Initiation of mucin-type O-glycosylation is controlled by a large family of UDP GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Most GalNAc-transferases contain a ricin-like lectin domain in the C-terminal end, which may confer GalNAc-glycopeptide substrate specificity to the enzyme. We have previously shown that the lectin domain of GalNAc-T4 modulates its substrate specificity to enable unique GalNAc-glycopeptide specificities and that this effect is selectively inhibitable by GalNAc; however, direct evidence of carbohydrate binding of GalNAc-transferase lectins has not been previously presented. Here, we report the direct carbohydrate binding of two GalNAc-transferase lectin domains, GalNAc-T4 and GalNAc-T2, representing isoforms reported to have distinct glycopeptide activity (GalNAc-T4) and isoforms without apparent distinct GalNAc-glycopeptide specificity (GalNAc-T2). Both lectins exhibited specificity for binding of free GalNAc. Kinetic and time-course analysis of GalNAc-T2 demonstrated that the lectin domain did not affect transfer to initial glycosylation sites, but selectively modulated velocity of transfer to subsequent sites and affected the number of acceptor sites utilized. The results suggest that GalNAc-transferase lectins serve to modulate the kinetic properties of the enzymes in the late stages of the initiation process of O-glycosylation to accomplish dense or complete O-glycan occupancy.

The lectin from the common mushroom Agaricus bisporus, the most popular edible species in Western countries, has potent antiproliferative effects on human epithelial cancer cells, without any apparent cytotoxicity. This property confers to it an important therapeutic potential as an antineoplastic agent. The three-dimensional structure of the lectin was determined by x-ray diffraction. The protein is a tetramer with 222 symmetry, and each monomer presents a novel fold with two ␤ sheets connected by a helix-loop-helix motif. Selectivity was studied by examining the binding of four monosaccharides and seven disaccharides in two different crystal forms. The T-antigen disaccharide, Gal␤1-3GalNAc, mediator of the antiproliferative effects of the protein, binds at a shallow depression on the surface of the molecule. The binding of N-acetylgalactosamine overlaps with that moiety of the T antigen, but surprisingly, Nacetylglucosamine, which differs from N-acetylgalactosamine only in the configuration of epimeric hydroxyl 4, binds at a totally different site on the opposite side of the helix-loop-helix motif. The lectin thus has two distinct binding sites per monomer that recognize the different configuration of a single epimeric hydroxyl. The structure of the protein and its two carbohydrate-binding sites are described in detail in this study.

No abstract

Galbeta1-3GalNAc (T-disaccharide) and related molecules were assayed to describe the structural requirements of carbohydrates to bind Agaricus bisporus lectin (ABL). Results provide insight into the most relevant regions of T-disaccharide involved in the binding of ABL. It was found that monosaccharides bind ABL weakly indicating a more extended carbohydrate-binding site as compared to those involvedin the T-disaccharide specific lectins such as jacalin and peanut agglutinin. Lacto-N-biose (Galbeta1-3GlcNAc) unlike T-disaccharide, is unable to inhibit the ABL interaction, thus showing the great importance of the position of the axial C-4 hydroxyl group of GalNAc in T-disaccharide. This finding could explain the inhibitory ability of Galbeta1-6GlcNAc and lactose because C-4 and C-3 hydroxyl groups of reducing Glc, respectively, occupy a similar position as reported by conformational analysis. From the comparison of different glycolipids bearing terminal T-disaccharide bound to different linkages, it can be seen than ABL binding is even more impaired by an adjacent C-6 residual position than by the anomeric influence of T-disaccharide. Furthermore, the addition of beta-GlcNAc to the terminal T-disaccharide in C-3 position of Gal does not affect the ABL binding whereas if an anionic group such as glucuronic acid is added to C-3, the binding is partially affected. These findings demonstrate that ABL holds a particular binding nature different from that of other T-disaccharide specific lectins.

Epithelial cancer cells show increased cell surface expression of mucin antigens with aberrant O-glycosylation, notably type I core (Galbeta1-3GalNAcalpha), termed Thomsen-Friedenreich disaccharide (TFD), a chemically well-defined carbohydrate antigen with a proven link to malignancy. Several TFD-binding proteins influence the proliferation of cells to which they bind. We studied the fine specificity of TFD-binding proteins and its relationship with epithelial tumor cell proliferation. Competitive binding assays against asialoglycophorin showed that Agaricus bisporus lectin (ABL) and human anti-TFD monoclonal antibody (mAb) TF1 were inhibited only by TFD and its alpha-derivatives. Peanut agglutinin (PNA), mAb TF2, and mAb TF5 were also inhibited by other carbohydrates such as lacto-N-biose (Galbeta1-3GlcNAc), lactose, and (Mealpha or beta) Gal, indicating lower recognition of the axial C-4 hydroxyl group position of GalNAc from TFD, and the major relevance of the terminal Gal on interaction of these three TFD-binding proteins. In the direct glycolipid-binding assay, ABL bound mostly to alpha-anomeric TFD-bearing glycolipids, whereas PNA interacted mainly with beta-linked TFD. Of the three anti-TFD mAbs analyzed, all bound N5b (terminal beta-TFD), but only TF2 interacted with N6 (terminal alpha-TFD). These findings indicate that TFD-binding proteins that stimulate the proliferation of epithelial tumor cell lines recognize mainly a terminal beta-Gal region of beta-linked TFD, whereas ABL, which inhibits the proliferation of these tumor cells, binds mainly to subterminal GalNAc of alpha-anomeric TFD.

Differences in fine specificity of antibodies are strong indications that different regions of the GM(1)-oligosaccharide are involved in antibody binding. High titres of specific anti-GM(1) antibody binding to cellular GM(1) can be explained by antigen exposure, that is, GM(1) exposes or forms mainly epitopes recognised by specific antibodies, and 'hides' those involved in binding of cross-reacting antibodies. Thus, the fine specificity of anti-GM(1) antibodies may influence disease severity by affecting antibody binding to cellular targets. Additionally, since antibody specificity studies are relatively easy to implement, fine specificity could be considered a useful predictor of disease severity.

Biological functions of nuclear proteins are regulated by posttranslational modifications (PTMs) that modulate gene expression and cellular physiology. However, the role of O-linked glycosylation (O-GalNAc) as a PTM of nuclear proteins in the human cell has not been previously reported. Here, we examined in detail the initiation of O-GalNAc glycan biosynthesis, representing a novel PTM of nuclear proteins in the nucleus of human cells, with an emphasis on HeLa cells. Using soluble nuclear fractions from purified nuclei, enzymatic assays, fluorescence microscopy, affinity chromatography, MS, and FRET analyses, we identified all factors required for biosynthesis of O-GalNAc glycans in nuclei: the donor substrate (UDP-GalNAc), nuclear polypeptide GalNAc-transferase activity, and a GalNAc transferase (polypeptide GalNAc-T3). Moreover, we identified O-GalNAc glycosylated proteins in the nucleus and present solid evidence for O-GalNAc glycan synthesis in this organelle. The demonstration of O-GalNAc glycosylation of nuclear proteins in mammalian cells reported here has important implications for cell and chemical biology. The nucleus is one of the most important structures of eukaryotic cells. This complex organelle stores the chromosomes and also regulates their duplication, segregation, repair, and expression through a series of specific processes. The cell's biological information is saved and transferred within the nucleus by three types of biopolymer molecules: DNA, RNA, and proteins (1). Proteins play crucial roles in nuclear scaffolding, DNA assembly, replication, transcription, and transport of molecules. The biological activity of proteins is directly modulated by their conformation, and changes in protein conformation are controlled mainly by post-translational modifications (PTMs). 3 The common PTMs of nuclear proteins are acetyla-This work was supported by CONICET Grant PIP 11220150100226 and SeCyT, UNC Grant 05/C422 (to F. J. I.). The authors declare that they have no conflicts of interest with the contents of this article. This work is dedicated to the memory of Mafalda C. Pellegrini-Irazoqui. This article contains Figs. S1-S5 and Table S1.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerge during the last months of 2019, expanding throughout the world as a highly transmissible infectious illness designated as COVID-19. Vaccines have now appeared, but the challenges in producing sufficient material and distributing them around the world means that effective treatments to limit infection and improve recovery are still urgently needed. This review is focused on the relevance of different glycobiological molecules that could potentially serve as or inspire therapeutic tools during SARS-CoV-2 infection. As such, we highlight the glycobiology of the SARS-CoV-2 infection process, where glycans on viral proteins and on host glycosaminoglycans have critical roles in efficient infection. We also take notice of the glycan-binding proteins involved in the infective capacity of virus and in human defense. In addition, we critically evaluate the glycobiological contribution of candidate drugs for COVID-19 therapy such as glycans for vaccines, anti-glycan antibodies, recombinant lectins, lectin inhibitors, glycosidase inhibitors, polysaccharides, and numerous glycosides, emphasizing some opportunities to repurpose FDA-approved drugs. For the next generation drugs suggested here, biotechnological engineering making new probes to block the SARS-CoV-2 infection might be based in the essential glycobiological insight on glycosyltransferases, glycans, glycan-binding proteins and glycosidases related to this pathology.