Specific Glycosidases Research Articles

Analysis of glycosylated cardiolipins from thermophilic bacteria using GC–MS and LC–ESI–MS/MS methods

Unusual glucose-substituted cardiolipins (Glcx-CLs) in three genera of thermophilic bacteria, having more than one glycosidically linked glucose to the hydroxyl of the central glycerol of Glcx-CLs were identified for the first time in thermophilic bacteria of the genera Geobacillus, Meiothermus, and Thermus. The number of glucoses reached up to five units. The structure of glycosidically linked oligosaccharides was determined based on shotgun analysis MS (electrospray high-resolution tandem mass spectrometry), partially methylated alditol acetates were identified by GC–MS, both electron ionization (EI) and positive chemical ionization (PCI), hydrophilic interaction liquid chromatography (HILIC) separation and identification of CLs glycosides by high resolution MS–ESI, and digestion by specific glycosidases.

N-linked glycans: an underappreciated key determinant of T cell development, activation, and function.

N-linked glycosylation is a post-translational modification that results in the decoration of newly synthesized proteins with diverse types of oligosaccharides that originate from the amide group of the amino acid asparagine. The sequential and collective action of multiple glycosidases and glycosyltransferases are responsible for determining the overall size, composition, and location of N-linked glycans that become covalently linked to an asparagine during and after protein translation. A growing body of evidence supports the critical role of N-linked glycan synthesis in regulating many features of T cell biology, including thymocyte development and tolerance, as well as T cell activation and differentiation. Here, we provide an overview of how specific glycosidases and glycosyltransferases contribute to the generation of different types of N-linked glycans and how these post-translational modifications ultimately regulate multiple facets of T cell biology.

Hybrid Organomineral Carriers for Therapeutic Proteins

—Drug delivery systems are developed to provide a necessary concentration and prolonged effect of the active substance in the organism. Orally administered protein preparations require a protection from the proteolysis in the gastrointestinal tract. Biocompatible hydrophilic polysaccharides in the composition of the matrix are especially promising, since they do not irritate the intestine and are gradually cleaved by specific glycosidases, releasing a therapeutic agent. The introduction of an insoluble porous mineral matrix into the composition of the carrier allows us to increase the concentration of the therapeutic agent in the matrix without a significant increase in the volume of the drug tablet form. In this work, a new original organomineral carrier was created based on heat-treated crushed clinoptilolite zeolite in combination with natural polysaccharides of red algae (agar–agar, agarose, and carrageenan). Granular and finely dispersed clinoptilolites in the composition of the matrix are loaded with a promising therapeutic agent (Bacillus pumilus ribonuclease (binase)), which shows a selective cytotoxicity to tumor cells. It was established that both granular and finely dispersed zeolites in a complex with polysaccharides retain the protein better as compared with pure zeolites and provide a gradual complete release of the enzyme in 18 h; at the same time, binase retains a catalytic activity and causes apoptosis in up to 23.8% of the population of HuTu80 human duodenal adenocarcinoma cells. Data obtained substantiate the prospects of designing dosage forms based on the studied organomineral carriers.

Hybrid Organomineral Carriers for Therapeutic Proteins

Drug delivery systems are designed to provide the necessary concentration and prolonged effect of the active substance in the body. Orally administered protein preparations require protection from proteolysis in the gastrointestinal tract. Biocompatible hydrophilic polysaccharides in the matrix are especially promising, since they do not irritate the intestines and are gradually cleaved by specific glycosidases, releasing the therapeutic agent. The introduction of an insoluble porous mineral matrix into composition of carrier makes it possible to increase the concentration of therapeutic in matrix without a significant increase in the volume of drug tablet form. In this work, a new original organic mineral carrier based on heat-treated crushed clinoptiolite zeolite in combination with natural polysaccharides from red seaweeds – agar-agar, agarose, and carrageenan was created. Granular and finely dispersed clinoptiolites in the matrix are loaded with a promising therapeutic agent, Bacillus pumilus ribonuclease (binase), which exhibits selective cytotoxicity to tumor cells. It has been established that both granular and finely dispersed zeolites in combination with polysaccharides retain protein better than pure zeolites and provide a gradual complete release of enzyme in 18 hours. At the same time, the enzyme retains its catalytic activity and induces apoptosis up to 23.8% cells of the human duodenal adenocarcinoma HuTu80. The data obtained substantiate the prospects of dosage forms based on the used organomineral carriers designing.

Characterization and Spatial Mapping of the Human Gut Metasecretome.

Bacterially secreted proteins play an important role in microbial physiology and ecology in many environments, including the mammalian gut. While gut microbes have been extensively studied over the past decades, little is known about the proteins that they secrete into the gastrointestinal tract. In this study, we developed and applied a computational pipeline to a comprehensive catalog of human-associated metagenome-assembled genomes in order to predict and analyze the bacterial metasecretome of the human gut, i.e., the collection of proteins secreted out of the cytoplasm by human gut bacteria. We identified the presence of large and diverse families of secreted carbohydrate-active enzymes and assessed their phylogenetic distributions across different taxonomic groups, which revealed an enrichment in Bacteroidetes and Verrucomicrobia. By mapping secreted proteins to available metagenomic data from endoscopic sampling of the human gastrointestinal tract, we specifically pinpointed regions in the upper and lower intestinal tract along the lumen and mucosa where specific glycosidases are secreted by resident microbes. The metasecretome analyzed in this study constitutes the most comprehensive list of secreted proteins produced by human gut bacteria reported to date and serves as a useful resource for the microbiome research community. IMPORTANCE Bacterially secreted proteins are necessary for the proper functioning of bacterial cells and communities. Secreted proteins provide bacterial cells with the ability to harvest resources from the exterior, import these resources into the cell, and signal to other bacteria. In the human gut microbiome, these actions impact host health and allow the maintenance of a healthy gut bacterial community. We utilized computational tools to identify the major components of human gut bacterially secreted proteins and determined their spatial distribution in the gastrointestinal tract. Our analysis of human gut bacterial secreted proteins will allow a better understanding of the impact of gut bacteria on human health and represents a step toward identifying new protein functions with interesting applications in biomedicine and industry.

Immunological Compatibility of Novel Enzyme-Converted Universal Blood Donor Cells

Introduction: In transfusion, unintentional mismatching of blood groups remains one of the most common causes of serious and sometimes fatal adverse reactions. Scarcity of certain blood types is another major transfusion challenge. The ABO blood group specificity is determined by the terminal monosaccharides, N-acetylgalactosamine on A and galactose on B RBCs. To create a universal blood supply, cell surface engineering has been studied as a method to minimize the immunological rejection of donor cells. Enzymatic removal of immunodominant sugars would uncover universally acceptable H-antigen (O group RBC). However, enzymatic conversion of A-type RBCs was lagging due to a lack of very active and specific glycosidases and the complex nature of the A blood group antigen. In this research, the performance of two novel enzymes that act together extremely efficiently to convert A-type RBCs to universal donor O RBCs, with high specificity for A antigen, is evaluated. Optimal conversion conditions are explored, as well as the immunological compatibility of the converted RBCs. Materials & Methods: Whole blood samples are collected from consenting A donors (with approval from the University of British Columbia clinical research ethics board), washed and diluted to 10 % hematocrit in PBS buffer (pH7.4). The washed RBCs were treated with the newly developed glycosidases for 1 h at 37 oC. The efficiency of the newly developed glycosidases and the characterization of enzyme converted O (ECO)-RBCs were evaluated by gel-column based micro typing system (MTS) cards against A antigen and flow cytometry using anti A and H antibodies. Safety and compatibility of ECO-RBCs were assessed by blood group serology studies. In brief, ECO-RBCs were incubated in Rh-matched sera collected from diverse donors and any serum reactivity against ECO-RBCs was assessed using anti IgG MTS cards and flow cytometry using anti C3d (complement protein) antibody. To further confirm the cross-matching of ECO-RBCs, the monocyte monolayer assay (MMA), a clinically used method, was performed using monocytes collected from B and O blood donors to assess the phagocytic activity against ECO-RBCs pre-incubated with serum samples. Phagocytic activity against ECO-RBCs was calculated using the monocyte index (MI). Results: Anti A MTS cards and flow cytometry analysis of antigen A and H expression, after enzymatic treatment, confirmed successful conversion of A blood group to universal O group. Our studies showed no significant hemolysis and no complement protein (C3d) deposition on ECO-RBCs pre-incubated with diverse sera, demonstrating the compatibility. Serology studies showed that ECO-RBCs are compatible with most sera (~50 donors) studied under transfusion conditions, though serum reactivity was shown by some O and B sera. A MI value of ≤ 6 % was obtained for all ECO-RBCs indicating minimal risk of an acute hemolytic reaction associated with ECO-RBC transfusion. Conclusions: We have developed novel glycosidases that efficiently remove immunodominant A antigen from RBC surfaces with high specificity to create universal blood donor cells at neutral pH and at enzyme concentrations that are much lower than those previously required. Based on preliminary results, ECO-RBCs have the potential to increase donor blood supply and thereby greatly advance the field of transfusion.

Linking oxidative DNA lesion 8-OxoG to tumor development and progression.

Cells of the aerobic metabolic organism are inevitably subjected to the damage from reactive oxygen species (ROS). ROS cause multiple forms of DNA damage, among which the oxidation product of guanine G 8-hydroxyguanine (8-oxoG) is the most frequent DNA oxidative damage, recognized by the specific glycosidase OGG1 that initiates the base excision repair pathway. If left unrepaired, 8-oxoG may pair with A instead of C, leading to a mutation of G: C to T: A during replication. Thus, the accumulation of 8-oxoG or the abnormal OGG1 repair is thought to affect gene function, which in turn leads to the development of tumor or aging-related diseases. However, a series of recent studies have shown that 8-oxoG tends to be produced in regulatory regions of the genome. 8-oxoG can be regarded as an epigenetic modification, while OGG1 is a specific reader of this information. Substrate recognition, binding or resection by OGG1 can cause DNA conformation changes or affect histone modifications, causing up-regulation or down-regulation of genes with different properties. Thus, in addition to the potential genotoxicity, the association of guanine oxidative damage with development of tumors is closely related to its aberrant initiation of gene expression through epigenetic mechanisms. In this review, we summarize the underlying mechanism of 8-oxoG and repair enzyme OGG1 in tumor development and progression, with aims to interpret the relationship between DNA oxidative damage and tumor from a new perspective, and provide new ideas and targets for tumor treatment.

One of the two N-glycans on the human Gb3/CD77 synthase is essential for its activity and allosterically regulates its function

N-glycosylation is a posttranslational modification that influences many protein properties, such as bioactivity, folding or solubility. The same principles apply to key enzymes in glycosylation pathways, including glycosyltransferases, that also undergoing N-glycosylation, changes in which may affect their activity. Human Gb3/CD77 synthase (encoded by A4GALT) is a Golgi-resident glycosyltransferase, which catalyzes the synthesis of Galα1→4Gal disaccharide on glycosphingolipid- and glycoprotein-derived acceptors, creating Gb3 or P1 antigens and P1 glycotopes (Galα1→4Galβ1→4GlcNAc-R), respectively. The molecules that contain Galα1→4Gal serve as receptors for pathogens and Shiga toxins, which are the major virulence factors of Shiga toxin-producing Escherichia coli (STEC). Human Gb3/CD77 synthase contains two N-glycosylation sites at positions N121 and N203. Using the recombinant soluble glycovariants of human Gb3/CD77 synthase with mutated N-glycosylation sequons expressed in HEK293E cells, we show that the glycovariants devoid of N-glycan at position N203 or simultaneously at N121 and N203 sites reveal no enzymatic activity. In contrast, the N-glycan at position N121 plays a negligible role, whereas the presence of both N-glycans is required for efficient secretion of the enzyme. Moreover, utilizing specific glycosidases, we have found that the fully N-glycosylated enzyme contains one complex and one hybrid/oligomannose N-glycan, while single mutants contain only the complex type. Finally, in silico analysis using the AlphaFold enzyme model showed that N-glycan attached to N203 sequon is located in a protein motif near the active site and may allosterically influence the activity. All these findings highlight the prerequisite role of N-glycosylation in human Gb3/CD77 synthase activity (N203 sequon) and solubility (both N121 and N203), with a particularly prominent role of N-glycan at position N203 in the regulation of enzyme activity.

Participation of Carbohydrate Structures in the Formation of Complex Structure of Hemocyanins from Haliotis tuberculata

Glycosylation plays an important role in many important processes in organisms. Their participation in the construction of the tertiary structure and immunotherapeutic activity of the hemocyanin Rapana venosa (RvH) from the Black Sea has been presented. Based on these results, the importance of glycans in organization of the structure of hemocyanin from the abdominal mollusk Haliotis tuberculata (HtH) was also studied.
 After incubation of both isoforms HtH1 and HtH2 with yeast Saccharomyces cerevisiae and enzyme Zymolyase, fractions with lower molecular weight (MW) between 45 and 65 kDa, which corresponds to MW of functional units (FUs) of hemocyanins, was identified. A comparative analysis of 10% SDS PAGE assays confirmed the depolymerization of the subunits HtH1 and HtH2 due to the specific glycosidase in the yeast S. cerevisiae and the enzyme Zymolyase. The presented results confirm our hypothesis that glycans are involved in the formation of the structural subunits of HtH.

Isolation and identification of Enterococcus gallinarum P581a, a strain of intestinal bacteria deglycosylating flavone C-glycosides.

Flavone C-glycosides are not easily degraded because of their strong C-C bond between sugar moieties and aglycones. However, some bacteria such as intestinal species can produce specific enzymes to degrade them. In this study, a bacterial strain P581a, which is capable of deglycosylating flavone C-glycosides, was isolated from human intestinal bacteria and was identified as Enterococcus gallinarum by morphological examination, physiological and biochemical analysis and 16S rRNA gene sequencing. This strain may produce a specific flavonoside glycosidase. The activity of the enzyme in the culture medium containing different quantity of carbon sources was also studied, and it was found that the content of carbon sources is negatively correlated with the deglycosylation efficiency of this strain.

Encapsulation of Combi-CLEAs of Glycosidases in Alginate Beads and Polyvinyl Alcohol for Wine Aroma Enhancement

The aromatic expression of wines can be enhanced by the addition of specific glycosidases, although their poor stability remains a limitation. Coimmobilization of glycosidases as cross-linked enzyme aggregates (combi-CLEAs) offers a simple solution yielding highly stable biocatalysts. Nevertheless, the small particle size of combi-CLEAs hinders their recovery, preventing their industrial application. Encapsulation of combi-CLEAs of glycosidases in alginate beads and in polyvinyl alcohol is proposed as a solution. Combi-CLEAS of β-d-glucosidase and α-l-arabinofuranosidase were prepared and encapsulated. The effects of combi-CLEA loading and particle size on the expressed specific activity (IU/gbiocatalyst) of the biocatalysts were evaluated. Best results were obtained with 2.6 mm diameter polyvinyl alcohol particles at a loading of 60 mgcombi-CLEA/gpolyvinyl alcohol, exhibiting activities of 1.9 and 1.0 IU/gbiocatalyst for β-d-glucosidase and α-l-arabinofuranosidase, respectively. Afterwards, the stability of the biocatalysts was tested in white wine. All the encapsulated biocatalysts retained full activity after 140 incubation days, outperforming both free enzymes and nonencapsulated combi-CLEAs. Nevertheless, the alginate-encapsulated biocatalysts showed a brittle consistency, making recovery unfeasible. Conversely, the polyvinyl-encapsulated biocatalyst remained intact throughout the assay. The encapsulation of combi-CLEAs in polyvinyl alcohol proved to be a simple methodology that allows their recovery and reuse to harness their full catalytic potential.

Novel Acinetobacter baumannii Myovirus TaPaz Encoding Two Tailspike Depolymerases: Characterization and Host-Recognition Strategy.

Acinetobacter baumannii, one of the most significant nosocomial pathogens, is capable of producing structurally diverse capsular polysaccharides (CPSs) which are the primary receptors for A. baumannii bacteriophages encoding polysaccharide-degrading enzymes. To date, bacterial viruses specifically infecting A. baumannii strains belonging to more than ten various capsular types (K types) were isolated and characterized. In the present study, we investigate the biological properties, genomic organization, and virus–bacterial host interaction strategy of novel myovirus TaPaz isolated on the bacterial lawn of A. baumannii strain with a K47 capsular polysaccharide structure. The phage linear double-stranded DNA genome of 93,703 bp contains 178 open reading frames. Genes encoding two different tailspike depolymerases (TSDs) were identified in the phage genome. Recombinant TSDs were purified and tested against the collection of A. baumannii strains belonging to 56 different K types. One of the TSDs was demonstrated to be a specific glycosidase that cleaves the K47 CPS by the hydrolytic mechanism.

A guide to the composition and functions of the extracellular matrix.

Extracellular matrix (ECM) is a dynamic 3-dimensional network of macromolecules that provides structural support for the cells and tissues. Accumulated knowledge clearly demonstrated over the last decade that ECM plays key regulatory roles since it orchestrates cell signaling, functions, properties and morphology. Extracellularly secreted as well as cell-bound factors are among the major members of the ECM family. Proteins/glycoproteins, such as collagens, elastin, laminins and tenascins, proteoglycans and glycosaminoglycans, hyaluronan, and their cell receptors such as CD44 and integrins, responsible for cell adhesion, comprise a well-organized functional network with significant roles in health and disease. On the other hand, enzymes such as matrix metalloproteinases and specific glycosidases including heparanase and hyaluronidases contribute to matrix remodeling and affect human health. Several cell processes and functions, among them cell proliferation and survival, migration, differentiation, autophagy, angiogenesis, and immunity regulation are affected by certain matrix components. Structural alterations have been also well associated with disease progression. This guide on the composition and functions of the ECM gives a broad overview of the matrisome, the major ECM macromolecules, and their interaction networks within the ECM and with the cell surface, summarizes their main structural features and their roles in tissue organization and cell functions, and emphasizes the importance of specific ECM constituents in disease development and progression as well as the advances in molecular targeting of ECM to design new therapeutic strategies.

Efficient Preparation and Bioactivity Evaluation of Glycan-Defined Glycoproteins.

Owing to the generation of heterogeneous glycoproteins in cells, it is highly difficult to study glycoprotein-mediated biological events and to develop biomedical agents. Thus, general and efficient methods to prepare homogeneous glycoproteins are in high demand. Herein, we report a general method for the efficient preparation of homogeneous glycoproteins that utilizes a combination of genetic code expansion and chemoselective ligation techniques. In the protocol to produce glycan-defined glycoproteins, an alkyne tag-containing protein, generated by genetic encoding of an alkynylated unnatural amino acid, was quantitatively coupled via click chemistry to versatile azide-appended glycans. The glycoproteins produced by the present strategy were found to recognize mammalian cell-surface lectins and enter the cells through lectin-mediated internalization. Also, cell studies exhibited that the glycoprotein containing multiple mannose-6-phosphate residues enters diseased cells lacking specific lysosomal glycosidases by binding to the cell-surface M6P receptor, and subsequently migrates to lysosomes for efficient degradation of stored glycosphingolipids.

Enzymatic Synthesis of a Fluorogenic Reporter Substrate and the Development of a High-Throughput Assay for Fucosyltransferase VIII Provide a Toolkit to Probe and Inhibit Core Fucosylation.

We report a straightforward enzymatic synthesis of the 4-methylumbelliferyl glycoside of a complex-type oligosaccharide substrate for core fucosylation. We demonstrate the use of this synthetic glycoconjugate in a newly developed enzyme assay to probe the activity and inhibition of fucosyltransferase VIII, which catalyzes the core fucosylation of N-glycans on eukaryotic glycoproteins. In this fucosyltransferase assay, we use the fluorogenic probe and a specific glycosidase in a sequentially coupled enzyme reaction to distinguish an unmodified 4-methylumbelliferyl oligosaccharide probe from a fucosylated probe. Our findings show that this strategy is very sensitive and specific in its detection of enzyme activity and can even be used for analyzing impure tissue lysate samples.

Mechanisms of Acinetobacter baumannii Capsular Polysaccharide Cleavage by Phage Depolymerases

Aerobic gram-negative bacterium Acinetobacter baumannii has recently become one of the most relevant pathogens associated with hospital-acquired infections worldwide. A. baumannii produces a capsule around the cell, which represents a thick viscous layer of structurally variable capsular polysaccharide (CPS). The capsule protects the bacteria against unfavorable environmental factors and biological systems, including bacteriophages and host immune system. Many A. baumannii phages have structural depolymerases (tailspikes) that specifically recognize and digest bacterial CPS. In this work, we studied the interaction of tailspike proteins of four lytic depolymerase-carrying phages with A. baumannii CPS. Depolymerases of three bacteriophages (Fri1, AS12, and BS46) were identified as specific glycosidases that cleave the CPS of A. baumannii strains 28, 1432, and B05, respectively, by the hydrolytic mechanism. The gp54 depolymerase from bacteriophage AP22 was characterized as a polysaccharide lyase that cleaves the CPS of A. baumannii strain 1053 by β-elimination at hexuronic acid (ManNAcA) residues.

Plant Glycosides and Glycosidases: A Treasure-Trove for Therapeutics.

Plants contain numerous glycoconjugates that are metabolized by specific glucosyltransferases and hydrolyzed by specific glycosidases, some also catalyzing synthetic transglycosylation reactions. The documented value of plant-derived glycoconjugates to beneficially modulate metabolism is first addressed. Next, focus is given to glycosidases, the central theme of the review. The therapeutic value of plant glycosidases is discussed as well as the present production in plant platforms of therapeutic human glycosidases used in enzyme replacement therapies. The increasing knowledge on glycosidases, including structure and catalytic mechanism, is described. The novel insights have allowed the design of functionalized highly specific suicide inhibitors of glycosidases. These so-called activity-based probes allow unprecedented visualization of glycosidases cross-species. Here, special attention is paid on the use of such probes in plant science that promote the discovery of novel enzymes and the identification of potential therapeutic inhibitors and chaperones.

Influence of a 4'-substituent on the efficiency of flavonol-based fluorescent indicators of β-glycosidase activity.

This article presents novel fluorescent probes, based on the excited-state intramolecular proton transfer (ESIPT) phenomenon and flavonols, sensitive to the action of specific glycosidases. 4'-Substituted flavonols were synthesized, using various approaches, and glycosylated with d-glucose, N-acetyl-d-glucosamine and d-glucuronic acid. Evaluation of the β-glycosidase activities was performed in neutral and acidic pH. In all the cases examined, an acidic environment accelerated enzymatic hydrolysis. It was demonstrated that the 4'-chloroflavonyl glycosides of all sugars tested, both in neutral and acidic pH, are the ones most sensitive to the presence of hydrolase. In turn, 4'-dimethylaminoflavonyl glucoside is not sensitive to glucosidase action at all. Generally, the rate of enzymatic hydrolysis increases as the electron-withdrawing nature of the 4'-substituent increases. An exception is the trifluoromethyl group which, in spite of having the most favourable Hammett constant, does not contribute enough to increase the rate of hydrolysis of its glucoside. The presented experimental results are supported by the electrostatic potential (ESP) analysis and related to the mechanisms of glycoside bond enzymatic hydrolysis.

Differential Recognition of Deacetylated PNAG Oligosaccharides by a Biofilm Degrading Glycosidase.

Exopolysaccharides consisting of partially de-N-acetylated poly-β-d-(1→6)-N-acetyl-glucosamine (dPNAG) are key structural components of the biofilm extracellular polymeric substance of both Gram-positive and Gram-negative human pathogens. De-N-acetylation is required for the proper assembly and function of dPNAG in biofilm development suggesting that different patterns of deacetylation may be preferentially recognized by proteins that interact with dPNAG, such as Dispersin B (DspB). The enzymatic degradation of dPNAG by the Aggregatibacter actinomycetemcomitans native β-hexosaminidase enzyme DspB plays a role in biofilm dispersal. To test the role of substrate de-N-acetylation on substrate recognition by DspB, we applied an efficient preactivation-based one-pot glycosylation approach to prepare a panel of dPNAG trisaccharide analogs with defined acetylation patterns. These analogs served as effective DspB substrates, and the rate of hydrolysis was dependent on the specific substrate de-N-acetylation pattern, with glucosamine (GlcN) located +2 from the site of cleavage being preferentially hydrolyzed. The product distributions support a primarily exoglycosidic cleavage activity following a substrate assisted cleavage mechanism, with the exception of substrates containing a nonreducing GlcN that were cleaved endo leading to the exclusive formation of a nonreducing disaccharide product. These observations provide critical insight into the substrate specificity of dPNAG specific glycosidase that can help guide their design as biocatalysts.

Abstract 235: Preclinical activity of an antibody drug conjugate targeting tumor specificmuc1 structural peptide-glycotope

Abstract SAR566658 antibody drug conjugate (ADC) is a humanized DS6 (huDS6) antibody conjugated through a cleavable linker to the cytotoxic maytansinoid derivative DM4 that has been evaluated in the clinical setting. The purpose of our work was; 1) characterize the epitope targeted by anti DS6 on mucin1, 2) determine the prevalence of antigen expression in a patient population and 3) further explore preclinical activities of the ADC. Murine DS6 monoclonal antibody (muDS6) was generated from serous ovary adenocarcinoma immunization of immunocompetent mice. It specifically recognizes a MUC-1 tandem repeat domain in the context of cancer associated glycosylation. CA6-positive MUC-1 carries mucin-type O-linked glycans with α2,3-sialylated and β1,4-galactosylated termini, and antibody binding was abrogated by treating MUC-1 with specific glycosidases that remove either one of these glycan structures. However, the antibody did not bind to synthetic glycans modeled according to the major O-glycans of MUC-1. Our characterization of the peptide-glycotope leads us to conclude that tumor associated glycosylation is essential for the formation of the epitope on the peptide sequence of the MUC-1 tandem repeat domain. CA6 expression was evaluated by immunohistochemistry in paraffin embedded tumor tissue samples: 35.2% of breast cancer patients, 70,1% of ovarian cancer patients and 58,5% of bladder cancer patients have at least 30% of CA6 positive cells with an intensity of 2+/3+ in a multinational population-based study. In pre-clinical in vivo models, SAR566658 induced anti-tumor activity against CA6 positive tumor models of human pancreas, cervix and bladder cancer as well as and 3 Breast Patient-Derived Xenografts (PDX). Efficacy was correlated with MUC1-CA6 expression levels. In 3 additional models, SAR566658 showed anti-tumor activity that was more potent when compared to 3 conventional tubulin cytotoxic agents, docetaxel, vinorelbine and vinblastine. Citation Format: Marc Trombe, Anne Caron, Alexia Tellier, Chantal Carrez, Stephane Guérif, Severine Clavier, Nathalie Karst, Juhani Saarinen, Tero Satomaa, Virve Pitkänen, Olli Aitio, Annamari Heiskanen, Matteo Fassan, Jan Pinkas, Raffaele Baffa, Veronique Blanc, Celine Nicolazzi. Preclinical activity of an antibody drug conjugate targeting tumor specificmuc1 structural peptide-glycotope [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 235.