking any one of these glycan units is found in an intracellular high‐molecular mass species resolvable by blue native gel electrophoresis. Individually, the remaining glycan moieties are not required for ER exit; however, serial deletion of these by N → A substitution correlates with these desglycan species failure to exit the ER. Desglycan Fet3 proteins that localize to the PM are wild type in function indicating that the missing carbohydrate is not required for native structure and biologic activity. This native function includes the interaction with the iron permease, Ftr1p, and wild type high‐affinity iron uptake activity. The four essential sequons are found within relatively nonpolar regions located in surface recesses and are strongly conserved among fungal Fet3 proteins. The remaining N‐linked sites are found in more surface exposed, less nonpolar environments, and their conservation is weak or absent. The data indicate that in Fet3p the N‐linked glycan has little effect on the enzyme's molecular activity but is critical to its cellular activity by maximizing the protein's exit from the ER and assembly into a functional iron uptake complex.     

A stereoselective 1,2-cis glycosylation toward the synthesis of a novel N-linked glycan from the Gram-negative bacterium, Campylobacter jejuni

It has been shown that certain prokaryotes, such as Campylobacter jejuni, have asparagine (Asn)-linked glycoproteins. However, the structures of their glycans are distinct from those of eukaryotic origin. They consist of a bacillosamine residue linked to Asn, an alpha-(1-->4)-GalpNAc repeat, and a branching beta-Glcp residue. In this paper, we describe a strategy for the stereoselective construction of the alpha-(1-->4)-GalpNAc repeat of a C. jejuni N-glycan, utilizing a pentafluoropropionyl (PFP) group as a temporary protective group of the C-4 OH group of the GalpN donor. The strategy was applied to the synthesis of the hexasaccharide alpha-GalpNAc-(1-->4)-alpha-GalpNAc-(1-->4)-[beta-Glcp-(1-->3)]-alpha-GalpNAc(1-->4)-alpha-GalpNAc-(1-->4)-GalpNAc.     

An alpha-numeric code for representing N-linked glycan structures in secreted glycoproteins

Advances in high-throughput techniques have led to the creation of increasing amounts of glycome data. The storage and analysis of this data would benefit greatly from a compact notation for describing glycan structures that can be easily stored and interpreted by computers. Towards this end, we propose a fixed-length alpha-numeric code for representing N-linked glycan structures commonly found in secreted glycoproteins from mammalian cell cultures. This code, GlycoDigit, employs a pre-assigned alpha-numeric index to represent the monosaccharides attached in different branches to the core glycan structure. The present branch-centric representation allows us to visualize the structure while the numerical nature of the code makes it machine readable. In addition, a difference operator can be defined to quantitatively differentiate between glycan structures for further analysis. The usefulness and applicability of GlycoDigit were demonstrated by constructing and visualizing an N-linked glycosylation network.     

Secretion and N-linked glycosylation are required for prostatic acid phosphatase catalytic and antinociceptive activity

Secretory human prostatic acid phosphatase (hPAP) is glycosylated at three asparagine residues (N62, N188, N301) and has potent antinociceptive effects when administered to mice. Currently, it is unknown if these N-linked residues are required for hPAP protein stability and activity in vitro or in animal models of chronic pain. Here, we expressed wild-type hPAP and a series of Asn to Gln point mutations in the yeast Pichia pastoris X33 then analyzed protein levels and enzyme activity in cell lysates and in conditioned media. Pichia secreted wild-type recombinant (r)-hPAP into the media (6-7 mg protein/L). This protein was as active as native hPAP in biochemical assays and in mouse models of inflammatory pain and neuropathic pain. In contrast, the N62Q and N188Q single mutants and the N62Q, N188Q double mutant were expressed at lower levels and were less active than wild-type r-hPAP. The purified N62Q, N188Q double mutant protein was also 1.9 fold less active in vivo. The N301Q mutant was not expressed, suggesting a critical role for this residue in protein stability. To explicitly test the importance of secretion, a construct lacking the signal peptide of hPAP was expressed in Pichia and assayed. This "cellular" construct was not expressed at levels detectable by western blotting. Taken together, these data indicate that secretion and post-translational carbohydrate modifications are required for PAP protein stability and catalytic activity. Moreover, our findings indicate that recombinant hPAP can be produced in Pichia--a yeast strain that is used to generate biologics for therapeutic purposes.     

Polyisoprenol specificity in the Campylobacter jejuni N-linked glycosylation pathway

Campylobacter jejuni contains a general N-linked glycosylation pathway in which a heptasaccharide is sequentially assembled onto a polyisoprenyl diphosphate carrier and subsequently transferred to the asparagine side chain of an acceptor protein. The enzymes in the pathway function at a membrane interface and have in common amphiphilic membrane-bound polyisoprenyl-linked substrates. Herein, we examine the potential role of the polyisoprene component of the substrates by investigating the relative substrate efficiencies of polyisoprene-modified analogues in individual steps of the pathway. Chemically defined substrates for PglC, PglJ, and PglB are prepared via semisynthetic approaches. The substrates included polyisoprenols of varying length, double bond geometry, and degree of saturation for probing the role of the hydrophobic polyisoprene in substrate specificity. Kinetic analysis reveals that all three enzymes exhibit distinct preferences for the polyisoprenyl carrier whereby cis-double bond geometry and alpha-unsaturation of the native substrate are important features, while the precise polyisoprene length may be less critical. These findings suggest that the polyisoprenyl carrier plays a specific role in the function of these enzymes beyond a purely physical role as a membrane anchor. These studies underscore the potential of the C. jejuni N-linked glycosylation pathway as a system for investigating the biochemical and biophysical roles of polyisoprenyl carriers common to prokaryotic and eukaryotic glycosylation.     

Cell surface glycoproteins from Thermoplasma acidophilum are modified with an N-linked glycan containing 6-C-sulfofucose

Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at pH 2 and 59°C. This extremophile is remarkable by the absence of a cell wall or an S-layer. Treating the cells with Triton X-100 at pH 3 allowed the extraction of all of the cell surface glycoproteins while keeping cells intact. The extracted glycoproteins were partially purified by cation-exchange chromatography, and we identified five glycoproteins by N-terminal sequencing and mass spectrometry of in-gel tryptic digests. These glycoproteins are positive for periodic acid-Schiff staining, have a high content of Asn including a large number in the Asn-X-Ser/Thr sequon and have apparent masses that are 34-48% larger than the masses deduced from their amino acid sequences. The pooled glycoproteins were digested with proteinase K and the purified glycopeptides were analyzed by NMR. Structural determination showed that the carbohydrate part was represented by two structures in nearly equal amounts, differing by the presence of one terminal mannose residue. The larger glycan chain consists of eight residues: six hexoses, one heptose and one sugar with an unusual residue mass of 226 Da which was identified as 6-deoxy-6-C-sulfo-D-galactose (6-C-sulfo-D-fucose). Mass spectrometry analyses of the peptides obtained by trypsin and chymotrypsin digestion confirmed the principal structures to be those determined by NMR and identified 14 glycopeptides derived from the main glycoprotein, Ta0280, all containing the Asn-X-Ser/Thr sequons. Thermoplasma acidophilum appears to have a "general" protein N-glycosylation system that targets a number of cell surface proteins.     

Asparagine-linked protein glycosylation: from eukaryotic to prokaryotic systems

Asparagine-linked protein glycosylation is a prevalent protein modification reaction in eukaryotic systems. This process involves the co-translational transfer of a pre-assembled tetradecasaccharide from a dolichyl-pyrophosphate donor to the asparagine side chain of nascent proteins at the endoplasmic reticulum (ER) membrane. Recently, the first such system of N-linked glycosylation was discovered in the Gram-negative bacterium, Campylobacter jejuni. Glycosylation in this organism involves the transfer of a heptasaccharide from an undecaprenyl-pyrophosphate donor to the asparagine side chain of proteins at the bacterial periplasmic membrane. Here we provide a detailed comparison of the machinery involved in the N-linked glycosylation systems of eukaryotic organisms, exemplified by the yeast Saccharomyces cerevisiae, with that of the bacterial system in C. jejuni. The two systems display significant similarities and the relative simplicity of the bacterial glycosylation process could provide a model system that can be used to decipher the complex eukaryotic glycosylation machinery.     

Stromal interaction molecule 1 (STIM1), a transmembrane protein with growth suppressor activity, contains an extracellular SAM domain modified by N-linked glycosylation

Stromal interaction molecule 1 (STIM1) is a cell surface transmembrane glycoprotein implicated in tumour growth control and stromal-haematopoietic cell interactions. A single sterile alpha motif (SAM) protein-protein interaction domain is modelled within its extracellular region, a subcellular localisation not previously described for other SAM domain-containing proteins. We have defined the transmembrane topology of STIM1 by determining the sites of N-linked glycosylation. We have confirmed that STIM1 is modified by N-linked glycosylation at two sites within the SAM domain itself, deduced as asparagine residues N131 and N171, demonstrating that STIM1 is translocated across the membrane of the endoplasmic reticulum such that the SAM domain resides within the endoplasmic reticulum (ER) lumen. Both N-linked oligosaccharides remain endoglycosidase H-sensitive, indicating absence of full processing within the ER and Golgi. This immature modification is nevertheless sufficient and critical for cell surface expression of STIM1. We show that STIM1-STIM1 homotypic interactions are mediated via the cytoplasmic rather than the extracellular region of STIM1, excluding an essential role for the SAM domain in these protein interactions. These studies provide the first evidence for an extracellular localisation of a SAM domain within any protein, and the first example of a SAM domain modified by N-linked glycosylation.     

N-linked oligosaccharides affect the enzymatic activity of CD39: diverse interactions between seven N-linked glycosylation sites

Rat CD39, a membrane-bound ectonucleoside triphosphate diphosphohydrolase that hydrolyzes extracellular nucleoside tri- and diphosphates, has seven potential N-glycosylation sites at asparagine residues 73, 226, 291, 333, 375, 429, and 458. To determine their roles in the structure and function of CD39, we mutated these sites individually or in combination by replacing asparagine with serine or glutamine and analyzed the surface expression and the enzymatic activity of the mutants. The results indicate that rat CD39 can be glycosylated at all seven sites when expressed in COS7 cells. Glycosylation sites 73 at the N terminus, 333 in the middle, and 429 and 458 at the C terminus were principally required for cell surface appearance of enzymatically active CD39. Whereas deletion of these sites individually had modest effects on surface ATPase activity, some double deletions of these sites had major effects on both surface activity and expression. The importance of these N-glycosylation sites is recognizable in other members of the ectonucleoside triphosphate diphosphohydrolase family.     

Inhibition of N-linked glycosylation in Dictyostetium discoideum: Effects of aggregate formation

The aggregation program of Dictyostelium discoideum is extremely sensitive to the effects of tunicamycin when the drug is added to cells during the first few hours of starvation. Inhibition of development is observed with concentrations as low as 0.5 micrograms/ml, which cause only a 25%-30% inhibition of general N-linked glycosylation. However, 0.5 micrograms/ml tunicamycin can result in the total inhibition of N-linked glycosylation of specific, developmentally regulated, proteins, as exemplified by the glycoprotein 117 antigen. If added after the first hours of starvation, tunicamycin cannot inhibit aggregation even when present at 10 micrograms/ml, which maximally inhibits N-linked glycosylation. cAMP pulses can override the inhibitory effects of tunicamycin on cell aggregation. The data support the hypothesis that there is an early developmental pathway that is dependent on the N-linked glycosylation of one, or a small set of developmentally regulated proteins and that this pathway may involve the biogenesis of the chemotactic signalling system. In addition, the data raise questions as to the role of N-linked oligosaccharides in cell cohesion.     

Role of the peripheral myelin protein 22 N-linked glycan in oligomer stability

Peripheral myelin protein 22 (PMP22) is a 22-kDa glycoprotein containing a single N-linked carbohydrate moiety. This posttranslational modification is conserved in PMP22 across species and within members of the PMP22 gene family; however, the function of the oligosaccharide is not known. To study the role of the PMP22 carbohydrate, site-directed mutagenesis was used to alter the glycosylation consensus sequence and produce a glycosylation-deficient mutant protein. This modified PMP22 was expressed in primary Schwann cells (SCs), and the effect of the N-glycan on the turnover rate, oligomerization, and intracellular trafficking of PMP22 was determined. Our data show a slight decrease in turnover rate from a half-life of approximately 70 min for the wild-type (wt) protein to 100 min for the glycosylation mutant. Although the presence of glycosylation-deficient PMP22 oligomers could be detected in SCs, we observed a decrease in oligomer stability compared with the wt oligomers. Both wt and mutant proteins showed similar localization in the endoplasmic reticulum and Golgi compartments and were transported to the SC surface. These results suggest that the N-glycan of PMP22 facilitates, in part, the stability of the PMP22 oligomer; however, the implications of PMP22 oligomerization remain unknown.     

Dynamic developmental elaboration of N-linked glycan complexity in the Drosophila melanogaster embryo

The structural diversity of glycoprotein N-linked oligosaccharides is determined by the expression and regulation of glycosyltransferase activities and by the availability of the appropriate acceptor/donor substrates. Cells in different tissues and in different developmental stages utilize these control points to manifest unique glycan expression patterns in response to their surroundings. The activity of a Toll-like receptor, called Tollo/Toll-8, induces a pattern of incompletely defined, but neural specific, glycan expression in the Drosophila embryo. Understanding the full extent of the changes in glycan expression that result from altered Tollo/Toll-8 signaling requires characterization of the complete N-linked glycan profile of both wild-type and mutant embryos. N-Linked glycans harvested from wild-type or mutant embryos were subjected to direct structural analysis by analytic and preparative high pressure liquid chromatography, by multidimensional mass spectrometry, and by exoglycosidase digestion, revealing a predominance of high mannose and paucimannose glycans. Di-, mono-, and nonfucosylated forms of hybrid, complex biantennary, and triantennary glycans account for 12% of the total wild-type glycan profile. Two sialylated glycans bearing N-acetylneuraminic acid were detected, the first direct demonstration of this modification in Drosophila. Glycan profiles change during normal development consistent with increasing alpha-mannosidase II and core fucosyl-transferase enzyme activities, and with decreasing activity of the Fused lobes processing hexosaminidase. In tollo/toll-8 mutants, a dramatic, expected loss of difucosylated glycans is accompanied by unexpected decreases in monofucosylated and nonfucosylated hybrid glycans and increases in some nonfucosylated paucimannose and biantennary glycans. Therefore, tollo/toll-8 signaling influences flux through several processing steps that affect the maturation of N-linked glycans.     

Role of N-linked glycan in the unfolding pathway of Erythrina corallodendron lectin

Erythrina corallodendron lectin (ECorL) exhibits an exquisitely structured oligosaccharide chain. Interestingly, the bacterially expressed, nonglycosylated counterpart, rECorL, possesses an essentially identical carbohydrate specificity and agglutinating activity as the glycosylated lectin, thus suggesting that the overall structure of the two are identical. This paper reports the unfolding behavior of E. corallodendron lectin in its glycosylated (EcorL) and nonglycosylated (rECorL) forms. ECorL shows a two-state unfolding pattern during isothermal melts and differential scanning calorimetry (DSC). The T(g) of ECorL as obtained from isothermal melts is 74 degrees C at pH 7.4. The T(p) obtained from DSC studies is between 74.8 to 68.1 degrees C in the pH range of 5.26-7.77. The recombinant lectin (rECorL), which is devoid of carbohydrate, shows, in contrast to the glycosylated protein, a non-two-state unfolding profile as measured by both probes mentioned, but the number of intermediates during unfolding could not be ascertained. Simulated annealing on ECorL, with the sugars removed, reveals that the protein Calpha backbones overlap, indicating that the overall structure, including the mode of dimerization, of rECorL is insignificantly altered as compared to ECorL. The alterations in the folding behavior of rECorL as compared to that observed in ECorL may be due to the fact that, unlike most other glycoproteins, one of the glycans in ECorL is unusually structured and forms many hydrogen bonds with the protein. It therefore appears that while the covalently linked sugar does not contribute appreciably to the final folded structure of ECorL, it does alter its folding process in a significant manner.     

Quantitative assessment of the preferences for the amino acid residues flanking archaeal N-linked glycosylation sites

Oligosaccharyltransferase (OST) catalyzes the transfer of an oligosaccharide to an asparagine residue in polypeptide chains. Using positional scanning peptide libraries, we assessed the effects of amino acid variations on the in vitro glycosylation efficiency within and adjacent to an N-glycosylation consensus, Asn-X-Ser/Thr, with an archaeal OST from Pyrococcus furiosus. The amino acid variations at the X(-2), X(-1) and X(+1) positions in the sequence X(-2)-X(-1)-Asn-X-Ser/Thr-X(+1) strongly influenced the glycosylation efficiency to a similar extent at position X. The rank orders of the amino acid preferences were unique at each site. We experimentally confirmed that the archaeal OST does not require an acidic residue at the -2 position, unlike the eubacterial OSTs. Pro was disfavored at the -1 and +1 positions, although the exclusion was not as strict as that at X, whereas Pro was the most favored amino acid residue among those studied at the -2 position. The overall amino acid preferences are correlated with a conformational propensity to extend around the sequon. The results of the library experiments revealed that the optimal acceptor sequence was PYNVTK, with a K(m) of 10 μM. The heat-stable, single-subunit OST of P. furiosus is a potential candidate enzyme for the production of recombinant glycoproteins in bacterial cells. Quantitative assessment of the amino acid preferences of the OST enzyme will facilitate the proper design of a production system.     

Functional glycosylation of human podoplanin: glycan structure of platelet aggregation-inducing factor

Podoplanin (Aggrus) is a mucin-type sialoglycoprotein that plays a key role in tumor cell-induced platelet aggregation. Podoplanin possesses a platelet aggregation-stimulating (PLAG) domain, and Thr52 in the PLAG domain of human podoplanin is important for its activity. Endogenous or recombinant human podoplanin were purified, and total glycosylation profiles were surveyed by lectin microarray. Analyses of glycopeptides produced by Edman degradation and mass spectrometry revealed that the disialyl-corel (NeuAc alpha2-3Gal beta l-3(NeuAc alpha2-6)GalNAc alpha l-O-Thr) structure was primarily attached to a glycosylation site at residue Thr52. Sialic acid-deficient podoplanin recovered its activity after additional sialylation. These results indicated that the sialylated Corel at Thr52 is critical for podoplanin-induced platelet aggregation.     

Glycoprotein expression in mouse cerebellum: effects of inhibitors of N-linked glycosylation.

1. Explants of cerebellum from foetal mouse were cultured in vitro for 10 days in the presence of one of 4 inhibitors of N-linked glycosylation (castanospermine, deoxymannojirimycin, swainsonine, and tunicamycin). 2. The effects of the inhibitors were compared with respect to: (a) the activity of enzymes involved in glycoprotein biosynthesis and degradation; (b) the expression of N-linked glycoproteins; (c) the morphology and ultrastructure of the treated cerebellar explants.     

The significance of N-linked glycosylation in pig endogenous retrovirus infectivity

The significance of the envelope glycoprotein in the transmission of pig endogenous retrovirus (PERV) to human cells was investigated. Pig endothelial cells (PEC) were transduced with the LacZ gene by a pseudotype infection and then infected with PERV subtype B. Culture supernatants of the infected PEC previously incubated with several types of drugs were inoculated into HEK293 cells. The inoculated cells were then stained and the number of LacZ-positive foci was counted. PERV from tunicamycin treated PEC was not transmitted to human cells, indicating the importance of N-linked sugars in this process. Moreover, while inhibition of the terminal alpha-glucose residues from the precursor N-glycan by castanospermine and 1-deoxynojirimycin attenuated PERV infectivity, the mannosidase inhibitors, 1-deoxymannojirimycin and swainsonine, upregulated the infectivity. In addition, treatment with alpha-mannosidase and incubation with concanavalin A completely abrogated the transmission of PERV to HEK293. These data imply that the high-mannose type of N-glycan plays a key role in PERV infectivity.