Protein O-glycosylation Research Articles

Comprehensive analysis of O-glycosylation of amyloid precursor protein (APP) using targeted and multi-fragmentation MS strategy

BackgroundThe aberrant proteolytic processing of amyloid precursor protein (APP) into amyloid β peptide (Aβ) in brain is a critical step in the pathogenesis of Alzheimer's disease (AD). As an O-glycosylated protein, O-glycosylation of APP is considered to be related to Aβ generation. Therefore, comprehensive analysis of APP O-glycosylation is important for understanding its functions. MethodsWe developed a Targeted MS approach with Multi-Fragmentation techniques (TMMF strategy), and successfully characterized O-glycosylation profiling of APP695 expressed in HEK-293 T cells. We calculated relative abundance of glycopeptides with various O-glycosites and O-glycans, and further investigated the alteration of APP O-glycosylation upon TNF-α treatment. ResultsA total of 14 O-glycosites were identified on three glycopeptides of APP, and at least four O-glycans including GalNAc (Tn antigen), core 1, and mono−/di-sialylated core 1 glycans were determinant at the residues of Thr576 and Thr577. We found a dense cluster of truncated O-glycans on the region nearby beginning of E2 domain and high abundance of sialylated O-glycans on the region close to β-cleavage site. Moreover, we also observed that TNF-α could upregulate the expression of APP and the truncated O-glycans on APP in HEK-293 T cell. ConclusionOur study established an intact O-glycopeptide MS analysis strategy for APP O-glycopeptide identification with enhanced fragmentation efficiency and detection sensitivity. These results provide a comprehensive O-glycosylation map of APP expressed in HEK-293 T cell. General significanceThe accurate O-glycosites and O-glycan structures on APP may lead to a better understanding of the roles O-glycosylation plays in the processing and functions of APP.

A literature review on lactopontin and its roles in early life.

ObjectiveOur study aims to review the functions and possible mechanisms of lactopontin (LPN) in early life.BackgroundHuman milk proteins provide a variety of protection and health benefits in early life. One of these multifunctional proteins is LPN, which is osteopontin (OPN) derived from milk.MethodsInformation used to write this paper was collected from Uniprot, PubMed, and Google Scholar, including in vitro, in vivo, and clinical studies.ConclusionsLPN is a highly phosphorylated, O-glycosylated acidic protein and a unique type of OPN, as it presents at the highest concentration and a higher degree of posttranslational modifications (PTMs) in human milk than other tissues and excretions. LPN is present in milk and the intestinal tracts of infants after consumption as a mixture of intact protein and peptides, which can bind diverse integrin and receptors in the target cell and drive downstream signaling pathways. LPN is found to play important roles in developing the immune, intestinal and nervous systems in early life. Moreover, LPN has also shown to support preterm infants’ health when they are especially vulnerable after delivery via animal studies. Additionally, LPN can form protein complex with another milk bioactive protein, lactoferrin (LF), to withstand proteolysis and perform more efficient biological activity. Therefore, LPN showed great potential for early life while more clinical trials and evidence are still emergying.

Display of the human mucinome with defined O-glycans by gene engineered cells

Mucins are a large family of heavily O-glycosylated proteins that cover all mucosal surfaces and constitute the major macromolecules in most body fluids. Mucins are primarily defined by their variable tandem repeat (TR) domains that are densely decorated with different O-glycan structures in distinct patterns, and these arguably convey much of the informational content of mucins. Here, we develop a cell-based platform for the display and production of human TR O-glycodomains (~200 amino acids) with tunable structures and patterns of O-glycans using membrane-bound and secreted reporters expressed in glycoengineered HEK293 cells. Availability of defined mucin TR O-glycodomains advances experimental studies into the versatile role of mucins at the interface with pathogenic microorganisms and the microbiome, and sparks new strategies for molecular dissection of specific roles of adhesins, glycoside hydrolases, glycopeptidases, viruses and other interactions with mucin TRs as highlighted by examples.

Cellular O-Glycome Reporter/Amplification (CORA): Analytical and Preparative Tools to Study Mucin-Type O-Glycans of Living Cells.

Mucin-type O-glycosylation (O-glycans, O-glycome) is among the most biologically important post-translational modification in glycoproteins but O-glycan structural diversity and expression are poorly understood due to the inadequacy of current analytical methods. We recently developed a new tool termed cellular O-glycome reporter/amplification (CORA), which uses O-glycan precursors, benzyl-α-GalNAc (Bn-α-GalNAc) or azido-Bn-α-GalNAc (N3 -Bn-α-GalNAc), as surrogates of protein O-glycosylation. Living cells metabolically convert these precursors to all types of O-GalNAc glycans representative of the cells' capabilities. The amplification and secretion of the O-glycome products greatly facilitates their analysis and functional studies. Here we describe protocols for analytical and preparative applications. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Cellular O-glycome reporter/amplification for the analysis of mucin-type O-glycans from living cells Basic Protocol 2: Preparation of cellular O-glycans from living cells for functional glycomics and glycan microarrays Basic Protocol 3: Conjugation of cellular O-glycans with a bifunctional fluorescent tag Basic Protocol 4: 2D-HPLC purification and MALDI-TOF/MS identification of individual PYAB-Bn-O-glycan.

Novel “mucinomics” platform reveals molecular signatures of cancer in cellular systems and ovarian cancer patient ascites fluid

Mucin domains are densely O-glycosylated modular protein domains found in a wide variety of cell surface and secreted proteins. When strung together in “tandem repeats”, mucin domains can form the large structures characteristic of mucin family proteins, such as MUC1 and MUC16. These canonical mucins are known to be key players in a host of human diseases, especially cancer. However, several other glycoproteins are known to contain mucin domains, such as: CD43, PSGL-1, and CD45, and their mucin domains are key to their molecular functions. However, a comprehensive list of mucin-domain glycoproteins does not exist, and this knowledge is critical in unraveling the role of mucin domains in health and disease. Recently, we characterized a bacterial mucinase, StcE, and used its unique properties to improve analysis of mucin-domain glycoproteins by mass spectrometry. Given StcE's selectivity for mucins, we reasoned that it could be employed to purify mucins from complex mixtures. Thus, the inactivated point mutant of StcE was conjugated to beads and we demonstrated that we could selectively enrich canonical mucins from HeLa cell lysate. However, we were faced with the issue of how to define a mucin domain. Thus, we developed a “mucin candidate algorithm” in order to calculate which human proteins have a high probability of bearing a mucin domain. With this program in-hand, we expanded our “mucinomics” platform to several other cell lines and crude ovarian cancer patient ascites fluid. We demonstrated high mucin overlap between ovarian cancer patients, and the enrichment strategy allowed us to detect hundreds of glycopeptides from the mucin proteins. Finally, we used a KRAS dox-inducible system to show which mucins contribute to molecular bulk at the cell surface. Ultimately, we demonstrate this mucinomics platform can be used in a wide variety of systems to define key molecular signatures of cancer.

A Genetic Approach to Display and Dissect the Cancer‐Associated O‐Glycoepitome

A hallmark of cancer is aberrant protein O-glycosylation with expression of truncated immature O-glycans including the classical Tn, STn, and T pancarcinoma antigens. The truncation of O-glycans not only present epitopes recognized by the natural low affinity antibodies to the TTn glycan haptens, which are the basis for the polyagglutination phenomena, but also expose composite aberrant O-glycopeptide epitopes that are not normally expressed and can elicit high affinity IgG antibodies. Several examples of monoclonal antibodies with high selectivity for truncated O-glycopeptide epitopes, which in effect selectively target the cancer form of O-glycoproteins, have been produced in the past. Studies demonstrate that such epitopes serve as highly cancer-specific targets potentially amenable for potent T-cell engaging strategies, however, the current insight into the abundance and distribution of aberrant O-glycopeptide epitopes and the optimal glycoforms for these remain limited. To systematically explore antibody epitopes available in the cancer-associated O-glycoproteome, we have developed a cell-based platform for display and production of human O-glycoproteins with homogeneous truncated glycoforms. This platform includes CHO and HEK293 cells stably engineered with homogeneous glycosylation capacities for the Tn, STn, and T glycoforms, and a panel of reporter expression constructs containing segments of human mucins and mucin-like domains from O-glycoproteins for display of these with different O-glycans. An overview of these efforts will be presented.

Protective Roles of O‐GlcNAc in Neurodegenerative Diseases

O-linked N-acetylglucosamine (O-GlcNAc) is an abundant form of protein O-glycosylation found in the nucleus, cytoplasm, and mitochondria of all multicellular eukaryotes. This modification is found on several hundred proteins but is surprisingly regulated by only two enzymes; the glycosyltransferase O-GlcNAc transferase (OGT) and the glycoside hydrolase O-GlcNAcase (OGA). Because the substrate of OGT is derived from glucose, the levels of O-GlcNAc within cells reflect environmental glucose availability. O-GlcNAc has also emerged as part of a coordinated response to cellular stresses and levels of this modification increase markedly on exposure to diverse stresses ranging from heat shock to oxidative stress. These observations have stimulated interest in the potential protective roles of increased O-GlcNAc in a range of tissues including in brain, where impaired nutrient utilization has been linked to various neurodegenerative diseases. Remarkably, brain permeable small molecule inhibitors of OGA have been shown to be surprisingly well tolerated in a range of animal models. Moreover, pharmacological agents that increase O-GlcNAc levels in the brains of various transgenic models of Alzheimer Disease (AD) strikingly reduce the progression of disease pathology and neurodegeneration. While these effects have been shown to be remarkably reproducible, and OGA inhibitors have now been found to be generally well tolerated in early clinical trials, the mechanisms by which O-GlcNAc protect against these pathologies and neurodegeneration remain less clear. Here we will discuss new advances in the biochemistry of OGA inhibitors, efforts to create improved inhibitors, and the protective effects of OGA in transgenic models of neurodegenerative diseases. Finally, we will discuss recent progress on various possible protective mechanisms by which increased O-GlcNAc levels confer protection against various neurodegenerative diseases ranging from blockade of aggregation of toxic proteins to the induction of autophagy.

Subcellular coordination of plant cell wall synthesis.

Organelles of the plant cell cooperate to synthesize and secrete a strong yet flexible polysaccharide-based extracellular matrix: the cell wall. Cell wall composition varies among plant species, across cell types within a plant, within different regions of a single cell wall, and in response to intrinsic or extrinsic signals. This diversity in cell wall makeup is underpinned by common cellular mechanisms for cell wall production. Cellulose synthase complexes function at the plasma membrane and deposit their product into the cell wall. Matrix polysaccharides are synthesized by a multitude of glycosyltransferases in hundreds of mobile Golgi stacks, and an extensive set of vesicle trafficking proteins govern secretion to the cell wall. In this review, we discuss the different subcellular locations at which cell wall synthesis occurs, review the molecular mechanisms that control cell wall biosynthesis, and examine how these are regulated in response to different perturbations to maintain cell wall homeostasis.

Plant Protein O-Arabinosylation.

A wide range of proteins with diverse functions in development, defense, and stress responses are O-arabinosylated at hydroxyprolines (Hyps) within distinct amino acid motifs of continuous stretches of Hyps, as found in the structural cell wall extensins, or at non-continuous Hyps as, for example, found in small peptide hormones and a variety of plasma membrane proteins involved in signaling. Plant O-glycosylation relies on hydroxylation of Prolines to Hyps in the protein backbone, mediated by prolyl-4-hydroxylase (P4H) which is followed by O-glycosylation of the Hyp C4-OH group by either galactosyltransferases (GalTs) or arabinofuranosyltranferases (ArafTs) yielding either Hyp-galactosylation or Hyp-arabinosylation. A subset of the P4H enzymes with putative preference to hydroxylation of continuous prolines and presumably all ArafT enzymes needed for synthesis of the substituted arabinose chains of one to four arabinose units, have been identified and functionally characterized. Truncated root-hair phenotype is one common denominator of mutants of Hyp formation and Hyp-arabinosylation glycogenes, which act on diverse groups of O-glycosylated proteins, e.g., the small peptide hormones and cell wall extensins. Dissection of different substrate derived effects may not be regularly feasible and thus complicate translation from genotype to phenotype. Recently, lack of proper arabinosylation on arabinosylated proteins has been shown to influence their transport/fate in the secretory pathway, hinting to an additional layer of functionality of O-arabinosylation. Here, we provide an update on the prevalence and types of O-arabinosylated proteins and the enzymatic machinery responsible for their modifications.

Highly glycosylated MUC1 mediates high affinity L-selectin binding at the human endometrial surface

BackgroundSialyl-Lewis X/L-selectin high affinity binding interactions between transmembrane O-glycosylated mucins proteins and the embryo have been implicated in implantation processes within the human reproductive system. However, the adhesive properties of these mucins at the endometrial cell surface are difficult to resolve due to known discrepancies between in vivo models and the human reproductive system and a lack of sensitivity in current in vitro models. To overcome these limitations, an in vitro model of the human endometrial epithelial was interrogated with single molecule force spectroscopy (SMFS) to delineate the molecular configurations of mucin proteins that mediate the high affinity L-selectin binding required for human embryo implantation.ResultsThis study reveals that MUC1 contributes to both the intrinsic and extrinsic adhesive properties of the HEC-1 cellular surface. High expression of MUC1 on the cell surface led to a significantly increased intrinsic adhesion force (148 pN vs. 271 pN, p < 0.001), whereas this adhesion force was significantly reduced (271 pN vs. 118 pN, p < 0.001) following siRNA mediated MUC1 ablation. Whilst high expression of MUC1 displaying elevated glycosylation led to strong extrinsic (> 400 pN) L-selectin binding at the cell surface, low expression of MUC1 with reduced glycosylation resulted in significantly less (≤200 pN) binding events.ConclusionsAn optimal level of MUC1 together with highly glycosylated decoration of the protein is critical for high affinity L-selectin binding. This study demonstrates that MUC1 contributes to cellular adhesive properties which may function to facilitate trophoblast binding to the endometrial cell surface through the L-selectin/sialyl-Lewis x adhesion system subsequent to implantation.

Effect of decorin and selected glycosylation inhibitors on the adhesion of caruncular epithelial cells of pregnant cows-part I.

Adhesion process ensures the formation of the appropriate connection between mother and foetus during placentation and further placental development, which determines physiological pregnancy course. Extracellular matrix of foetal membranes are a rich source of biologically active proteins, the synthesis of which is regulated by hormones. Depending on the stage of pregnancy, the protein profile of the placenta changes, thanks to which its remodelling is possible. The aim of the study was to evaluate the effect of decorin, as well as selected glycosylation inhibitors on the adhesion of caruncular epithelial cells derived from cows during pregnancy. Placental cells were isolated from healthy, pregnant (2nd and 4th month) cows after slaughter, which allowed for the establishment of 4 primary cell cultures without visible cells of fibroblast morphology. The presence of decorin in cell monolayer and cell lysates was determined by the use of immunocytochemistry and Western blotting, respectively. The viability of cells was evaluated by MTT assay. The adhesion of cells to fibronectin was measured spectrophotometrically. Protein N-glycosylation and O-glycosylation have a modulating effect on the adhesion and viability of placental cells during early-mid pregnancy. Decorin and tunicamycin were shown to have anti-adhesive properties with respect to caruncular cells of the pregnant bovine uterus.

OGP: A Repository of Experimentally Characterized O-glycoproteins to Facilitate Studies on O-glycosylation

Numerous studies on cancers, biopharmaceuticals, and clinical trials have necessitated comprehensive and precise analysis of protein O-glycosylation. However, the lack of updated and convenient databases deters the storage of and reference to emerging O-glycoprotein data. To resolve this issue, an O-glycoprotein repository named OGP was established in this work. It was constructed with a collection of O-glycoprotein data from different sources. OGP contains 9354 O-glycosylation sites and 11,633 site-specific O-glycans mapping to 2133 O-glycoproteins, and it is the largest O-glycoprotein repository thus far. Based on the recorded O-glycosylation sites, an O-glycosylation site prediction tool was developed. Moreover, an OGP-based website is already available (https://www.oglyp.org/). The website comprises four specially designed and user-friendly modules: statistical analysis, database search, site prediction, and data submission. The first version of OGP repository and the website allow users to obtain various O-glycoprotein-related information, such as protein accession Nos., O-glycosylation sites, O-glycopeptide sequences, site-specific O-glycan structures, experimental methods, and potential O-glycosylation sites. O-glycosylation data mining can be performed efficiently on this website, which will greatly facilitate related studies. In addition, the database is accessible from OGP website (https://www.oglyp.org/download.php).

Identification of potential glycoprotein biomarkers in oral squamous cell carcinoma using sweet strategies.

The prevalence of oral squamous cell carcinoma (OSCC) is high in South and Southeast Asia regions. Most OSCC patients are detected at advanced stages low 5-year survival rates. Aberrant expression of glycosylated proteins was found to be associated with malignant transformation and cancer progression. Hence, identification of cancer-associated glycoproteins could be used as potential biomarkers that are beneficial for diagnosis or clinical management of patients. This study aims to identify the differentially expressed glycoproteins using lectin-based glycoproteomics approaches. Serum samples of 40 patients with OSCC, 10 patients with oral potentially malignant disorder (OPMD), and 10 healthy individuals as control group were subjected to two-dimensional gel electrophoresis (2-DE) coupled with lectin Concanavalin A and Jacalin that specifically bind to N- and O-glycosylated proteins, respectively. Five differentially expressed N- and O-glycoproteins with various potential glycosylation sites were identified, namely N-glycosylated α1-antitrypsin (AAT), α2-HS-glycoprotein (AHSG), apolipoprotein A-I (APOA1), and haptoglobin (HP); as well as O-glycosylated AHSG and clusterin (CLU). Among them, AAT and APOA1 were further validated using enzyme-linked immunosorbent assay (ELISA) (n = 120). It was found that AAT and APOA1 are significantly upregulated in OSCC and these glycoproteins are independent risk factors of OSCC. The clinical utility of AAT and APOA1 as potential biomarkers of OSCC is needed for further evaluation.

A glycoengineered antigen exploiting a conserved protein O-glycosylation pathway in the Burkholderia genus for detection of glanders infections

ABSTRACT We recently described a protein O-glycosylation pathway conserved in all species of the Burkholderia genus that results in the synthesis and incorporation of a trisaccharide glycan to membrane-exported proteins. Here, we exploited this system to construct and evaluate a diagnostic tool for glanders. Burkholderia mallei, the causative agent of glanders, is a highly infectious and fatal zoonotic pathogen that infects horses, mules, donkeys, and occasionally humans. A highly sensitive and specific diagnostic tool is crucial for the control, elimination, and eradication of B. mallei infections. We constructed plasmids carrying synthetic genes encoding a modified, previously unannotated Burkholderia glycoprotein containing three glycosylation sequons fused to the cholera toxin B-subunit. The resulting proteins were glycosylated in the B. cenocepacia K56-2 parental strain, but not in glycosylation-deficient mutants, as determined by SDS-PAGE and fluorescent lectin blots. One of these glycoproteins was used as an antigen in ELISA and western blots to screen a panel of serum samples collected from glanders-infected and healthy horses, which were previously investigated by complement fixation test and indirect ELISA based on a semi-purified fraction of B. mallei. We show that ELISA and western blot assays based on our glycoprotein antigen provide 100% specificity, with a sensitivity greater than 88%. The glycoprotein antigen was recognized by serum samples collected from patients infected with B. pseudomallei, B. mallei, B. multivorans, and B. cenocepacia. Our results indicate that protein O-glycosylation in Burkholderia can be exploited as a biomarker for diagnosis of Burkholderia-associated infections.

Free CA125 promotes ovarian cancer cell migration and tumor metastasis by binding Mesothelin to reduce DKK1 expression and activate the SGK3/FOXO3 pathway.

Objective: CA125/MUC16 is an O-glycosylated protein that is expressed on the surfaces of ovarian epithelial cells. This molecule is a widely used tumor-associated marker for diagnosis of ovarian cancer. Recently, CA125 was shown to be involved in ovarian cancer metastasis. The purpose of this study was to investigate the mechanism of CA125 during ovarian cancer metastasis.Methods: We analyzed the Oncomine and CSIOVDB databases to determine the expression levels of DKK1 in ovarian cancer. DKK1 expression levels were upregulated or downregulated and applied with CA125 to Transwell and Western blot assays to ascertain the underlying mechanism by which CA125 stimulates cell migration via the SGK3/FOXO3 pathway. Anti-mesothelin antibodies (anti-MSLN) were used to block CA125 stimulation. Then the expression levels of DKK1were tested by enzyme-linked immunosorbent assay (ELISA) to eliminate the blocking effect of anti-MSLN to CA125 stimulation. Xenograft mouse models were used to detect the effects of CA125 and anti-MSLN on ovarian cancer metastasis in vivo.Results: DKK1 levels were downregulated in ovarian tumor tissues according to the analyses of two databases and significantly correlated with FIGO stage, grade and disease-free survival in ovarian cancer patients. DKK1 levels were downregulated by CA125 stimulation in vitro. Overexpression of DKK1 reversed the ability of exogenous CA125 to mediate cell migration by activating the SGK3/FOXO3 signaling pathway. Anti-MSLN abrogated the DKK1 reduction and increased the apoptosis of ovarian cancer cells. The use of anti-MSLN in xenograft mouse models significantly reduced tumor growth and metastasis accelerated by CA125.Conclusions: These experiments revealed that the SGK3/FOXO3 pathway was activated, wherein decreased expression of DKK1 was caused by CA125, which fuels ovarian cancer cell migration. Mesothelin is a potential therapeutic target for the treatment of ovarian cancer metastasis.

O-glycosylated clusterin as a sensitive marker for diagnosing early stages of prostate cancer.

Prostate-specific antigen (PSA) has been the most popular diagnostic marker for prostate cancer. The frequent occurrence of low PSA values (<10 ng/ml) in patients with highly suspicious prostate cancer, however, has undermined the accuracy of clinical examinations. The aim of this study was to develop a better resolution for diagnosing prostate cancer to overcome the disadvantage of PSA. We focused on the glycosylation status of patients' serum proteins and conducted comprehensive lectin microarray analyses to characterize N- and O-glycans using sera from prostate cancer and benign prostatic diseases. Next, we retrieved candidate serum proteins with characteristic glycan structures using lectin-immobilized beads and identified them by quantitative mass spectrometry using a technique referred to as isobaric tag for relative and absolute quantitation (iTRAQ)labeling. Finally, we constructed a new assay to quantify a candidate glycoprotein with the newly identified glycans. Lectin microarray analyses revealed that sera from patients with prostate cancer had a higher affinity for Jacalin, Amaranthus caudatus (ACA) lectin, and Maclura pomifera (MPA) lectin, compared with that from patients with benign prostatic diseases and normal subjects, suggesting that O-glycosylated proteins are more abundant in sera from patients with prostate cancer. Then, serum glycoproteins preferentially adsorbed onto Jacalin-Agarose as well as biotin-ACA/and biotin-MPA/streptavidin-immobilized magnetic beads were isolated, labeled with iTRAQ, and identified using quantitative mass spectrometry. It was found that the ACA- and MPA-recognizable clusterin was more enriched in patients' sera from prostate cancer compared with those from benign prostatic diseases. Following this discovery, we constructed a Luminex-based assay to quantify O-glycosylated clusterin, in which total serum clusterin was first captured on anti-clusterin antibody-immobilized beads, and then clusterin-associated O-glycans were determined by the pair of biotin-MPA and streptavidin-phycoerythrin. When PSA values registered less than 10 ng/ml, the corresponding serum level of MPA-recognized clusterin determined by this assay was beneficial for distinguishing the patients with prostate cancer from the patients with benign prostatic disease. For PSA values that measure less than 10 ng/ml, the serum O-glycosylated clusterin level can be a complementary indicator for the malignancy of prostate cancer.

Potential of Anti-MUC1 Antibodies as a Targeted Therapy for Gastrointestinal Cancers.

Gastrointestinal cancers (GI) account for 26% of cancer incidences globally and 35% of all cancer-related deaths. The main challenge is to target cancer specific antigens. Mucins are heavily O-glycosylated proteins overexpressed in different cancers. The transmembrane glycoprotein MUC1 is the most likeable target for antibodies, owing to its specific overexpression and aberrant glycosylation in many types of cancers. For the past 30 years, MUC1 has remained a possible diagnostic marker and therapeutic target. Despite initiation of numerous clinical trials, a comprehensively effective therapy with clinical benefit is yet to be achieved. However, the interest in MUC1 as a therapeutic target remains unaltered. For all translational studies, it is important to incorporate updated relevant research findings into therapeutic strategies. In this review we present an overview of the antibodies targeting MUC1 in GI cancers, their potential role in immunotherapy (i.e., antibody-drug and radioimmunoconjugates, CAR-T cells), and other novel therapeutic strategies. We also present our perspectives on how the mechanisms of action of different anti-MUC1 antibodies can target specific hallmarks of cancer and therefore be utilized as a combination therapy for better clinical outcomes.

Glycoproteomic analysis identifies cryptdin-related sequence 1 as O-glycosylated protein modified with α1,2-fucose in the small intestine

The modification of galactose with α1,2-fucose is involved in symbiosis with intestinal bacteria and elimination of pathogenic bacteria. It is postulated that α1,2-fucosylated mucin secreted from goblet cells is involved in defending an organism against infections, but the detailed molecular mechanisms are yet to be elucidated. It was previously reported that Paneth cells of the small intestine were positive for UEA-1 lectin staining. However, glycoproteins in Paneth cells carrying α1,2-fucose have not yet been identified. Glycoproteomic analysis of ileal lysates identified 3212 O-linked and 2962 N-linked glycopeptides. In particular, cryptdin-related sequence 1 (CRS1) expressed in Paneth cells was found to be α1,2-fucosylated. Unlike other antimicrobial α-defensin proteins, CRS1 contains unique Thr residues, which are modified with O-glycans, with 3HexNAc2Hex1Fuc1NeuAc being the main glycoform. Identification of α1,2-fucose on the O-glycans of CRS1 expressed in Paneth cells will pave the way for a mechanistic understanding of α1,2-fucose-dependent symbiosis with intestinal bacteria and elimination of pathogenic bacteria in the intestine.

ER-resident oxidoreductases are glycosylated and trafficked to the cell surface to promote matrix degradation by tumour cells.

Tumour growth and invasiveness require extracellular matrix (ECM) degradation and are stimulated by the GALA pathway, which induces protein O-glycosylation in the endoplasmic reticulum (ER). ECM degradation requires metalloproteases, but whether other enzymes are required is unclear. Here, we show that GALA induces the glycosylation of the ER-resident calnexin (Cnx) in breast and liver cancer. Glycosylated Cnx and its partner ERp57 are trafficked to invadosomes, which are sites of ECM degradation. We find that disulfide bridges are abundant in connective and liver ECM. Cell surface Cnx-ERp57 complexes reduce these extracellular disulfide bonds and are essential for ECM degradation. In vivo, liver cancer cells but not hepatocytes display cell surface Cnx. Liver tumour growth and lung metastasis of breast and liver cancer cells are inhibited by anti-Cnx antibodies. These findings uncover a moonlighting function of Cnx-ERp57 at the cell surface that is essential for ECM breakdown and tumour development.

Antibacterial potential of donkey’s milk disclosed by untargeted proteomics

Donkey's milk (DM) has been extensively investigated as a valuable substitute of breast milk, often suitable to manage cow's milk protein allergy in infants. DM exhibits potent inhibitory properties against numerous microbial species. Although oligosaccharides and lipids might contribute to the antimicrobial potential, the current inventory of proteins is not able to justify the low count of microorganisms generally observed in DM.The shotgun proteomic analysis of fractionated DM disclosed a set of 94 gene products, 41% of which have documented antimicrobial activity or are involved in transferring the passive immunity to the donkey offspring. The concerted action of lysozyme, lactoferrin, immunoglobulins provides the molecular basis for part of the DM antibacterial potential. The pH -4.6 insoluble fraction contained significant levels of L-amino acid oxidase, identified with 11 unique peptides matching the horse homologue gene product. This enzyme catalyses the oxidative deamination of amino acids into ketoacids, producing ammonia and H2O2. κ-casein, likely occurring as a fully O-glycosylated protein, may concur to inhibit the adhesion of pathogenic microorganisms, along with other glycoproteins.Proteomics supports the alimentary use of DM not only as a substitute of human milk in early infancy, but also for growing children, convalescent, elderly people and general population. SignificanceDonkey's milk (DM) is acquiring increasing popularity because it is a suitable substitute of the human milk, when breastfeeding is not possible and infants suffer from cow's milk allergy.DM is characterized by a much lower microbial load compared to ruminants' milk. This feature has been traditionally attributed to the high content of lysozyme. DM exhibits potent activity against a broad range of bacteria, viruses and fungi, suggesting that other protein components can be responsible of the antimicrobial potential. The gel-free proteomic analysis of pH 4.6-insoluble and soluble (whey) fractions demonstrated that DM contains a large number of gene products involved in antimicrobial mechanisms and in transferring passive immunity to the donkey offspring.DM contains relatively high levels of L-amino acid oxidase that catalyses the oxidative deamination of amino acid substrates into ketoacids, with production of ammonia and H2O2. In combination with lysozyme, lactoferrin and immunoglobulins, the presence of L-amino acid oxidase provides the molecular basis of the antibacterial potential observed for DM. Considered the low microbial load, DM can be sanitated at mild conditions, thereby preserving many of the native nutritional traits.Thus, DM can be considered a safe and nutritionally valid alimentary resource for growing children, convalescent, elderly people and general population.Data of this study represent the largest inventory of proteins identified in Equidae milk, so far.