Detection of Tumor-Associated Autoantibodies in the Sera of Pancreatic Cancer Patients Using Engineered MUC1 Glycopeptide Nanoparticle Probes.

Pancreatic cancer is one of the deadliest cancers worldwide, mainly due to late diagnosis. Therefore, there is an urgent need for novel diagnostic approaches to identify the disease as early as possible. We have developed a diagnostic assay for pancreatic cancer based on the detection of naturally occurring tumor associated autoantibodies against Mucin-1 (MUC1) using engineered glycopeptides on nanoparticle probes. We used a structure-guided approach to develop unnatural glycopeptides as model antigens for tumor-associated MUC1. We designed a collection of 13 glycopeptides to bind either SM3 or 5E5, two monoclonal antibodies with distinct epitopes known to recognize tumor associated MUC1. Glycopeptide binding to SM3 or 5E5 was confirmed by surface plasmon resonance and rationalized by molecular dynamics simulations. These model antigens were conjugated to gold nanoparticles and used in a dot-blot assay to detect autoantibodies in serum samples from pancreatic cancer patients and healthy volunteers. Nanoparticle probes with glycopeptides displaying the SM3 epitope did not have diagnostic potential. Instead, nanoparticle probes displaying glycopeptides with high affinity for 5E5 could discriminate between cancer patients and healthy controls. Remarkably, the best-discriminating probes show significantly better true and false positive rates than the current clinical biomarkers CA19-9 and carcinoembryonic antigen (CEA).

Identification of FasL as a crucial host factor driving COVID-19 pathology and lethality.

The dysregulated immune response and inflammation resulting in severe COVID-19 are still incompletely understood. Having recently determined that aberrant death-ligand-induced cell death can cause lethal inflammation, we hypothesized that this process might also cause or contribute to inflammatory disease and lung failure following SARS-CoV-2 infection. To test this hypothesis, we developed a novel mouse-adapted SARS-CoV-2 model (MA20) that recapitulates key pathological features of COVID-19. Concomitantly with occurrence of cell death and inflammation, FasL expression was significantly increased on inflammatory monocytic macrophages and NK cells in the lungs of MA20-infected mice. Importantly, therapeutic FasL inhibition markedly increased survival of both, young and old MA20-infected mice coincident with substantially reduced cell death and inflammation in their lungs. Intriguingly, FasL was also increased in the bronchoalveolar lavage fluid of critically-ill COVID-19 patients. Together, these results identify FasL as a crucial host factor driving the immuno-pathology that underlies COVID-19 severity and lethality, and imply that patients with severe COVID-19 may significantly benefit from therapeutic inhibition of FasL.

Structure-Guided Approach for the Development of MUC1-Glycopeptide-Based Cancer Vaccines with Predictable Responses.

Mucin-1 (MUC1) glycopeptides are exceptional candidates for potential cancer vaccines. However, their autoantigenic nature often results in a weak immune response. To overcome this drawback, we carefully engineered synthetic antigens with precise chemical modifications. To be effective and stimulate an anti-MUC1 response, artificial antigens must mimic the conformational dynamics of natural antigens in solution and have an equivalent or higher binding affinity to anti-MUC1 antibodies than their natural counterparts. As a proof of concept, we have developed a glycopeptide that contains noncanonical amino acid (2S,3R)-3-hydroxynorvaline. The unnatural antigen fulfills these two properties and effectively mimics the threonine-derived antigen. On the one hand, conformational analysis in water shows that this surrogate explores a landscape similar to that of the natural variant. On the other hand, the presence of an additional methylene group in the side chain of this analog compared to the threonine residue enhances a CH/π interaction in the antigen/antibody complex. Despite an enthalpy-entropy balance, this synthetic glycopeptide has a binding affinity slightly higher than that of its natural counterpart. When conjugated with gold nanoparticles, the vaccine candidate stimulates the formation of specific anti-MUC1 IgG antibodies in mice and shows efficacy comparable to that of the natural derivative. The antibodies also exhibit cross-reactivity to selectively target, for example, human breast cancer cells. This investigation relied on numerous analytical (e.g., NMR spectroscopy and X-ray crystallography) and biophysical techniques and molecular dynamics simulations to characterize the antigen-antibody interactions. This workflow streamlines the synthetic process, saves time, and reduces the need for extensive, animal-intensive immunization procedures. These advances underscore the promise of structure-based rational design in the advance of cancer vaccine development.

Polypeptide N-acetylgalactosaminyltransferase (GalNAc-T) isozyme surface charge governs charge substrate preferences to modulate mucin type O-glycosylation.

A large family of polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) initiate mucin type O-glycosylation transferring α-GalNAc from a UDP-GalNAc donor to the hydroxyl groups of Ser and Thr residues of peptides and proteins, thereby defining sites of O-glycosylation. Mutations and differential expression of several GalNAc-Ts are associated with many disease states including cancers. The mechanisms by which these isozymes choose their targets and their roles in disease are not fully understood. We previously showed that the GalNAc-Ts possess common and unique specificities for acceptor type, peptide sequence and prior neighboring, and/or remote substrate GalNAc glycosylation. In the present study, the role of flanking charged residues was investigated using a library of charged peptide substrates containing the central -YAVTPGP- acceptor sequence. Eleven human and one bird GalNAc-T were initially characterized revealing a range of preferences for net positive, net negative, or unique combinations of flanking N- and/or C-terminal charge, correlating to each isozyme's different electrostatic surface potential. It was further found that isoforms with high sequence identity (>70%) within a subfamily can possess vastly different charge specificities. Enzyme kinetics, activities obtained at elevated ionic strength, and molecular dynamics simulations confirm that the GalNAc-Ts differently recognize substrate charge outside the common +/-3 residue binding site. These electrostatic interactions impact how charged peptide substrates bind/orient on the transferase surface, thus modulating their activities. In summary, we show the GalNAc-Ts utilize more extended surfaces than initially thought for binding substrates based on electrostatic, and likely other hydrophobic/hydrophilic interactions, furthering our understanding of how these transferases select their target.

Identification of global inhibitors of cellular glycosylation.

Small molecule inhibitors of glycosylation enzymes are valuable tools for dissecting glycan functions and potential drug candidates. Screening for inhibitors of glycosyltransferases are mainly performed by in vitro enzyme assays with difficulties moving candidates to cells and animals. Here, we circumvent this by employing a cell-based screening assay using glycoengineered cells expressing tailored reporter glycoproteins. We focused on GalNAc-type O-glycosylation and selected the GalNAc-T11 isoenzyme that selectively glycosylates endocytic low-density lipoprotein receptor (LDLR)-related proteins as targets. Our screen of a limited small molecule compound library did not identify selective inhibitors of GalNAc-T11, however, we identify two compounds that broadly inhibited Golgi-localized glycosylation processes. These compounds mediate the reversible fragmentation of the Golgi system without affecting secretion. We demonstrate how these inhibitors can be used to manipulate glycosylation in cells to induce expression of truncated O-glycans and augment binding of cancer-specific Tn-glycoprotein antibodies and to inhibit expression of heparan sulfate and binding and infection of SARS-CoV-2.

Molecular Recognition of GalNAc in Mucin-Type O-Glycosylation.

Acetylgalactosamine (GalNAc)-type O-glycosylation is an essential posttranslational modification (PTM) that plays fundamental roles in biology. Malfunction of this PTM is exemplified by the presence of truncated O-glycans in cancer. For instance, the glycoprotein MUC1 is overexpressed in many tumor tissues and tends to carry simple oligosaccharides that allow for the presentation of different tumor-associated antigens, such as the Tn or sTn antigens (GalNAc-α-1-O-Thr/Ser and Neu5Acα2-6GalNAcα1-O-Ser/Thr, respectively). In other cases, such as tumoral calcinosis associated with O-glycosylation of the fibroblast growth factor 23, O-glycans are absent or less abundant. Significant progress has been made in determining the three-dimensional structures of biomolecules that recognize GalNAc, such as antibodies, lectins, mucinases, GalNAc-transferases, and other glycosyltransferases. Analysis of the complexes between these entities and GalNAc-containing glycopeptides, in most cases derived from crystallographic or NMR analysis, provides an understanding of the key structural elements that control molecular recognition of these glycopeptides. Here, we describe and compare the binding sites of these proteins in detail, focusing on how the GalNAc moieties interact selectively with them. We also summarize the differences and similarities in GalNAc recognition. In general, the recognition of GalNAc-containing glycopeptides is determined by hydrogen bonds between hydroxyl groups and the N-acetyl group of GalNAc with proteins, as well as CH-π contacts in which the hydrophobic α-face of the sugar and the methyl group of NHAc can be involved. The latter interaction usually provides the basis for selectivity. It is worth noting that binding of these glycopeptides depends primarily on recognition of the sugar moiety, with some exceptions such as a few anti-MUC1 antibodies that primarily recognize the peptide backbone and use the sugar to facilitate shape complementarity or to establish a limited number of interactions with the protein. Focusing specifically on the GalNAc moiety, we can observe that there is some degeneracy of interactions within the same protein families, likely due to substrate flexibility. However, when all studied proteins are considered together, despite the commonalities within each protein family, no pattern can be discerned between the different families, apart from the presence of common residues such as Tyr, His, or Asp, which are responsible for hydrogen bonds. The lack of a pattern can be anticipated, given the diverse functions of mucinases, glycosyltransferases, antibodies, and lectins. Finally, it is important to point out that the conformational differences observed in solution in glycopeptides bearing GalNAc-α-1-O-Ser or GalNAc-α-1-O-Thr also can be found in the bound state. This unique characteristic is exploited, for instance, by the enzyme C1GalT1 to broadly glycosylate both acceptor substrates. The findings summarized in this review may contribute to the rational structure-guided development of therapeutic vaccines, novel diagnostic tools for early cancer detection, and new cancer treatments for cancer with tailored anti-Tn or anti-STn antibodies or new drugs to inhibit GalNAc-T isoenzymes.

A cross-sectional serosurvey of SARS-CoV-2 and co-infections in stray cats from the second wave to the sixth wave of COVID-19 outbreaks in Spain.

Severe Acute Respiratory Syndrome Coronavirus 2 is the causative agent of Coronavirus Disease 2019 in humans. Among domestic animals, cats are more susceptible to SARS-CoV-2 than dogs. The detection of anti-SARS-CoV-2 antibodies in seemingly healthy cats and/or infected cats which are in close contact with infected humans has been described. The presence of animals that tested positive by serology or molecular techniques could represent a potential transmission pathway of SARS-CoV-2 that can spill over into urban wildlife. This study analyses the seroprevalence variation of SARS-CoV-2 in stray cats from different waves of outbreaks in a geographical area where previous seroepidemiological information of SARS-CoV-2 was available and investigate if SARS-CoV-2-seropositive cats were exposed to other co-infections causing an immunosuppressive status and/or a chronic disease that could lead to a SARS-CoV-2 susceptibility. For this purpose, a total of 254 stray cats from Zaragoza (Spain) were included. This analysis was carried out by the enzyme-linked immunosorbent assay using the receptor binding domain of Spike antigen and confirmed by serum virus neutralization assay. The presence of co-infections including Toxoplasma gondii, Leishmania infantum, Dirofilaria immitis, feline calicivirus, feline herpesvirus type 1, feline leukemia virus and feline immunodeficiency virus, was evaluated using different serological methods. A seropositivity of 1.57% was observed for SARS-CoV-2 including the presence of neutralizing antibodies in three cats. None of the seropositive to SARS-CoV-2 cats were positive to feline coronavirus, however, four SARS-CoV-2-seropositive cats were also seropositive to other pathogens such as L. infantum, D. immitis and FIV (n = 1), L. infantum and D. immitis (n = 1) and L. infantum alone (n = 1).Considering other pathogens, a seroprevalence of 16.54% was detected for L. infantum, 30.31% for D. immitis, 13.78%, for T. gondii, 83.86% for feline calicivirus, 42.52% for feline herpesvirus type 1, 3.15% for FeLV and 7.87% for FIV.Our findings suggest that the epidemiological role of stray cats in SARS-CoV-2 transmission is scarce, and there is no increase in seropositivity during the different waves of COVID-19 outbreaks in this group of animals. Further epidemiological surveillances are necessary to determine the risk that other animals might possess even though stray cats do not seem to play a role in transmission.

Preclinical Studies of Granulysin-Based Anti-MUC1-Tn Immunotoxins as a New Antitumoral Treatment.

Two granulysin (GRNLY) based immunotoxins were generated, one containing the scFv of the SM3 mAb (SM3GRNLY) and the other the scFv of the AR20.5 mAb (AR20.5GRNLY). These mAb recognize different amino acid sequences of aberrantly O-glycosylated MUC1, also known as the Tn antigen, expressed in a variety of tumor cell types. We first demonstrated the affinity of these immunotoxins for their antigen using surface plasmon resonance for the purified antigen and flow cytometry for the antigen expressed on the surface of living tumor cells. The induction of cell death of tumor cell lines of different origin positive for Tn antigen expression was stronger in the cases of the immunotoxins than that induced by GRNLY alone. The mechanism of cell death induced by the immunotoxins was studied, showing that the apoptotic component demonstrated previously for GRNLY was also present, but that cell death induced by the immunotoxins included also necroptotic and necrotic components. Finally, we demonstrated the in vivo tumor targeting by the immunotoxins after systemic injection using a xenograft model of the human pancreatic adenocarcinoma CAPAN-2 in athymic mice. While GRNLY alone did not have a therapeutic effect, SM3GRNLY and AR20.5GRNLY reduced tumor volume by 42 and 60%, respectively, compared with untreated tumor-bearing mice, although the results were not statistically significant in the case of AR20.5GRNLY. Histological studies of tumors obtained from treated mice demonstrated reduced cellularity, nuclear morphology compatible with apoptosis induction and active caspase-3 detection by immunohistochemistry. Overall, our results exemplify that these immunotoxins are potential drugs to treat Tn-expressing cancers.

O-fucosylation stabilizes the TSR3 motif in thrombospondin-1 by interacting with nearby amino acids and protecting a disulfide bond.

Thrombospondin type-1 repeats (TSRs) are small protein motifs containing six conserved cysteines forming three disulfide bonds that can be modified with an O-linked fucose. Protein O-fucosyltransferase 2 (POFUT2) catalyzes the addition of O-fucose to TSRs containing the appropriate consensus sequence, and the O-fucose modification can be elongated to a Glucose-Fucose disaccharide with the addition of glucose by ß3-glucosyltransferase (B3GLCT). Elimination of Pofut2 in mice results in embryonic lethality in mice, highlighting the biological significance of O-fucose modification on TSRs. Knockout of POFUT2 in HEK293T cells has been shown to cause complete or partial loss of secretion of many proteins containing O-fucosylated TSRs. In addition, POFUT2 is localized to the endoplasmic reticulum (ER) and only modifies folded TSRs, stabilizing their structures. These observations suggest that POFUT2 is involved in an ER quality control mechanism for TSR folding and that B3GLCT also participates in quality control by providing additional stabilization to TSRs. However, the mechanisms by which addition of these sugars result in stabilization are poorly understood. Here, we conducted molecular dynamics (MD) simulations and provide crystallographic and NMR evidence that the Glucose-Fucose disaccharide interacts with specific amino acids in the TSR3 domain in thrombospondin-1 that are within proximity to the O-fucosylation modification site resulting in protection of a nearby disulfide bond. We also show that mutation of these amino acids reduces the stabilizing effect of the sugars in vitro. These data provide mechanistic details regarding the importance of O-fucosylation and how it participates in quality control mechanisms inside the ER.

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.

Serological evidence of SARS-CoV-2 and co-infections in stray cats in Spain.

A new coronavirus known as SARS-CoV-2 emerged in Wuhan in 2019 and spread rapidly to the rest of the world causing the pandemic disease named coronavirus disease of 2019 (COVID-19). Little information is known about the impact this virus can cause upon domestic and stray animals. The potential impact of SARS-CoV-2 has become of great interest in cats due to transmission among domestic cats and the severe phenotypes described recently in a domestic cat. In this context, there is a public health warning that needs to be investigated in relation with the epidemiological role of this virus in stray cats. Consequently, in order to know the impact of the possible transmission chain, blood samples were obtained from 114 stray cats in the city of Zaragoza (Spain) and tested for SARS-CoV-2 and other selected pathogens susceptible to immunosuppression including Toxoplasma gondii, Leishmania infantum, feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV) from January to October 2020. Four cats (3.51%), based on enzyme-linked immunosorbent assay (ELISA) using the receptor binding domain (RBD) of Spike antigen, were seroreactive to SARS-CoV-2. T. gondii, L. infantum, FeLV and FIV seroprevalence was 12.28%, 16.67%, 4.39% and 19.30%, respectively. Among seropositive cats to SARS-CoV-2, three cats were also seropositive to other pathogens including antibodies detected against T. gondii and FIV (n = 1); T. gondii (n = 1); and FIV and L. infantum (n = 1). The subjects giving positive for SARS-CoV-2 were captured in urban areas of the city in different months: January 2020 (2/4), February 2020 (1/4) and July 2020 (1/4). This study revealed, for the first time, the exposure of stray cats to SARS-CoV-2 in Spain and the existence of concomitant infections with other pathogens including T. gondii, L. infantum and FIV, suggesting that immunosuppressed animals might be especially susceptible to SARS-CoV-2 infection.

Anticancer and Structure Activity Relationship of Non-Symmetrical Choline Kinase Inhibitors.

Choline kinase inhibitors are an outstanding class of cytotoxic compounds useful for the treatment of different forms of cancer since aberrant choline metabolism is a feature of neoplastic cells. Here, we present the most in-depth structure-activity relationship studies of an interesting series of non-symmetric choline kinase inhibitors previously reported by our group: 3a-h and 4a-h. They are characterized by cationic heads of 3-aminophenol bound to 4-(dimethylamino)- or 4-(pyrrolidin-1-yl)pyridinium through several linkers. These derivatives were evaluated both for their inhibitory activity on the enzyme and their antiproliferative activity in a panel of six human tumor cell lines. The compounds with the N-atom connected to the linker (4a-h) show the best inhibitory results, in the manner of results supported by docking studies. On the contrary, the best antiproliferative compounds were those with the O-atom bounded to the linker (3a-h). On the other hand, as was predictable in both families, the inhibitory effect on the enzyme is better the shorter the length of the linker. However, in tumor cells, lipophilicity and choline uptake inhibition could play a decisive role. Interestingly, compounds 3c and 4f, selected for both their ability to inhibit the enzyme and good antiproliferative activity, are endowed with low toxicity in non-tumoral cells (e.g., human peripheral lymphocytes) concerning cancer cells. These compounds were also able to induce apoptosis in Jurkat leukemic cells without causing significant variations of the cell cycle. It is worth mentioning that these derivatives, besides their inhibitory effect on choline kinase, displayed a modest ability to inhibit choline uptake thus suggesting that this mechanism may also contribute to the observed cytotoxicity.

Fucosyltransferase-specific inhibition via next generation of fucose mimetics.

The ability to custom-modify cell surface glycans holds great promise for treatment of a variety of diseases. We propose a glycomimetic of l-fucose that markedly inhibits the creation of sLeX by FTVI and FTVII, but has no effect on creation of LeX by FTIX. Our findings thus indicate that selective suppression of sLex display can be achieved, and STD-NMR studies surprisingly reveal that the mimetic does not compete with GDP-fucose at the enzymatic binding site.

Recent advances in the design of choline kinase α inhibitors and the molecular basis of their inhibition.

Upregulated choline metabolism, characterized by an increase in phosphocholine (PCho), is a hallmark of oncogenesis and tumor progression. Choline kinase (ChoK), the enzyme responsible for PCho synthesis, has consequently become a promising drug target for cancer therapy and as such a significant number of ChoK inhibitors have been developed over the last few decades. More recently, due to the role of this enzyme in other pathologies, ChoK inhibitors have also been used in new therapeutic approaches against malaria and rheumatoid arthritis. Here, we review research results in the field of ChoKα inhibitors from their synthesis to the molecular basis of their binding mode. Strategies for the development of inhibitors and their selectivity on ChoKα over ChoKß, the plasticity of the choline-binding site, the discovery of new exploitable binding sites, and the allosteric properties of this enzyme are highlighted. The outcomes summarized in this review will be a useful guide to develop new multifunctional potent drugs for the treatment of various human diseases.

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.

Synthesis, biological evaluation, in silico modeling and crystallization of novel small monocationic molecules with potent antiproliferative activity by dual mechanism.

Seeking for new anticancer drugs with strong antiproliferative activity and simple molecular structure, we designed a novel series of compounds based on our previous reported pharmacophore model composed of five moieties. Antiproliferative assays on four tumoral cell lines and evaluation of Human Choline Kinase CKα1 enzymatic activity was performed for these compounds. Among tested molecules, those ones with biphenyl spacer showed betters enzymatic and antiproliferative activities (n-v). Docking and crystallization studies validate the hypothesis and confirm the results. The most active compound (t) induces a significant arrest of the cell cycle in G0/G1 phase that ultimately lead to apoptosis, following the mitochondrial pathway, as demonstrated for other choline kinase inhibitors. However additional assays reveal that the inhibition of choline uptake could also be involved in the antiproliferative outcome of this class of compounds.

Production of a Granulysin-Based, Tn-Targeted Cytolytic Immunotoxin Using Pulsed Electric Field Technology.

Granulysin is a protein present in the granules of human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, with cytolytic activity against microbes and tumors. Previous work demonstrated the therapeutic effect of the intratumoral injection of recombinant granulysin and of the systemic injection of an immunotoxin between granulysin and the anti-carcinoembryonic antigen single-chain Fv antibody fragment MFE23, which were produced in the yeast Pichia pastoris. In the present work, we developed a second immunotoxin combining granulysin and the anti-Tn antigen single-chain Fv antibody fragment SM3, that could have a broader application in tumor treatment than our previous immunotoxin. In addition, we optimized a method based on electroporation by pulsed electric field (PEF) to extract the remaining intracellular protein from yeast, augmenting the production and purificiation yield. The immunotoxin specifically recognized the Tn antigen on the cell surface. We also compared the thermal stability and the cytotoxic potential of the extracellular and intracellular immunotoxins on Tn-expressing human cell lines, showing that they were similar. Moreover, the bioactivity of both immunotoxins against several Tn+ cell lines was higher than that of granulysin alone.

Ser and Thr acceptor preferences of the GalNAc-Ts vary among isoenzymes to modulate mucin-type O-glycosylation.

A family of polypeptide GalNAc-transferases (GalNAc-Ts) initiates mucin-type O-glycosylation, transferring GalNAc onto hydroxyl groups of Ser and Thr residues of target substrates. The 20 GalNAc-T isoenzymes in humans are classified into nine subfamilies according to sequence similarity. GalNAc-Ts select their sites of glycosylation based on weak and overlapping peptide sequence motifs, as well prior substrate O-GalNAc glycosylation at sites both remote (long-range) and neighboring (short-range) the acceptor. Together, these preferences vary among GalNAc-Ts imparting each isoenzyme with its own unique specificity. Studies on the first identified GalNAc-Ts showed Thr acceptors were preferred over Ser acceptors; however studies comparing Thr vs. Ser glycosylation across the GalNAc-T family are lacking. Using a series of identical random peptide substrates, with single Thr or Ser acceptor sites, we determined the rate differences (Thr/Ser rate ratio) between Thr and Ser substrate glycosylation for 12 isoenzymes (representing 7 GalNAc-T subfamilies). These Thr/Ser rate ratios varied across subfamilies, ranging from ~2 to ~18 (for GalNAc-T4/GalNAc-T12 and GalNAc-T3/GalNAc-T6, respectively), while nearly identical Thr/Ser rate ratios were observed for isoenzymes within subfamilies. Furthermore, the Thr/Ser rate ratios did not appreciably vary over a series of fixed sequence substrates of different relative activities, suggesting the ratio is a constant for each isoenzyme against single acceptor substrates. Finally, based on GalNAc-T structures, the different Thr/Ser rate ratios likely reflect differences in the strengths of the Thr acceptor methyl group binding to the active site pocket. With this work, another activity that further differentiates substrate specificity among the GalNAc-Ts has been identified.

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.

Synthesis, conformational analysis and in vivo assays of an anti-cancer vaccine that features an unnatural antigen based on an sp2-iminosugar fragment.

The Tn antigen (GalNAc-α-1-O-Thr/Ser) is a well-known tumor-associated carbohydrate determinant. The use of glycopeptides that incorporate this structure has become a significant and promising niche of research owing to their potential use as anticancer vaccines. Herein, the conformational preferences of a glycopeptide with an unnatural Tn antigen, characterized by a threonine decorated with an sp2-iminosugar-type α-GalNAc mimic, have been studied both in solution, by combining NMR spectroscopy and molecular dynamics simulations, and in the solid state bound to an anti-mucin-1 (MUC1) antibody, by X-ray crystallography. The Tn surrogate can mimic the main conformer sampled by the natural antigen in solution and exhibits high affinity towards anti-MUC1 antibodies. Encouraged by these data, a cancer vaccine candidate based on this unnatural glycopeptide and conjugated to the carrier protein Keyhole Limpet Hemocyanin (KLH) has been prepared and tested in mice. Significantly, the experiments in vivo have proved that this vaccine elicits higher levels of specific anti-MUC1 IgG antibodies than the analog that bears the natural Tn antigen and that the elicited antibodies recognize human breast cancer cells with high selectivity. Altogether, we compile evidence to confirm that the presentation of the antigen, both in solution and in the bound state, plays a critical role in the efficacy of the designed cancer vaccines. Moreover, the outcomes derived from this vaccine prove that there is room for exploring further adjustments at the carbohydrate level that could contribute to designing more efficient cancer vaccines.