N-linked protein glycosylation is the most abundant posttranslation modification of secretory proteins in eukaryotes. A wide range of functions are attributed to glycan structures covalently linked to asparagine residues within the asparagine-X-serine/threonine consensus sequence (Asn-Xaa-Ser/Thr). We found an N-linked glycosylation system in the bacterium Campylobacter jejuni and demonstrate that a functional N-linked glycosylation pathway could be transferred into Escherichia coli. Although the bacterial N-glycan differs structurally from its eukaryotic counterparts, the cloning of a universal N-linked glycosylation cassette in E. coli opens up the possibility of engineering permutations of recombinant glycan structures for research and industrial applications.

The Htm1/EDEM protein has been proposed to act as a "degradation lectin" for endoplasmic reticulum-associated degradation (ERAD) of misfolded glycoproteins. In this study, we provide genetic and biochemical evidence that Yos9 protein in Saccharomyces cerevisiae is essential for efficient degradation of mutant glycoproteins. Yos9 is a member of the OS-9 protein family, which is conserved among eukaryotes and shows similarities with mannose-6-phosphate receptors (MPRs). We found that amino acids conserved among OS-9 family members and MPRs were essential for Yos9 protein function. Immunoprecipitation showed that Yos9 specifically associated with misfolded carboxypeptidase Y (CPY*), an ERAD substrate, but only when it carried Man8GlcNAc2 or Man5GlcNAc2 N-glycans. Our experiments further suggested that Yos9 acts in the same pathway as Htm1/EDEM. Yos9 protein is important for glycoprotein degradation and may act via its MRH domain as a degradation lectin-like protein in the glycoprotein degradation pathway.

Cancer cells are frequently characterized by aberrant increases in protein N-glycosylation and by disruption of E-cadherin-mediated adherens junctions. The relationship between altered N-glycosylation and loss of E-cadherin adhesion in cancer, however, remains unclear. Previously, we reported that complex N-glycans on the extracellular domains of E-cadherin inhibited the formation of mature adherens junctions. Here, we examined whether dysregulated N-glycosylation was one of the underlying causes for cellular discohesion in oral cancer. We show that dense cultures of human salivary epidermoid carcinoma A253 cells exhibited elevated expression of DPAGT1, the gene that initiates protein N-glycosylation. Overexpression of DPAGT1 correlated with the production of E-cadherin-bearing complex N-glycans in nascent adherens junctions. Partial inhibition of DPAGT1 with small interfering RNA reduced the complex N-glycans of E-cadherin and increased the abundance of A-catenin and stabilizing proteins in adherens junctions. This was associated with the assembly of functional tight junctions. The inverse relationship between DPAGT1 expression and intercellular adhesion was a feature of oral squamous cell carcinoma. Oral squamous cell carcinomas displayed overexpression of DPAGT1 that correlated with diminished localization of E-cadherin and A-catenin at the sites of adherens junctions. Our studies show for the first time that DPAGT1 is an upstream regulator of E-cadherin N-glycosylation status and adherens junction composition and suggest that dysregulation of DPAGT1 causes disturbances in intercellular adhesion in oral cancer.

Galectins are characterized by their binding affinity for β-galactosides, a unique binding site sequence motif, and wide taxonomic distribution and structural conservation in vertebrates, invertebrates, protista, and fungi. Since their initial description, galectins were considered to bind endogenous (“self”) glycans and mediate developmental processes and cancer. In the past few years, however, numerous studies have described the diverse effects of galectins on cells involved in both innate and adaptive immune responses, and the mechanistic aspects of their regulatory roles in immune homeostasis. More recently, however, evidence has accumulated to suggest that galectins also bind exogenous (“non-self”) glycans on the surface of potentially pathogenic microbes, parasites, and fungi, suggesting that galectins can function as pattern recognition receptors (PRRs) in innate immunity. Thus, a perplexing paradox arises by the fact that galectins also recognize lactosamine-containing glycans on the host cell surface during developmental processes and regulation of immune responses. According to the currently accepted model for non-self recognition, PRRs recognize pathogens via highly conserved microbial surface molecules of wide distribution such as LPS or peptidoglycan (pathogen-associated molecular patterns; PAMPs), which are absent in the host. Hence, this would not apply to galectins, which apparently bind similar self/non-self molecular patterns on host and microbial cells. This paradox underscores first, an oversimplification in the use of the PRR/PAMP terminology. Second, and most importantly, it reveals significant gaps in our knowledge about the diversity of the host galectin repertoire, and the subcellular targeting, localization, and secretion. Furthermore, our knowledge about the structural and biophysical aspects of their interactions with the host and microbial carbohydrate moieties is fragmentary, and warrants further investigation.

In the Gram-negative bacterium Campylobacter jejuni there is a pgl (protein glycosylation) locus-dependent general N-glycosylation system of proteins. One of the proteins encoded by pgl locus, PglB, a homolog of the eukaryotic oligosaccharyltransferase component Stt3p, is proposed to function as an oligosaccharyltransferase in this prokaryotic system. The sequence requirements of the acceptor polypeptide for N-glycosylation were analyzed by reverse genetics using the reconstituted glycosylation of the model protein AcrA in Escherichia coli. As in eukaryotes, the N-X-S/T sequon is an essential but not a sufficient determinant for N-linked protein glycosylation. This conclusion was supported by the analysis of a novel C. jejuni glycoprotein, HisJ. Export of the polypeptide to the periplasm was required for glycosylation. Our data support the hypothesis that eukaryotic and bacterial N-linked protein glycosylation are homologous processes.

Epithelial cell-cell adhesion is controlled by multiprotein complexes that include E-cadherinmediated adherens junctions (AJs) and ZO-1-containing tight junctions (TJs). Previously, we reported that reduction of E-cadherin N-glycosylation in normal and cancer cells promoted stabilization of AJs through changes in the composition and cytoskeletal association of E-cadherin scaffolds. Here, we show that enhanced interaction of hypoglycosylated E-cadherin-containing AJs with protein phosphatase 2A (PP2A) represents a mechanism for promoting TJ assembly. In MDCK cells, attenuation of cellular N-glycosylation with siRNA to DPAGT1, the first gene in the Nglycosylation pathway, reduced N-glycosylation of surface E-cadherin and resulted in increased recruitment of stabilizing proteins γ-catenin, α-catenin, vinculin and PP2A to AJs. Greater association of PP2A with AJs correlated with diminished binding of PP2A to ZO-1 and claudin-1 and with increased pools of serine-phosphorylated ZO-1 and claudin-1. More ZO-1 was found in complexes with occludin and claudin-1, and this corresponded to enhanced transepithelial resistance (TER), indicating physiological assembly of TJs. Similar maturation of AJs and TJs was detected after transfection of MDCK cells with the hypoglycosylated E-cadherin variant, V13. Our data indicate that E-cadherin N-glycans coordinate the maturity of AJs with the assembly of TJs by affecting the association of PP2A with these junctional complexes.

Protein–carbohydrate interactions mediated by lectins have been recognized as key components of innate immunity in vertebrates and invertebrates, not only for recognition of potential pathogens, but also for participating in downstream effector functions, such as their agglutination, immobilization, and complement-mediated opsonization and killing. More recently, lectins have been identified as critical regulators of mammalian adaptive immune responses. Fish are endowed with virtually all components of the mammalian adaptive immunity, and are equipped with a complex lectin repertoire. In this review, we discuss evidence suggesting that: (a) lectin repertoires in teleost fish are highly diversified, and include not only representatives of the lectin families described in mammals, but also members of lectin families described for the first time in fish species; (b) the tissue-specific expression and localization of the diverse lectin repertoires and their molecular partners is consistent with their distinct biological roles in innate and adaptive immunity; (c) although some lectins may bind endogenous ligands, others bind sugars on the surface of potential pathogens; (d) in addition to pathogen recognition and opsonization, some lectins display additional effector roles, such as complement activation and regulation of immune functions; (e) some lectins that recognize exogenous ligands mediate processes unrelated to immunity: they may act as anti-freeze proteins or prevent polyspermia during fertilization.

The economic factor of the microalgae harvesting step acts as a barrier to scaling up microalgae-based technology designed for wastewater treatment. In view of that, this study presents an alternative microalgae-bacteria system, which is proposed for eliminating the economic obstacle. Instead of the microalgae-bacteria (activated algae) flocs, the study aimed to develop activated algae granules comprising the microalgae Chlorella sp. as a target species. The presence of the filamentous microalgae (Phormidium sp.) was necessary for the occurrence of the granulation processes. A progressive decrease in frequency of the free Chlorella sp. cells was achieved once with the development of the activated algae granules as a result of the target microalgae being captured in the dense and tangled network of filaments. The mature activated algae granules ranged between 600 and 2,000 µm, and were characterized by a compact structure and significant settling ability (21.6 ± 0.9 m/h). In relation to the main aim of this study, a microalgae recovery efficiency of higher than 99% was achieved only by fast sedimentation of the granules; this performance highlighted the viability of the granular activated algae system for sustaining a microalgae harvesting procedure with neither cost nor energy inputs.