Sun X, Ju T, Cummings RD. Differential expression of Cosmc, T-synthase and mucins in Tn-positive colorectal cancers. BMC Cancer. 2018;18 (1) :827. Publisher's VersionAbstract


The Tn neoantigen (GalNAcα1-O-Ser/Thr) is an O-glycan expressed in various types of human cancers. Studies in several Tn-expressing cancer cell lines and pancreatic tumors have identified loss of Cosmc expression caused by either mutations or promoter hypermethylation. In this study, we explored the mechanism(s) for Tn expression in human colorectal cancers (CRC).


Tn-expressing cell populations were isolated from CRC cell lines by Fluorescence-associated cell sorting (FACS). The expression of the Tn and sialylated Tn (STn) antigens, Cosmc, T-synthase, and mucins was characterized in paired specimens with CRC and in CRC cell lines by immunostaining, western blot, and qPCR.


Using well-defined monoclonal antibodies, we confirmed prevalent Tn/STn expression in CRC samples. However, a majority of these tumors had elevated T-synthase activity and expression of both Cosmc and T-synthase proteins. Meanwhile, Tn antigen expression was not caused by mucin overproduction. In addition, we found that Tn-expressing CRC cell lines had either loss-of-function mutations in Cosmc or reversible Tn antigen expression, which was not caused by the deficiency of T-synthase activity.


Our results demonstrate multiple mechanisms for Tn expression in CRCs.


Colorectal carcinoma; Cosmc; Mutation; STn antigen; T-synthase; Tn antigen

Ferreira RG, Rodrigues LC, Nascimento DC, Kanashiro A, Melo PH, Borges VF, Gozzi A, da Silva Prado D, Borges MC, Ramalho FS, et al. Galectin-3 aggravates experimental polymicrobial sepsis by impairing neutrophil recruitment to the infectious focus. J Infect. 2018;77 (5) :391-397. Publisher's VersionAbstract
Sepsis is an overwhelming systemic inflammation resulting from an uncontrolled infection that causes extensive tissue damage, organ dysfunction and eventually death. A growing body of evidence indicates that impaired neutrophil migration to the site of infection is associated with poor outcome in sepsis. Here we show that galectin-3 (Gal-3), an endogenous glycan-binding protein, plays a critical role in sepsis outcome. We found that serum Gal-3 concentration increased in patients with septic shock and mice undergoing sepsis induced by cecal ligation and puncture (CLP). Mice deficient in Gal-3 (Gal-3 KO) are more resistant to sepsis induced by CLP, showing lower levels of biochemical markers and neutrophil infiltration for organ injury/dysfunction than those observed in wild-type mice (WT). Furthermore, Gal-3 KO mice show an increased number of neutrophils in the primary focus of infection and reduced bacterial loads in the peritoneal cavity, blood, and lungs. Mechanistically, blood neutrophils from septic mice show higher levels of surface-bound Gal-3 than neutrophils from naive mice. The deficiency of Gal-3 was associated with increased rolling and adhesion of these cells in mesenteric venules. Our results indicate that Gal-3, secreted during sepsis, inhibits neutrophil migration into the infectious focus, which promotes the bacterial spread and worsens the outcome of sepsis.
Giovannone N, Antonopoulos A, Liang J, Geddes Sweeney J, Kudelka MR, King SL, Lee GS, Cummings RD, Dell A, Barthel SL, et al. Human B Cell Differentiation Is Characterized by Progressive Remodeling of O-Linked Glycans. Front Immunol. 2018;9 (2857). Publisher's VersionAbstract
Germinal centers (GC) are microanatomical niches where B cells proliferate, undergo antibody affinity maturation, and differentiate to long-lived memory B cells and antibody-secreting plasma cells. For decades, GC B cells have been defined by their reactivity to the plant lectin peanut agglutinin (PNA), which binds serine/threonine (O-linked) glycans containing the asialylated disaccharide Gal-β1,3-GalNAc-Ser/Thr (also called T-antigen). In T cells, acquisition of PNA binding by activated T cells and thymocytes has been linked with altered tissue homing patterns, cell signaling, and survival. Yet, in GC B cells, the glycobiological basis and significance of PNA binding remains surprisingly unresolved. Here, we investigated the basis for PNA reactivity of GC B cells. We found that GC B cell binding to PNA is associated with downregulation of the α2,3 sialyltransferase, ST3GAL1 (ST3Gal1), and overexpression of ST3Gal1 was sufficient to reverse PNA binding in B cell lines. Moreover, we found that the primary scaffold for PNA-reactive O-glycans in B cells is the B cell receptor-associated receptor-type tyrosine phosphatase CD45, suggesting a role for altered O-glycosylation in antigen receptor signaling. Consistent with similar reports in T cells, ST3Gal1 overexpression in B cells in vitro induced drastic shortening in O-glycans, which we confirmed by both antibody staining and mass spectrometric O-glycomic analysis. Unexpectedly, ST3Gal1-induced changes in O-glycan length also correlated with altered binding of two glycosylation-sensitive CD45 antibodies, RA3-6B2 (more commonly called B220) and MEM55, which (in humans) have previously been reported to favor binding to naïve/GC subsets and memory/plasmablast subsets, respectively. Analysis of primary B cell binding to B220, MEM55, and several plant lectins suggested that B cell differentiation is accompanied by significant loss of O-glycan complexity, including loss of extended Core 2 O-glycans. To our surprise, decreased O-glycan length from naïve to post-GC fates best correlated not with ST3Gal1, but rather downregulation of the Core 2 branching enzyme GCNT1. Thus, our data suggest that O-glycan remodeling is a feature of B cell differentiation, dually regulated by ST3Gal1 and GCNT1, that ultimately results in expression of distinct O-glycosylation states/CD45 glycoforms at each stage of B cell differentiation.
Purohit S, Li T, Guan W, Song X, Song J, Tian Y, Li L, Sharma A, Dun B, Mysona D, et al. Multiplex glycan bead array for high throughput and high content analyses of glycan binding proteins. Nat Commun. 2018;9 (1) :258. Publisher's VersionAbstract
Glycan-binding proteins (GBPs) play critical roles in diverse cellular functions such as cell adhesion, signal transduction and immune response. Studies of the interaction between GBPs and glycans have been hampered by the availability of high throughput and high-content technologies. Here we report multiplex glycan bead array (MGBA) that allows simultaneous analyses of 384 samples and up to 500 glycans in a single assay. The specificity, sensitivity and reproducibility of MGBA are evaluated using 39 plant lectins, 13 recombinant anti-glycan antibodies, and mammalian GBPs. We demonstrate the utility of this platform by the analyses of natural anti-glycan IgM and IgG antibodies in 961 human serum samples and the discovery of anti-glycan antibody biomarkers for ovarian cancer. Our data indicate that the MGBA platform is particularly suited for large population-based studies that require the analyses of large numbers of samples and glycans.
DeCicco RePass MA, Bhat N, Heimburg-Molinaro J, Bunnell S, Cummings RD, Ward HD. Molecular cloning, expression, and characterization of UDP N-acetyl-α-d-galactosamine: Polypeptide N-acetylgalactosaminyltransferase 4 from Cryptosporidium parvum. Mol Biochem Parasitol. 2018;221 :56-65. Publisher's VersionAbstract
Cryptosporidium spp. are the causative agents of diarrheal disease worldwide, but effective treatments are lacking. Cryptosporidium employs mucin-like glycoproteins with O-glycans to attach to and infect host intestinal epithelial cells. The Tn antigen (GalNAcα1-Ser/Thr) is an O-glycan essential for these processes, as Tn-specific lectins and a Tn-specific monoclonal antibody block attachment to and infection of host cells in vitro. The enzymes in Cryptosporidium catalyzing their synthesis, however, have not been studied. Previously, we identified four genes encoding putative UDP N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts) in the genomes of three Cryptosporidium spp. Here we report the in silico analysis, cloning, expression, purification, and characterization of one of the four enzymes Cryptosporidium parvum (Cp)-ppGalNAc-T4. This enzyme contains the characteristic domains and motifs conserved in ppGalNAc-Ts and is expressed at multiple time points during in vitro infection. Recombinant soluble Cp-ppGalNAc-T4 was enzymatically active against an unmodified EA2 peptide suggesting that it may function as an "initiating" ppGalNAc-T. Cp-ppGalNAc-T4 also exhibited a strong preference for UDP-GalNAc over other nucleotide sugar donors and was active against unmodified and O-glycosylated versions of the C. parvum gp40-derived peptide, with a preference for the former, suggesting it may play a role in modifying this glycoprotein in vivo. Given the importance of mucin-type O-glycosylation in Cryptosporidium spp., the enzymes that catalyze their synthesis may serve as potential therapeutic targets.
Sardar MYR, Mandhapati AR, Park S, Wever WJ, Cummings RD, Chaikof EL. Convergent Synthesis of Sialyl LewisX- O-Core-1 Threonine. J Org Chem. 2018;83 (9) :4963-4972. Publisher's VersionAbstract
Selectins are a class of cell adhesion molecules that play a critical role during the initial steps of inflammation. The N-terminal domain of P-selectin glycoprotein ligand-1 (PSGL-1) binds to all selectins, but with the highest affinity to P-selectin. Recent evidence suggests that the blockade of P-selectin/PSGL-1 interactions provides a viable therapeutic option for the treatment of many inflammatory diseases. Herein, we report the total synthesis of threonine bearing sialyl LewisX (sLeX) linked to a Core-1- O-hexasaccharide 1, as a key glycan of the N-terminal domain of PSGL-1. A convergent synthesis using α-selective sialylation and a regioselective [4+2] glycosylation are the key features of this synthesis.
Hu L, Sankaran B, Laucirica DR, Patil K, Salmen W, Ferreon ACM, Tsoi PS, Lasanajak Y, Smith DF, Ramani S, et al. Glycan recognition in globally dominant human rotaviruses. Nat Commun. 2018;9 (1) :2631. Publisher's VersionAbstract
Rotaviruses (RVs) cause life-threatening diarrhea in infants and children worldwide. Recent biochemical and epidemiological studies underscore the importance of histo-blood group antigens (HBGA) as both cell attachment and susceptibility factors for the globally dominant P[4], P[6], and P[8] genotypes of human RVs. How these genotypes interact with HBGA is not known. Here, our crystal structures of P[4] and a neonate-specific P[6] VP8*s alone and in complex with H-type I HBGA reveal a unique glycan binding site that is conserved in the globally dominant genotypes and allows for the binding of ABH HBGAs, consistent with their prevalence. Remarkably, the VP8* of P[6] RVs isolated from neonates displays subtle structural changes in this binding site that may restrict its ability to bind branched glycans. This provides a structural basis for the age-restricted tropism of some P[6] RVs as developmentally regulated unbranched glycans are more abundant in the neonatal gut.
Behrens AJ, Duke RM, Petralia LMC, Lehoux S, Carlow CKS, Taron CH, Foster JM. Changes in canine serum N-glycosylation as a result of infection with the heartworm parasite Dirofilaria immitis. Sci Rep. 2018;8 (1) :16625. Publisher's VersionAbstract
Filariases are diseases caused by infection with filarial nematodes and transmitted by insect vectors. The filarial roundworm Dirofilaria immitis causes heartworm disease in dogs and other carnivores. D. immitis is closely related to Onchocerca volvulus, Wuchereria bancrofti and Brugia malayi, which cause onchocerciasis (river blindness) and lymphatic filariasis (elephantiasis) in humans and are neglected tropical diseases. Serum N-glycosylation is very sensitive to both pathological infections and changes in mammalian biology due to normal aging or lifestyle choices. Here, we report significant changes in the serum N-glycosylation profiles of dogs infected with D. immitis. Our data derive from analysis of serum from dogs with established patent infections and from a longitudinal infection study. Overall, galactosylation and core fucosylation increase, while sialylation decreases in infected dog sera. We also identify individual glycan structures that change significantly in their relative abundance during infection. Notably, the abundance of the most dominant N-glycan in canine serum (biantennary, disialylated A2G2S2) decreases by over 10 percentage points during the first 6 months of infection in each dog analyzed. This is the first longitudinal study linking changes in mammalian serum N-glycome to progression of a parasitic infection
Behrens AJ, Duke RM, Petralia LM, Harvey DJ, Lehoux S, Magnelli PE, Taron CH, Foster JM. Glycosylation profiling of dog serum reveals differences compared to human serum. Glycobiology. 2018;28 (11) :825-831. Publisher's VersionAbstract
Glycosylation is the most common post-translational modification of serum proteins, and changes in the type and abundance of glycans in human serum have been correlated with a growing number of human diseases. While the glycosylation pattern of human serum is well studied, little is known about the profiles of other mammalian species. Here, we report detailed glycosylation profiling of canine serum by hydrophilic interaction chromatography-ultraperformance liquid chromatography (HILIC-UPLC) and mass spectrometry. The domestic dog (Canis familiaris) is a widely used model organism and of considerable interest for a large veterinary community. We found significant differences in the serum N-glycosylation profile of dogs compared to that of humans, such as a lower abundance of galactosylated and sialylated glycans. We also compare the N-glycan profile of canine serum to that of canine IgG - the most abundant serum glycoprotein. Our data will serve as a baseline reference for future studies when performing serum analyses of various health and disease states in dogs.
Koelsch KA, Cavett J, Smith K, Moor JS, Lehoux SD, Jia N, Mather T, Quadri SMS, Rasmussen A, Kaufman EC, et al. Evidence for Alternate Modes of B cell Activation Involving Fab Acquired-N-Glycosylations in Antibody Secreting Cells Infiltrating the Labial Salivary Glands of Sjögren's Syndrome Patients. Arthritis Rheumatol. 2018. Publisher's VersionAbstract


To better understand the role of B cells, potential mechanisms for their aberrant activation, and the production of autoantibodies in the pathogenesis of Sjögren's Syndrome (SS), we explored selection pressures and N-glycosylation acquired by somatic mutation (acN-glyc) in the immunoglobulin (Ig) variable regions (V-regions) of antibody secreting cells (ASCs) isolated from the minor salivary glands of SS patients and non-SS controls with sicca symptoms.


We report a novel method to produce and characterize recombinant monoclonal antibodies (mAbs) from SS patient and control labial salivary gland single-cell sorted ASC infiltrates that can be utilized to concurrently probe any other expressed genes. V-regions were amplified by RT-PCR, sequenced, and analyzed for incidence of N-glycosylation and selection pressure, then expressed as the native mAbs, or mutant mAbs lacking the acN-glyc for specificity testing. Protein modeling was used to demonstrate how even acN-glycs outside of the complementarity-determining region (CDR) could participate in, or inhibit, antigen binding.


V-region sequence analyses revealed clonal expansions and evidence for secondary light chain editing and allelic inclusion not previously reported in SS. We found increased acN-glycs in the sequences from SS patients and that acN-glycs were associated with increased replacement mutations and lowered selection pressure. We also identified a clonal set of polyreactive mAbs with differential FWR1 acN-glycs and demonstrated that removal of the acN-glyc could nearly abolish binding to the autoantigens.


Our findings support an alternative mechanism involving V-region N-glycosylation for the selection and proliferation of some autoreactive B cells in SS patients.

Kudelka MR, Nairn AV, Sardar MY, Sun X, Chaikof EL, Ju T, Moremen KW, Cummings RD. Isotopic Labeling with Cellular O-glycome Reporter/Amplification (ICORA) for Comparative O-glycomics of Cultured Cells. Glycobiology. 2018.Abstract
Mucin-type O-glycans decorate >80% of secretory and cell surface proteins and contribute to health and disease. However, dynamic alterations in the O-glycome are poorly understood because current O-glycomic methodologies are not sufficiently sensitive nor quantitative. Here we describe a novel isotope labeling approach termed Isotope-Cellular O-glycome Reporter Amplification (ICORA) to amplify and analyze the O-glycome from cells. In this approach, cells are incubated with Ac3GalNAc-Bn (Ac3GalNAc-[1H7]Bn) or a heavy labeled Ac3GalNAc-BnD7 (Ac3GalNAc-[2D7]Bn) O-glycan precursor (7 Da mass difference), which enters cells and upon de-esterification is modified by Golgi enzymes to generate Bn-O-glycans secreted into the culture media. After recovery, heavy and light Bn-O-glycans from two separate conditions are mixed, analyzed by MS, and statistically interrogated for changes in O-glycan abundance using a semi-automated approach. ICORA enables ~100–1000 fold enhanced sensitivity and increased throughput compared to traditional O-glycomics. We validated ICORA with model cell lines and used it to define alterations in the O-glycome in colorectal cancer. ICORA is a useful tool to explore the dynamic regulation of the O-glycome in health and disease.
Purohit S, Li T, Guan W, Song X, Song J, Yanna T, Li L, Sharma A, Dun B, Mysona D, et al. Multiplex glycan bead array for high throughput and high content analyses of glycan binding proteins. Nature Commun. 2018;9 (1) :258. Publisher's VersionAbstract
Glycan-binding proteins (GBPs) play critical roles in diverse cellular functions such as cell adhesion, signal transduction and immune response. Studies of the interaction between GBPs and glycans have been hampered by the availability of high throughput and high-content technologies. Here we report multiplex glycan bead array (MGBA) that allows simultaneous analyses of 384 samples and up to 500 glycans in a single assay. The specificity, sensitivity and reproducibility of MGBA are evaluated using 39 plant lectins, 13 recombinant anti-glycan antibodies, and mammalian GBPs. We demonstrate the utility of this platform by the analyses of natural anti-glycan IgM and IgG antibodies in 961 human serum samples and the discovery of anti-glycan antibody biomarkers for ovarian cancer. Our data indicate that the MGBA platform is particularly suited for large population-based studies that require the analyses of large numbers of samples and glycans.
Jankowska E, Parsons LM, Song X, Smith DF, Cummings RD, Cipollo JF. A Comprehensive Caenorhabditis elegans N-glycan Shotgun Array. Glycobiology. 2018. Publisher's VersionAbstract
Here we present a Caenorhabditis elegans N-glycan shotgun array. This nematode serves as a model organism for many areas of biology including but not limited to tissue development, host–pathogen interactions, innate immunity, and genetics. Caenorhabditis elegans N-glycans contain structural motifs that are also found in other nematodes as well as trematodes and lepidopteran species. Glycan binding toxins that interact with C. elegansglycoconjugates also do so with some agriculturally relevant species, such as Haemonchus contortusAscaris suumOesophagostomum dentatum and Trichoplusia ni. This situation implies that protein–carbohydrate interactions seen with C. elegans glycans may also occur in other species with related glycan structures. Therefore, this array may be useful to study these relationships in other nematodes as well as trematode and insect species. The array contains 134 distinct glycomers spanning a wide range of C. elegans N-glycans including the subclasses high mannose, pauci mannose, high fucose, mammalian-like complex and phosphorylcholine substituted forms. The glycans presented on the array have been characterized by two-dimensional separation, ion trap mass spectrometry, and lectin affinity. High fucose glycans were well represented and contain many novel core structures found in C. elegans as well as other species. This array should serve as an investigative platform for carbohydrate binding proteins that interact with N-glycans of C. elegans and over a range of organisms that contain glycan motifs conserved with this nematode.
Pantophlet R, Trattnig N, Murrell S, Lu N, Chau D, Rempel C, Wilson IA, Kosma P. Bacterially derived synthetic mimetics of mammalian oligomannose prime antibody responses that neutralize HIV infectivity. Nat Commun. 2017;8 (1) :1601.Abstract
Oligomannose-type glycans are among the major targets on the gp120 component of the HIV envelope protein (Env) for broadly neutralizing antibodies (bnAbs). However, attempts to elicit oligomannose-specific nAbs by immunizing with natural or synthetic oligomannose have so far not been successful, possibly due to B cell tolerance checkpoints. Here we design and synthesize oligomannose mimetics, based on the unique chemical structure of a recently identified bacterial lipooligosaccharide, to appear foreign to the immune system. One of these mimetics is bound avidly by members of a family of oligomannose-specific bnAbs and their putative common germline precursor when presented as a glycoconjugate. The crystal structure of one of the mimetics bound to a member of this bnAb family confirms the antigenic resemblance. Lastly, immunization of human-antibody transgenic animals with a lead mimetic evokes nAbs with specificities approaching those of existing bnAbs. These results provide evidence for utilizing antigenic mimicry to elicit oligomannose-specific bnAbs to HIV-1.
Collins BC, Nakahara H, Acharya S, Cooper MD, Herrin BR, Wilson IA. Crystal structure of an anti-idiotype variable lymphocyte receptor. Acta Crystallogr F Struct Biol Commun. 2017;73 (Pt 12) :682-687.Abstract
Variable lymphocyte receptors (VLRs), the leucine-rich repeat (LRR)-based antigen receptors of jawless fish, have great utility in a wide variety of biochemical and biological applications, similar to classical Ig-based antibodies. VLR-based reagents may be particularly useful when traditional antibodies are not available. An anti-idiotype lamprey VLR, VLR39, has previously been identified that recognizes the heavy-chain CDR3 of the B-cell receptor (BCR) of a leukemic clone from a patient with chronic lymphocytic leukemia (CLL). VLR39 was used successfully to track the re-emergence of this clone in the patient following chemotherapy. Here, the crystal structure of VLR39 is presented at 1.5 Å resolution and compared with those of other protein-specific VLRs. VLR39 adopts a curved solenoid fold and exhibits substantial structural similarity to other protein-binding VLRs. VLR39 has a short LRRCT loop that protrudes outwards away from the concave face and is similar to those of its protein-specific VLR counterparts. Analysis of the VLR39-BCR interaction by size-exclusion chromatography and biolayer interferometry using the scFv version of the BCR confirms that VLR39 recognizes the BCR Fv region. Such VLR-based reagents may be useful for identifying and monitoring leukemia in CLL patients and in other clinical diagnostic assays.
Zhang P, Li K, Yang G, Xia C, Polston JE, Li G, Li S, Lin Z, Yang L-J, Bruner SD, et al. Cytotoxic protein from the mushroom possesses a unique mode for glycan binding and specificity. Proc Natl Acad Sci U S A. 2017;114 (34) :8980-8985.Abstract
Glycans possess significant chemical diversity; glycan binding proteins (GBPs) recognize specific glycans to translate their structures to functions in various physiological and pathological processes. Therefore, the discovery and characterization of novel GBPs and characterization of glycan-GBP interactions are significant to provide potential targets for therapeutic intervention of many diseases. Here, we report the biochemical, functional, and structural characterization of a 130-amino-acid protein, Y3, from the mushroom Biochemical studies of recombinant Y3 from a yeast expression system demonstrated the protein is a unique GBP. Additionally, we show that Y3 exhibits selective and potent cytotoxicity toward human T-cell leukemia Jurkat cells compared with a panel of cancer cell lines via inducing caspase-dependent apoptosis. Screening of a glycan array demonstrated GalNAcβ1-4(Fucα1-3)GlcNAc (LDNF) as a specific Y3-binding ligand. To provide a structural basis for function, the crystal structure was solved to a resolution of 1.2 Å, revealing a single-domain αβα-sandwich motif. Two monomers were dimerized to form a large 10-stranded, antiparallel β-sheet flanked by α-helices on each side, representing a unique oligomerization mode among GBPs. A large glycan binding pocket extends into the dimeric interface, and docking of LDNF identified key residues for glycan interactions. Disruption of residues predicted to be involved in LDNF/Y3 interactions resulted in the significant loss of binding to Jurkat T-cells and severely impaired their cytotoxicity. Collectively, these results demonstrate Y3 to be a GBP with selective cytotoxicity toward human T-cell leukemia cells and indicate its potential use in cancer diagnosis and treatment.
Zhang W, van Eijk M, Guo H, van Dijk A, Bleijerveld OB, Verheije HM, Wang G, Haagsman HP, Veldhuizen EJA. Expression and characterization of recombinant chicken mannose binding lectin. Immunobiology. 2017;222 (3) :518-528.Abstract
Mannose binding lectin (MBL) is a serum collagenous C-type lectin that plays an important role in the innate immune protection against pathogens. Previously, human and mouse studies have demonstrated that MBL binds a broad range of pathogens that results in their neutralization through agglutination, enhanced phagocytosis, and/or complement activation via the lectin pathway. The role of MBL in chicken is not well understood although the MBL concentration in serum seems to correlate with protection against infections. To investigate the role of MBL in chicken further, recombinant chicken MBL (RcMBL) was produced in HeLa R19 cells and purified using mannan affinity chromatography followed by gel filtration. RcMBL was shown to be structurally and functionally similar to native chicken MBL (NcMBL) isolated from serum. RcMBL is expressed as an oligomeric protein (mixture of trimers and oligomerized trimers) with a monomeric mass of 26kDa as determined by mass spectrometry, corresponding to the predicted mass. Glycan array analysis indicated that RcMBL bound most strongly to high-mannose glycans but also glycans with terminal fucose and GlcNac residues. The biological activity of RcMBL was demonstrated via its capacity to agglutinate Salmonella Typhimurium and to inhibit the hemagglutination activity of influenza A virus. The production of a structurally well-characterized and functionally active RcMBL will facilitate detailed studies into the protective role of MBL in innate defense against pathogens in chicken and other avian species.
Tati S, Fisk JC, Abdullah J, Karacosta L, Chrisikos T, Philbin P, Morey S, Ghazal D, Zazala F, Jessee J, et al. Humanization of JAA-F11, a Highly Specific Anti-Thomsen-Friedenreich Pancarcinoma Antibody and InVitro Efficacy Analysis. Neoplasia. 2017;19 (9) :716-733.Abstract
JAA-F11 is a highly specific mouse monoclonal to the Thomsen-Friedenreich Antigen (TF-Ag) which is an alpha-O-linked disaccharide antigen on the surface of ~80% of human carcinomas, including breast, lung, colon, bladder, ovarian, and prostate cancers, and is cryptic on normal cells. JAA-F11 has potential, when humanized, for cancer immunotherapy for multiple cancer types. Humanization of JAA-F11, was performed utilizing complementarity determining regions grafting on a homology framework. The objective herein is to test the specificity, affinity and biology efficacy of the humanized JAA-F11 (hJAA-F11). Using a 609 target glycan array, 2 hJAA-F11 constructs were shown to have excellent chemical specificity, binding only to TF-Ag alpha-linked structures and not to TF-Ag beta-linked structures. The relative affinity of these hJAA-F11 constructs for TF-Ag was improved over the mouse antibody, while T20 scoring predicted low clinical immunogenicity. The hJAA-F11 constructs produced antibody-dependent cellular cytotoxicity in breast and lung tumor lines shown to express TF-Ag by flow cytometry. Internalization of hJAA-F11 into cancer cells was also shown using a surface binding ELISA and confirmed by immunofluorescence microscopy. Both the naked hJAA-F11 and a maytansine-conjugated antibody (hJAA-F11-DM1) suppressed in vivo tumor progression in a human breast cancer xenograft model in SCID mice. Together, our results support the conclusion that the humanized antibody to the TF-Ag has potential as an adjunct therapy, either directly or as part of an antibody drug conjugate, to treat breast cancer, including triple negative breast cancer which currently has no targeted therapy, as well as lung cancer.
Mahajan S, Khairnar A, Bishnoi R, Ramya TNC. Microbial F-type lectin domains with affinity for blood group antigens. Biochem Biophys Res Commun. 2017;491 (3) :708-713.Abstract
F-type lectins are fucose binding lectins with characteristic fucose binding and calcium binding motifs. Although they occur with a selective distribution in viruses, prokaryotes and eukaryotes, most biochemical studies have focused on vertebrate F-type lectins. Recently, using sensitive bioinformatics search techniques on the non-redundant database, we had identified many microbial F-type lectin domains with diverse domain organizations. We report here the biochemical characterization of F-type lectin domains from Cyanobium sp. PCC 7001, Myxococcus hansupus and Leucothrix mucor. We demonstrate that while all these three microbial F-type lectin domains bind to the blood group H antigen epitope on fucosylated glycans, there are fine differences in their glycan binding specificity. Cyanobium sp. PCC 7001 F-type lectin domain binds exclusively to extended H type-2 motif, Myxococcus hansupus F-type lectin domain binds to B, H type-1 and Lewis motifs, and Leucothrix mucor F-type lectin domain binds to a wide range of fucosylated glycans, including A, B, H and Lewis antigens. We believe that these microbial lectins will be useful additions to the glycobiologist's toolbox for labeling, isolating and visualizing glycans.
Bunker JJ, Erickson SA, Flynn TM, Henry C, Koval JC, Meisel M, Jabri B, Antonopoulos DA, Wilson PC, Bendelac A. Natural polyreactive IgA antibodies coat the intestinal microbiota. Science. 2017;358 (6361).Abstract
Large quantities of immunoglobulin A (IgA) are constitutively secreted by intestinal plasma cells to coat and contain the commensal microbiota, yet the specificity of these antibodies remains elusive. Here we profiled the reactivities of single murine IgA plasma cells by cloning and characterizing large numbers of monoclonal antibodies. IgAs were not specific to individual bacterial taxa but rather polyreactive, with broad reactivity to a diverse, but defined, subset of microbiota. These antibodies arose at low frequencies among naïve B cells and were selected into the IgA repertoire upon recirculation in Peyer's patches. This selection process occurred independent of microbiota or dietary antigens. Furthermore, although some IgAs acquired somatic mutations, these did not substantially influence their reactivity. These findings reveal an endogenous mechanism driving homeostatic production of polyreactive IgAs with innate specificity to microbiota.