Structure aided design of a Neu5Gc specific lectin

Day, Christopher J.; Paton, Adrienne W.; Higgins, M.A.; Shewell, Lucy K.; Jen, Freda E.-C.; Schulz, Benjamin L.; Herdman, Brock P; Paton, James C.; Jennings, Michael P.

doi:10.1038/s41598-017-01522-9

Cited by 18 publications

(17 citation statements)

References 26 publications

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“…A glycan search engine (Bruker, Germany) and GlycomeDB (https://glytoucan.org) were used for glycan identification. As expected, the glycan Hex5HexNAc4NeuGc2dHex1, similar to N003G in the N-glycan microarray [39], was found in MCF-7 and K562 cells (Fig. 4b), in contrast to the observation in human normal leukocytes where no glycosidically bound Neu5Gc was found.…”

Section: Resultssupporting

confidence: 79%

Crystal structure of a cytocidal protein from lamprey and its mechanism of action in the selective killing of cancer cells

et al. 2019

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Background In previous research, we found that lamprey immune protein (LIP) possessed cytocidal activity against tumor cells, but the mechanism of the selective recognition and killing of tumor cells by LIP was not identified. Methods Superresolution microscopy, crystallographic structural analysis, glycan chip assay, SPR experiments, FACS assays, computational studies and mass spectrometric analysis firmly establish the mode of action of LIP, which involves dual selective recognition and efficient binding. Results We determined the overall crystallographic structure of LIP at a resolution of 2.25 Å. LIP exhibits an elongated structure with dimensions of 105 Å × 30 Å × 30 Å containing an N-terminal lectin module and a C-terminal aerolysin module. Moreover, the Phe 209 -Gly 232 region is predicted to insert into the lipid bilayer to form a transmembrane β-barrel, in which the hydrophobic residues face the lipid bilayer, and the polar residues constitute the hydrophilic lumen of the pore. We found that LIP is able to kill various human cancer cells with minimal effects on normal cells. Notably, by coupling biochemical and computational studies, we propose a hypothetical mechanism that involves dual selective recognition and efficient binding dependent on both N-linked glycans on GPI-anchored proteins (GPI-APs) and sphingomyelin (SM) in lipid rafts. Furthermore, specific binding of the lectin module with biantennary bisialylated nonfucosylated N-glycan or sialyl Lewis X-containing glycan structures on GPI-APs triggers substantial conformational changes in the aerolysin module, which interacts with SM, ultimately resulting in the formation of a membrane-bound oligomer in lipid rafts. Conclusions LIP holds great potential for the application of a marine protein towards targeted cancer therapy and early diagnosis in humans. Electronic supplementary material The online version of this article (10.1186/s12964-019-0358-y) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 79%

Crystal structure of a cytocidal protein from lamprey and its mechanism of action in the selective killing of cancer cells

et al. 2019

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“…Flow cell 1 was made as a blank cell (ethanolamine-blocked surface) to allow for double reference subtraction. Sugars were initially flowed over the 6E4 antibody from 0.01 to 100 µM, and the concentration for each sample was adjusted on the basis of observed binding as previously described ( 36 ). KDO was run at a maximum concentration of 500 nM, Neu5Ac was run at a maximum concentration of 10 µM, and N -glycolylneuraminic acid (Neu5Gc) was reduced to 50 µM.…”

Section: Methodsmentioning

confidence: 99%

Nontypeable Haemophilus influenzae Lipooligosaccharide Expresses a Terminal Ketodeoxyoctanoate In Vivo , Which Can Be Used as a Target for Bactericidal Antibody

Apicella

Coffin

Ketterer

et al. 2018

mBio

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Nontypeable Haemophilus influenzae (NTHi) is an important pathogen in individuals of all ages. The lipooligosaccharide (LOS) of NTHi has evolved a complex structure that can be attributed to a multiplicity of glycosyltransferases, the random switching of glycosyltransferase gene expression via phase variation, and the complex structure of its core region with multiple glycoform branch points. This article adds to that complexity by describing a multifunctional enzyme (LsgB) which optimally functions when the species is grown on a solid surface and which can add either a ketodeoxyoctanoate (KDO) or an N-acetylneuramic acid (Neu5Ac) moiety to a terminal N-acetyllactosamine structure of LOS. Our studies show that expression of lsgB is reduced four- to sixfold when NTHi is grown in broth. The substrate that the enzyme utilizes is dependent upon the concentration of free Neu5Ac (between 1 and 10 µg/ml) in the environment. In environments in which Neu5Ac is below that level, the enzyme utilizes endogenous CMP-KDO as the substrate. Our studies show that during in vivo growth in an NTHi biofilm, the KDO moiety is expressed by the organism. Monoclonal antibody 6E4, which binds KDO, is bactericidal for NTHi strains that express the KDO epitope at high levels. In a survey of 33 NTHi strains isolated from healthy and diseased individuals, the antibody was bactericidal (>90% kill) for 12 strains (36%). These studies open up the possibility of using a KDO-based glycoconjugate vaccine as part of a multicomponent vaccine against NTHi.

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“…Subsequently, the Stx binding specificity of the B pentamer is elucidated, followed by a short survey of the remarkable interaction of the toxin's A subunit with Toll-like receptor 4 (TLR4) and the intracellular retrograde routing of Stx. [140,[281][282][283][284] similar to the subtilase cytotoxin (SubAB), which represents the prototype of a "new" family of potent AB 5 cytotoxins produced by STEC strains [285][286][287][288]. The 32 kDa A subunit of Stx is made of a large enzymatically active 27.5 kDa A1 and a small 4.5 kDa A2 fragment, which are linked via a disulfide bond [136].…”

Section: Shiga Toxin and Toxin-mediated Cell Damagementioning

confidence: 99%

Valid Presumption of Shiga Toxin-Mediated Damage of Developing Erythrocytes in EHEC-Associated Hemolytic Uremic Syndrome

Detzner

Pohlentz

Müthing

2020

Toxins

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The global emergence of clinical diseases caused by enterohemorrhagic Escherichia coli (EHEC) is an issue of great concern. EHEC release Shiga toxins (Stxs) as their key virulence factors, and investigations on the cell-damaging mechanisms toward target cells are inevitable for the development of novel mitigation strategies. Stx-mediated hemolytic uremic syndrome (HUS), characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute renal injury, is the most severe outcome of an EHEC infection. Hemolytic anemia during HUS is defined as the loss of erythrocytes by mechanical disruption when passing through narrowed microvessels. The formation of thrombi in the microvasculature is considered an indirect effect of Stx-mediated injury mainly of the renal microvascular endothelial cells, resulting in obstructions of vessels. In this review, we summarize and discuss recent data providing evidence that HUS-associated hemolytic anemia may arise not only from intravascular rupture of erythrocytes, but also from the extravascular impairment of erythropoiesis, the development of red blood cells in the bone marrow, via direct Stx-mediated damage of maturing erythrocytes, leading to “non-hemolytic” anemia.

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Structure aided design of a Neu5Gc specific lectin

Cited by 18 publications

References 26 publications

Crystal structure of a cytocidal protein from lamprey and its mechanism of action in the selective killing of cancer cells

Crystal structure of a cytocidal protein from lamprey and its mechanism of action in the selective killing of cancer cells

Nontypeable Haemophilus influenzae Lipooligosaccharide Expresses a Terminal Ketodeoxyoctanoate In Vivo , Which Can Be Used as a Target for Bactericidal Antibody

Valid Presumption of Shiga Toxin-Mediated Damage of Developing Erythrocytes in EHEC-Associated Hemolytic Uremic Syndrome

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