Revisiting the immunopathology of congenital disorders of glycosylation: an updated review.

Glycosylation is a critical post-translational modification that plays a pivotal role in several biological processes, such as the immune response. Alterations in glycosylation can modulate the course of various pathologies, such as the case of congenital disorders of glycosylation (CDG), a group of more than 160 rare and complex genetic diseases. Although the link between glycosylation and immune dysfunction has already been recognized, the immune involvement in most CDG remains largely unexplored and poorly understood. In this study, we provide an update on the immune dysfunction and clinical manifestations of the 12 CDG with major immune involvement, organized into 6 categories of inborn errors of immunity according to the International Union of Immunological Societies (IUIS). The immune involvement in phosphomannomutase 2 (PMM2)-CDG - the most frequent CDG - was comprehensively reviewed, highlighting a higher prevalence of immune issues during infancy and childhood and in R141H-bearing genotypes. Finally, using PMM2-CDG as a model, we point to links between abnormal glycosylation patterns in host cells and possibly favored interactions with microorganisms that may explain the higher susceptibility to infection. Further characterizing immunopathology and unusual host-pathogen adhesion in CDG can not only improve immunological standards of care but also pave the way for innovative preventive measures and targeted glycan-based therapies that may improve quality of life for people living with CDG.

Unraveling Molecular Recognition of Glycan Ligands by Siglec-9 via NMR Spectroscopy and Molecular Dynamics Modeling.

Human sialic-acid-binding immunoglobulin-like lectin-9 (Siglec-9) is a glycoimmune checkpoint receptor expressed on several immune cells. Binding of Siglec-9 to sialic acid containing glycans (sialoglycans) is well documented to modulate its functions as an inhibitory receptor. Here, we first assigned the amino acid backbone of the Siglec-9 V-set domain (Siglec-9d1), using well-established triple resonance three-dimensional nuclear magnetic resonance (NMR) methods. Then, we combined solution NMR and molecular dynamic simulation methods to decipher the molecular details of the interaction of Siglec-9 with the natural ligands α2,3 and α2,6 sialyl lactosamines (SLN), sialyl Lewis X (sLeX), and 6-O sulfated sLeX and with two synthetically modified sialoglycans that bind with high affinity. As expected, Neu5Ac is accommodated between the F and G ß-strands at the canonical sialic acid binding site. Addition of a heteroaromatic scaffold 9N-5-(2-methylthiazol-4-yl)thiophene sulfonamide (MTTS) at the C9 position of Neu5Ac generates new interactions with the hydrophobic residues located at the G-G' loop and the N-terminal region of Siglec-9. Similarly, the addition of the aromatic substituent (5-N-(1-benzhydryl-1H-1,2,3-triazol-4-yl)methyl (BTC)) at the C5 position of Neu5Ac stabilizes the conformation of the long and flexible B'-C loop present in Siglec-9. These results expose the underlying mechanism responsible for the enhanced affinity and specificity for Siglec-9 for these two modified sialoglycans and sheds light on the rational design of the next generation of modified sialoglycans targeting Siglec-9.

SARS-CoV-2 Rapid Antigen Test Based on a New Anti-Nucleocapsid Protein Monoclonal Antibody: Development and Real-Time Validation.

SARS-CoV-2 diagnostic tests have become an important tool for pandemic control. Among the alternatives for COVID-19 diagnosis, antigen rapid diagnostic tests (Ag-RDT) are very convenient and widely used. However, as SARS-CoV-2 variants may continuously emerge, the replacement of tests and reagents may be required to maintain the sensitivity of Ag-RDTs. Here, we describe the development and validation of an Ag-RDT during an outbreak of the Omicron variant, including the characterization of a new monoclonal antibody (anti-DTC-N 1B3 mAb) that recognizes the Nucleocapsid protein (N). The anti-DTC-N 1B3 mAb recognized the sequence TFPPTEPKKDKKK located at the C-terminus of the N protein of main SARS-CoV-2 variants of concern. Accordingly, the Ag-RDT prototypes using the anti-DTC-N 1B3 mAB detected all the SARS-CoV-2 variants-Wuhan, Alpha, Gamma, Delta, P2 and Omicron. The performance of the best prototype (sensitivity of 95.2% for samples with Ct ≤ 25; specificity of 98.3% and overall accuracy of 85.0%) met the WHO recommendations. Moreover, results from a patients' follow-up study indicated that, if performed within the first three days after onset of symptoms, the Ag-RDT displayed 100% sensitivity. Thus, the new mAb and the Ag-RDT developed herein may constitute alternative tools for COVID-19 point-of-care diagnosis and epidemiological surveillance.

Structural insights into Siglec-15 reveal glycosylation dependency for its interaction with T cells through integrin CD11b.

Sialic acid-binding Ig-like lectin 15 (Siglec-15) is an immune modulator and emerging cancer immunotherapy target. However, limited understanding of its structure and mechanism of action restrains the development of drug candidates that unleash its full therapeutic potential. In this study, we elucidate the crystal structure of Siglec-15 and its binding epitope via co-crystallization with an anti-Siglec-15 blocking antibody. Using saturation transfer-difference nuclear magnetic resonance (STD-NMR) spectroscopy and molecular dynamics simulations, we reveal Siglec-15 binding mode to α(2,3)- and α(2,6)-linked sialic acids and the cancer-associated sialyl-Tn (STn) glycoform. We demonstrate that binding of Siglec-15 to T cells, which lack STn expression, depends on the presence of α(2,3)- and α(2,6)-linked sialoglycans. Furthermore, we identify the leukocyte integrin CD11b as a Siglec-15 binding partner on human T cells. Collectively, our findings provide an integrated understanding of the structural features of Siglec-15 and emphasize glycosylation as a crucial factor in controlling T cell responses.

Long non-coding RNAs are essential for Schistosoma mansoni pairing-dependent adult worm homeostasis and fertility.

The trematode parasite Schistosoma mansoni causes schistosomiasis, which affects over 200 million people worldwide. Schistosomes are dioecious, with egg laying depending on the females' obligatory pairing with males. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides with low or no protein-coding potential that have been involved in other species with reproduction, stem cell maintenance, and drug resistance. In S. mansoni, we recently showed that the knockdown of one lncRNA affects the pairing status of these parasites. Here, we re-analyzed public RNA-Seq data from paired and unpaired adult male and female worms and their gonads, obtained from mixed-sex or single-sex cercariae infections, and found thousands of differentially expressed pairing-dependent lncRNAs among the 23 biological samples that were compared. The expression levels of selected lncRNAs were validated by RT-qPCR using an in vitro unpairing model. In addition, the in vitro silencing of three selected lncRNAs showed that knockdown of these pairing-dependent lncRNAs reduced cell proliferation in adult worms and their gonads, and are essential for female vitellaria maintenance, reproduction, and/or egg development. Remarkably, in vivo silencing of each of the three selected lncRNAs significantly reduced worm burden in infected mice by 26 to 35%. Whole mount in situ hybridization experiments showed that these pairing-dependent lncRNAs are expressed in reproductive tissues. These results show that lncRNAs are key components intervening in S. mansoni adult worm homeostasis, which affects pairing status and survival in the mammalian host, thus presenting great potential as new therapeutic target candidates.

Structures of the Inhibitory Receptor Siglec-8 in Complex with a High-Affinity Sialoside Analogue and a Therapeutic Antibody.

Human sialic acid binding immunoglobulin-like lectin-8 (Siglec-8) is an inhibitory receptor that triggers eosinophil apoptosis and can inhibit mast cell degranulation when engaged by specific monoclonal antibodies (mAbs) or sialylated ligands. Thus, Siglec-8 has emerged as a critical negative regulator of inflammatory responses in diverse diseases, such as allergic airway inflammation. Herein, we have deciphered the molecular recognition features of the interaction of Siglec-8 with the mAb lirentelimab (2C4, under clinical development) and with a sialoside mimetic with the potential to suppress mast cell degranulation. The three-dimensional structure of Siglec-8 and the fragment antigen binding (Fab) portion of the anti-Siglec-8 mAb 2C4, solved by X-ray crystallography, reveal that 2C4 binds close to the carbohydrate recognition domain (V-type Ig domain) on Siglec-8. We have also deduced the binding mode of a high-affinity analogue of its sialic acid ligand (9-N-napthylsufonimide-Neu5Ac, NSANeuAc) using a combination of NMR spectroscopy and X-ray crystallography. Our results show that the sialoside ring of NSANeuAc binds to the canonical sialyl binding pocket of the Siglec receptor family and that the high affinity arises from the accommodation of the NSA aromatic group in a nearby hydrophobic patch formed by the N-terminal tail and the unique G-G' loop. The results reveal the basis for the observed high affinity of this ligand and provide clues for the rational design of the next generation of Siglec-8 inhibitors. Additionally, the specific interactions between Siglec-8 and the N-linked glycans present on the high-affinity receptor FcεRIα have also been explored by NMR.

Tracking the turnover of SARS-CoV-2 VOCs Gamma to Delta in a Brazilian state (Minas Gerais) with a high-vaccination status.

The emergence and global dissemination of Severe Acute Respiratory Syndrome virus 2 (SARS-CoV-2) variants of concern (VOCs) have been described as the main factor driving the Coronavirus Disease 2019 pandemic. In Brazil, the Gamma variant dominated the epidemiological scenario during the first period of 2021. Many Brazilian regions detected the Delta variant after its first description and documented its spread. To monitor the introduction and spread of VOC Delta, we performed Polymerase Chain Reaction (PCR) genotyping and genome sequencing in ten regional sentinel units from June to October 2021 in the State of Minas Gerais (MG). We documented the introduction and spread of Delta, comprising 70 per cent of the cases 8 weeks later. Comparing the viral loads of the Gamma and Delta dominance periods, we provide additional evidence that the latter is more transmissible. The spread and dominance of Delta did not culminate in the increase in cases and deaths, suggesting that the vaccination may have restrained the epidemic growth. Analysis of 224 novel Delta genomes revealed that Rio de Janeiro state was the primary source for disseminating this variant in the state of MG. We present the establishment of Delta, providing evidence of its enhanced transmissibility and showing that this variant shift did not aggravate the epidemiological scenario in a high immunity setting.

Structural basis for the synthesis of the core 1 structure by C1GalT1.

C1GalT1 is an essential inverting glycosyltransferase responsible for synthesizing the core 1 structure, a common precursor for mucin-type O-glycans found in many glycoproteins. To date, the structure of C1GalT1 and the details of substrate recognition and catalysis remain unknown. Through biophysical and cellular studies, including X-ray crystallography of C1GalT1 complexed to a glycopeptide, we report that C1GalT1 is an obligate GT-A fold dimer that follows a SN2 mechanism. The binding of the glycopeptides to the enzyme is mainly driven by the GalNAc moiety while the peptide sequence provides optimal kinetic and binding parameters. Interestingly, to achieve glycosylation, C1GalT1 recognizes a high-energy conformation of the α-GalNAc-Thr linkage, negligibly populated in solution. By imposing this 3D-arrangement on that fragment, characteristic of α-GalNAc-Ser peptides, C1GalT1 ensures broad glycosylation of both acceptor substrates. These findings illustrate a structural and mechanistic blueprint to explain glycosylation of multiple acceptor substrates, extending the repertoire of mechanisms adopted by glycosyltransferases.

Atomic and Specificity Details of Mucin 1 O-Glycosylation Process by Multiple Polypeptide GalNAc-Transferase Isoforms Unveiled by NMR and Molecular Modeling.

The large family of polypeptide GalNAc-transferases (GalNAc-Ts) controls with precision how GalNAc O-glycans are added in the tandem repeat regions of mucins (e.g., MUC1). However, the structural features behind the creation of well-defined and clustered patterns of O-glycans in mucins are poorly understood. In this context, herein, we disclose the full process of MUC1 O-glycosylation by GalNAc-T2/T3/T4 isoforms by NMR spectroscopy assisted by molecular modeling protocols. By using MUC1, with four tandem repeat domains as a substrate, we confirmed the glycosylation preferences of different GalNAc-Ts isoforms and highlighted the importance of the lectin domain in the glycosylation site selection after the addition of the first GalNAc residue. In a glycosylated substrate, with yet multiple acceptor sites, the lectin domain contributes to orientate acceptor sites to the catalytic domain. Our experiments suggest that during this process, neighboring tandem repeats are critical for further glycosylation of acceptor sites by GalNAc-T2/T4 in a lectin-assisted manner. Our studies also show local conformational changes in the peptide backbone during incorporation of GalNAc residues, which might explain GalNAc-T2/T3/T4 fine specificities toward the MUC1 substrate. Interestingly, we postulate that a specific salt-bridge and the inverse γ-turn conformation of the PDTRP sequence in MUC1 are the main structural motifs behind the GalNAc-T4 specificity toward this region. In addition, in-cell analysis shows that the GalNAc-T4 isoform is the only isoform glycosylating the Thr of the immunogenic epitope PDTRP in vivo, which highlights the relevance of GalNAc-T4 in the glycosylation of this epitope. Finally, the NMR methodology established herein can be extended to other glycosyltransferases, such as C1GalT1 and ST6GalNAc-I, to determine the specificity toward complex mucin acceptor substrates.

Long non-coding RNAs as possible therapeutic targets in protozoa, and in Schistosoma and other helminths.

Long non-coding RNAs (lncRNAs) emerged in the past 20 years due to massive amounts of scientific data regarding transcriptomic analyses. They have been implicated in a plethora of cellular processes in higher eukaryotes. However, little is known about lncRNA possible involvement in parasitic diseases, with most studies only detecting their presence in parasites of human medical importance. Here, we review the progress on lncRNA studies and their functions in protozoans and helminths. In addition, we show an example of knockdown of one lncRNA in Schistosoma mansoni, SmLINC156349, which led to in vitro parasite adhesion, motility, and pairing impairment, with a 20% decrease in parasite viability and 33% reduction in female oviposition. Other observed phenotypes were a decrease in the proliferation rate of both male and female worms and their gonads, and reduced female lipid and vitelline droplets that are markers for well-developed vitellaria. Impairment of female worms' vitellaria in SmLINC156349-silenced worms led to egg development deficiency. All those results demonstrate the great potential of the tools and methods to characterize lncRNAs as potential new therapeutic targets. Further, we discuss the challenges and limitations of current methods for studying lncRNAs in parasites and possible solutions to overcome them, and we highlight the future directions of this exciting field.

Molecular Recognition Insights of Sialic Acid Glycans by Distinct Receptors Unveiled by NMR and Molecular Modeling.

All cells are decorated with a highly dense and complex structure of glycan chains, which are mostly attached to proteins and lipids. In this context, sialic acids are a family of nine-carbon acidic monosaccharides typically found at the terminal position of glycan chains, modulating several physiological and pathological processes. Sialic acids have many structural and modulatory roles due to their negative charge and hydrophilicity. In addition, the recognition of sialic acid glycans by mammalian cell lectins, such as siglecs, has been described as an important immunological checkpoint. Furthermore, sialic acid glycans also play a pivotal role in host-pathogen interactions. Various pathogen receptors exposed on the surface of viruses and bacteria are responsible for the binding to sialic acid sugars located on the surface of host cells, becoming a critical point of contact in the infection process. Understanding the molecular mechanism of sialic acid glycans recognition by sialic acid-binding proteins, present on the surface of pathogens or human cells, is essential to realize the biological mechanism of these events and paves the way for the rational development of strategies to modulate sialic acid-protein interactions in diseases. In this perspective, nuclear magnetic resonance (NMR) spectroscopy, assisted with molecular modeling protocols, is a versatile and powerful technique to investigate the structural and dynamic aspects of glycoconjugates and their interactions in solution at the atomic level. NMR provides the corresponding ligand and protein epitopes, essential for designing and developing potential glycan-based therapies. In this review, we critically discuss the current state of knowledge about the structural features behind the molecular recognition of sialic acid glycans by different receptors, naturally present on human cells or pathogens, disclosed by NMR spectroscopy and molecular modeling protocols.

Survey of SARS-CoV-2 genetic diversity in two major Brazilian cities using a fast and affordable Sanger sequencing strategy.

Genetic variants of SARS-CoV-2 have been emerging and circulating in many places across the world. Rapid detection of these variants is essential since their dissemination can impact transmission rates, diagnostic procedures, disease severity, response to vaccines or patient management. Sanger sequencing has been used as the preferred approach for variant detection among circulating human immunodeficiency and measles virus genotypes. Using primers to amplify a fragment of the SARS-CoV-2 genome encoding part of the Spike protein, we showed that Sanger sequencing allowed us to rapidly detect the introduction and spread of three distinct SARS-CoV-2 variants in two major Brazilian cities. In both cities, after the predominance of variants closely related to the virus first identified in China, the emergence of the P.2 variant was quickly followed by the detection of the P1 variant, which became dominant in less than one month after it was first detected.

Assessment of reference genes at six different developmental stages of Schistosoma mansoni for quantitative RT-PCR.

Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the most used, fast, and reproducible method to confirm large-scale gene expression data. The use of stable reference genes for the normalization of RT-qPCR assays is recognized worldwide. No systematic study for selecting appropriate reference genes for usage in RT-qPCR experiments comparing gene expression levels at different Schistosoma mansoni life-cycle stages has been performed. Most studies rely on genes commonly used in other organisms, such as actin, tubulin, and GAPDH. Therefore, the present study focused on identifying reference genes suitable for RT-qPCR assays across six S. mansoni developmental stages. The expression levels of 25 novel candidates that we selected based on the analysis of public RNA-Seq datasets, along with eight commonly used reference genes, were systematically tested by RT-qPCR across six developmental stages of S. mansoni (eggs, miracidia, cercariae, schistosomula, adult males and adult females). The stability of genes was evaluated with geNorm, NormFinder and RefFinder algorithms. The least stable candidate reference genes tested were actin, tubulin and GAPDH. The two most stable reference genes suitable for RT-qPCR normalization were Smp_101310 (Histone H4 transcription factor) and Smp_196510 (Ubiquitin recognition factor in ER-associated degradation protein 1). Performance of these two genes as normalizers was successfully evaluated with females maintained unpaired or paired to males in culture for 8 days, or with worm pairs exposed for 16 days to double-stranded RNAs to silence a protein-coding gene. This study provides reliable reference genes for RT-qPCR analysis using samples from six different S. mansoni life-cycle stages.

Structural Insights into the Molecular Recognition Mechanism of the Cancer and Pathogenic Epitope, LacdiNAc by Immune-Related Lectins.

Interactions of glycan-specific epitopes to human lectin receptors represent novel immune checkpoints for investigating cancer and infection diseases. By employing a multidisciplinary approach that combines isothermal titration calorimetry, NMR spectroscopy, molecular dynamics simulations, and X-ray crystallography, we investigated the molecular determinants that govern the recognition of the tumour and pathogenic glycobiomarker LacdiNAc (GalNAcß1-4GlcNAc, LDN), including their comparison with the ubiquitous LacNAc epitope (Galß1-4GlcNAc, LN), by two human immune-related lectins, galectin-3 (hGal-3) and the macrophage galactose C-type lectin (hMGL). A different mechanism of binding and interactions was observed for the hGal-3/LDN and hMGL/LDN complexes, which explains the remarkable difference in the binding specificity of LDN and LN by these two lectins. The new structural clues reported herein are fundamental for the chemical design of mimetics targeting hGal-3/hMGL recognition process.

Crystal Structure of the Carbohydrate Recognition Domain of the Human Macrophage Galactose C-Type Lectin Bound to GalNAc and the Tumor-Associated Tn Antigen.

The human macrophage galactose lectin (MGL) is an endocytic type II transmembrane receptor expressed on immature monocyte-derived dendritic cells and activated macrophages and plays a role in modulating the immune system in response to infections and cancer. MGL contains an extracellular calcium-dependent (C-type) carbohydrate recognition domain (CRD) that specifically binds terminal N-acetylgalactosamine glycan residues such as the Tn and sialyl-Tn antigens found on tumor cells, as well as other N- and O-glycans displayed on certain viruses and parasites. Even though the glycan specificity of MGL is known and several binding glycoproteins have been identified, the molecular basis for substrate recognition has remained elusive due to the lack of high-resolution structures. Here we present crystal structures of the MGL CRD at near endosomal pH and in several complexes, which reveal details of the interactions with the natural ligand, GalNAc, the cancer-associated Tn-Ser antigen, and a synthetic GalNAc mimetic ligand. Like the asialoglycoprotein receptor, additional calcium atoms are present and contribute to stabilization of the MGL CRD fold. The structure provides the molecular basis for preferential binding of N-acetylgalactosamine over galactose and prompted the re-evaluation of the binding modes previously proposed in solution. Saturation transfer difference nuclear magnetic resonance data acquired using the MGL CRD and interpreted using the crystal structure indicate a single binding mode for GalNAc in solution. Models of MGL1 and MGL2, the mouse homologues of MGL, explain how these proteins might recognize LewisX and GalNAc, respectively.

Structural characterization of an unprecedented lectin-like antitumoral anti-MUC1 antibody.

The molecular basis of antibody 5E5, which recognizes the entire GalNAc unit as a primary epitope is disclosed. The antibody's contacts with the peptide are mostly limited to two residues, allowing it to show some degree of promiscuity. These findings open the door to the chemical design of peptide-mimetics for developing efficient anti-cancer vaccines and diagnostic tools.

Novel Alkyl(aryl)-Substituted 2,2-Difluoro-6-(trichloromethyl)-2H-1,3,2-oxazaborinin-3-ium-2-uides: Synthesis, Antimicrobial Activity, and CT-DNA Binding Evaluations.

The synthesis, antimicrobial activity evaluations, biomolecule-binding properties (DNA), and absorption and emission properties of a new series of (Z)-1,1,1-trichloro-4-alkyl(aryl)amino-4-arylbut-3-en-2-ones (4, 5) and 2,2-difluoro-3-alkyl(aryl)amino-4-aryl-6-(trichloromethyl)-2H-1,3,2-oxazaborinin-3-ium-2-uides (6, 7) in which 3(4)-alkyl(aryl) = H, Me, iso-propyl, n-butyl, C6H5, 4-CH3C6H4, 4-CH3OC6H4, 4-NO2C6H4, 4-FC6H4, 4-BrC6H4, 2-naphthyl, is reported. A series of ß-enaminoketones (4, 5) is synthesized from the O,N-exchange reaction of some amines (3) with (Z)-1,1,1-trichloro-4-methoxy-4-aryl-but-3-en-2-ones (1, 2) at 61-90% yields. Subsequently, reactions of the resulting ß-enaminoketones with an appropriate source of boron (BF3.OEt2) gave the corresponding oxazaborinine derivatives (6, 7) at 50-91% yields. UV-Vis and emission properties of biomolecule-binding properties for the DNA of these new BF2-ß-enamino containing CCl3 units were also evaluated. Some compounds from the present series also exhibited potent antimicrobial effects on various pathogenic microorganisms at concentrations below those that showed cytotoxic effects. Compounds 4d, 4e, 6e, and 6f showed the best results and are very significant against P. zopfii, which causes diseases in humans and animals.

Structural Characterization of N-Linked Glycans in the Receptor Binding Domain of the SARS-CoV-2 Spike Protein and their Interactions with Human Lectins.

The glycan structures of the receptor binding domain of the SARS-CoV2 spike glycoprotein expressed in human HEK293F cells have been studied by using NMR. The different possible interacting epitopes have been deeply analysed and characterized, providing evidence of the presence of glycan structures not found in previous MS-based analyses. The interaction of the RBD 13 C-labelled glycans with different human lectins, which are expressed in different organs and tissues that may be affected during the infection process, has also been evaluated by NMR. In particular, 15 N-labelled galectins (galectins-3, -7 and -8 N-terminal), Siglecs (Siglec-8, Siglec-10), and C-type lectins (DC-SIGN, MGL) have been employed. Complementary experiments from the glycoprotein perspective or from the lectin's point of view have permitted to disentangle the specific interacting epitopes in each case. Based on these findings, 3D models of the interacting complexes have been proposed.

Molecular basis for fibroblast growth factor 23 O-glycosylation by GalNAc-T3.

Polypeptide GalNAc-transferase T3 (GalNAc-T3) regulates fibroblast growth factor 23 (FGF23) by O-glycosylating Thr178 in a furin proprotein processing motif RHT178R↓S. FGF23 regulates phosphate homeostasis and deficiency in GALNT3 or FGF23 results in hyperphosphatemia and familial tumoral calcinosis. We explored the molecular mechanism for GalNAc-T3 glycosylation of FGF23 using engineered cell models and biophysical studies including kinetics, molecular dynamics and X-ray crystallography of GalNAc-T3 complexed to glycopeptide substrates. GalNAc-T3 uses a lectin domain mediated mechanism to glycosylate Thr178 requiring previous glycosylation at Thr171. Notably, Thr178 is a poor substrate site with limiting glycosylation due to substrate clashes leading to destabilization of the catalytic domain flexible loop. We suggest GalNAc-T3 specificity for FGF23 and its ability to control circulating levels of intact FGF23 is achieved by FGF23 being a poor substrate. GalNAc-T3's structure further reveals the molecular bases for reported disease-causing mutations. Our findings provide an insight into how GalNAc-T isoenzymes achieve isoenzyme-specific nonredundant functions.

The Plasticity of the Carbohydrate Recognition Domain Dictates the Exquisite Mechanism of Binding of Human Macrophage Galactose-Type Lectin.

The human macrophage galactose-type lectin (MGL), expressed on macrophages and dendritic cells (DCs), modulates distinct immune cell responses by recognizing N-acetylgalactosamine (GalNAc) containing structures present on pathogens, self-glycoproteins, and tumor cells. Herein, NMR spectroscopy and molecular dynamics (MD) simulations were used to investigate the structural preferences of MGL against different GalNAc-containing structures derived from the blood group A antigen, the Forssman antigen, and the GM2 glycolipid. NMR spectroscopic analysis of the MGL carbohydrate recognition domain (MGL-CRD, C181-H316) in the absence and presence of methyl α-GalNAc (α-MeGalNAc), a simple monosaccharide, shows that the MGL-CRD is highly dynamic and its structure is strongly altered upon ligand binding. This plasticity of the MGL-CRD structure explains the ability of MGL to accommodate different GalNAc-containing molecules. However, key differences are observed in the recognition process depending on whether the GalNAc is part of the blood group A antigen, the Forssman antigen, or GM2-derived structures. These results are in accordance with molecular dynamics simulations that suggest the existence of a distinct MGL binding mechanism depending on the context of GalNAc moiety presentation. These results afford new perspectives for the rational design of GalNAc modifications that fine tune MGL immune responses in distinct biological contexts, especially in malignancy.