Functional binding of E-selectin to its ligands is enhanced by structural features beyond its lectin domain.

Selectins are key to mediating interactions involved in cellular adhesion and migration, underlying processes such as immune responses, metastasis, and transplantation. Selectins are composed of a lectin domain, an epidermal growth factor (EGF)-like domain, multiple short consensus repeats (SCRs), a transmembrane domain, and a cytoplasmic tail. It is well-established that the lectin and EGF domains are required to mediate interactions with ligands; however, the contributions of the other domains in mediating these interactions remain obscure. Using various E-selectin constructs produced in a newly developed silkworm-based expression system and several assays performed under both static and physiological flow conditions, including flow cytometry, glycan array analysis, surface plasmon resonance, and cell-rolling assays, we show here that a reduction in the number of SCR domains is correlated with a decline in functional E-selectin binding to hematopoietic cell E- and/or L-selectin ligand (HCELL) and P-selectin glycoprotein ligand-1 (PSGL-1). Moreover, the binding was significantly improved through E-selectin dimerization and by a substitution (A28H) that mimics an extended conformation of the lectin and EGF domains. Analyses of the association and dissociation rates indicated that the SCR domains, conformational extension, and dimerization collectively contribute to the association rate of E-selectin-ligand binding, whereas just the lectin and EGF domains contribute to the dissociation rate. These findings provide the first evidence of the critical role of the association rate in functional E-selectin-ligand interactions, and they highlight that the SCR domains have an important role that goes beyond the structural extension of the lectin and EGF domains.

Using Eukaryotic Expression Systems to Generate Human α1,3-Fucosyltransferases That Effectively Create Selectin-Binding Glycans on Stem Cells.

Recruitment of circulating cells toward target sites is primarily dependent on selectin/ligand adhesive interactions. Glycosyltransferases are involved in the creation of selectin ligands on proteins and lipids. α1,3-Fucosylation is imperative for the creation of selectin ligands, and a number of fucosyltransferases (FTs) can modify terminal lactosamines on cells to create these ligands. One FT, fucosyltransferase VI (FTVI), adds a fucose in an α1,3 configuration to N-acetylglucosamine to generate sialyl Lewis X (sLex) epitopes on proteins of live cells and enhances their ability to bind E-selectin. Although a number of recombinant human FTVIs have been purified, apart from limited commercial enzymes, they were not characterized for their activity on live cells. Here we focused on establishing a robust method for producing FTVI that is active on living cells (hematopoietic cells and mesenchymal stromal cells). To this end, we used two expression systems, Bombyx mori (silkworm) and Pichia pastoris (yeast), to produce significant amounts of N-terminally tagged FTVI and demonstrated that these enzymes have superior activity when compared to currently available commercial enzymes that are produced from various expression systems. Overall, we outline a scheme for obtaining large amounts of highly active FTVI that can be used for the application of FTVI in enhancing the engraftment of cells lacking the sLex epitopes.

Dynamic structure mediates halophilic adaptation of a DNA polymerase from the deep-sea brines of the Red Sea.

The deep-sea brines of the Red Sea are remote and unexplored environments characterized by high temperatures, anoxic water, and elevated concentrations of salt and heavy metals. This environment provides a rare system to study the interplay between halophilic and thermophilic adaptation in biologic macromolecules. The present article reports the first DNA polymerase with halophilic and thermophilic features. Biochemical and structural analysis by Raman and circular dichroism spectroscopy showed that the charge distribution on the protein's surface mediates the structural balance between stability for thermal adaptation and flexibility for counteracting the salt-induced rigid and nonfunctional hydrophobic packing. Salt bridge interactions via increased negative and positive charges contribute to structural stability. Salt tolerance, conversely, is mediated by a dynamic structure that becomes more fixed and functional with increasing salt concentration. We propose that repulsive forces among excess negative charges, in addition to a high percentage of negatively charged random coils, mediate this structural dynamism. This knowledge enabled us to engineer a halophilic version of Thermococcus kodakarensis DNA polymerase.-Takahashi, M., Takahashi, E., Joudeh, L. I., Marini, M., Das, G., Elshenawy, M. M., Akal, A., Sakashita, K., Alam, I., Tehseen, M., Sobhy, M. A., Stingl, U., Merzaban, J. S., Di Fabrizio, E., Hamdan, S. M. Dynamic structure mediates halophilic adaptation of a DNA polymerase from the deep-sea brines of the Red Sea.

Quantitative Characterization of E-selectin Interaction with Native CD44 and P-selectin Glycoprotein Ligand-1 (PSGL-1) Using a Real Time Immunoprecipitation-based Binding Assay.

Selectins (E-, P-, and L-selectins) interact with glycoprotein ligands to mediate the essential tethering/rolling step in cell transport and delivery that captures migrating cells from the circulating flow. In this work, we developed a real time immunoprecipitation assay on a surface plasmon resonance chip that captures native glycoforms of two well known E-selectin ligands (CD44/hematopoietic cell E-/L-selectin ligand and P-selectin glycoprotein ligand-1) from hematopoietic cell extracts. Here we present a comprehensive characterization of their binding to E-selectin. We show that both ligands bind recombinant monomeric E-selectin transiently with fast on- and fast off-rates, whereas they bind dimeric E-selectin with remarkably slow on- and off-rates. This binding requires the sialyl Lewis x sugar moiety to be placed on both O- and N-glycans, and its association, but not dissociation, is sensitive to the salt concentration. Our results suggest a mechanism through which monomeric selectins mediate initial fast on and fast off kinetics to help capture cells out of the circulating shear flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to significantly slow rolling.

Identification and characterization of Polycomb group genes in the silkworm, Bombyx mori.

Polycomb group (PcG) proteins are involved in chromatin modifications for maintaining gene repression that play important roles in the regulation of gene expression, tumorigenesis, chromosome X-inactivation, and genomic imprinting in Drosophila melanogaster, mammals, and even plants. To characterize the orthologs of PcG genes in the silkworm, Bombyx mori, 13 candidates were identified from the updated silkworm genome sequence by using the fruit fly PcG genes as queries. Comparison of the silkworm PcG proteins with those from other insect species revealed that the insect PcG proteins shared high sequence similarity. High-level expressions of all the silkworm PcG genes were maintained through day 2 to day 7 of embryogenesis, and tissue microarray data on day 3 of the fifth instar larvae showed that their expression levels were relatively low in somatic tissues, except for Enhancer of zeste (E(Z)). In addition, knockdown of each PRC2 component, such as E(Z), Extra sex combs (ESC), and Suppressor of zeste 12 (SU(Z)12), considerably decreased the global levels of H3K27me3 but not of H3K27me2. Taken together, these results suggest that insect PcG proteins are highly conserved during evolution and might play similar roles in embryogenesis.

Quick and Easy Assembly of a One-Step qRT-PCR Kit for COVID-19 Diagnostics Using In-House Enzymes.

One-step reverse-transcription quantitative polymerase chain reaction (qRT-PCR) is the most widely applied method for COVID-19 diagnostics. Notwithstanding the facts that one-step qRT-PCR is well suited for the diagnosis of COVID-19 and that there are many commercially available one-step qRT-PCR kits in the market, their high cost and unavailability due to airport closures and shipment restriction became a major bottleneck that had driven the desire to produce the key components of such kits locally. Here, we provide a simple, economical, and powerful one-step qRT-PCR kit based on patent-free, specifically tailored versions of Moloney murine leukemia virus reverse transcriptase and Thermus aquaticus DNA polymerase and termed R3T (Rapid Research Response Team) one-step qRT-PCR. We also demonstrate the robustness of our enzyme production strategies and provide the optimal reaction conditions for their efficient augmentation in a one-step approach. Our kit was routinely able to reliably detect as low as 10 copies of the synthetic RNAs of SARS-CoV-2. More importantly, our kit successfully detected COVID-19 in clinical samples of broad viral titers with similar reliability and selectivity to that of the Invitrogen SuperScript III Platinum One-step qRT-PCR and TaqPath one-step RT-qPCR kits. Overall, our kit has shown robust performance in both laboratory settings and the Saudi Ministry of Health-approved testing facility.

The telomere-specific non-LTR retrotransposons SART1 and TRAS1 are suppressed by Piwi subfamily proteins in the silkworm, Bombyx mori.

The telomere structures in Bombyx mori are thought to be maintained mainly by the transposition of the specialized telomeric retroelements SART and TRAS. The silkworm genome has telomeric TTAGG repeats and telomerase, but this telomerase displays little or no activity. Here, we report that the transcription of the telomeric retroelements SART1 and TRAS1 is suppressed by the silkworm Piwi subfamily proteins BmAgo3 and Siwi. The silkworm Piwi subfamily was found to be expressed predominantly in the gonads and early embryo, as in other model organisms, but in BmN4 cultured cells, these proteins formed granules that were separate from the nuage, which is a different behaviour pattern. The expression of TRAS1 was increased in BmN4 cells when BmAgo3 or Siwi were silenced by RNAi. Our results suggest that B. mori Piwi proteins are involved in regulating the transposition of telomeric retroelements, and that the functional piRNA pathway is conserved in BmN4 cultured cells.

Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System.

The KOD DNA polymerase from Thermococcus kodakarensis (Tkod-Pol) has been preferred for PCR due to its rapid elongation rate, extreme thermostability and outstanding fidelity. Here in this study, we utilized silkworm-baculovirus expression vector system (silkworm-BEVS) to express the recombinant Tkod-Pol (rKOD) with N-terminal (rKOD-N) or C-terminal (rKOD-C) tandem fusion tags. By using BEVS, we produced functional rKODs with satisfactory yields, about 1.1 mg/larva for rKOD-N and 0.25 mg/larva for rKOD-C, respectively. Interestingly, we found that rKOD-C shows higher thermostability at 95 °C than that of rKOD-N, while that rKOD-N is significantly unstable after exposing to long period of heat-shock. We also assessed the polymerase activity as well as the fidelity of purified rKODs under various conditions. Compared with commercially available rKOD, which is expressed in E. coli expression system, rKOD-C exhibited almost the same PCR performance as the commercial rKOD did, while rKOD-N did lower performance. Taken together, our results suggested that silkworm-BEVS can be used to express and purify efficient rKOD in a commercial way.