Glycan-Protein Interactions Determine Kinetics of N-Glycan Remodeling

AbstractA hallmark of N-linked glycosylation in the secretory compartments of eukaryotic cells is the sequential remodeling of an initially uniform oligosaccharide to a site-specific, heterogeneous ensemble of glycostructures on mature proteins. To understand site-specific processing, we used protein disulfide isomerase (PDI), a model protein with five glycosylation sites, for molecular dynamics (MD) simulations and compared the result to a biochemical in vitro analysis with four different glycan processing enzymes. As predicted by an analysis of the accessibility of the N-glycans for their processing enzymes derived from the MD simulations, N-glycans at different glycosylation sites showed different kinetic properties for the processing enzymes. In addition, altering the tertiary structure context of N-glycan substrates affected N-glycan remodeling in a site-specific way. We propose that differential, tertiary structure context dependent N-glycan reactivities lead to different glycan structures in the same protein through kinetically controlled processing pathways.

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The potential of a site-specific delivery of thiamine hydrochloride as a novel insect repellent exerting long-term protection on human skin: In-vitro, ex-vivo study and clinical assessment

Journal of Pharmaceutical Sciences ◽

10.1016/j.xphs.2021.07.017 ◽

2021 ◽

Author(s):

Mai El Halawany ◽

Randa Latif ◽

Alia Badawi

Keyword(s):

Human Skin ◽

Clinical Assessment ◽

Ex Vivo ◽

Thiamine Hydrochloride ◽

Insect Repellent ◽

Site Specific ◽

A Site ◽

Ex Vivo Study

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Phase Variation of Campylobacter jejuni 81-176 Lipooligosaccharide Affects Ganglioside Mimicry and Invasiveness In Vitro

Infection and Immunity ◽

10.1128/iai.70.2.787-793.2002 ◽

2002 ◽

Vol 70 (2) ◽

pp. 787-793 ◽

Cited By ~ 154

Author(s):

Patricia Guerry ◽

Christine M. Szymanski ◽

Martina M. Prendergast ◽

Thomas E. Hickey ◽

Cheryl P. Ewing ◽

...

Keyword(s):

Campylobacter Jejuni ◽

Phase Variation ◽

Outer Core ◽

Gene Encoding ◽

Site Specific ◽

Reading Frame ◽

Specific Mutation ◽

Normal Human ◽

A Site

ABSTRACT The outer cores of the lipooligosaccharides (LOS) of many strains of Campylobacter jejuni mimic human gangliosides in structure. A population of cells of C. jejuni strain 81-176 produced a mixture of LOS cores which consisted primarily of structures mimicking GM2 and GM3 gangliosides, with minor amounts of structures mimicking GD1b and GD2. Genetic analyses of genes involved in the biosynthesis of the outer core of C. jejuni 81-176 revealed the presence of a homopolymeric tract of G residues within a gene encoding CgtA, an N-acetylgalactosaminyltransferase. Variation in the number of G residues within cgtA affected the length of the open reading frame, and these changes in cgtA corresponded to a change in LOS structure from GM2 to GM3 ganglioside mimicry. Site-specific mutation of cgtA in 81-176 resulted in a major LOS core structure that lacked GalNAc and resembled GM3 ganglioside. Compared to wild-type 81-176, the cgtA mutant showed a significant increase in invasion of INT407 cells. In comparison, a site-specific mutation of the neuC1 gene resulted in the loss of sialic acid in the LOS core and reduced resistance to normal human serum but had no affect on invasion of INT407 cells.

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The Hrq1 helicase stimulates Pso2 translesion nuclease activity to promote DNA inter-strand crosslink repair

10.1101/773267 ◽

2019 ◽

Cited By ~ 2

Author(s):

Cody M. Rogers ◽

Chun-Ying Lee ◽

Samuel Parkins ◽

Nicholas J. Buehler ◽

Sabine Wenzel ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage Response ◽

Nuclease Activity ◽

Site Specific ◽

Damage Response ◽

Physical Barriers ◽

A Site ◽

Translesion Polymerases ◽

Stimulation Of

AbstractDNA inter-strand crosslink (ICL) repair requires a complicated network of DNA damage response pathways. Removal of these lesions is vital as they are physical barriers to essential DNA processes that require the separation of duplex DNA, such as replication and transcription. The Fanconi anemia (FA) pathway is the principle mechanism for ICL repair in metazoans and is coupled to replication. In Saccharomyces cerevisiae, a degenerate FA pathway is present, but ICLs are predominantly repaired by a pathway involving the Pso2 nuclease that is hypothesized to digest through the lesion to provide access for translesion polymerases. However, Pso2 lacks translesion nuclease activity in vitro, and mechanistic details of this pathway are lacking, especially relative to FA. We recently identified the Hrq1 helicase, a homolog of the disease-linked RECQL4, as a novel component of Pso2- mediated ICL repair. Here, we show that Hrq1 stimulates the Pso2 nuclease in a mechanism that requires Hrq1 catalytic activity. Importantly, Hrq1 also stimulates Pso2 translesion nuclease activity through a site- specific ICL in vitro. Stimulation of Pso2 nuclease activity is specific to eukaryotic RecQ4 subfamily helicases, and Hrq1 likely interacts with Pso2 through their N-terminal domains. These results advance our understanding of FA-independent ICL repair and establish a role for the RecQ4 helicases in the repair of these dangerous lesions.

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Involvement of a site-specific trans-acting factor and a common RNA-binding protein in the editing of chloroplast mRNAs: development of a chloroplast in vitro RNA editing system

The EMBO Journal ◽

10.1093/emboj/20.5.1144 ◽

2001 ◽

Vol 20 (5) ◽

pp. 1144-1152 ◽

Cited By ~ 109

Author(s):

T. Hirose

Keyword(s):

Rna Editing ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Site Specific ◽

A Site

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Thermo-Sensitive Vesicles in Controlled Drug Delivery for Chemotherapy

Pharmaceutics ◽

10.3390/pharmaceutics10030150 ◽

2018 ◽

Vol 10 (3) ◽

pp. 150 ◽

Cited By ~ 14

Author(s):

Elisabetta Mazzotta ◽

Lorena Tavano ◽

Rita Muzzalupo

Keyword(s):

Drug Delivery ◽

Cancer Treatment ◽

Controlled Drug Delivery ◽

Solid Tumours ◽

Site Specific ◽

Mild Hyperthermia ◽

Promising Tool ◽

A Site

Thermo-sensitive vesicles are a promising tool for triggering the release of drugs to solid tumours when used in combination with mild hyperthermia. Responsivity to temperature makes them intelligent nanodevices able to provide a site-specific chemotherapy. Following a brief introduction concerning hyperthermia and its advantageous combination with vesicular systems, recent investigations on thermo-sensitive vesicles useful for controlled drug delivery in cancer treatment are reported in this review. In particular, the influence of bilayer composition on the in vitro and in vivo behaviour of thermo-sensitive formulations currently under investigation have been extensively explored.

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Engineering an acetyllysine reader with photocrosslinking amino acid for interactome profiling

Chemical Communications ◽

10.1039/d1cc04611j ◽

2021 ◽

Author(s):

Babu Sudhamalla ◽

Anirban Roy ◽

Soumen Barman ◽

Jyotirmayee Padhan

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Protein Interactions ◽

Unnatural Amino Acids ◽

Protein Protein Interactions ◽

Site Specific ◽

Powerful Approach

The site-specific installation of light-activable crosslinker unnatural amino acids offers a powerful approach to trap transient protein-protein interactions both in vitro and in vivo. Herein, we engineer a bromodomain to...

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Purification and Characterization of the R64 Shufflon-Specific Recombinase

Journal of Bacteriology ◽

10.1128/jb.182.10.2787-2792.2000 ◽

2000 ◽

Vol 182 (10) ◽

pp. 2787-2792 ◽

Cited By ~ 14

Author(s):

Atsuko Gyohda ◽

Teruya Komano

Keyword(s):

Electrophoretic Analysis ◽

Recombination Reaction ◽

Site Specific ◽

Dna Inversion ◽

Repeat Sequences ◽

In Vitro Recombination ◽

Recombinase Gene ◽

A Site

ABSTRACT The shufflon, a multiple DNA inversion system in plasmid R64, consists of four invertible DNA segments which are separated and flanked by seven 19-bp repeat sequences. The product of a site-specific recombinase gene, rci, promotes site-specific recombination between any two of the inverted 19-bp repeat sequences of the shufflon. To analyze the molecular mechanism of this recombination reaction, Rci protein was overproduced and purified. The purified Rci protein promoted the in vitro recombination reaction between the inverted 19-bp repeats of supercoiled DNA of a plasmid carrying segment A of the R64 shufflon. The recombination reaction was enhanced by the bacterial host factor HU. Gel electrophoretic analysis indicated that the Rci protein specifically binds to the DNA segments carrying the 19-bp sequences. The binding affinity of the Rci protein to the four shufflon segments as well as four synthetic 19-bp sequences differed greatly: among the four 19-bp repeat sequences, the repeat-a and -d sequences displayed higher affinity to Rci protein. These results suggest that the differences in the affinity of Rci protein for the 19-bp repeat sequences determine the inversion frequencies of the four segments.

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4-Cyanoindole-2′-deoxyribonucleoside as a Dual Fluorescence and Infrared Probe of DNA Structure and Dynamics

Molecules ◽

10.3390/molecules24030602 ◽

2019 ◽

Vol 24 (3) ◽

pp. 602 ◽

Cited By ~ 3

Author(s):

Ismail A. Ahmed ◽

Arusha Acharyya ◽

Christina M. Eng ◽

Jeffrey M. Rodgers ◽

William F. DeGrado ◽

...

Keyword(s):

Protein Interactions ◽

Rna Structure ◽

High Throughput Screening ◽

Local Environment ◽

Spectroscopic Properties ◽

Site Specific ◽

Structure And Dynamics ◽

High Fluorescence ◽

Fluorescence Quencher ◽

A Site

Unnatural nucleosides possessing unique spectroscopic properties that mimic natural nucleobases in both size and chemical structure are ideally suited for spectroscopic measurements of DNA/RNA structure and dynamics in a site-specific manner. However, such unnatural nucleosides are scarce, which prompts us to explore the utility of a recently found unnatural nucleoside, 4-cyanoindole-2′-deoxyribonucleoside (4CNI-NS), as a site-specific spectroscopic probe of DNA. A recent study revealed that 4CNI-NS is a universal nucleobase that maintains the high fluorescence quantum yield of 4-cyanoindole and that among the four natural nucleobases, only guanine can significantly quench its fluorescence. Herein, we further show that the C≡N stretching frequency of 4CNI-NS is sensitive to the local environment, making it a useful site-specific infrared probe of oligonucleotides. In addition, we demonstrate that the fluorescence-quencher pair formed by 4CNI-NS and guanine can be used to quantitatively assess the binding affinity of a single-stranded DNA to the protein system of interest via fluorescence spectroscopy, among other applications. We believe that this fluorescence binding assay is especially useful as its potentiality allows high-throughput screening of DNA–protein interactions.

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