Lectin Activity Research Articles

Abstract 13789: Identification of a Proteomic Signature Showing Ongoing Inflammation in Severe Rheumatic Heart Disease

Introduction: Rheumatic heart disease (RHD) remains a major source of morbidity and mortality in developing countries. A deeper insight into the pathogenetic mechanisms underlying RHD could provide opportunities for drug repurposing, guide recommendations for secondary penicillin prophylaxis, and/or inform development of near-patient diagnostics. Methods: We conducted a proteomic study in 215 African patients with severe RHD and 230 controls, using the SWATH-MS technique. We applied a machine learning (ML) approach to feature selection among the 366 proteins quantifiable in at least 40% of samples, using the Boruta wrapper algorithm. The case-control differences and contribution to AUC of the ROC for each of the 56 proteins identified by the Boruta algorithm were calculated by Logistic Regression adjusted for age, sex and BMI. Biological pathways and functions enriched for proteins were identified using ClueGo pathway analyses. Results: Adiponectin, complement component C7 and fibulin-1, a component of heart valve matrix, were each higher in cases when compared with controls. Ficolin-3, a protein with calcium-independent lectin activity that activates the complement pathway, was lower in cases than controls. The top 6 biomarkers from the Boruta analyses conferred an AUC of 0.90 indicating excellent discriminatory capacity between RHD cases and controls. Conclusions: These results support the presence of an ongoing inflammatory response in RHD, at a time when severe valve disease has developed, and distant from previous episodes of acute rheumatic fever. This biomarker signature could have potential utility in recognizing different degrees of ongoing inflammation in RHD patients, which may in turn be related to prognostic severity.

Lectin Activity in Commonly Consumed Plant-Based Foods: Calling for Method Harmonization and Risk Assessment.

Lectins are ubiquitous proteins characterized through their ability to bind different types of carbohydrates. It is well known that active lectins from insufficiently prepared legumes can cause adverse human health effects. The objective of this study was to determine the activity of lectins in samples across plant families representing commercially available edible plants, and the feasibility of inactivating lectins through soaking and boiling. Lectins were extracted from the plant families Adoxaceae, Amaranthaceae, Cannabaceae, Fabaceae, Gramineae, Lamiaceae, Linaceae, Pedaliaceae, and Solanaceae. A hemagglutination assay based on non-treated or trypsin treated rabbit erythrocytes was used to measure the lectin activity. The results showed the highest lectin activity in species from the Fabaceae family and demonstrated that soaking and boiling have an effect on the levels of active lectins. This is the first large study that combines lectin activity obtained from two different assays with raw and processed edible plants. In addition, we examined the current risk assessment, and regulations necessary for an adequate official reporting of results. We encourage the scientific community to further explore this field and agree on harmonized methods for analysis and interpretation, and hope that our methodology can initiate this development.

The Use of Lectins as Tools to Combat SARS-CoV-2.

In December 2019, China announced the first case of an infection caused by an, until then, unknown virus: SARS-CoV-2. Since then, researchers have been looking for viable alternatives for the treatment and/or cure of viral infection. Among the possible complementary solutions are lectins, proteins that are reversibly bound to different carbohydrates. The Spike protein, present on the viral surface, can interact with different cell receptors: ACE2, CD147, and DC-SIGNR. Since lectins have an affinity for different carbohydrates, the binding with the glycosylated cell receptors represents a possibility of preventing the virus from binding to the receptors of host cells. In this review, we discuss the main lectins that are possible candidates for use in the treatment of Covid-19, highlighting those that have already demonstrated antiviral activity in vivo and in vitro, including mannose-binding lectin, Griffithsin, BanLec, and others. We also aim to discuss the possible mechanism of action of lectins, which appears to occur through the mediation of viral fusion in host cells, by binding of lectins to glycosylated receptors found in human cells and/or binding of these proteins with the spike glycoprotein, present in virus surface. Moreover, we discuss the use of lectins in clinical practice. Even with the development of effective vaccines, new cases of viral infection with the same virus, or new outbreaks with different viruses can occur; so, the development of new treatments should not be discarded. Moreover, the discussions made in this work are relevant regarding the anti-viral properties of lectins.

Anti-infective activity of Cratylia argentea lectin (CFL) against experimental infection with virulent Listeria monocytogenes in Swiss mice

BackgroundThe lectin from Cratylia argentea (CFL) is able to modulate the immune system response and is thus a potential phytotherapeutic substance. Hypothesis/PurposeIn this study, we investigated the role of CFL on control of bacterial infection caused by Listeria monocytogenes, the causative agent of human listeriosis. Study designSwiss mice were infected with L. monocytogenes and then treated with CFL. MethodsAdult Swiss mice weighing with 30–40 g were infected intraperitoneally with a bacterial suspension (0.2 ml; 1 × 107 CFU/ml). After 30 min, the mice were treated with CFL intravenously at concentrations of 0.1 or 10 mg/kg. Control mice received phosphate-buffered saline (PBS). The animals were euthanized 24 h after infection. ResultsWe observed that i.v. administration of CFL to Swiss mice did not cause acute toxicity, and reduced the leukocyte counts in the bloodstream 24 h after infection with virulent L. monocytogenes. There was a reduction in the bacterial burden within peritoneal macrophages after infection in CFL-treated mice. Accordingly, the bacterial counts in the bloodstream, spleen and liver also decreased in comparison with the PBS group. Histological damage in the spleen and liver was lower in mice that received CFL treatment. In vitro antimicrobial assays demonstrated that CFL does not inhibit the growth of L. monocytogenes. The mRNA expression of the anti-inflammatory cytokine IL-10 was enhanced with CFL treatment after infection. ConclusionThe lectin from C. argentea (CFL) has immunomodulatory and anti-infective properties of pharmacological interest for control of infectious diseases.

Molecular dynamics simulations elucidate oligosaccharide recognition pathways by galectin-3 at atomic resolution

The recognition of carbohydrates by lectins plays key roles in diverse cellular processes such as cellular adhesion, proliferation, and apoptosis, which makes it a therapeutic target of significance against cancers. One of the most functionally active lectins, galectin-3 is distinctively known for its specific binding affinity toward β-galactoside. However, despite the prevalence of high-resolution crystallographic structures, the mechanistic basis and more significantly, the dynamic process underlying carbohydrate recognition by galectin-3 are currently elusive. To this end, we employed extensive Molecular Dynamics simulations to unravel the complete binding event of human galectin-3 with its native natural ligand N-acetyllactosamine (LacNAc) at atomic precision. The simulation trajectory demonstrates that the oligosaccharide diffuses around the protein and eventually identifies and binds to the biologically designated binding site of galectin-3 in real time. The simulated bound pose correlates with the crystallographic pose with atomic-level accuracy and recapitulates the signature stabilizing galectin-3/oligosaccharide interactions. The recognition pathway also reveals a set of transient non-native ligand poses in its course to the receptor. Interestingly, kinetic analysis in combination with a residue-level picture revealed that the key to the efficacy of a more active structural variant of the LacNAc lay in the ligand’s resilience against disassociation from galectin-3. By catching the ligand in the act of finding its target, our investigations elucidate the detailed recognition mechanism of the carbohydrate-binding domain of galectin-3 and underscore the importance of ligand–target binary complex residence time in understanding the structure–activity relationship of cognate ligands.

A Linkage-specific Sialic Acid Labeling Strategy Reveals Different Site-specific Glycosylation Patterns in SARS-CoV-2 Spike Protein Produced in CHO and HEK Cell Substrates.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus utilizes the extensively glycosylated spike (S) protein protruding from the viral envelope to bind to angiotensin-converting enzyme-related carboxypeptidase (ACE2) as its primary receptor to mediate host-cell entry. Currently, the main recombinant S protein production hosts are Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells. In this study, a recombinant S protein truncated at the transmembrane domain and engineered to express a C-terminal trimerization motif was transiently produced in CHO and HEK cell suspensions. To further evaluate the sialic acid linkages presenting on S protein, a two-step amidation process, employing dimethylamine and ammonium hydroxide reactions in a solid support system, was developed to differentially modify the sialic acid linkages on the glycans and glycopeptides from the S protein. The process also adds a charge to Asp and Glu which aids in ionization. We used MALDI-TOF and LC-MS/MS with electron-transfer/higher-energy collision dissociation (EThcD) fragmentation to determine global and site-specific N-linked glycosylation patterns. We identified 21 and 19 out of the 22 predicted N-glycosites of the SARS-CoV-2 S proteins produced in CHO and HEK, respectively. It was found that the N-glycosite at 1,158 position (N1158) and at 122, 282 and 1,158 positions (N122, N282 and N1158) were absent on S from CHO and HEK cells, respectively. The structural mapping of glycans of recombinant human S proteins reveals that CHO-Spike exhibits more complex and higher sialylation (α2,3-linked) content while HEK-Spike exhibits more high-mannose and a small amount of α2,3- and α2,6-linked sialic acids. The N74 site represents the most abundant glycosite on both spike proteins. The relatively higher amount of high-mannose abundant sites (N17, N234, N343, N616, N709, N717, N801, and N1134) on HEK-Spike suggests that glycan-shielding may differ among the two constructs. HEK-Spike can also provide different host immune system interaction profiles based on known immune system active lectins. Collectively, these data underscore the importance of characterizing the site-specific glycosylation of recombinant human spike proteins from HEK and CHO cells in order to better understand the impact of the production host on this complex and important protein used in research, diagnostics and vaccines.

A proposed lectin-mediated mechanism to explain the in Vivo antihyperglycemic activity of γ-conglutin from Lupinus albus seeds.

Experiments conducted in vitro and in vivo, as well as clinical trials for hypoglycemic therapeutics, support the hypoglycemic properties of the lectin γ‐conglutin, a Lupinus seed storage protein, by a mechanism not yet been clarified.Structural studies established that binding of γ‐conglutin, in native and denatured form, to insulin occurs by a strong binding that resists rupture when 0.4 M NaCl and 0.4 M galactose are present, suggesting that strong electrostatic interactions are involved. Studies on binding of γ‐conglutin in native and denatured form to HepG2 membrane glycosylated receptors were conducted, which reveal that only the native form of γ‐conglutin with lectin activity is capable of binding to these receptors.Glycosylated insulin receptors were detected on purified HepG2 cell membranes and characterized by 1D and 2D analyses.Preclinical assays with male mice (CD‐1) indicated that native and denatured γ‐conglutins display antihyperglycemic effect, decreasing glucose in blood comparable after 120 min to that exhibited by the animal group treated with metformin, used to treat T2D and used as a positive control. Measurement of organ injury/functional biomarkers (hepatic, pancreatic, renal, and lipid profile) was comparable to that of metformin treatment or even better in terms of safety endpoints (pancreatic and hepatic biomarkers).

Trefoil Factor Family (TFF) Peptides

Trefoil factor family (TFF) peptides mainly consist of characteristic TFF domains, which contain about 40 amino acid residues, including 6 conserved cysteine residues. TFF peptides possess a single (mammalian TFF1 and TFF3), two (mammalian TFF2, Xenopus laevis xP2) or four TFF domains (X. laevis xP4). They exhibit lectin activities and are characteristic exocrine products of the mucous epithelia. Here, they play different roles for mucosal protection and the innate immune defense: TFF1 is a gastric tumor suppressor; TFF2 builds a lectin complex with the mucin MUC6, physically stabilizing the inner gastric mucus layer; and TFF3 forms a disulfide-linked heterodimer with IgG Fc binding protein (FCGBP), probably preventing the infiltration of microorganisms. Minor amounts of TFF peptides are endocrine products of the immune and nervous systems. Pathologically, TFF peptides are linked to inflammation. There are increasing indications that TFF peptides can antagonize cytokine receptors, such as receptors for IL-1β, IL-6, and TNFα (thereby acting as anti-inflammatory peptides). TFF peptides can probably also activate a variety of receptors, such as CXCR4. The TFF domain is a unique shuffled module which is also present in a number of mosaic proteins, such as zona pellucida proteins, sugar degrading enzymes and frog skin mucins. Here, their function seems to be defined by a lectin activity, which might even allow a role in fertilization.

Complement Alternative and Mannose-Binding Lectin Pathway Activation Is Associated With COVID-19 Mortality.

BackgroundThe SARS-CoV-2 infection triggers excessive immune response resulting in increased levels of pro-inflammatory cytokines, endothelial injury, and intravascular coagulopathy. The complement system (CS) activation participates to this hyperinflammatory response. However, it is still unclear which activation pathways (classical, alternative, or lectin pathway) pilots the effector mechanisms that contribute to critical illness. To better understand the immune correlates of disease severity, we performed an analysis of CS activation pathways and components in samples collected from COVID-19 patients hospitalized in Grenoble Alpes University Hospital between 1 and 30 April 2020 and of their relationship with the clinical outcomes.MethodsWe conducted a retrospective, single-center study cohort in 74 hospitalized patients with RT-PCR-proven COVID-19. The functional activities of classical, alternative, and mannose-binding lectin (MBL) pathways and the antigenic levels of the individual components C1q, C4, C3, C5, Factor B, and MBL were measured in patients’ samples during hospital admission. Hierarchical clustering with the Ward method was performed in order to identify clusters of patients with similar characteristics of complement markers. Age was included in the model. Then, the clusters were compared with the patient clinical features: rate of intensive care unit (ICU) admission, corticoid treatment, oxygen requirement, and mortality.ResultsFour clusters were identified according to complement parameters. Among them, two clusters revealed remarkable profiles: in one cluster (n = 15), patients exhibited activation of alternative and lectin pathways and low antigenic levels of MBL, C4, C3, Factor B, and C5 compared to all the other clusters; this cluster had the higher proportion of patients who died (27%) and required oxygen support (80%) or ICU care (53%). In contrast, the second cluster (n = 19) presented inflammatory profile with high classical pathway activity and antigenic levels of complement components; a low proportion of patients required ICU care (26%) and no patient died in this group.ConclusionThese findings argue in favor of prominent activation of the alternative and MBL complement pathways in severe COVID-19, but the spectrum of complement involvement seems to be heterogeneous requiring larger studies.

Purification, Characterization, and Assessment of Antimicrobial Activity and Toxicity of Portulaca elatior Leaf Lectin (PeLL).

Lectins are carbohydrate-binding proteins with several bioactivities, including antimicrobial properties. Portulaca elatior is a species found at Brazilian Caatinga and data on the biochemical composition of this plant are scarce. The present work describes the purification of P. elatior leaf lectin (PeLL) as well as the assessment of its antimicrobial activity and toxicity. PeLL, isolated by chromatography on a chitin column, had native liquid charge and subunit composition evaluated by electrophoresis. Hemagglutinating activity (HA) of PeLL was determined in the presence of carbohydrates or divalent cations, as well as after heating and incubation at different pH values. Changes in the lectin conformation were monitored by evaluating intrinsic tryptophan fluorescence and using the extrinsic probe bis-ANS. Antimicrobial activity was evaluated against Pectobacterium strains and Candida species. The minimal inhibitory (MIC), bactericidal (MBC), and fungicidal (MFC) concentrations were determined. Finally, PeLL was evaluated for in vitro hemolytic activity in human erythrocytes and in vivo acute toxicity in mice (5 and 10mg/kg b.w. per os). PeLL (pI 5.4; 20kDa) had its HA was inhibited by mannose, galactose, Ca2+, Mg2+, and Mn2+. PeLL HA was resistant to heating at 100°C, although conformational changes were detected. PeLL was more active in the acidic pH range, in which no conformational changes were observed. The lectin presented MIC and MBC of 0.185 and 0.74μg/mL for all Pectobacterium strains, respectively; MIC of 1.48μg/mL for C. albicans, C. tropicalis, and C. krusei; MIC and MFC of 0.74 and 2.96μg/mL for C. parapsilosis. No hemolytic activity or signs of acute toxicity were observed in the mice. In conclusion, a new, low-toxic, and thermostable lectin was isolated from P. elatior leaves, being the first plant compound to show antibacterial activity against Pectobacterium.

Fermented goat's milk modulates immune response during iron deficiency anemia recovery.

Iron deficiency and iron overload can affect the normal functioning of the innate and adaptive immune responses. Fermented milk products may enhance immune functions, but little is known about the effect of fermented milks on modulation of the immune response during iron deficiency anemia and recovery with normal or high dietary iron intake. Eighty male Wistar rats were randomly assigned to a control group fed a standard diet or to an anemic group fed a diet deficit in iron. Control and anemic groups were fed for 30 days with diets based on a fermented goat's or cow's milk product, with normal iron content or iron overload. In general, during anemia recovery lectin and alternative complement pathway activity and lactoferrin decreased, because it improves iron homeostasis, which is critically important in immune system functions. Fermented goat's milk diet enhanced immune function during iron deficiency recovery, suppressed oxidant-induced eotaxin and fractalkine expression due to the concurrent reduction of free radical production and pro-inflammatory cytokines, and decreased monocyte chemoattractant protein-1 and monocyte migration and adhesion. The increase in interferon-γ can confer immunological colonization of gut microbiota and downregulate inflammation. Fermented goat's milk consumption enhances immune function, modifying complement pathway activity and decreasing pro-inflammatory cytokines as well as lactoferrin concentration, due to the improvement of iron homeostasis, which is critically important in the normal function of the immune system. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The Acinetobacter baumannii Autotransporter Adhesin Ata Recognizes Host Glycans as High-Affinity Receptors.

Acinetobacter baumannii is a significant opportunistic pathogen responsible for infections of the lung, blood, skin, urinary tract, and soft tissues, with some strains exhibiting almost complete resistance to commonly used antibiotics. This multidrug resistance, together with a dearth of new antibiotic development, mean novel methods of treatment and prevention are urgently needed. Although many A. baumannii factors required to colonize the host have been identified, little is known about the specific host molecules recognized by these factors. A. baumannii produces a trimeric autotransporter adhesin known as Ata that has been previously demonstrated to bind components of the host cell's extracellular matrix, which are often heavily glycosylated. We hypothesized that Ata would exhibit lectin activity which would play a role in adherence to the host cell surface. Our biophysical analysis using glycan arrays and surface plasmon resonance demonstrated that Ata binds galactose, N-acetylglucosamine, and galactose (β1-3/4) N-acetylglucosamine with high-affinity. These structures are present on many of the proteins which were previously reported to be bound by Ata. We also demonstrated that the recognition of human plasma fibronectin by Ata requires this ability to bind glycans, as the interaction between Ata and fibronectin does not occur when fibronectin is deglycosylated. This strongly suggests a key role for Ata lectin activity during host adherence. This information will assist in directing the development of new and effective treatments to block host interactions using glycans and/or novel compounds in multidrug resistant A. baumannii infections.

Ficolin activation as a potential biomarker of the severity of schizophrenia

Several studies have examined the complement system in schizophrenia, suggesting an involvement of the lectin pathway. We analyzed 49 patients with schizophrenia and explored the association between psychopathology of schizophrenia and complement component 3 (C3) serum levels, C-reactive protein (CRP) serum levels, ficolin activation, and mannose-binding lectin (MBL) activation. In the multiple regression analysis, a negative association was observed between the Positive and Negative Syndrome Scale (PANSS) total score and ficolin activation. Body mass index (BMI) was positively associated with the serum levels of C3 and CRP. MBL activation was not associated with any independent variables. Our findings facilitate a better understanding of the complement system in schizophrenia. Additional studies with a large sample population are needed to confirm our results.

BIOCHEMICAL CHARACTERISTIC OF SOLUBLE LECTINS IN CORN UNDER THE IMPACT OF WATER DEFICIT AND HYPERTHERMIA

Introduction. Drought and high temperature are two of the key factors of the environment limiting crop capacity of grains. Response of the plants to drought and high temperature is very complex and includes interaction between various molecular, physiological and biochemical processes. Synthesis of a number of proteins present under normal conditions, including lectins, increases along with synthesis of stress proteins under adverse conditions.Aim. The goal of our study is to identify the changes in the activity and biochemical characteristics of soluble lectins in maize seedlings with different drought tolerance under water and heat stress in order to create new biochemical methods for assessing drought tolerance.Methods. Three-day young sprouts of corn lines (Zea mays L.) with different levels of drought tolerance were used in the research: drought-tolerant lines Od 329, IK107 zM, non-drought tolerant lines GK 26, IK107VS3 / 66. Lectin activity was defined on the basis of their ability to agglutinate trypsinized erythrocytes of white rats. Electrophoresis was performed in 10 % PAGE following the Laemmli method.Results. The study enabled us to identify increase in soluble lectin activity (244-281 % of the reference value) under the given stress factors in drought-tolerant lines, and decrease in soluble lectin activity (39 - 79 % of reference value) under the given stress factors in non-drought-tolerant lines. Soluble lectins were isolated and purifid using salting out with ammonium sulfate, dialysis and affiity chromatography. The molecular weight of the isolated soluble lectins is in the range of 50-60 kDa. The isolated lectins had a high affiity for N-acetylglucosamine and D-fructose-6-phosphate.Conclusion. Therefore, it has been established that corn lines with positively different levels of drought tolerance are characterized by varying activity of soluble lectins.

Assessment of lectinic activity potentials in extracts of some tropical Euphorbiaceace

Lectins bind a variety of cells having cell surface glycoprotein or glycolipid, such as erythrocytes, leukemic cells, yeast and several types of bacteria. Several specificity groups have been identified such as mannose, galactose, N-acetyl glucoseamine, N-acetyl galactoseamine, L-fucose and N-acetyl neuraminic acid. The presence of two or more binding sites for each Lectin molecule allows the agglutination of many cell type. Sixteen (16) species of some tropical Euphorbiaceae plants were assessed for the presence of Lectins. The leaves of Acalypha torta, Acalypha wiskesiana, Acalypha hispida, Codiaeum variegatum, Euphorbia milli, Euphorbia pulcherima, Jatropha curcas and Jatropha gossypifola; the seeds of Croton tiglium, Ricinus comminus and Tetracarpidium conophorum; the stem of Euphorbia tirucalli and the tubers of Manihot esculenta (Cassava, vitamin A variety), Manihot esculenta (cassava, NR 8082 variety), Manihot esculenta (Cassava, TMX 419 variety), Manihot esculenta (Cassava, TMX 4(2) 1425 variety) were used for sourcing the Lectins. A. torta, A. wiskesiana, C. variegatum, C. tiglium, E. milli, E. pulcherima, E. tirucalli, J. curcas, J. gossypifola, R. comminus and T. conophorum agglutinated pooled washed human ABO cells in saline (direct haemagglutination) while A. hispida and the four varieties of M. esculenta showed no agglutination reaction. E. pulcherima showed specificity for B cells only while E. tirucalli showed specificity for O cells only, hence could be rightly indicated by referring to them as anti-B Ep and anti-H Et Lectins respectively (where Ep=Euphorbia pulcherima and Et= Euphorbia tirucalli). However A. torta and T. conophorum cross-reacted with pooled washed ABO cells in differing strengths and when standardized, showed that A. torta at a titre of 16 reacted specifically with O cells and T. conophorum at a titre of 128 reacted specifically with B cells. Based on this, these Lectins could be indicated as anti-H At and anti-B Tc respectively (Where At= Acalypha torta and Tc= Tetracarpidium conophorum). The protein content of the crude extracts of the sixteen (16) species were also assayed using Biuret protein assay method and the results revealed that there are no relation or association between the quantity of protein content and agglutination patterns of the extracts. This research has therefore succeeded in revealing presence of Lectinic properties in extracts of some tropical Euphorbiaceae.

Assessment of lectinic activity potentials in extracts of some tropical Euphorbiaceace

Assessment of lectinic activity potentials in extracts of some tropical Euphorbiaceace

Functional and Molecular Basis of Thrombopoietin Receptor Activation By Mutant Calreticulin

▪The thrombopoietin receptor MPL is required for oncogenic transformation by mutant CALR (CALRMUT). We and others have previously shown that CALRMUT binds to the thromobopoietin receptor, MPL, to activate the MPL-JAK-STAT signaling axis, and that the positive charge of the CALRMUT C-terminus is required to mediate this interaction.To provide additional understanding of the mechanism by which CALRMUT is oncogenic, we sought to further resolve which amino acids within the CALRMUT C-terminus are required for CALRMUT to bind MPL, activate MPL signaling, and transform hematopoietic cells. To do this, we generated serial truncations of the mutant C-terminal tail. We found that even the most severely truncated form of the CALRMUT C-terminus retained the ability to bind MPL, but failed to activate JAK-STAT signaling and transform Ba/F3-MPL cells to IL-3 independence. This provides the first evidence that mutant CALR binding to MPL does not uniformly correlate with activation of JAK-STAT signaling and cellular transformation, and suggests a model whereby different thresholds of positive charge in the CALRMUT C-terminus are required to enable binding of CALRMUT to MPL and to activate MPL signaling.We next sought to elucidate the components of MPL that are required to bind CALRMUT and to support oncogenic transformation. We observed that CALRMUT bound to the extracellular domain of MPL, but not the intracellular domain, but that neither domain alone was capable of supporting CALRMUT-associated transformation. We therefore hypothesized that CALRMUT interaction with the MPL extracellular domain still required the intracellular domain of MPL to transmit canonical MPL-associated downstream signaling. We focused on three tyrosine residues within the carboxy-terminal of MPL (Tyr-591, Tyr-626 and Tyr-631), and generated MPL variants where these residues were systematically mutated individually, in tandem, or altogether. We observed no changes in the ability of any of the MPL variants to physically bind to CALRMUT. In contrast, MPL variants harboring mutation of all 3 tyrosine residues were unable to support cytokine-independent growth. Specifically, substitution of Tyr-626 significantly impaired the ability of MPL to support CALRMUT-associated transformation, whereas substitution of Tyr-591/Tyr-31 only had minor effects.Finally, we sought to gain functional insight into how CALRMUT interacts with MPL and how this interaction drives cellular transformation. Wild-type calreticulin is an ER-resident chaperone that interacts with newly synthesized glycoproteins as well as with the polypeptide chain to enable proper protein folding. We created variants of CALRMUT harboring mutations in critical residues implicated in three key functionalities of wild-type CALR - (i) lectin activity, (ii) polypeptide binding and (iii) chaperone activity - and tested their capacity to bind to MPL and confer cytokine-independence. We observed that lectin-deficient CALRMUT were unable to bind to MPL or confer cytokine independence. In contrast, polypeptide binding-deficient variants of CALRMUT retained MPL binding ability and conferred cytokine-independent growth. Finally, we observed that CALRMUT harboring mutations in residues implicated in wild-type CALR chaperone activity retained the ability to physically interact with MPL and confer cytokine-independent growth. Consistent with this, an in vitro binding assay demonstrated that CALRWT exhibits strong, direct binding to the co-chaperone molecule ERp57, whereas CALRMUT does not. Cumulatively, these data suggest that the interaction of CALRMUT with MPL is dependent on the CALRMUT lectin activity but not on its chaperone functionality.In conclusion, our data provide additional insights into the molecular mechanism by which CALRMUT interacts with MPL to induce MPN. Specifically, we uncouple the binding of CALRMUT to MPL from MPL activation and define the key properties of MPL required for CALRMUT binding and for its activation. Together, our findings consolidate the critical role of the positive electrostatic charge of the CALRMUT C-terminus in the oncogenic activity of CALRMUT and suggest a model of transformation whereby CALRMUT acquires a neomorphic function involving pathogenic binding to MPL and possibly to other proteins. DisclosuresNo relevant conflicts of interest to declare.

Purification and erythrocyte-membrane perturbing activity of a ketose-specific lectin from Moringa oleifera seeds

This study purified a hemagglutinating protein (MoL) from Moringa oleifera seed, and investigated its hemolytic activity. Molecular weight and stability of MoL were also determined. Modification of some amino acid residues was carried out and the effect on MoL hemagglutinating activity determined. Other investigated parameters are the effects of temperature, concentration, incubation period, pH, and sugars on the protein's hemagglutinating and hemolytic activities. The native and subunit molecular weights were estimated as 30 and 27.5 kDa respectively. Hemagglutinating activity of MoL was slightly inhibited by fructose and sucrose, stable at temperature up to 90°C and within pH range of 2–4. Modification of tryptophan and arginine residues resulted in partial loss of hemagglutinating activity. The hemolytic activity of MoL was concentration, temperature, pH, and time-dependent. The study concluded that MoL showed hemolytic (membrane-perturbing) activity in moderate acidic conditions. This suggests its potential exploitation in improved intracellular delivery of bioactive compounds.

Identification and Characterization of Genes for the Allo A Lectins in Japanese Rhinoceros Beetle (Trypoxylus dichotomus [Allomyrina dichotoma]).

The Japanese rhinoceros beetle (Trypoxylus dichotomus [Allomyrina dichotoma]) produces the lectins allo A-I and allo A-II, which have strong N-acetyllactosamine (Galβ1-4GlcNAc)-binding activity. It has been suggested that the two lectins are formed from three subunits (α, β, and γ), with allo A-I comprising α and γ subunits and allo A-II comprising β and γ subunits. Here, we determined the cDNA sequences of these subunits using both conventional polymerase chain reaction (PCR)-cloning-sequencing and transcriptome-sequencing analyses. For the α and β subunits, one gene (locus) for each was predicted, whereas for the γ subunit, two types of cDNA sequences were obtained, which we named γ1 and γ2. These two types probably have distinct loci. Average nucleotide sequence identities among the subunits ranged from 87.6% (between α and γ1) to 92.6% (between γ1 and γ2), suggesting that they form a gene family. Although no homology was found between the sequences of allo A and other known lectin proteins in a protein database search, some unknown proteins containing the DUF3421 domain were identified. Those DUF3421 domain-encoding proteins are upregulated in the insect larval midgut. Thus, we infer that allo A genes also play an important role in larvae and that their lectin activity may have been obtained collaterally.

Dynamics of Germination Behaviour, Protein Secondary Structure, Technofunctional Properties, Antinutrients, Antioxidant Capacity and Mineral Elements in Germinated Dhaincha.

SUMMARYResearch backgroundDhaincha (Sesbania aculeata) is a forage legume primarily used for green manuring and animal feed. Good nutritional profile of dhaincha makes it a potential alternative legume in human nutrition. However, the presence of high amount of antinutrients poses a problem in its utilisation for food applications. The present investigation intends to germinate dhaincha seeds at different time-temperature regimes and to evaluate the process of germination to ascertain optimal conditions and improve its potential for utilisation.Experimental approachDhaincha seeds were germinated at 24, 28 and 32 °C for 24, 48 and 72 h. Germination characteristics and germination loss, spectral characteristics, technofunctionality, antinutrients, bioactive constituents, antioxidant capacity and mineral element content of germinated dhaincha were evaluated. Optimal balance of technobiofunctionality of germinated dhaincha seeds was validated by principal component analysis.Results and conclusionsSprout length and germination loss increased with the higher germination temperature and prolonged germination time. Seeds showed similar germination rate at 28 and 32 °C and it was markedly higher than at 24 °C. Germination for 24 h resulted in mild conformational changes in the secondary structure of proteins, whereas germination for 48 and 72 h exhibited major conformational changes in the β-sheets, resulting in the improvement in the hydration and foaming properties. Progression of germination (72 h) caused the decrease of tannin (24.47%), phytic acid (16.38%) and saponin (24.58%) mass fractions, and of trypsin inhibitor (40.33%) and lectin activity (62.50%). Slight decrease of DPPH˙ (3.7%) and ABTS˙+ (18.5%) values was also observed, whereas total flavonoid content (36.14%) and metal chelating activity (26.76%) increased. Total phenolics, FRAP, and reducing power decreased after 24 h, followed by a gradual increase. Zinc extractability increased drastically with germination. Germination at 28 °C for 72 h resulted in higher reduction of antinutrients with optimal retention of antioxidant activity and better functional characteristics, as validated by principal component analysis.Novelty and scientific contributionDhaincha is an unknown crop in Europe, and even in Asia it is predominantly used as green manure and animal feed. This research demonstrated that the intervention in germination can transform dhaincha into a promising crop for food industry. Germinated dhaincha exhibited enhanced technobiofunctionality for utilisation in various food formulations.