by
Sujata Halder;
Susan Cotmore;
Jamie Heimburg-Molinaro;
David Smith;
Richard Cummings;
Xi Chen;
Alana J. Trollope;
Simon J. North;
Stuart M. Haslam;
Anne Dell;
Peter Tattersall;
Robert McKenna;
Mavis Agbandje-McKenna
The recognition of sialic acids by two strains of minute virus of mice (MVM), MVMp (prototype) and MVMi (immunosuppressive), is an essential requirement for successful infection. To understand the potential for recognition of different modifications of sialic acid by MVM, three types of capsids, virus-like particles, wild type empty (no DNA) capsids, and DNA packaged virions, were screened on a sialylated glycan microarray (SGM). Both viruses demonstrated a preference for binding to 9-O-methylated sialic acid derivatives, while MVMp showed additional binding to 9-O-acetylated and 9-O-lactoylated sialic acid derivatives, indicating recognition differences. The glycans recognized contained a type-2 Galβ1-4GlcNAc motif (Neu5Acα2-3Galβ1-4GlcNAc or 3′SIA-LN) and were biantennary complex-type N-glycans with the exception of one. To correlate the recognition of the 3′SIA-LN glycan motif as well as the biantennary structures to their natural expression in cell lines permissive for MVMp, MVMi, or both strains, the N- and O-glycans, and polar glycolipids present in three cell lines used for in vitro studies, A9 fibroblasts, EL4 T lymphocytes, and the SV40 transformed NB324K cells, were analyzed by MALDI-TOF/TOF mass spectrometry. The cells showed an abundance of the sialylated glycan motifs recognized by the viruses in the SGM and previous glycan microarrays supporting their role in cellular recognition by MVM. Significantly, the NB324K showed fucosylation at the non-reducing end of their biantennary glycans, suggesting that recognition of these cells is possibly mediated by the Lewis X motif as in 3′SIA-Le X identified in a previous glycan microarray screen.
Human milk glycans (HMGs) are prebiotics, pathogen receptor decoys, and regulators of host physiology and immune responses. Mechanistically, human lectins (glycan-binding proteins, hGBPs) expressed by dendritic cells (DC) are of major interest, as these cells directly contact HMGs. To explore such interactions, we screened many C-type lectins and Siglecs expressed by DC for glycan binding on microarrays presenting over 200 HMGs. Unexpectedly, DC-SIGN showed robust binding to many HMGs, whereas other C-type lectins failed to bind, and Siglecs-5 and -9 showed weak binding to a few glycans. By contrast, most hGBPs bound to multiple glycans on other microarrays lacking HMGs. An α-linked fucose residue was characteristic of HMGs bound by DC-SIGN. Binding of DC-SIGN to the simple HMGs 2′-fucosyllactose (2′-FL) and 3-fucosyllactose (3-FL) was confirmed by flow cytometry to beads conjugated with 2′-FL or 3-FL, as well as the ability of the free glycans to inhibit DC-SIGN binding. 2′-FL had an IC50 of ~1 mM for DC-SIGN, which is within the physiological concentration of 2′-FL in human milk. These results demonstrate that DC-SIGN among the many hGBPs expressed by DC binds to α-fucosylated HMGs, and suggest that such interactions may be important in influencing immune responses in the developing infant.
by
Liya Hu;
Banumathi Sankaran;
Daniel R. Laucirica;
Ketki Patil;
Wilhelm Salmen;
Allan Chris M Ferreon;
Phoebe S. Tsoi;
Yi Lasanajak;
David Smith;
Sasirekha Ramani;
Robert L. Atmar;
Mary K. Estes;
Josephine C. Ferreon;
B.V. Venkataram Prasad
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.
Tandem zinc finger (ZF) proteins are the largest and most rapidly diverging family of DNA-binding transcription regulators in mammals. ZFP568 represses a transcript of placental-specific insulin like growth factor 2 (Igf2-P0) in mice. ZFP568 binds a 24-base pair sequence-specific element upstream of Igf2-P0 via the eleven-ZF array. Both DNA and protein conformations deviate from the conventional one finger-three bases recognition, with individual ZFs contacting 2, 3, or 4 bases and recognizing thymine on the opposite strand. These interactions arise from a shortened minor groove caused by an AT-rich stretch, suggesting adaptability of ZF arrays to sequence variations. Despite conservation in mammals, mutations at Igf2 and ZFP568 reduce their binding affinity in chimpanzee and humans. Our studies provide important insights into the evolutionary and structural dynamics of ZF-DNA interactions that play a key role in mammalian development and evolution. Evolutionary and structure-function dynamics of zinc finger-DNA interactions reveal unconventional recognition codes and co-evolution of ZFP568 and its target gene Igf2 in mammals.
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Mammalian cells are constantly and unavoidably exposed to DNA damage from endogenous and exogenous sources, frequently to the detriment of genomic integrity and biological function. Cells acquire a large number of chemically diverse lesions per day, and each can have a different genetic fate and biological consequences. However, our knowledge of how and when specific lesions are repaired or how they may compromise the fidelity of DNA replication or transcription and lead to deleterious biological endpoints in mammalian cells is limited. Studying individual lesions requires technically challenging approaches for the targeted introduction of defined lesions into relevant DNA sequences of interest. Here, we present a systematic analysis of factors influencing yield and an improved, efficient and reliable protocol for the production of mammalian expression phagemid vectors containing defined DNA base modifications in any sequence position of either complementary DNA strand. We applied our improved protocol to study the transcriptional mutagenesis-mediated phenotypic consequences of the common oxidative lesion 5-hydroxyuracil, placed in the G12 mutational hotspot of the KRAS oncogene. 5-OHU induced sustained oncogenic signaling in Neil1 Neil1-/-Neil2 -/- mouse cells. The resulting advance in technology will have broad applicability for investigation of single lesion DNA repair, mutagenesis, and DNA damage responses in mammalian cells.
PRDM9 is the only mammalian gene that has been associated with speciation. The PR/SET domain 9 (PRDM9) protein is a major determinant of meiotic recombination hot spots and acts through sequence-specific DNA binding via its C2H2 zinc finger (ZF) tandem array, which is highly polymorphic within and between species. The most common human variant, PRDM9 allele A (PRDM9a), contains 13 fingers (ZF1–13). Allele C (PRDM9c) is the second-most common among African populations and differs from PRDM9a by an arginine-to-serine change (R764S) in ZF9 and by replacement of ZF11 with two other fingers, yielding 14 fingers in PRDM9c. Here we co-crystallized the six-finger fragment ZF8 –13 of PRDM9c, in complex with an oligonucleotide representing a known PRDM9c-specific hot spot sequence, and compared the structure with that of a characterized PRDM9a-specific complex. There are three major differences. First, Ser764in ZF9 allows PRDM9c to accommodate a variable base, whereas PRDM9a Arg764recognizes a conserved guanine. Second, the two-finger expansion of ZF11 allows PRDM9c to recognize three-base-pair-longer sequences. A tryptophan in the additional ZF interacts with a conserved thymine methyl group. Third, an Arg–Asp dipeptide immediately preceding the ZF helix, conserved in two PRDM9a fingers and three PRDM9c fingers, permits adaptability to variations from a C:G base pair (G–Arg interaction) to a G:C base pair (C–Asp interaction). This Arg–Asp conformational switch allows identical ZF modules to recognize different sequences. Our findings illuminate the molecular mechanisms for flexible and conserved binding of human PRDM9 alleles to their cognate DNA sequences.
The advances in healthcare over the past several decades have resulted in populations now living longer. With this increase in longevity, a wider prevalence of cardiovascular diseases is more common and known to be a major factor in rising healthcare costs. A wealth of scientific evidence has implicated cell senescence as an important component in the etiology of these age-dependent pathologies. A number of studies indicate that an excess of reactive oxygen species (ROS) contributes to trigger and accelerate the cardiac senescence processes, and a new role of monoamine oxidases, MAO-A and MAO-B, is emerging in this context. These mitochondrial enzymes regulate the level of catecholamines and serotonin by catalyzing their oxidative deamination in the heart. MAOs' expression substantially increases with ageing (6-fold MAO-A in the heart and 4-fold MAO-B in neuronal tissue), and their involvement in cardiac diseases is supposedly related to the formation of ROS, via the hydrogen peroxide produced during the substrate degradation. Here, we will review the most recent advances in this field and describe why MAOs could be effective targets in order to prevent age-associated cardiovascular disease.
by
Yan Liu;
Ryan McBride;
Mark Stoll;
Angelina S Palma;
Lisete Silva;
Sanjay Agravat;
Kiyoko F Aoki-Kinoshita;
Matthew P Campbell;
Catherine E Costello;
Anne Dell;
Stuart M Haslam;
Niclas G Karlsson;
Kay-Hooi Khoo;
Daniel Kolarich;
Milos V Novotny;
Nicolle H Packer;
Rene Ranzinger;
Erdmann Rapp;
Pauline M Rudd;
Weston B Struwe;
Michael Tiemeyer;
Lance Wells;
William S York;
Joseph Zaia;
Carsten Kettner;
James C Paulson;
Ten Feizi;
David Smith
MIRAGE (Minimum Information Required for A Glycomics Experiment) is an initiative that was created by experts in the fields of glycobiology, glycoanalytics and glycoinformatics to produce guidelines for reporting results from the diverse types of experiments and analyses used in structural and functional studies of glycans in the scientific literature. As a sequel to the guidelines for sample preparation (Struwe et al. 2016, Glycobiology, 26:907-910) and mass spectrometry data (Kolarich et al. 2013, Mol. Cell Proteomics, 12:991-995), here we present the first version of guidelines intended to improve the standards for reporting data from glycan microarray analyses. For each of eight areas in the workflow of a glycan microarray experiment, we provide guidelines for the minimal information that should be provided in reporting results. We hope that the MIRAGE glycan microarray guidelines proposed here will gain broad acceptance by the community, and will facilitate interpretation and reproducibility of the glycan microarray results with implications in comparison of data from different laboratories and eventual deposition of glycan microarray data in international databases.
Glycan microarrays prepared by immobilization of amino-functionalized glycans on NHS-activated glass slides have been successfully used to study protein-glycan interactions. Fluorescently tagged glycans with an amino functional group can be prepared from natural glycans released from glycoproteins. These tagged glycans can be enzymatically modified with various glycosyltransferases, phosphotransferases, sulfotransferases, etc., to quickly expand the size and diversity of the tagged glycan libraries (TGLs). The TGLs, presented in the format of microarrays, provide a convenient platform for identifying the glycan ligands of glycan-binding proteins (GBPs). The chapter provides the background to prepare a defined glycan microarray and uses as an example glycans generated as phosphodiesters and phosphomonoesters of high-mannose type N-glycans. The method describes the preparation of high-mannose type glycan-AEAB conjugates (GAEABs), the purification of their phosphodiesters, and the subsequent mild acid hydrolysis to obtain corresponding phosphomonoesters. These GAEABs are covalently printed as a phosphorylated glycan microarray and used for analysis of the glycan ligand specificities of P-type lectins, such as the mannose-6-phosphate receptors (Man-6-P receptors or MPRs).