Publications by authors named "Ola Blixt"

93 Publications

Host Range of Influenza A Virus H1 to H16 in Eurasian Ducks Based on Tissue and Receptor Binding Studies.

J Virol 2021 Feb 24;95(6). Epub 2021 Feb 24.

Department of Viroscience, Erasmus Medical Center, Rotterdam, the Netherlands.

Dabbling and diving ducks partly occupy shared habitats but have been reported to play different roles in wildlife infectious disease dynamics. Influenza A virus (IAV) epidemiology in wild birds has been based primarily on surveillance programs focused on dabbling duck species, particularly mallard (). Surveillance in Eurasia has shown that in mallards, some subtypes are commonly (H1 to H7 and H10), intermediately (H8, H9, H11, and H12), or rarely (H13 to H16) detected, contributing to discussions on virus host range and reservoir competence. An alternative to surveillance in determining IAV host range is to study virus attachment as a determinant for infection. Here, we investigated the attachment patterns of all avian IAV subtypes (H1 to H16) to the respiratory and intestinal tracts of four dabbling duck species ( and spp.), two diving duck species ( spp.), and chicken, as well as to a panel of 65 synthetic glycan structures. We found that IAV subtypes generally showed abundant attachment to colon of the duck species, mallard, and Eurasian teal (), supporting the fecal-oral transmission route in these species. The reported glycan attachment profile did not explain the virus attachment patterns to tissues but showed significant attachment of duck-originated viruses to fucosylated glycan structures and H7 virus tropism for Neu5Gc-LN. Our results suggest that ducks play an important role in the ecology and epidemiology of IAV. Further knowledge on virus tissue attachment, receptor distribution, and receptor binding specificity is necessary to understand the mechanisms underlying host range and epidemiology of IAV. Influenza A viruses (IAVs) circulate in wild birds worldwide. From wild birds, the viruses can cause outbreaks in poultry and sporadically and indirectly infect humans. A high IAV diversity has been found in mallards (), which are most often sampled as part of surveillance programs; meanwhile, little is known about the role of other duck species in IAV ecology and epidemiology. In this study, we investigated the attachment of all avian IAV hemagglutinin (HA) subtypes (H1 to H16) to tissues of six different duck species and chicken as an indicator of virus host range. We demonstrated that the observed virus attachment patterns partially explained reported field prevalence. This study demonstrates that dabbling ducks of the genus are potential hosts for most IAV subtypes, including those infecting poultry. This knowledge is useful to target the sampling of wild birds in nature and to further study the interaction between IAVs and birds.
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http://dx.doi.org/10.1128/JVI.01873-20DOI Listing
February 2021

PODO447: a novel antibody to a tumor-restricted epitope on the cancer antigen podocalyxin.

J Immunother Cancer 2020 11;8(2)

The Biomedical Research Centre and School of Biomedical Engineering, The University of British Columbia, Vancouver, British Columbia, Canada

Background: The success of new targeted cancer therapies has been dependent on the identification of tumor-specific antigens. Podocalyxin (Podxl) is upregulated on tumors with high metastatic index and its presence is associated with poor outcome, thus emerging as an important prognostic and theragnostic marker in several human cancers. Moreover, in human tumor xenograft models, Podxl expression promotes tumor growth and metastasis. Although a promising target for immunotherapy, the expression of Podxl on normal vascular endothelia and kidney podocytes could hamper efforts to therapeutically target this molecule. Since pathways regulating post-translational modifications are frequently perturbed in cancer cells, we sought to produce novel anti-Podxl antibodies (Abs) that selectively recognize tumor-restricted glycoepitopes on the extracellular mucin domain of Podxl.

Methods: Splenic B cells were isolated from rabbits immunized with a Podxl-expressing human tumor cell line. Abs from these B cells were screened for potent reactivity to Podxl neoplastic cell lines but not Podxl primary endothelial cells. Transcripts encoding heavy and light chain variable regions from promising B cells were cloned and expressed as recombinant proteins. Tumor specificity was assessed using primary normal tissue and an ovarian cancer tissue microarray (TMA). Mapping of the tumor-restricted epitope was performed using enzyme-treated human tumor cell lines and a glycan array.

Results: One mAb (PODO447) showed strong reactivity with a variety of Podxl+ tumor cell lines but not with normal primary human tissue including Podxl+ kidney podocytes and most vascular endothelia. Screening of an ovarian carcinoma TMA (219 cases) revealed PODO447 reactivity with the majority of tumors, including 65% of the high-grade serous histotype. Subsequent biochemical analyses determined that PODO447 reacts with a highly unusual terminal N-acetylgalactosamine beta-1 (GalNAcβ1) motif predominantly found on the Podxl protein core. Finally, Ab-drug conjugates showed specific efficacy in killing tumor cells .

Conclusions: We have generated a novel and exquisitely tumor-restricted mAb, PODO447, that recognizes a glycoepitope on Podxl expressed at high levels by a variety of tumors including the majority of life-threatening high-grade serous ovarian tumors. Thus, tumor-restricted PODO447 exhibits the appropriate specificity for further development as a targeted immunotherapy.
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http://dx.doi.org/10.1136/jitc-2020-001128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692987PMC
November 2020

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

Chem Commun (Camb) 2020 Dec;56(96):15137-15140

Institute of Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, Mariano Esquillor s/n, Campus Rio Ebro, Edificio I+D, Zaragoza, Spain.

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.
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http://dx.doi.org/10.1039/d0cc06349eDOI Listing
December 2020

Evaluation of Sialyl-Lactotetra as a Marker for Epithelial Ovarian Tumors.

Front Oncol 2020 23;10:561888. Epub 2020 Sep 23.

Department of Obstetrics and Gynecology, Sahlgrenska Cancer Centre, Institute of Clinical Sciences, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.

Ovarian carcinoma is a heterogeneous disease with distinct molecular and histological profiles, ranging from low grade atypia to highly aggressive tumors associated with a poor prognosis. In the present study, glycosphingolipids were isolated from human high-grade serous ovarian carcinoma, whereby the novel stem cell marker Sialyl-lactotetra (S-Lc) was characterized in two out of three cases. The presence and level of S-Lc was further evaluated immunohistochemically in a cohort of patients with ovarian tumors ranging from benign lesions to high grade serous carcinoma ( = 478). Its expression was assessed in association with tumor grade, stage, histology, and survival. The data showed that S-Lc is most common and highly expressed in borderline type tumors and carcinomas with low levels of aggressiveness, such as mucinous, endometrioid, and low grade serous. Accordingly, S-Lc-positivity was associated with better disease-free survival. The expression of S-Lc was seemingly associated with lineage continuity and could be traced from premalignant lesions to carcinoma, suggesting inheritance by a stem cell lineage that gives rise to generally indolent tumors.
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http://dx.doi.org/10.3389/fonc.2020.561888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7539041PMC
September 2020

Specificity of human natural antibodies referred to as anti-Tn.

Mol Immunol 2020 04 19;120:74-82. Epub 2020 Feb 19.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya, Moscow, 117997, Russian Federation. Electronic address:

To understand the role of human natural IgM known as antibodies against the carbohydrate epitope Tn, the antibodies were isolated using GalNAcα-Sepharose affinity chromatography, and their specificity was profiled using microarrays (a glycan array printed with oligosaccharides and bacterial polysaccharides, as well as a glycopeptide array), flow cytometry, and inhibition ELISA. The antibodies bound a restricted number of GalNAcα-terminated oligosaccharides better than the parent monosaccharide, e.g., 6-O-Su-GalNAcα and GalNAcα1-3Galβ1-3(4)GlcNAcβ. The binding with several bacterial polysaccharides that have no structural resemblance to the affinity ligand GalNAcα was quite unexpected. Given that GalNAcα is considered the key fragment of the Tn antigen, it is surprising that these antibodies bind weakly GalNAcα-OSer and do not bind a wide variety of GalNAcα-OSer/Thr-containing mucin glycopeptides. At the same time, we have observed specific binding to cells having Tn-positive glycoproteins containing similar glycopeptide motifs in a conformationally rigid macromolecule. Thus, specific recognition of the Tn antigen apparently requires that the naturally occurring "anti-Tn" IgM recognize a complex epitope comprising the GalNAcα as an essential component and a fairly long amino acid sequence where the amino acids adjacent to GalNAcα do not contact the antibody paratope; i.e., the antibodies recognize a spatial epitope or a molecular pattern rather than a classical continuous sequence. In addition, we have not found any increase in the binding of natural antibodies when GalNAcα residues were clustered. These results may help in further development of anticancer vaccines based on synthetic Tn constructs.
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http://dx.doi.org/10.1016/j.molimm.2020.02.005DOI Listing
April 2020

A Graphene-Based Glycan Biosensor for Electrochemical Label-Free Detection of a Tumor-Associated Antibody.

Sensors (Basel) 2019 Dec 9;19(24). Epub 2019 Dec 9.

Department of Glycobiotechnology, Institute of Chemistry, Slovak Academy of Sciences, Dubravska cesta 9, 845 38 Bratislava, Slovakia.

The study describes development of a glycan biosensor for detection of a tumor-associated antibody. The glycan biosensor is built on an electrochemically activated/oxidized graphene screen-printed electrode (GSPE). Oxygen functionalities were subsequently applied for covalent immobilization of human serum albumin (HSA) as a natural nanoscaffold for covalent immobilization of Thomsen-nouvelle (Tn) antigen (GalNAc--Ser/Thr) to be fully available for affinity interaction with its analyte-a tumor-associated antibody. The step by step building process of glycan biosensor development was comprehensively characterized using a battery of techniques (scanning electron microscopy, atomic force microscopy, contact angle measurements, secondary ion mass spectrometry, surface plasmon resonance, Raman and energy-dispersive X-ray spectroscopy). Results suggest that electrochemical oxidation of graphene SPE preferentially oxidizes only the surface of graphene flakes within the graphene SPE. Optimization studies revealed the following optimal parameters: activation potential of +1.5 V Ag/AgCl/3 M KCl, activation time of 60 s and concentration of HSA of 0.1 g L. Finally, the glycan biosensor was built up able to selectively and sensitively detect its analyte down to low aM concentration. The binding preference of the glycan biosensor was in an agreement with independent surface plasmon resonance analysis.
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http://dx.doi.org/10.3390/s19245409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6960651PMC
December 2019

Combining Click Reactions for the One-Pot Synthesis of Modular Biomolecule Mimetics.

Org Lett 2019 09 10;21(18):7544-7548. Epub 2019 Sep 10.

Department of Chemistry , Aarhus University , Langelandsgade 140 , 8000 Aarhus C, Denmark.

Here, we report on the first combined one-pot use of the two so-called "click reactions": the thiol-ene coupling and the copper-catalyzed alkyne-azide cycloaddition. These reactions were employed in an alternating and one-pot fashion to combine appropriately functionalized monomeric carbohydrate building blocks to create mimics of trisaccharides and tetrasaccharides as single anomers, with only minimal purification necessary. The deprotected oligosaccharide mimics were found to bind both plant lectins and human galectin-3.
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http://dx.doi.org/10.1021/acs.orglett.9b02811DOI Listing
September 2019

Clustering of Giant Unilamellar Vesicles Promoted by Covalent and Noncovalent Bonding of Functional Groups at Membrane-Embedded Peptides.

Bioconjug Chem 2019 08 7;30(8):2156-2164. Epub 2019 Aug 7.

Department of Chemistry, Chemical Biology , University of Copenhagen , Thorvaldsensvej 40 , 1871 Frederiksberg C , Denmark.

Access to clusters of cell-sized globular objects such as giant unilamellar vesicles (GUVs) is of increasing interest due to their potential applications in prototissue and cell-cell adhesion studies. Aggregations of GUVs by four different approaches were observed via covalent as well as noncovalent bond participations of functional groups at membrane embedded cholesterylpeptides using optical microscopy. Passive air oxidation of GUV-surface thiols into trans-GUV disulfide bonds promoted multivesicle aggregation. Aggregations of GUVs into multiclusters were also achieved by introduction of bispyridyl-ligand substituted peptides into GUV-membranes succeeded by rhodium diacetate mediated vesicle clustering and, furthermore, by coinstalling a biotin moiety streptavidin addition attenuating the clustering effect visualized by formation of compact superaggregated GUV-multiclusters. Contacting between two different GUV-populations, i.e., GUV-heteroconnection, was achieved by trans-GUV phenyl ester-hydrazine ligations producing GUV-heteroclusters. Indirectly, GUV-clustering was achieved by strain-promoted azide-alkyne cycloaddition (SPAAC) reacting bicyclononyne (BCN)-GUVs with azido-GlcNAc succeeded by biotinylated wheat germ agglutinin (WGA)-lectin/streptavidin incubation arousing cross-binding of GUVs.
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http://dx.doi.org/10.1021/acs.bioconjchem.9b00394DOI Listing
August 2019

Recombinant Glycoprotein E of Varicella Zoster Virus Contains Glycan-Peptide Motifs That Modulate B Cell Epitopes into Discrete Immunological Signatures.

Int J Mol Sci 2019 Feb 22;20(4). Epub 2019 Feb 22.

Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy at the University of Gothenburg, 41346 Gothenburg, Sweden.

A recombinant subunit vaccine (Shingrix) was recently licensed for use against herpes zoster. This vaccine is based on glycoprotein E (gE) of varicella zoster virus (VZV), the most abundantly expressed protein of VZV, harboring sites for N- and O-linked glycosylation. The subunit vaccine elicits stronger virus-specific CD4+ T cell response as well as antibody B cell response to gE, compared to the currently used live attenuated vaccine (Zostavax). This situation is at variance with the current notion since a live vaccine, causing an active virus infection, should be far more efficient than a subunit vaccine based on only one single viral glycoprotein. We previously found gE to be heavily glycosylated, not least by numerous clustered O-linked glycans, when it was produced in human fibroblasts. However, in contrast to Zostavax, which is produced in fibroblasts, the recombinant gE of Shingrix is expressed in Chinese hamster ovary (CHO) cells. Hence, the glycan occupancy and glycan structures of gE may differ considerably between the two vaccine types. Here, we aimed at (i) defining the glycan structures and positions of recombinant gE and (ii) identifying possible features of the recombinant gE O-glycosylation pattern contributing to the vaccine efficacy of Shingrix. Firstly, recombinant gE produced in CHO cells ("Shingrix situation") is more scarcely decorated by O-linked glycans than gE from human fibroblasts ("Zostavax situation"), with respect to glycan site occupancy. Secondly, screening of immunodominant B cell epitopes of gE, using a synthetic peptide library against serum samples from VZV-seropositive individuals, revealed that the O-linked glycan signature promoted binding of IgG antibodies via a decreased number of interfering O-linked glycans, but also via specific O-linked glycans enhancing antibody binding. These findings may, in part, explain the higher protective efficacy of Shingrix, and can also be of relevance for development of subunit vaccines to other enveloped viruses.
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http://dx.doi.org/10.3390/ijms20040954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6412795PMC
February 2019

Characterization of avian influenza virus attachment patterns to human and pig tissues.

Sci Rep 2018 08 15;8(1):12215. Epub 2018 Aug 15.

Zoonosis Science Centre, Department of Medical Sciences, Uppsala University, Uppsala, SE, 751 23, Sweden.

Wild birds of Anseriformes and Charadriiformes are natural reservoirs of influenza A viruses (IAVs). Occasionally, IAVs transmit and adapt to mammalian hosts, and are maintained as epidemic strains in their new hosts. Viral adaptions to mammalian hosts include altered receptor preference of host epithelial sialylated oligosaccharides from terminal α2,3-linked sialic acid (SA) towards α2,6-linked SA. However, α2,3-linked SA has been found in human respiratory tract epithelium, and human infections by avian IAVs (AIVs) have been reported. To further explore the attachment properties of AIVs, four AIVs of different subtypes were investigated on human and pig tissues using virus histochemistry. Additionally, glycan array analysis was performed for further characterization of IAVs' receptor structure tropism. Generally, AIV attachment was more abundant to human tissues than to pig tissues. The attachment pattern was very strong to human conjunctiva and upper respiratory tract, but variable to the lower respiratory tract. AIVs mainly attached to α2,3-linked SA, but also to combinations of α2,3- and α2,6-linked SA. The low attachment of these AIV isolates to pig tissues, but high attachment to human tissues, addresses the question whether AIVs in general require passage through pigs to obtain adaptions towards mammalian receptor structures.
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http://dx.doi.org/10.1038/s41598-018-29578-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6093914PMC
August 2018

ABO Blood Group Antigen Decorated Giant Unilamellar Vesicles Exhibit Distinct Interactions with Plasmodium falciparum Infected Red Blood Cells.

ACS Chem Biol 2018 09 13;13(9):2421-2426. Epub 2018 Aug 13.

Department of Chemistry, Chemical Biology , University of Copenhagen , Thorvaldsensvej 40 , 1871 Frederiksberg C , Denmark.

Severe malaria is considered to be the deadliest disease of this century, primarily among children in sub-Saharan Africa. It stems from infection by the virulent parasite Plasmodium falciparum. The pathogenesis of the disease is based on the rosetting phenomenon, which occurs during the life cycle of the parasite in red blood cells (RBCs) and promotes the binding of parasitized RBCs to healthy ones. The role of the ABO blood group antigens in relation to the phenomenon has previously only been investigated in clinical isolates obtained from malaria patients. Here, we aim to clarify their role using synthetic ABO-decorated giant unilamellar vesicles (GUVs), which serve as simple biomimetic models of RBC-size cell membranes. Our results suggest clearly and for the first time that the blood group A and O antigens have a direct impact on receptor-specific rosetting phenomena when compared to the B antigen, which only participates in rosetting to an insignificant degree. Thus, glycodecorated GUVs represent a practical tool for studying cell-surface interactions.
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http://dx.doi.org/10.1021/acschembio.8b00635DOI Listing
September 2018

Epitope-mapping of the glycoprotein from Crimean-Congo hemorrhagic fever virus using a microarray approach.

PLoS Negl Trop Dis 2018 07 9;12(7):e0006598. Epub 2018 Jul 9.

Department of Chemistry, University of Copenhagen, Frederiksberg, Denmark.

Crimean-Congo hemorrhagic fever virus (CCHFV) causes severe acute human disease with lethal outcome. The knowledge about the immune response for this human health threat is highly limited. In this study, we have screened the glycoprotein of CCHFV for novel linear B-cell epitopic regions using a microarray approach. The peptide library consisted of 168 synthesized 20mer peptides with 10 amino acid overlap covering the entire glycoprotein. Using both pooled and individual human sera from survivors of CCHF disease in Turkey five peptide epitopes situated in the mucin-like region and GP 38 (G15-515) and GN G516-1037 region of the glycoprotein were identified as epitopes for a CCHF immune response. An epitope walk of the five peptides revealed a peptide sequence located in the GN region with high specificity and sensitivity. This peptide sequence, and a sequence downstream, reacted also against sera from survivors of CCHF disease in South Africa. The cross reactivity of these peptides with samples from a geographically distinct region where genetically diverse strains of the virus circulate, enabled the identification of unique peptide epitopes from the CCHF glycoprotein that could have application in development of diagnostic tools. In this study clinical samples from geographically distinct regions were used to identify conserved linear epitopic regions of the glycoprotein of CCHF.
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http://dx.doi.org/10.1371/journal.pntd.0006598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6053253PMC
July 2018

Effect of Noncanonical Amino Acids on Protein-Carbohydrate Interactions: Structure, Dynamics, and Carbohydrate Affinity of a Lectin Engineered with Fluorinated Tryptophan Analogs.

ACS Chem Biol 2018 08 12;13(8):2211-2219. Epub 2018 Jun 12.

Austrian Centre of Industrial Biotechnology , Petersgasse 14 , 8010 Graz , Austria.

Protein-carbohydrate interactions play crucial roles in biology. Understanding and modifying these interactions is of major interest for fighting many diseases. We took a synthetic biology approach and incorporated noncanonical amino acids into a bacterial lectin to modulate its interactions with carbohydrates. We focused on tryptophan, which is prevalent in carbohydrate binding sites. The exchange of the tryptophan residues with analogs fluorinated at different positions resulted in three distinctly fluorinated variants of the lectin from Ralstonia solanacearum. We observed differences in stability and affinity toward fucosylated glycans and rationalized them by X-ray and modeling studies. While fluorination decreased the aromaticity of the indole ring and, therefore, the strength of carbohydrate-aromatic interactions, additional weak hydrogen bonds were formed between fluorine and the ligand hydroxyl groups. Our approach opens new possibilities to engineer carbohydrate receptors.
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http://dx.doi.org/10.1021/acschembio.8b00377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102642PMC
August 2018

Improved spot morphology for printed glycan arrays.

Biotechniques 2018 03 1;64(3):110-116. Epub 2018 Mar 1.

Russian Academy of Sciences, Miklukho-Maklaya 16/10, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997, Moscow, Russia.

Despite considerable success studying glycan-binding proteins using printed glycan arrays (PGAs), unambiguous quantitation of spot intensities by fluorescent readers remains a challenge. The main obstacles are the varying spot shape and size and in-spot fluorescence distribution caused by uneven drying of the printed drops. Two methods have been suggested for solving this problem: using polymeric glycoconjugates, which makes it possible to equalize the physicochemical properties (hydrophobicity, charge, and size) of different glycans, and applying a glycan solution on a slide coated with a thin oil mask, which hinders evaporation of the drop. Both approaches yield spots with similar sizes and an even distribution of the signal across the spot and are likely to be useful for improving the prints of other classes of molecules.
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http://dx.doi.org/10.2144/btn-2017-0111DOI Listing
March 2018

Repertoire of Abs primed by bacteria in gnotobiotic mice.

Innate Immun 2018 04 16;24(3):180-187. Epub 2018 Mar 16.

1 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Russia.

Innate immunity natural Abs (NAbs) execute a number of functions, including protection and surveillance. Despite active research, the stimuli that induce the formation of NAbs are still described only hypothetically. Here, we compared repertoires of anti-glycan Abs in the peripheral blood of mice that received per os various bacteria. The repertoires of Abs of mice primed in this way were compared using a microarray that included about 350 glycans, as well as 150 bacterial polysaccharides. Sterile mice did not possess anti-glycan Abs. Oral inoculation of a single strain or combination of two to four strains of bacteria, as well as putting the animals on short-term nutrition with non-sterile food, did not contribute significantly to the formation of Abs, whereas a single gavage of digested food of non-sterile mice induced the formation of a repertoire close to the natural ones. Interestingly, the priming with polysaccharide Ags (in a composition of the bacterial cell envelope), that is, dominant Ags of bacteria, led to the induction of Abs against typical glycans of mammalian glycoproteins and glycolipids (e.g. Abs of the ABH blood group system) that do not have a structural similarity to the polysaccharides. The results support the importance of early contact with a naïve immune system with microorganisms of the environment to form a normal NAbs repertoire.
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http://dx.doi.org/10.1177/1753425918763524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6852387PMC
April 2018

A novel monoclonal antibody targeting carboxymethyllysine, an advanced glycation end product in atherosclerosis and pancreatic cancer.

PLoS One 2018 8;13(2):e0191872. Epub 2018 Feb 8.

Chemical Glyco-Biology Laboratory, Department of Chemistry, Faculty of Science, University of Copenhagen, Copenhagen, Denmark.

Advanced glycation end products are formed by non-enzymatic reactions between proteins and carbohydrates, causing irreversible lysine and arginine alterations that severely affect protein structure and function. The resulting modifications induce inflammation by binding to scavenger receptors. An increase in advanced glycation end products is observed in a number of diseases e.g. atherosclerosis and cancer. Since advanced glycation end products also are present in healthy individuals, their detection and quantification are of great importance for usage as potential biomarkers. Current methods for advanced glycation end product detection are though limited and solely measure total glycation. This study describes a new epitope-mapped single chain variable fragment, D1-B2, against carboxymethyllysine, produced from a phage library that was constructed from mouse immunizations. The phage library was selected against advanced glycation end product targets using a phage display platform. Characterization of its binding pattern was performed using large synthetic glycated peptide and protein libraries displayed on microarray slides. D1-B2 showed a preference for an aspartic acid, three positions N-terminally from a carboxymethyllysine residue and also bound to a broad collection of glycated proteins. Positive immunohistochemical staining of mouse atherosclerotic plaques and of a tissue microarray of human pancreatic tumors confirmed the usability of the new scFv for advanced glycation end product detection in tissues. This study demonstrates a promising methodology for high-throughput generation of epitope-mapped monoclonal antibodies against AGE.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0191872PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5805250PMC
March 2018

Synthesis of Oligo-(alkyne-triplet)peptide Constructs.

Org Lett 2017 12 5;19(24):6522-6525. Epub 2017 Dec 5.

Department of Chemistry, Chemical Biology, University of Copenhagen , Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.

Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click synthesis of an Fmoc-(trispropargyl)amino acid building block for solid phase peptide synthesis (SPPS) of oligo-(trialkyne)peptide constructs is reported. These can carry potentially indefinite numbers of inherent alkyne-triplets, which are click derivatized with GlcNAc-azide into the corresponding glycopeptides.
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http://dx.doi.org/10.1021/acs.orglett.7b03231DOI Listing
December 2017

Synthesis of BODIPY-Labeled Cholesterylated Glycopeptides by Tandem Click Chemistry for Glycocalyxification of Giant Unilamellar Vesicles (GUVs).

Chemistry 2017 Jul 22;23(40):9472-9476. Epub 2017 Jun 22.

Department of Chemistry, Chemical Biology, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark.

The glycocalyx cover membrane surfaces of all living cells. These complex architectures render their interaction mechanisms on the membrane surface difficult to study. Artificial cell-sized membranes with selected and defined glycosylation patterns may serve as a minimalistic approach to systematically study cell surface glycan interactions. The development of a facile general synthetic procedure for the synthesis of BODIPY-labeled cholesterylated glycopeptides, which can coat cell-size giant unilamellar vesicles (GUVs), is described. These peptide constructs were synthesized by: 1) solid-phase peptide synthesis (SPPS) using cholesterylated Fmoc-amino acids (Fmoc=9-fluorenylmethoxycarbonyl) followed by tandem click reactions, 2) attachment of a BODIPY-bicyclononyne (BCN) (prepared by Mitsunobu chemistry via novel aryl BCN-ethers) in the absence of a catalyst, and 3) glycosylation by means of copper(I)-catalyzed click reaction of an azidoglycan. Seven different GUV-glycoforms were prepared and four of these were evaluated with their corresponding four specific anti-glycan binding lectins.
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http://dx.doi.org/10.1002/chem.201702104DOI Listing
July 2017

Epitope mapping of a new anti-Tn antibody detecting gastric cancer cells.

Glycobiology 2017 07;27(7):635-645

Chemical Glyco-Biology Laboratory, Department of Chemistry, University of Copenhagen, 2100 Copenhagen, Denmark.

Here, we introduce a novel scFv antibody, G2-D11, specific for two adjacent Tn-antigens (GalNAc-Ser/Thr) binding equally to three dimeric forms of the epitope, Ser-Thr, Thr-Thr and Thr-Ser. Compared to other anti-Tn reagents, the binding of G2-D11 is minimally influenced by the peptide structure, which indicates a high degree of carbohydrate epitope dominance and a low influence from the protein backbone. With a high affinity (KDapp = 1.3 × 10-8 M) and no cross-reactivity to either sialyl-Tn epitope or blood group A antigens, scFv G2-D11 is an excellent candidate for a well-defined anti-Tn-antigen reagent. Detailed immunohistochemical evaluation of tissue sections from a cohort of 80 patients with gastric carcinoma showed in all cases positive tumor cells. The observed staining was localized to the cytoplasm and in some cases to the membrane, whereas the surrounding tissue was completely negative demonstrating the usefulness of the novel Tn-antigen binding antibody.
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http://dx.doi.org/10.1093/glycob/cwx033DOI Listing
July 2017

Immunization with DNA Plasmids Coding for Crimean-Congo Hemorrhagic Fever Virus Capsid and Envelope Proteins and/or Virus-Like Particles Induces Protection and Survival in Challenged Mice.

J Virol 2017 05 28;91(10). Epub 2017 Apr 28.

Folkhälsomyndigheten, Stockholm, Sweden

Crimean-Congo hemorrhagic fever virus (CCHFV) is a bunyavirus causing severe hemorrhagic fever disease in humans, with high mortality rates. The requirement of a high-containment laboratory and the lack of an animal model hampered the study of the immune response and protection of vaccine candidates. Using the recently developed interferon alpha receptor knockout (IFNAR) mouse model, which replicates human disease, we investigated the immunogenicity and protection of two novel CCHFV vaccine candidates: a DNA vaccine encoding a ubiquitin-linked version of CCHFV Gc, Gn, and N and one using transcriptionally competent virus-like particles (tc-VLPs). In contrast to most studies that focus on neutralizing antibodies, we measured both humoral and cellular immune responses. We demonstrated a clear and 100% efficient preventive immunity against lethal CCHFV challenge with the DNA vaccine. Interestingly, there was no correlation with the neutralizing antibody titers alone, which were higher in the tc-VLP-vaccinated mice. However, the animals with a lower neutralizing titer, but a dominant cell-mediated Th1 response and a balanced Th2 response, resisted the CCHFV challenge. Moreover, we found that in challenged mice with a Th1 response (immunized by DNA/DNA and boosted by tc-VLPs), the immune response changed to Th2 at day 9 postchallenge. In addition, we were able to identify new linear B-cell epitope regions that are highly conserved between CCHFV strains. Altogether, our results suggest that a predominantly Th1-type immune response provides the most efficient protective immunity against CCHFV challenge. However, we cannot exclude the importance of the neutralizing antibodies as the surviving immunized mice exhibited substantial amounts of them. Crimean-Congo hemorrhagic fever virus (CCHFV) is responsible for hemorrhagic diseases in humans, with a high mortality rate. There is no FDA-approved vaccine, and there are still gaps in our knowledge of the immune responses to infection. The recently developed mouse models mimic human CCHF disease and are useful to study the immunogenicity and the protection by vaccine candidates. Our study shows that mice vaccinated with a specific DNA vaccine were fully protected. Importantly, we show that neutralizing antibodies are not sufficient for protection against CCHFV challenge but that an extra Th1-specific cellular response is required. Moreover, we describe the identification of five conserved B-cell epitopes, of which only one was previously known, that could be of great importance for the development of diagnostics tools and the improvement of vaccine candidates.
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http://dx.doi.org/10.1128/JVI.02076-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5411611PMC
May 2017

Optimization of the Small Glycan Presentation for Binding a Tumor-Associated Antibody: Application to the Construction of an Ultrasensitive Glycan Biosensor.

Langmuir 2017 03 8;33(11):2709-2716. Epub 2017 Mar 8.

Department of Glycobiotechnology, Institute of Chemistry, Slovak Academy of Sciences , Dubravska cesta 9, 845 38 Bratislava, Slovakia.

The main aim of the study was to optimize the interfacial presentation of a small antigen-a Tn antigen (N-acetylgalactosamine)-for binding to its analyte anti-Tn antibody. Three different methods for the interfacial display of a small glycan are compared here, including two methods based on the immobilization of the Tn antigen on a mixed self-assembled monolayer (SAM) (2D biosensor) and the third one utilizing a layer of a human serum albumin (HSA) for the immobilization of a glycan forming a 3D interface. Results showed that the 3D interface with the immobilized Tn antigen is the most effective bioreceptive surface for binding its analyte. The 3D impedimetric glycan biosensor exhibited a limit of detection of 1.4 aM, a wide linear range (6 orders of magnitude), and high assay reproducibility with an average relative standard deviation of 4%. The buildup of an interface was optimized using various techniques with the visualization of the glycans on the biosensor surface by atomic force microscopy. The study showed that the 3D biosensor is not only the most sensitive compared to other two biosensor platforms but that the Tn antigen on the 3D biosensor surface is more accessible for antibody binding with better kinetics of binding (t = 137 s, t = the time needed to attain 50% of a steady-state signal) compared to the 2D biosensor configuration with t = 354 s. The 3D glycan biosensor was finally applied for the analysis of a human serum sample spiked with an analyte.
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http://dx.doi.org/10.1021/acs.langmuir.6b04021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5659382PMC
March 2017

Linear Multiepitope (Glyco)peptides for Type-Specific Serology of Herpes Simplex Virus (HSV) Infections.

ACS Infect Dis 2017 05 6;3(5):360-367. Epub 2017 Mar 6.

Department of Chemistry, Faculty of Science, University of Copenhagen , Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.

Detection of type-specific antibodies is an important and essential part of accurate diagnosis, even in silent carriers of herpes simplex virus (HSV)-1 (oral) and HSV-2 (genital) infections. Serologic assays that identify HSV-1 and HSV-2 type-specific antibodies have been commercially available for more than a decade but often face problems related to cross-reactivity and similar issues. Attempts to identify type-specific peptide epitopes for use in serology for both HSV-1 and HSV-2 have been limited. We recently demonstrated epitope mapping of envelope glycoprotein G2 and identified a type-specific glycopeptide epitope that broadly recognized HSV-2 infected individuals. In the present work we have performed a comprehensive glycopeptide synthesis and microarray epitope mapping of 14 envelope proteins from HSV-1 and HSV-2, namely, gB, gC, gD, gE, gG, gH, and gI, using sera from HSV-1- and HSV-2-infected individuals and control sera. Several unique type-specific peptide epitopes with high sensitivity were identified and synthesized as one large linear multiepitope sequence using microwave-assisted solid-phase (glyco)peptide synthesis. Microarray validation with clinically defined HSV and Varicella Zoster (VZV) sera confirmed excellent cumulative specificities and sensitivities.
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http://dx.doi.org/10.1021/acsinfecdis.7b00001DOI Listing
May 2017

A Combinatory Antibody-Antigen Microarray Assay for High-Content Screening of Single-Chain Fragment Variable Clones from Recombinant Libraries.

PLoS One 2016 21;11(12):e0168761. Epub 2016 Dec 21.

Chemical Glyco-Biology Laboratory, Department of Chemistry, Copenhagen University, Copenhagen, Denmark.

We have developed a combinatory antibody-antigen microarray for direct screening of multiple single-chain fragment variable (scFv) clones with no need for pre-purification or enrichment before screening. The straightforward workflow allows for early selection of binders to predefined peptide and glycopeptide targets. A capture antibody is contact printed on microarray slides, side by side with the antigens of interest. A large number of scFv clones, in supernatants, are printed on top of the capture antibody and the antigen in a "spot-on-spot" print. The printed scFv clones, which bind to the capture antibody, are detected using biotinylated antigen, while the binding of scFv clones to the printed antigen is detected through a mouse anti-tag antibody. Two different analyses are thus performed on the same slide, generating two kinds of information: one on the ability of an individual scFv clone to bind to the soluble form of the antigen, which may favour selection for higher affinity rather than avidity, while the other allows the identification of large numbers of clones, simultaneously, due to the binding of scFv clones to densely presented antigens, thus providing an overall increased hit rate. The functionality of the new screening approach was illustrated through the generation of antibodies against peptides from the chaperone complex Ku70/Ku80 and the GalNAcα-serine/threonine epitope on the IgA1 alpha chain hinge region. In total, 659 scFv clones were screened with a hit rate of approximately 20%. This approach allowed the identification of functional antibodies in both cases, illustrating the usefulness and capacity of this combinatory microarray screening technique for efficient analysis and validation of antibodies at an early stage of antibody generation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0168761PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5176327PMC
July 2017

Intra-tumour IgA1 is common in cancer and is correlated with poor prognosis in bladder cancer.

Heliyon 2016 Aug 16;2(8):e00143. Epub 2016 Aug 16.

Division of Oncology and Pathology, Dept. of Clinical Sciences Lund, Lund University, Lund, Sweden.

A high frequency of IgA1-positive tumour cells was found in tissue micro-arrays of oesophagus, colon, testis, lung, breast, bladder and ovarian cancer. IgA1 was observed in the cytoplasm and the plasma membrane. A correlation was found between intra-tumour IgA1 and poor overall survival in a large cohort of bladder cancer patients (n = 99, p = 0.011, log-rank test). The number of IgA1-positive tumour cells was also found to be higher in female than male bladder cancer patients. The presence of IgA1 was confirmed in formalin-fixed paraffin-embedded ovarian carcinoma samples using LC-MS/MS analysis. Uptake of IgA1 was also observed in breast cancer and melanoma cell lines when cultivated in the presence of serum from healthy individuals, indicating a possible origin of the IgA1 antibodies in cancer cells.
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http://dx.doi.org/10.1016/j.heliyon.2016.e00143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4992093PMC
August 2016

Synthesis of Cholesterol-Substituted Glycopeptides for Tailor-Made Glycocalyxification of Artificial Membrane Systems.

Chembiochem 2016 08 17;17(15):1403-6. Epub 2016 Jun 17.

Department of Chemistry, Chemical Biology, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark.

Synthetic minimal membrane systems are extremely useful for better understanding of complex cellular structures and cell surface processes. We have developed a facile method for synthesis of cholesterylated peptides, each bearing a carbohydrate moiety and a fluorescent tag. The position of the cholesterol moiety on the peptide can be controlled by using a new Fmoc-protected cholesterol-triazole-lysine group, which we constructed by means of solid-phase peptide synthesis. We succeeded in integrating the glyco modules into giant unilamellar vesicles by electroformation or infusion in buffer solution. The glyco-decorated liposomes were recognized by a lectin and had unique topological membrane features. In conclusion, this work is a proof of principle for the functionalization of artificial membranes with a primitive synthetic glycocalyx useful for studying carbohydrate-protein interactions on a simplified cell-like membrane surface.
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http://dx.doi.org/10.1002/cbic.201600258DOI Listing
August 2016

Cytotoxic activity against human neuroblastoma and melanoma cells mediated by IgM antibodies derived from peripheral blood of healthy donors.

Int J Cancer 2016 Jun 23;138(12):2963-73. Epub 2016 Feb 23.

Translational Immunology, Deutsches Krebsforschungszentrum, Heidelberg, Germany.

A small percentage of healthy donors identified in the Western population carry antibodies in their peripheral blood which convey cytotoxic activity against certain human melanoma and neuroblastoma cell lines. We measured the cytotoxic activity of sera and plasmas from healthy donors on the human neuroblastoma cell line Kelly and various melanoma cell lines. Antibodies of IgM isotype, presumably belonging to the class of naturally occurring antibodies, exerted cytotoxic activity in a complement-dependent fashion. Apart from complement-dependent tumor cell lysis, we observed C3 opsonization in all tumor cell lines upon treatment with cytotoxic plasmas. Cell lines tested primarily expressed membrane complement regulatory proteins (mCRP) CD46, CD55 and CD59 to various extents. Blocking of mCRPs by monoclonal antibodies enhanced cell lysis and opsonization, though some melanoma cells remained resistant to complement attack. Epitopes recognized by cytotoxic antibodies were represented by gangliosides such as GD2 and GD3, as evidenced by cellular sialidase pretreatment and enhanced expression of distinct gangliosides. It remains to be clarified why only a small fraction of healthy persons carry these antitumor cytotoxic antibodies.
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http://dx.doi.org/10.1002/ijc.30025DOI Listing
June 2016

Viral O-GalNAc peptide epitopes: a novel potential target in viral envelope glycoproteins.

Rev Med Virol 2016 Jan 2;26(1):34-48. Epub 2015 Nov 2.

Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.

Viral envelope glycoproteins are major targets for antibodies that bind to and inactivate viral particles. The capacity of a viral vaccine to induce virus-neutralizing antibodies is often used as a marker for vaccine efficacy. Yet the number of known neutralization target epitopes is restricted owing to various viral escape mechanisms. We expand the range of possible viral glycoprotein targets, by presenting a previously unknown type of viral glycoprotein epitope based on a short peptide stretch modified with small O-linked glycans. Besides being immunologically active, these epitopes have a high potential for antigenic variation. Thus, sera from patients infected with EBV develop individual IgG responses addressing the different possible glycopeptide glycoforms of one short peptide backbone that reflect individual variations in the course of virus infection. In contrast, in HSV type 2 meningitis patients, CSF antibodies are focussed to only one single glycoform peptide of a major viral glycoprotein. Thus, dependent on the viral disease, the serological response may be variable or constant with respect to the number of targeted peptide glycoforms. Mapping of these epitopes relies on a novel three-step procedure that identifies any reactive viral O-glycosyl peptide epitope with respect to (i) relevant peptide sequence, (ii) the reactive glycoform out of several possible glycopeptide isomers of that peptide sequence, and (iii) possibly tolerated carbohydrate or peptide structural variations at glycosylation sites. In conclusion, the viral O-glycosyl peptide epitopes may be of relevance for development of subunit vaccines and for improved serodiagnosis of viral diseases. Copyright © 2015 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/rmv.1859DOI Listing
January 2016

A novel monoclonal antibody to a defined peptide epitope in MUC16.

Glycobiology 2015 Nov 22;25(11):1172-82. Epub 2015 Jul 22.

Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark

The MUC16 mucin is overexpressed and aberrantly glycosylated in ovarian carcinomas. Immunodetection of circulating MUC16 is one of the most used cancer biomarker assays, but existing antibodies to MUC16 fail to distinguish normal and aberrant cancer glycoforms. Although all antibodies react with the tandem-repeat region, their epitopes appear to be conformational dependent and not definable by a short peptide. Aberrant glycoforms of MUC16 may constitute promising targets for diagnostic and immunotherapeutic intervention, and it is important to develop well-defined immunogens for induction of potent MUC16 immunity. Here, we developed a MUC16 vaccine based on a 1.7TR (264 aa) expressed in Escherichia coli and in vitro enzymatically glycosylated to generate the aberrant cancer-associated glycoform Tn. This vaccine elicited a potent serum IgG response in mice and we identified two major immunodominant linear peptide epitopes within the tandem repeat. We developed one monoclonal antibody, 5E11, reactive with a minimum epitope with the sequence FNTTER. This sequence contains potential N- and O-glycosylation sites and, interestingly, glycosylation blocked binding of 5E11. In immunochemistry of ovarian benign and cancer lesions, 5E11 showed similar reactivity as traditional MUC16 antibodies, suggesting that the epitope is not efficiently glycosylated. The study provides a vaccine design and immunodominant MUC16 TR epitopes.
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http://dx.doi.org/10.1093/glycob/cwv056DOI Listing
November 2015

RIFINs are adhesins implicated in severe Plasmodium falciparum malaria.

Nat Med 2015 Apr 9;21(4):314-7. Epub 2015 Mar 9.

Center for Infectious Disease Research, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.

Rosetting is a virulent Plasmodium falciparum phenomenon associated with severe malaria. Here we demonstrate that P. falciparum-encoded repetitive interspersed families of polypeptides (RIFINs) are expressed on the surface of infected red blood cells (iRBCs), where they bind to RBCs--preferentially of blood group A--to form large rosettes and mediate microvascular binding of iRBCs. We suggest that RIFINs have a fundamental role in the development of severe malaria and thereby contribute to the varying global distribution of ABO blood groups in the human population.
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http://dx.doi.org/10.1038/nm.3812DOI Listing
April 2015

A diverse range of bacterial and eukaryotic chitinases hydrolyzes the LacNAc (Galβ1-4GlcNAc) and LacdiNAc (GalNAcβ1-4GlcNAc) motifs found on vertebrate and insect cells.

J Biol Chem 2015 Feb 5;290(9):5354-66. Epub 2015 Jan 5.

From the Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Grønnegaardsvej 10, 1870 Frederiksberg C., Denmark,

There is emerging evidence that chitinases have additional functions beyond degrading environmental chitin, such as involvement in innate and acquired immune responses, tissue remodeling, fibrosis, and serving as virulence factors of bacterial pathogens. We have recently shown that both the human chitotriosidase and a chitinase from Salmonella enterica serovar Typhimurium hydrolyze LacNAc from Galβ1-4GlcNAcβ-tetramethylrhodamine (LacNAc-TMR (Galβ1-4GlcNAcβ(CH2)8CONH(CH2)2NHCO-TMR)), a fluorescently labeled model substrate for glycans found in mammals. In this study we have examined the binding affinities of the Salmonella chitinase by carbohydrate microarray screening and found that it binds to a range of compounds, including five that contain LacNAc structures. We have further examined the hydrolytic specificity of this enzyme and chitinases from Sodalis glossinidius and Polysphondylium pallidum, which are phylogenetically related to the Salmonella chitinase, as well as unrelated chitinases from Listeria monocytogenes using the fluorescently labeled substrate analogs LacdiNAc-TMR (GalNAcβ1-4GlcNAcβ-TMR), LacNAc-TMR, and LacNAcβ1-6LacNAcβ-TMR. We found that all chitinases examined hydrolyzed LacdiNAc from the TMR aglycone to various degrees, whereas they were less active toward LacNAc-TMR conjugates. LacdiNAc is found in the mammalian glycome and is a common motif in invertebrate glycans. This substrate specificity was evident for chitinases of different phylogenetic origins. Three of the chitinases also hydrolyzed the β1-6 bond in LacNAcβ1-6LacNAcβ-TMR, an activity that is of potential importance in relation to mammalian glycans. The enzymatic affinities for these mammalian-like structures suggest additional functional roles of chitinases beyond chitin hydrolysis.
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http://dx.doi.org/10.1074/jbc.M114.607291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4342453PMC
February 2015