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Isolation and characterization of BanLec-I, a mannoside-binding lectin from Musa paradisiac (banana)
Abstract
A lectin (BanLec-I) from banana (Musa paradisiac) with a binding specificity for oligomannosidic glycans of size classes higher than (Man)6GlcNAc was isolated and purified by affinity chromatography on a Sephadex G-75 column. It did not agglutinate untreated human or sheep erythrocytes, but it did agglutinate rabbit erythrocytes. BanLec-I stimulated T-cell proliferation. On size-exclusion chromatography, BanLec-I has a molecular mass of approx. 27 kDa, and on SDS/PAGE the molecular mass is approx. 13 kDa. The isoelectric point is 7.2-7.5. BanLec-I was found to be very effective as a probe in detecting glycoproteins, e.g. on nitrocellulose blots.
... Banana is widely consumed all over the globe as a wholesome nutritional fruit containing 75% moisture, 23% carbohydrates, 1.1% protein and 0.33% fat (wet weight basis) (15). Among the proteins, BanLec is a minor protein present in relatively low amounts: 4 mg lectin/100 g fruit, edible portion (16,17). Banana lectin (BanLec) is a homodimeric plant lectin (subunit molecular weight: 15 kDa; isoelectric point: 7.2-7.5) ...
... Banana lectin (BanLec) is a homodimeric plant lectin (subunit molecular weight: 15 kDa; isoelectric point: 7.2-7.5) belonging to the jacalin-related lectin family (16,17). BanLec belongs to a subgroup of this family that binds to glucose/mannose, but is unique in recognizing internal a-1,3 linkages as well as b-1,3 linkages at the reducing termini (18,19). ...
... The present study is focused on studying the effect of purified BanLec on basophils and mast cells from different atopic subjects. Since lectins are often present in significant amounts in many plant foods (1,2,7,11,16), it appeared interesting to study the interactions of BanLec with mast cells and basophils of atopic and non-atopic subjects, to understand their physiological significance and role in non-allergic food hypersensitivity reactions. ...
Dietary lectins play a major role in the activation of mast cells / basophils by bridging cell surface IgE glycans to release histamine and other mediators. In the present study, the effect of mannose / glucose-specific banana lectin (BanLec) on the activation of mast cells / basophils from non-atopic and atopic subjects has been investigated. BanLec was purified from banana pulp in a yield of 7 mg/kg. Leukocytes isolated from heparinized blood of non-atopic / atopic subjects were used for quantitation of the released histamine. Approximately 28.2% of the atopics (n = 117) was positive by skin prick test (SPT) to purified BanLec (100 mg/mL concentration), and all the non-atopics (n = 20) were negative. Maximal release of histamine was seen at 2 g of BanLec. In percent histamine release, an increase of 35-40% is observed in case of atopics (n = 7) compared to non-atopics (n = 5), and the histamine release from atopic and non-atopic subjects correlates fairly well with the total serum IgE levels (R2 = 0.817). BanLec also induces release of histamine (26.7%) from mast cells present in rat peritoneal exudate cells. BanLec can significantly activate and degranulate mast cells and basophils by cross-linking the trimannosidic core mannose of IgE glycans in atopic population as compared to non-atopic population; the activation is marginal in the case of non-atopics.
... In contrast, the isolate became a 2-fold more susceptible for the plant lectin GNA (Table 2) [26]. Later studies showed that this resistant virus, compared to the wild-type NL4.3 strain, was also two to 5-fold more susceptible to other CBAs such as OAA and BanLec, In addition, the MVN-resistant strain showed a 14-fold decreased susceptibility to GRFT ( [54,55]. BanLec exists as a homodimeric protein with two subunits of 15 kDa. ...
... BanLec exists as a homodimeric protein with two subunits of 15 kDa. Cloning studies showed that it belongs to the group of mannose-specific jacalin-related lectins, and was found to be a T cell mitogen [54,55]. Each monomer has two carbohydrate-binding sites, of which the second was quite unique in the jacalin-related lectin family [56]. ...
... Each monomer has two carbohydrate-binding sites, of which the second was quite unique in the jacalin-related lectin family [56]. Sugar binding experiments indicated its specificity for high-mannose type glycans containing eight or nine mannose residues and no binding with Man6 or mono-and bi-antennary complex type glycans [54]. Later binding and crystal structure studies showed unique carbohydrate-binding properties to internal 3-O-α-D-glucopyranosyl units and α(1,3)-linked glucosyl residues as well as β(1,3)-linkages at the reducing termini [56][57][58]. ...
The glycoproteins on the surfaces of enveloped viruses, such as HIV, can be considered as a unique target for antiviral therapy. Different carbohydrate-binding agents (CBAs) target specific glycans present on viral glycoproteins of enveloped viruses. It has been shown that long-term CBA pressure in vitro can result in mutant HIV-1 isolates with several N-linked glycan deletions on gp120. These studies demonstrated that mainly high-mannose type glycans are deleted. However, interestingly, N241, N262 and N356 on gp120 have never been found to be affected after prolonged CBA exposure. Here, we review the mutation and (cross)-resistance profiles of eleven specific generated CBA-resistant HIV-1 strains. We observed that the broad-neutralizing anti-carbohydrate binding mAb 2G12 became completely inactive against all the generated CBA-resistant HIV-1 clade B isolates. In addition, all of the CBAs discussed in this review, with the exception of NICTABA, interfered with the binding of 2G12 mAb to gp120 expressed on HIV-1-infected T cells. The cross-resistance profiles of mutant HIV-1 strains are varying from increased susceptibility to very high resistance levels, even among different classes of CBAs with dissimilar sugar specificities or binding moieties [e.g., α(1,3), α(1,2), α(1,6)]. Recent studies demonstrated promising results in non-topical formulations (e.g., intranasally or subcutaneously), highlighting their potential for prevention (microbicides) and antiviral therapy.
... During an investigation of the human antibody response to various foods, Koshte et al. [12] observed marked binding of IgG4 to banana (Musa paradisiaca) extract from its fruit. In view of the mucilaginous nature of the banana extract, they considered the possibility of non-specific binding to IgG4 and decided to purify the active principle. ...
... That was how the banana lectin (BanLec or BL) was first isolated from Musa paradisiaca by Koshte and colleagues [12]. They also reported that BanLec is a homodimeric protein that binds mannose and mannose-containing oligosaccharides and functions as a potent T-cell mitogen [9,13]. ...
... Hence, potential applications could also be explored for rBanLec in view of its safety for human and animal consumption, its resistance to proteolytic degradation and binding to mucosal surfaces, which could increase the availability of therapeutics and enhance or redirect an immune response against target immunogens [26,37,38]. Moreover BanLec is an excellent tool for glycoprotein research [12]. ...
Lectins are a group of proteins of non-immune origin that recognize and bind to carbohydrates without modifying them. Banana is the common name for both herbaceous plants of the genus Musa and for the fruit they produce. They are indeed a promising source for many medicinal applications. Banana lectins have the potential for inhibiting HIV-1 reverse transcriptase activity, suppressing cancer cell proliferation and stimulating macrophage activities. Nevertheless, compared to other plant lectins, there is relatively little information in the literature on banana lectins, particularly with respect to their structure and biological functions. Herein we focus our review on the structure, functions and exploitable properties of banana lectins.
... The mannose/glucose binding lectin isolated from bananas, BanLec, was first identified through an investigation looking at antibody responses towards a variety of foods [66]. ...
... BanLec binds high-mannose structures that exist as N-linked glycans found on mammalian proteins with greater affinity than some other mJRL members [60,66]. This suggests that BanLec has potential uses in carbohydrate biology. ...
... The lectin termed BanLec, isolated from the ripened fruit of the banana (Musa acuminata cultivars), exists as a dimer with a molecular weight of approximately 30 kDa [67]. It is a member of the jacalin-related lectin (JRL) family and can recognize high-mannose structures [60,66,92]. Lectins in this family are characterized by the presence of a βprism 1 structure composed of three Greek Key turn motifs. ...
Despite years of research, a vaccine that can end the HIV/AIDS pandemic is not in the foreseeable future. Although the search for a vaccine is ongoing, novel methods to prevent HIV-1 infection should be pursued. We found that a lectin isolated from bananas, BanLec, is a potent inhibitor of HIV-1 infection. BanLec is able to bind to the high mannose structures found on the heavily glycosylated envelope protein of HIV-1, which allows it to recognize a broad range of different viral isolates. We determined that BanLec inhibits entry of the virus into the cell before cellular attachment of the virus. The anti-HIV-1 activity of BanLec compares well to the clinically approved anti-HIV drugs T-20 and maraviroc. These drugs and other anti-HIV lectins are being considered as components of a vaginally-applied anti-HIV microbicide for inhibition of sexual transmission. Therefore, we began to assess the potential of BanLec as a microbicide component.
Since pro-inflammatory conditions have been the bane of clinical trials of potential microbicides, we decided to assess whether BanLec has mitogenic activity that could lead to cellular activation and proliferation. These conditions could lead to inflammatory cytokine production that favor increased HIV-1 replication. We found that the addition of
BanLec to peripheral blood lymphocytes induced mitogenic activity. However, a lectin related to BanLec, griffithsin, has anti-HIV-1 activity and is not mitogenic. Therefore, we hypothesized that the mitogenic activity of BanLec could be reduced without abolishing its anti-viral activity. Using a recombinant version of BanLec, we found that substituting the histidine at position 84 leads to altered mitogenic activity. In particular, a histidine to threonine substitution (H84T) resulted in a loss of mitogenic activity, yet that mutant retained potent anti-HIV activity. In addition, the H84T mutant has reduced agglutination activity, suggesting that impaired cross-linking ability may be responsible for reduced mitogenicity. We have demonstrated that BanLec is a potent inhibitor of HIV-1 replication and that its mitogenic activity is separable from its anti-HIV-1 activity. Our results suggest that the BanLec mutant H84T is a promising candidate for future anti-HIV therapies.
... The banana lectin (Musa acuminata), first isolated by Koshte and colleagues [1], is a homotetramer, subunit M r 15 kDa. It was reported to induce the formation of IgG 4 antibodies, to be a T-cell mitogen, and to bind mannose and some of its oligosaccharides [1,2]. ...
... The banana lectin (Musa acuminata), first isolated by Koshte and colleagues [1], is a homotetramer, subunit M r 15 kDa. It was reported to induce the formation of IgG 4 antibodies, to be a T-cell mitogen, and to bind mannose and some of its oligosaccharides [1,2]. Peumans et al. [3] have cloned the gene that encodes the banana lectin and found it to belong to a family of jacalin-related lectins. ...
... isolichenan and pullulan). It also is worth noting that the banana lectin binds to Sephadex G-100, crossed linked dextran gel [1] and can be eluted with methyl a-glucoside. ...
This paper extends our knowledge of the rather bizarre carbohydrate binding poperties of the banana lectin (Musa acuminata). Although a glucose/mannose binding protein which recognizes α-linked gluco-and manno-pyranosyl groups of polysaccharide chain ends, the banana lectin was shown to bind to internal 3-O-α-d-glucopyranosyl units. Now we report that this lectin also binds to the reducing glucosyl groups of β-1,3-linked glucosyl oligosaccharides (e.g. laminaribiose oligomers). Additionally, banana lectin also recognizes β1,6-linked glucosyl end groups (gentiobiosyl groups) as occur in many fungal β1,3/1,6-linked polysaccharides. This behavior clearly distinguishes the banana lectin from other mannose/glucose binding lectins, such as concanavalin A and the pea, lentil and Calystegia sepium lectins.
... These glycosylation sites can serve as a targeting pattern for CARs. Banana Lectin (BanLec) is a lectin extracted from the fruit of bananas (Musa acuminate) that binds high mannose glycans (29,30). The binding of BanLec to virally expressed high mannose glycans has antiviral activity (30,31), but wild-type BanLec is also strongly mitogenic and induces nonspecific T cell activation (32). ...
... The vast majority of CAR constructs contain an extracellular single chain variable fragment derived from a monoclonal antibody for protein binding (23). Instead, we used a lectin with specific binding to high mannose, an altered glycosylation pattern common to viral envelopes (26)(27)(28)(29)(30). Our glycoprotein targeting mitigates the potential risk of antigen downregulation, a mechanism commonly employed by cancer cells to evade targeted immunotherapies (48). ...
H84T-Banana Lectin (BanLec) CAR-NK cells bind high mannose glycosites that decorate the SARS-CoV-2 envelope, thereby decreasing cellular infection in a model of SARS-CoV-2. H84T-BanLec CAR-NK cells are innate effector cells, activated by virus. This novel cellular agent is a promising therapeutic, capable of clearing circulating SARS-CoV-2 virus and infected cells. Banana Lectin (BanLec) binds high mannose glycans on viral envelopes, exerting an anti-viral effect. A point mutation (H84T) divorces BanLec mitogenicity from antiviral activity. SARS-CoV-2 contains high mannose glycosites in proximity to the receptor binding domain of the envelope Spike (S) protein. We designed a chimeric antigen receptor (CAR) that incorporates H84T-BanLec as the extracellular moiety. Our H84T-BanLec CAR was devised to specifically direct NK cell binding of SARS-CoV-2 envelope glycosites to promote viral clearance. The H84T-BanLec CAR was stably expressed at high density on primary human NK cells during two weeks of ex vivo expansion. H84T-BanLec CAR-NK cells reduced S-protein pseudotyped lentiviral infection of 293T cells expressing ACE2, the receptor for SARS-CoV-2. NK cells were activated to secrete inflammatory cytokines when in culture with virally infected cells. H84T-BanLec CAR-NK cells are a promising cell therapy for further testing against wild-type SARS-CoV-2 virus in models of SARS-CoV-2 infection. They may represent a viable off-the-shelf immunotherapy for patients suffering from COVID-19.
... Proliferation induced by Musa acuminata (Del Monte banana) lectin led to the expression of the cytokines interferon-γ, tumor necrosis factor-α, and interleukin-2 in mouse splenocytes [11]. BanLec from M. paradisiaca stimulated T cell proliferation in man in presence of IL-2 like Concanavalin A [13,14]. The immune pathway activated by BanLec-I was not identified in these studies [13,14]. ...
... BanLec from M. paradisiaca stimulated T cell proliferation in man in presence of IL-2 like Concanavalin A [13,14]. The immune pathway activated by BanLec-I was not identified in these studies [13,14]. BanLec also had strong mitogenic activity towards murine T-cells [15]. ...
The aim of the study was to gain deeper insights in the potential of polyclonal stimulation
of PBMC with banana lectin (BanLec) from Musa paradisiaca. BanLec induced a marked proliferative
response in cow and pig PBMC, but was strongest in pigs, where it induced an even higher proliferation
rate than Concanavalin A. Molecular processes associated with respective responses in porcine
PBMC were examined with differential proteome analyses. Discovery proteomic experiments was
applied to BanLec stimulated PBMC and cellular and secretome responses were analyzed with label
free LC-MS/MS. In PBMC, 3955 proteins were identified. After polyclonal stimulation with BanLec,
459 proteins showed significantly changed abundance in PBMC. In respective PBMC secretomes,
2867 proteins were identified with 231 differentially expressed candidates as reaction to BanLec
stimulation. The transcription factor “E74 like ETS transcription factor 1 (ELF1)” was solely enriched
in BanLec stimulated PBMC. BanLec induced secretion of several immune regulators, amongst them
positive regulators of activated T cell proliferation and Jak-STAT signaling pathway. Top changed
immune proteins were CD226, CD27, IFNG, IL18, IL2, CXCL10, LAT, ICOS, IL2RA, LAG3, and
CD300C. BanLec stimulates PBMC of cows and pigs polyclonally and induces IL2 pathway and further
proinflammatory cytokines. Proteomics data are available via ProteomeXchange with identifier
PXD027505.
... ConA in the cows we tested additional mitogens. The T cell mitogen BanLec (24), which activates T cells of man through the IL-2 pathway (25,26), led to similar hyperproliferation of PBL from BNP dams as observed with ConA ( Fig. 5A, reaction difference factor: 5.0, BNP to controls, **** p < 0.0001). PBL of ID cows responded to BanLec stimulation with a similar immune response intensity as BNP (Fig. 5B, reaction difference factor: 3.0 ID to controls, **** p < 0.0001). ...
... The immune deviant response described here clearly depends on IL-2. Since BanLec activates T cells in man (24,26) via the IL-2 pathway (25), the hyperproliferative response of BNP and ID animals to BanLec (Fig. 5B) indicated a role for IL-2. This was confirmed by sole application of IL-2 (Fig. 5C). ...
A BVD vaccine with novel production technology and adjuvant was introduced to improve immune responses against BVD. However, 5-10% of vaccinated cattle produced pathogenic antibodies, which transferred bovine neonatal pancytopenia (BNP) via colostrum to almost any calf given respective colostrum. Diseased calves had a very high lethality rate of 90%. Dysregulated immune response of dams to the novel vaccine is not fully understood to date. In this study, we clarified by in-depth characterization of immune capacities if the immune response was changed by this particular vaccine or if these cows were prone to a different type of immune response per se.
There was a marked difference in the response after polyclonal in vitro stimulation of peripheral blood derived lymphocytes (PBL). BNP dams showed a highly significant hyperproliferation to Pokeweed Mitogen (PWM), Concanavalin A (ConA), Musa paradisiaca banana lectin (BanLec) and interleukin-2 (IL-2). Subsequent differential proteome analyses revealed substantial qualitative differences in immune capacities between both cow groups. PBL of BNP dams reacted with a STAT3 regulated response compared to PBL of controls, which used STAT1 pathway after ConA stimulation. We could detect this immune response pattern also in cows that were never vaccinated at all. Therefore, our data prove that respective BVD vaccination did not cause this deviant immune response, but that a deviant immune phenotype (ID) already existed in cattle and still exists. PBL of these ID cows prefer the IL-2/JAK2/STAT3 pathway for immune responses. Since ID cows additionally have a two-fold increased mastitis incidence, our findings have clinical relevance.
... Yes (Boyd et al., 1997;Gustafson et al., 1997;Balzarini et al., 2006;Witvrouw et al., 2005;Alexandre et al., 2010;Barrientos et al., 2003;O'Keefe et al., 2003;Kachko et al., 2013;Helle et al., 2006;Takebe et al., 2013;Tsai et al., 2004;Tsai et al., 2003 Unk -Unknown. 1 or 2 CV-N-like oligomannose (D1 D3) gp120 (O'Keefe et al., 2000;Botos and Wlodawer, 2003;Boyd et al., 1997;Gustafson et al., 1997;Yang et al., 1999;Kachko et al., 2013;Helle et al., 2006;Bolmstedt et al., 2001;Shenoy et al., 2001;Bewley and Otero-Quintero, 2001;Bewley, 2001 (Koshte et al., 1990;Swanson et al., 2010;Swanson et al., 2015;Gavrovic-Jankulovic et al., 2008;Dimitrijevic et al., No (Alexandre et al., 2010;Jensen et al., 2014;Mori et al., 2005;Nixon et al., 2013;Ishag et al., 2013;Meuleman et al., 2011;Kouokam et al., 2011;Ferir et al., 2011;O'Keefe et al., 2010;Emau et al., 2007;Barton et al., 2014) SCL Scilla campanulata HIV (4.6e8 mM) ...
... The BanLec lectin was isolated from banana, Musa acuminate, and exists as a homodimer with a molecular weight of 30 kDa (Koshte et al., 1990). The lectin exhibits a Jacalin-like lectin fold with an RMSD of 0.934 Å over 92 a-carbons when superposed with the Jacalin structure (1JAC) (Fig. 5) (Singh et al., 2005;Meagher et al., 2005). ...
Many natural lectins have been reported to have antiviral activity. As some of these have been put forward as potential development candidates for preventing or treating viral infections, we have set out in this review to survey the literature on antiviral lectins. The review groups lectins by structural class and class of source organism we also detail their carbohydrate specificity and their reported antiviral activities. The review concludes with a brief discussion of several of the pertinent hurdles that heterologous proteins must clear to be useful clinical candidates and cites examples where such studies have been reported for antiviral lectins. Though the clearest path currently being followed is the use of antiviral lectins as anti-HIV microbicides via topical mucosal administration, some investigators have also found systemic efficacy against acute infections following subcutaneous administration.
... Although the purification of proteins from fruits is a challenging procedure, because of both the low abundance of proteins and the large amounts of interfering compounds, the natural BanLec was efficiently obtained from ripe banana. However, the average amount of BanLec extracted from banana fruit pulp was similar to data from literature [20]. According to the electrophoretic profile and MS sequencing, two almost identical proteins around 15 kDa were present in BanLec preparations orally administered to mice, which agglutinated rabbit erythrocytes and displayed specificity towards both mannose and glucose. ...
... The effect of BanLec as an immunomodulatory agent was first reported by Koshte et al., [20], as it stimulated T-cells from human peripheral blood in the presence of interleukin-2. After that, BanLec was reported as a relevant IgG4-inducing antigen in humans [10] and both the recombinant and the natural forms of BanLec were found to proliferate CD3+, CD4+ and CD8+ T-cells, but not B-cells or monocytes [9]. ...
Banana lectin (BanLec) is a dimeric protein occurring in fruit pulp that modulates immune cell functioning in vitro. In order to assess the immune response in vivo, BanLec from ripe banana (Musa acuminata) fruit was purified and orally given to mice for seven days. The analysis of cytokines in the mice peripheral blood revealed increased IL-10, IL-17 and TNFα, and a reduction of IFNγ and IL-6. In the thymus, an increase of CD4+ and a decrease of CD8+ T-cells were observed after oral administration of BanLec. The modulation of pro- and anti-inflammatory cytokines and T-cells in the peripheral blood and thymus of mice demonstrated the immunomodulatory properties of natural BanLec in vivo. This research brings new data on a protein from a fresh fruit consumed worldwide that may act as an immunomodulator, potentially affecting the host response to infections, immune diseases and cancer.
... Immunogenicity of biologic drugs is a widely acknowledged issue, and our data suggest that future efforts to deimmunize GRFT by structure-guided elimination of T-cell epitopes (31, 32) may be necessary before the product can be used for chronic treatment of viral infection in humans. Lectins are well known for their mitogenic and agglutinating properties (28,29,(33)(34)(35)(36) which prevent their use as therapeutics. We previously addressed the mitogenicity concern by showing that GRFT lacks T-cell mitogenic activity (14). ...
... There is substantial precedent for patient self-administration of drugs via the subcutaneous route; indeed, the peptide HIV fusion inhibitor Enfurtide (T-20) is administered in this fashion. Our data confirm that we can achieve HIV-1 Du156 50% serum neutralization indices in excess of 500 after 14 daily doses of GRFT at 10 mg/kg (Fig. 2), which should be sufficient to inhibit HIV replication and perhaps to promote viral evolution toward enhanced humoral antibody suppression (7,34,37). These levels were also more than sufficient to prevent JEV infection in a mouse infection model and HCV infection in in a mouse-humanized liver model (17,20). ...
Griffithsin (GRFT) is a red algae derived lectin that binds the terminal mannose residues of N-linked glycans found on the surface of HIV-1, HIV-2 and other enveloped viruses including HCV, SARS-CoV, and Ebola virus. GRFT displays no human T-cell mitogenic activity and does not induce production of pro-inflammatory cytokines in treated human cell lines. However, despite the growing evidence showing the broad spectrum nanomolar or better antiviral activity of GRFT, no study has reported a comprehensive assessment of GRFT safety as a potential systemic antiviral treatment. The results presented in this work show that minimal toxicity is induced by a range of single and repeated daily subcutaneous doses of GRFT in two rodent species, although we noted treatment-associated increases in spleen and liver mass, suggestive of an anti-drug immune response. The drug is systemically distributed, accumulating to high levels in the serum and plasma after subcutaneous delivery. Further, we showed that serum from GRFT treated animals retained anti-viral activity against HIV-1 enveloped pseudoviruses in a cell-based neutralization assay. Overall, our data presented here show that GRFT accumulates to relevant therapeutic concentrations which are tolerated with minimal toxicity. These studies support further development of GRFT as a systemic antiviral therapeutic agent against enveloped viruses, although de-immunizing the molecule may be necessary if it is to be used in long term treatment of chronic viral infections.
... That dosage extrapolated to humans would bẽ 0.04 mg rBanLec/kg BW/day [35]. That therapeutic dosage for a human weighing 70 kg (~2.8 mg BanLec) could be found in one banana (average BanLec content is~4 mg/100 g banana pulp [36]; a medium-sized banana contains~120 g of banana pulp [37]. As we observe certain differences in different genetic backgrounds, BanLec could be a part of a personalized application strategy in the future. ...
Compared to the general population, patients with inflammatory bowel disease (IBD) are less likely to be vaccinated, putting them at an increased risk of vaccine-preventable illnesses. This risk is further compounded by the immunosuppressive therapies commonly used in IBD management. Therefore, developing new treatments for IBD that maintain immune function is crucial, as successful management can lead to better vaccination outcomes and overall health for these patients. Here, we investigate the potential of recombinant banana lectin (rBanLec) as a supporting therapeutic measure to improve IBD control and possibly increase vaccination rates among IBD patients. By examining the therapeutic efficacy of rBanLec in a murine model of experimental colitis, we aim to lay the foundation for its application in improving vaccination outcomes. After inducing experimental colitis in C57BL/6 and BALB/c mice with 2,4,6-trinitrobenzene sulfonic acid, we treated animals orally with varying doses of rBanLec 0.1–10 µg/mL (0.01—1 µg/dose) during the course of the disease. We assessed the severity of colitis and rBanLec’s modulation of the immune response compared to control groups. rBanLec administration resulted in an inverse dose–response reduction in colitis severity (less pronounced weight loss, less shortening of the colon) and an improved recovery profile, highlighting its therapeutic potential. Notably, rBanLec-treated mice exhibited significant modulation of the immune response, favoring anti-inflammatory pathways (primarily reduction in a local [TNFα]/[IL-10]) crucial for effective vaccination. Our findings suggest that rBanLec could mitigate the adverse effects of immunosuppressive therapy on vaccine responsiveness in IBD patients. By improving the underlying immune response, rBanLec may increase the efficacy of vaccinations, offering a dual benefit of disease management and prevention of vaccine-preventable illnesses. Further studies are required to translate these findings into clinical practice.
... That dosage extrapolated to humans would be ~0.04 mg rBanLec/kg BW/day [34]. That therapeutic dosage for a human weighing 70 kg (~2.8 mg BanLec) could be found in one banana (average BanLec content is ~4 mg/100 g banana pulp [35]; a medium-sized banana contains ~120 g of banana pulp [36]. As we observe certain differences in different genetic backgrounds, BanLec could be a part of a personalized application strategy in the future. ...
Compared to the general population, patients with inflammatory bowel disease (IBD) are less likely to be vaccinated, putting them at an increased risk of vaccine-preventable illnesses. This risk is further compounded by the immunosuppressive therapies commonly used in IBD management. Therefore, developing new treatments for IBD that maintain immune function is crucial, as successful management can lead to better vaccination outcomes and overall health for these patients. Here, we investigate the potential of recombinant banana lectin (rBanLec) as a supporting therapeutic measure to improve IBD control and possibly increase vaccination rates among IBD patients. By examining the therapeutic efficacy of rBanLec in a murine model of experimental colitis, we aim to lay the foundation for its application in improving vaccination outcomes. After inducing experimental colitis in C57BL/6 and BALB/c mice with 2,4,6-trinitrobenzene sulfonic acid, we treated animals orally with varying doses of rBanLec 0.1-10 µg/ml (0.01 – 1 µg/dose) during the course of the disease. We assessed the severity of colitis and rBanLec’s modulation of the immune response compared to control groups. rBanLec administration resulted in a dose-dependent reduction in colitis severity (less pronounced weight loss, less shortening of the colon) and an improved recovery profile, highlighting its therapeutic potential. Notably, rBanLec-treated mice exhibited significant modulation of the immune response, favoring anti-inflammatory pathways (primarily reduction in a local [TNFα]/[IL-10]) crucial for effective vaccination. Our findings suggest that rBanLec could mitigate the adverse effects of immunosuppressive therapy on vaccine responsiveness in IBD patients. By improving the underlying immune response, rBanLec may increase the efficacy of vaccinations, offering a dual benefit of disease management and prevention of vaccine-preventable illnesses. Further studies are required to translate these findings into clinical practice.
... Banana lectin (BanLec) is a dimeric protein composed of two identical subunits approximately 15 kDa in size [5]. Purified BanLec showed β-prism I fold, a characteristic feature of jacalin-related lectins (JRLs) and a unique feature of two primary binding sites per subunit [6]. ...
Lectins are proteins that reversibly bind to carbohydrates and are commonly found across many species. The Banana Lectin (BanLec) is a member of the Jacalin-related Lectins, heavily studied for its immunomodulatory, antiproliferative, and antiviral activity. In this study, a novel sequence was generated in silico considering the native BanLec amino acid sequence and 9 other lectins belonging to JRL. Based on multiple alignment of these proteins, 11 amino acids of the BanLec sequence were modified because of their potential for interference in active binding site properties resulting in a new lectin named recombinant BanLec-type Lectin (rBTL). rBTL was expressed in E. coli and was able to keep biological activity in hemagglutination assay (rat erythrocytes), maintaining similar structure with the native lectin. Antiproliferative activity was demonstrated on human melanoma lineage (A375), evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT). rBTL was able to inhibit cellular growth in a concentration-dependent manner, in an 8-h incubation, 12 µg/mL of rBTL led to a 28.94% of cell survival compared to cell control with 100%. Through a nonlinear fit out log-concentration versus biological response, an IC50% of 3.649 µg/mL of rBTL was determined. In conclusion, it is possible to state that the changes made to the rBTL sequence maintained the structure of the carbohydrate-binding site without changing specificity. The new lectin is biologically active, with an improved carbohydrate recognition spectrum compared to nBanLec, and can also be considered cytotoxic for A375 cells.
... Mushroom [48] Ion exchange chromatography BanLec Banana [40] Exclusion chromatography in gel FSL (Fusarium sambucinum) Fungus [42] Ion exchange chromatography PTA e LRA Pinellia ternata and Lycoris radiata [43] Molecular exclusion (PD-10 Desalting) Swartzia laevicarpa Legume [49] Among the modes of operation of the various chromatographic processes are the fixed bed and the expanded bed. The fixed bed has high efficiency, being a technique of easy implementation in production processes. ...
Growing medical, engineering, biochemical, and biological interest has led to a steady pace of research and development into polymeric monolithic structures with densely interconnected pores for purifying bio compounds. Cryogels, which are generated by freezing a reactive polymerization mixture, are highlighted due to their versatility and low relative cost as macroporous, polymeric, monolithic adsorbents. The conversion of cryogels into affinity adsorbents is one possible alternative to their optimal application. Some of the most often utilized supports for immobilizing particular ligands are monolithic columns manufactured with epoxy radicals on their surfaces. The purification of biomolecules with a high degree of specificity, such as lectins and glycoproteins with an affinity for glycosylated groups, has garnered interest in the use of fixed non-traditional beds functionalized with ligands of particular interest. The interaction is both robust enough to permit the adsorption of glycoproteins and reversible enough to permit the dissociation of molecules in response to changes in the solution’s pH. When compared to other protein A-based approaches, this one has been shown to be more advantageous than its counterparts in terms of specificity, ease of use, and cost-effectiveness. Information on polymeric, macroporous, monolithic adsorbents used in the affinity chromatographic purification of lectins has been published and explored.
... The first report on the isolation of banana lectin (BanLec) was from Musa paradisiac [4]. It belongs to the jacalin-related superfamily (mJRL) of lectins, which recognizes and binds to high mannose glycans [5]. ...
Fluorescently labeled lectins are useful tools for in vivo and in vitro studies of the structure and function of tissues and various pathogens such as viruses, bacteria, and fungi. For the evaluation of high-mannose glycans present on various glycoproteins, a three-dimensional (3D) model of the chimera was designed from the crystal structures of recombinant banana lectin (BanLec, Protein Data Bank entry (PDB): 5EXG) and an enhanced green fluorescent protein (eGFP, PDB 4EUL) by applying molecular modeling and molecular mechanics and expressed in Escherichia coli. BanLec-eGFP, produced as a soluble cytosolic protein of about 42 kDa, revealed β-sheets (41%) as the predominant secondary structures, with the emission peak maximum detected at 509 nm (excitation wavelength 488 nm). More than 65% of the primary structure was confirmed by mass spectrometry. Competitive BanLec-eGFP binding to high mannose glycans of the influenza vaccine (Vaxigrip®) was shown in a fluorescence-linked lectin sorbent assay (FLLSA) with monosaccharides (mannose and glucose) and wild type BanLec and H84T BanLec mutant. BanLec-eGFP exhibited binding to mannose residues on different strains of Salmonella in flow cytometry, with especially pronounced binding to a Salmonella Typhi clinical isolate. BanLec-eGFP can be a useful tool for screening high-mannose glycosylation sites on different microorganisms.
... The effect of treatments (banana pulp extract, pulp extract + peel extract and peel powder) on total weight and average daily gain (Means ± SE) between trial groups calves of group 2. BanLec is a special lectin as it is highly immunogenic in man and causes a strong IgG4 antibody response (Koshte et al., 1990). In agreement with this, it was reported that BanLec is capable of inducing IgG4 formation, being as a T-cell mitogen, binding mannose and some of its oligosaccharides (Mo et al., 2001) and inducing Th1 cytokines production (Souza et al., 2013). ...
The present study investigated the effects of overripe pulp and green peel extract and powder of banana fruit (Musa. cavendish) on haematological, biochemical, immunological, health, and performance of Holstein dairy calves. In all, 40 newborn calves were randomly divided into four groups of 10 animals. In the control group, animals received no banana meal. In group 1, calves were supplemented with 2 g (dry matter)/kg body weight/day of overripe banana pulp extract. The calves in group 2 were supplemented with 1 g (dry matter) of overripe banana pulp extract/kg body weight/day and 1 g (dry matter) of green banana peel extract/kg body weight/day. The animals in group 3 were supplemented with 2 g/kg body weight/day of green banana peel powder. The feeding period of calves on the tested supplements was 5 days. Blood samples and other evaluations were taken on day 0 (at birth, before supplementation) and on days 7, 15 and 30. Just a trend towards better average daily weight gain was seen in groups 2 and 3 than others (p = 0.073). Significant group and sampling time interactions were seen for the quantities of RBC (group 1 was lower than other groups at day 30), MCV (group 3 was lower than other groups at day 30) and MCH (group 1 was higher than other groups at day 30) (p < 0.05). A trend towards significance in values of IgG (group 1 was lower than other groups at days 15 and 30) and bilirubin (higher values at day 7 in groups 1 and 2 than control, higher amounts at days 15 and 30 in groups 3 and 2 than control, respectively) was also observed. In conclusion, banana supplementation in neonatal calves had beneficial effects on the values of RBC, MCV, MCH, bilirubin, IgG and average daily weight gain in dairy calves.
... For comparison, PBLs were exposed to WT or H84T BanLec in parallel. BanLec is a known potent T cell mitogen 21 . In two different PBL donors, as expected, H84T-treated PBLs incorporated less BrdU than those that were treated with WT BanLec. ...
Lectins, carbohydrate-binding proteins, have been regarded as potential antiviral agents, as some can bind glycans on viral surface glycoproteins and inactivate their functions. However, clinical development of lectins has been stalled by the mitogenicity of many of these proteins, which is the ability to stimulate deleterious proliferation, especially of immune cells. We previously demonstrated that the mitogenic and antiviral activities of a lectin (banana lectin, BanLec) can be separated via a single amino acid mutation, histidine to threonine at position 84 (H84T), within the third Greek key. The resulting lectin, H84T BanLec, is virtually non-mitogenic but retains antiviral activity. Decreased mitogenicity was associated with disruption of pi–pi stacking between two aromatic amino acids. To examine whether we could provide further proof-of-principle of the ability to separate these two distinct lectin functions, we identified another lectin, Malaysian banana lectin (Malay BanLec), with similar structural features as BanLec, including pi–pi stacking, but with only 63% amino acid identity, and showed that it is both mitogenic and potently antiviral. We then engineered an F84T mutation expected to disrupt pi–pi stacking, analogous to H84T. As predicted, F84T Malay BanLec (F84T) was less mitogenic than wild type. However, F84T maintained strong antiviral activity and inhibited replication of HIV, Ebola, and other viruses. The F84T mutation disrupted pi–pi stacking without disrupting the overall lectin structure. These findings show that pi–pi stacking in the third Greek key is a conserved mitogenic motif in these two jacalin-related lectins BanLec and Malay BanLec, and further highlight the potential to rationally engineer antiviral lectins for therapeutic purposes.
... Banana lectin (BanLec): It is a lectin isolated from the banana fruit, Musa acuminata [82]. This lectin binds to glycoproteins with a high content of mannosylated carbohydrates, such as the human immunodeficiency virus type 1 (HIV-1). ...
When COVID-19 appeared in November 2019 in the city of Wuhan, in central China, no epidemiologist had predicted such a pandemic. Currently, no vaccine or drug is available, the treatment with azithromycin and hydroxychloroquine remains limited because it does not specifically target the viral pathogen. Most enveloped viruses express glycoproteins on their surface; lectins are proteins that specifically bind to glycosylated residues, many of which are proposed as a treatment for viral infections. Several antiviral lectins are successfully used against hepatitis C, influenza A / B, herpes, Japanese encephalitis, HIV and the Ebola virus. In this review, we will highlight the various viral infections treated with lectin-based drugs, as well as their modes of administration. In addition, and in order to inhibit the binding of COVID-19 to these receptors, we propose the use of some lectins as antiviral therapeutic agents, either by blocking receptors (glycoproteins) of the virus at the level of host cells or by masking the glycoproteins of the viral envelope.
... The colonization capacity of microorganism is also largely dependent on the presence of microbial adhesins, which are lectins as well. Banana lectin (BL) was isolated from Musa paradisiaca by Koshte et al. [7]. The recombinant lectin which is the object of this study was produced by Gavrovic-Jankulovic et al. [8]. ...
The surface of microorganisms is covered with polysaccharide structures which are in immediate contact with receptor structures on host’s cells and antibodies. The interaction between microorganisms and their host is dependent on surface glycosylation and in this study we have tested the interaction of plant lectins with different microorganisms. Enzyme-linked lectin sorbent assay - ELLSA was used to test the binding of recombinant Musa acuminata lectin - BL to 27 selected microorganisms and 7 other lectins were used for comparison: Soy bean agglutinin - SBA, Lens culinaris lectin - LCA, Wheat germ agglutinin - WGA, RCA120 - Ricinus communis agglutinin, Con A - from Canavalia ensiformis, Sambucus nigra agglutinin - SNA I and Maackia amurensis agglutinin - MAA. The goal was to define the microorganisms’ surface glycosylation by means of interaction with the selected plant lectins and to make a comparison with BL. Among the tested lectins most selective binding was observed for RCA120 which preferentially bound Lactobacillus casei DG. Recombinant banana lectin showed specific binding to all of the tested fungal species. The binding of BL to Candida albicans was further tested with fluorescence microscopy and flow cytometry and it was concluded that this lectin can differentiate ß-glucan rich surfaces. The binding of BL to S. boulardii could be inhibited with ß-glucan from yeast with IC50 1.81 μg mL⁻¹ and to P. roqueforti with 1.10 μg mL⁻¹. This unique specificity of BL could be exploited for screening purposes and potentially for the detection of ß-glucan in solutions.
... Subsequent studies by Shaper (1967) also determined that there is no evidence for implicating the banana/plantain as several lectins have been isolated from banana fruit, including BanLec, which belongs to the mannosespecific jacalin-related lectins (Peumans et al., 2000). This lectin is an important murine T-cell mitogen and can induce human T-cell proliferation (Koshte et al., 1990). The oral administration of kalakhar for 20 consecutive days did not show any noticeable effects on the targeted organs. ...
The ethnic communities of north east India are rich with their traditional delicacies. They make an alkaline preparation from parts of banana plants especially Musa bulbisiana species. Aquous extract of either dry peels of ripen banana or stem of the plant is used as common alkaline food additive among many of the ethnic communities. The preparation is commonly known as "Kalakhar" among the Assamese people. In the present study, it has been hypothesized that excess intake of this food additive may cause tissue injury. Thus, in vivo administration of this alkaline preparation was carried out in albino mice at a dose of 15ml/kg body weight per day. Administration through oral route was done for short duration (20 days) and long duration (40 days) in two separate groups of animals. The experimental mice were sacrified at the end of the period and collected liver and intestine for histological studies. The results showed cellular degeneration in different rate in liver tissue. Infiltration of neutrofils in hepatic tissue indicates cellular necrosis. Effects on the intestinal epithelial cells are recorded following long duration (40 days) treatment. It causes collapse of surface cell layer at certain areas of intestine. It is to be noted that the effects of the extract could be dose and duration dependent. A health friendly effects of this food additive prevailed in the society for centuries can not be ignored. The extract intake in specific dose could be helpful in correction of eating abnormaliries and thus, protect the system against metabolic disorder.
... This proves the existence of an immune deviant phenotype among a certain subpopulation of cattle, which is not triggered by vaccination with PregSure BVD but occurs naturally. The immune deviant response described here in these ID cows clearly depends on IL-2 ( Figure 6C), which we could prove through hyperproliferative response of BNP and ID lymphocytes to stimulation with BanLec ( Figure 6B), a mitogen that activates human T cells (28,30) via the IL-2 pathway (29). For bovine T cells, the specific impact of IL-2 itself was not described so far. ...
A novel vaccine against bovine viral diarrhea (BVD) induced pathogenic antibody production in 5–10% of BVD-vaccinated cows. Transfer of these antibodies via colostrum caused Bovine neonatal pancytopenia (BNP) in calves, with a lethality rate of 90%. The exact immunological mechanisms behind the onset of BNP are not fully understood to date. To gain further insight into these mechanisms, we analyzed the immune proteome from alloreactive antibody producers (BNP cows) and non-responders. After in vitro stimulation of peripheral blood derived lymphocytes (PBL), we detected distinctly deviant expression levels of several master regulators of immune responses in BNP cells, pointing to a changed immune phenotype with severe dysregulation of immune response in BNP cows. Interestingly, we also found this response pattern in 22% of non-BVD-vaccinated cows, indicating a genetic predisposition of this immune deviant (ID) phenotype in cattle. We additionally analyzed the functional correlation of the ID phenotype with 10 health parameters and 6 diseases in a retrospective study over 38 months. The significantly increased prevalence of mastitis among ID cows emphasizes the clinical relevance of this deviant immune response and its potential impact on the ability to fight infections.
... In contrast, AcmJRL readily hemagglutinates rat erythrocytes ( Supplementary Fig. 2) with a specific hemagglutinating activity of 62.5 µg/mL, a value higher compared with that (8.9 µg/mL) of the lectin purified from Chinese leek seeds 42 and the mannose specific lectin purified from mulberry seeds 43 . Other mannose binding lectins were reported to be unable to hemagglutinate sheep erythrocytes 44,45 , but the striking difference in the hemagglutinating activity of AcmJRL compared to the majority of mannose binding lectins described to date is the inability of AcmJRL to hemagglutinate rabbit erythrocytes 21,41,46,47 . However, comparing agglutination activity of lectins between laboratories has to be taken with caution, as the results can be highly dependent on the erythrocyte preparation and experimental protocol. ...
A mannose binding jacalin-related lectin from Ananas comosus stem (AcmJRL) was purified and biochemically characterized. This lectin is homogeneous according to native, SDS-PAGE and N-terminal sequencing and the theoretical molecular mass was confirmed by ESI-Q-TOF-MS. AcmJRL was found homodimeric in solution by size-exclusion chromatography. Rat erythrocytes are agglutinated by AcmJRL while no agglutination activity is detected against rabbit and sheep erythrocytes. Hemagglutination activity was found more strongly inhibited by mannooligomannosides than by D-mannose. The carbohydrate-binding specificity of AcmJRL was determined in some detail by isothermal titration calorimetry. All sugars tested were found to bind with low affinity to AcmJRL, with Ka values in the mM range. In agreement with hemagglutination assays, the affinity increased from D-mannose to di-, tri- and penta-mannooligosaccharides. Moreover, the X-ray crystal structure of AcmJRL was obtained in an apo form as well as in complex with D-mannose and methyl-α-D-mannopyranoside, revealing two carbohydrate-binding sites per monomer similar to the banana lectin BanLec. The absence of a wall separating the two binding sites, the conformation of β7β8 loop and the hemagglutinating activity are reminiscent of the BanLec His84Thr mutant, which presents a strong anti-HIV activity in absence of mitogenic activity.
... Banana lectin (BanLec) is a dimeric protein composed of two identical subunits of ~ 15 kDa [4,5]. Previous studies have shed light on its unique property of interaction with α-linked terminal gluco-and manno-pyranosyl residues, internal 3-o-α-D-glucopyranosyl as well as β1,3-linked glucosyl oligosaccharides [6,7]. ...
Banana lectin (BanLec) exhibits specificity to glucose/mannose residues present in oligo saccharides or glycoconjugates and has attracted a lot of attention recently as a potent therapeutic agent. Structural studies and molecular cloning methods has revealed the presence of three different BanLec proteins in two species. In our study, initial mass spectrometric analysis of affinity purified native BanLec from banana pulp (Musa paradisiaca) indicated the presence of proteins with different molecular mass. Through the bottom up and top down analysis we identified three major isoforms with acetylation at N terminus. Especially, top down analysis revealed one isoform being present as non acetylated species. The combination of mass spectrometry approaches provided insights on genetic variants and differential modifications in native BanLec.
... Banlec is a lectin isolated from the ripened fruit of the banana, Musa acuminata or Musa paradisiac (Koshte et al. 1990 ;Peumans et al. 2000 ). It is a ~30 kDa homodimeric lectin with each monomer containing 141 residues. ...
Carbohydrate-binding proteins constitute a growing class of antiviral agents. They prevent infection by blocking virus entry into the host target cells and also block virus transmission from virus-infected cells to non-infected cells. In order to illustrate the molecular basis of their antiviral activity towards human immunodeficiency virus (HIV), a comprehensive review of the three-dimensional structures of various antiviral lectins, as well as modes and atomic determinants of their high-affinity oligosaccharide recognition, is presented here. The collective information derived from these studies aids in the understanding of carbohydrate recognition of the gp120 envelope protein by these antiviral lectins and may lead to novel directions in the development of alternative drug-leads for the prevention of HIV transmission. © Springer Science+Business Media New York 2014. All rights are reserved.
... [10] BanLec-1, a glucose/mannose-specific lectin, was isolated from banana and partially characterized. [11,12] It is a dimeric protein composed of two identical 15 kDa subunits. BanLec-1 has sequence similarity to JRLs, and is not only expressed specifically in fruit, but is also expressed developmentally during fruit ripening. ...
It has been demonstrated that the lectin from Musa paradisiaca (BanLec-1) could inhibit the cellular entry of human immunodeficiency virus (HIV). In order to evaluate its effects on tobacco mosaic virus (TMV), the banlec-1 gene was cloned and transformed into Escherichia coli and tobacco, respectively. Recombinant BanLec-1 showed metal ions dependence, and higher thermal and pH stability. Overexpression of banlec-1 in tobacco resulted in decreased leaf size, and higher resistance to TMV infection, which includes reduced TMV cellular entry, more stable chlorophyll contents, and enhanced antioxidant enzymes. BanLec-1 was found to bind directly to the TMV capsid protein in vitro, and to inhibit TMV infection in a dose-dependent manner. In contrast to limited prevention in vivo, purified rBanLec-1 exhibited more significant effects on TMV infection in vitro. Taken together, our study indicated that BanLec-1 could prevent TMV infection in tobacco, probably through the interaction between BanLec-1 and TMV capsid protein.
... Ban-Lec: Ban-Lec, isolated from the banana fruit (Musca acuminata cultivators), is yet another high mannose binding lectin that recently has been linked to potent inhibition (sub-nanomolar EC 50 values) of HIV isolates of clades B and C [213,214]. ...
Although the development of a protective vaccine remains the most effective strategy for the global control of HIV/AIDS, another practical form of medical intervention would be a microbicide capable of preventing HIV-1 transmission at the mucosal level. A broad spectrum of antiviral molecules have demonstrated in vitro efficacy in proof-of-principle studies, and a selected few have already been tested in pre-clinical and clinical microbicide trials. Nevertheless, major hurdles remain to be overcome and there is still much uncertainty about the choice of inhibitors, formulations and administration vehicles for obtaining a safe and effective microbicide. A special category of HIV-1 microbicides are those based on proteins or peptides that interfere with the earliest steps in the viral infectious cycle. Besides a high degree of target specificity and a limited, if any, systemic absorption, protein-based microbicides offer the unique advantage of being suitable to in vivo expression by engineered bacteria or viral vectors, which might ensure prolonged protection without the need for planned, intercourse-coordinated application. In this respect, vaginal or rectal microbiota such as Lactobacillus spp. represent ideal expression systems as they would not only produce the inhibitor of choice at the mucosal surface, but also would easily blend within the resident microflora and offer additional valuable homeostatic effects. In this article, we review the current state of the art on protein-based microbicides.
... Banana lectin (Banlec) is a non-glycosylated homodimeric protein with specificity for mannose and high mannose oligosaccharides [20]. It is a member of the jacalin-related superfamily of lectins [21]. ...
The sequence of unfolding events of dimeric banana lectin (Banlec), as induced by guanidine hydrochloride (GdnHCl), has been investigated by size-exclusion HPLC, fluorescence, far-UV CD, low temperature phosphorescence and selective chemical modification. 8-Anilino-1-naphthalenesulfonate (ANS) binding indicates a structured unfolding intermediate which has been characterized as dissociated monomer by size-exclusion chromatography. Interestingly, the unfolding elution pattern reveals two distinct unfolded states. One is a usual random coil. The other represents a novel species having elution behavior and structural compactness (Stokes radius) similar to dissociated monomer but showing no regular secondary structure as determined by far-UV CD, thus resembling a natively unfolded state. N-bromosuccinimide (NBS) oxidation shows that single tryptophan residue remains unmodified in dissociated monomer intermediate while the same is oxidized in natively unfolded-like species. Such difference in tryptophan environment in these species is supported by acrylamide quenching studies, and phosphorescence results at 77 K which show a blue-shift of (0,0) band from 414.8 nm to 409.2 nm. The present results reveal subtlety of structural characteristics of unfolded states of Banlec in GdnHCl, which provide important insight in protein unfolding reaction.
... The ease with which a branched oligosaccharide could be modelled into the carbohydrate binding region of the lectin, as a direct consequence of the presence of two nearly equivalent primary binding sites, readily explains the unique specificity of banana lectin for branched mannans. The branched oligomannosides which have been shown to bind the lectin include the trisaccharide Man α1,6 (Manα1,3) Man and the pentasaccaride found in the core region of N-linked glycan chains ) as well as Man 8,9 GlcNAcAc 2 reported earlier (Koshte, et al., 1990). It is satisfying that all of them have branching involving 3 and 6 positions as in the model. ...
... BL was purified by the procedure of Koshte et. al. [21]. 250 g of banana pulp was soaked in 1 L distilled water containing 250 mM NaCl, 5 mM MgCl 2 and 5 mM CaCl 2. The mixture was subjected to homogenization followed by drop wise addition of 4 M NaOH to maintain pH upto 7.4. ...
Banana lectin (BL) is a homodimeric protein categorized among jacalin-related family of lectins. The effect of acidic pH was examined on conformational stability of BL by using circular dichroism, intrinsic fluorescence, 1-anilino-8-napthalene sulfonate (ANS) binding, size exclusion chromatography (SEC) and dynamic light scattering (DLS). During acid denaturation of BL, the monomerization of native dimeric protein was found at pH 2.0. The elution profile from SEC showed two different peaks (59.65 ml & 87.98 ml) at pH 2.0 while single peak (61.45 ml) at pH 7.4. The hydrodynamic radii (R h) of native BL was 2.9 nm while at pH 2.0 two species were found with R h of 1.7 and 3.7 nm. Furthermore at, pH 2.0 the secondary structures of BL remained unaltered while tertiary structure was significantly disrupted with the exposure of hydrophobic clusters confirming the existence of molten globule like state. The unfolding of BL with different subunit status was further evaluated by urea and temperature mediated denaturation to check their stability. As inferred from high Cm and ΔG values, the monomeric form of BL offers more resistance towards chemical denaturation than the native dimeric form. Besides, dimeric BL exhibited a Tm of 77°C while no loss in secondary structures was observed in monomers even up to 95°C. To the best of our knowledge, this is the first report on monomeric subunit of lectins showing more stability against denaturants than its native dimeric state.
... nivalus) contain reasonably high levels of the lectin (Van Damme et al., 1987). On the other hand, HIV inhibiting lectins such as those from prokaryotes and some plants are produced in low quantities and it is thus not feasible for direct isolation from the source (Koshte et al., 1990;. ...
Lectins are non-immune carbohydrate-binding proteins/glycoproteins that are found everywhere in nature, from bacteria to human cells. They have also been a valuable biological tool for the purification and subsequent characterisation of glycoproteins due to their carbohydrate binding recognition capacity. Antinociceptive, antiulcer, anti-inflammatory activities and immune modulatory properties have been discovered in several plant lectins, with these qualities varying depending on the lectin carbohydrate-binding site. The Coronavirus of 2019 (COVID-19) is a respiratory disease that has swept the globe, killing millions and infecting millions more. Despite the availability of COVID-19 vaccinations and the vaccination of a huge portion of the world's population, viral infection rates continue to rise, causing major concern. Part of the reason for the vaccine's ineffectiveness has been attributed to repeated mutations in the virus's epitope determinant elements. The surface of the Coronavirus envelope is heavily glycosylated, with approximately sixty N-linked oligomannose, composite, and hybrid glycans covering the core of Man3GlcNAc2Asn. Some O–linked glycans have also been discovered. Many of these glyco-chains have also been subjected to multiple mutations, with only a few remaining conserved. As a result, numerous plant lectins with specificity for these viral envelope sugars have been discovered to interact preferentially with them and are being investigated as a potential future tool to combat coronaviruses such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by preventing viral attachment to the host. The review will discuss the possible applications of plant lectins as anti-coronaviruses including SARS-CoV-2, antinociceptive, anti-inflammation and its immune modulating effect.
Lectins are defined as carbohydrate-binding proteins/glycoproteins of none immune origin, they are ubiquitous in nature, exist from bacteria to human cells. And due to their carbohydrate-binding recognition capacity, they have been a useful biological tool for the purification of glycoproteins and their subsequent characterization. Some plant lectins have also been revealed to own antinociceptive, antiulcer, and anti-inflammatory properties, where these features, in many instances, depending on the lectin carbohydrate-binding site. Coronavirus disease of 2019 (COVID-19) is a respiratory disease that struck the entire world leaving millions of people dead and more infected. Although COVID-19 vaccines have been made available, and quite a large number of world populations have already been immunized, the viral infection rates remained in acceleration, which continues to provoke major concern about the vaccines' efficacy. The belief in the ineffectiveness of the vaccine has been attributed in part to the recurrent mutations that occur in the epitope determinant fragments of the virus. Coronavirus envelope surface is extensively glycosylated being covered by more than sixty N-linked oligomannose, composite, and hybrid glycans with a core of Man3GlcNAc2Asn. In addition some O-linked glycans are also detected. Of these glyco-chains, many have also been exposed to several mutations, and a few remained conserved. Therefore, numerous plant lectins with a specificity directed towards these viral envelope sugars have been found to interact preferentially with them and are suggested to be scrutinized as a possible future tool to combat coronaviruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through blocking the viral attachment to the host cells. In this review, we will discuss the possible applications of plant lectins as anti-3 coronaviruses including SARS-CoV-2, antinociceptive, anti-inflammatory, and antiulcer agents with the proposed mechanism of their actions.
Lectins are defined as carbohydrate-binding proteins/glycoproteins of none immune origin, they are ubiquitous in nature, exist from bacteria to human cells. And due to their carbohydrate-binding recognition capacity, they have been a useful biological tool for the purification of glycoproteins and their subsequent characterization. Some plant lectins have also been revealed to own antinociceptive, antiulcer, and anti-inflammatory properties, where these features, in many instances, depending on the lectin carbohydrate-binding site. Coronavirus disease of 2019 (COVID-19) is a respiratory disease that struck the entire world leaving millions of people dead and more infected. Although COVID-19 vaccines have been made available, and quite a large number of world populations have already been immunized, the viral infection rates remained in acceleration, which continues to provoke major concern about the vaccines' efficacy. The belief in the ineffectiveness of the vaccine has been attributed in part to the recurrent mutations that occur in the epitope determinant fragments of the virus. Coronavirus envelope surface is extensively glycosylated being covered by more than sixty N-linked oligomannose, composite, and hybrid glycans with a core of Man3GlcNAc2Asn. In addition some O–linked glycans are also detected. Of these glyco-chains, many have also been exposed to several mutations, and a few remained conserved. Therefore, numerous plant lectins with a specificity directed towards these viral envelope sugars have been found to interact preferentially with them and are suggested to be scrutinized as a possible future tool to combat coronaviruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through blocking the viral attachment to the host cells. In this review, we will discuss the possible applications of plant lectins as anti-coronaviruses including SARS-CoV-2, antinociceptive, anti-inflammatory, and antiulcer agents with the proposed mechanism of their actions.
The aim of the study was to examine the anticancer activities of banana lectin (BanLec) from the ripen pulp of Musa paradisiaca. In this investigation, we have analyzed the cytotoxicity of BanLec against Vero, HepG2 and THP-1 cell lines. Among these, BanLec exhibited a potent cytotoxicity against THP-1cells, with an IC50 value of 244.38 µg/mL followed by HepG2 with an IC50 of 504.83 µg/mL and no cytotoxic activity towards Vero cells. Moreover, in this study we have evaluated the cell death mechanism induced by BanLec on THP-1cells. These cells were treated with 244.38 µg/mL concentration of BanLec to induce cell death at 24 h incubation. The apoptotic induction was carried out by Annexin V, DAPI staining, TUNEL assay and gene expression studies by qRT-PCR. BanLec induced apoptosis with morphological changes such as cell membrane blebbing, cell shrinkage, chromatin condensation, cell budding and nuclei broken into fragments. The caspase-3 gene expression was upregulated indicating the induction of apoptosis on treatment with BanLec. Using flow cytometry technique, the number of apoptotic cells in G0/G1 phase was evaluated. There was an accumulation and increase in the percentage of cells in G0/G1 phase stating the apoptosis induction against THP-1 cells. On conclusion BanLec showed an apoptosis induction in THP-1 cells in a caspase dependent manner. These collectively results evidently showed that the BanLec exerts cytotoxicity and apoptosis against THP-1 cells. It seems to be one of the potent anticancer compound having a possible therapeutic potential against the human leukemia derived diseases.
In the present study aimed to purify the lectin from the sap of Musa acuminata pseudostem and elucidate the apoptotic and angiogenic molecular mechanism in both in-vitro and in-vivo model. Mannose specific lectin was purified by using mannose affinity column chromatography and analyzed by RP-HPLC, SDS-PAGE, and PAS staining method. Furthermore, the protein was identified by MALDI-MS/MS. MAL effectively agglutinates trypsinized RBCs and showed effective cytotoxicity against various human cancer cell lines. MAL mitigates the cell proliferation, colony formation, cell migration, arrest the cell cycle in the G2/M phase, and induce apoptosis by altering the expression of apoptotic proteins/mRNA level (Bax and Bcl-2) via caspase 8/9, 3 dependent pathway in both in-vitro and in-vivo. Supporting this, in-vivo EAC tumor mice models prove the efficacy of MAL by inducing cell death and inhibiting the neovessel formation by targeting the MVD, inhibition of VEGF secretion, suppressing the expression of MMPs, HIF-1α, Flt-1, Akt, Jnk, and Erk1/2. More importantly, the MAL treatment leads to effective inhibition of tumor growth and an increase in the survivability of EAC mice. Our study summarizes that the MAL having a significant anticancer potential expressively degenerates the tumor development by inducing apoptosis and suppressing neoangiogenesis.
Bovine viral diarrhea virus (BVDV) and bovine alphaherpesvirus type 1 (BoHV-1) are responsible for major economic losses of livestock worldwide, making their eradication an important objective of veterinary research. Vaccines against these infectious agents are commercially available but have some limitations due to the specific features of these viral agents. The development of new antiviral drugs is therefore essential. Native banana lectin (BanLec) is a lectin isolated from banana fruit (Musa acuminata) and has a high affinity for mannose glycans found in several viral envelopes. The inhibitory properties of this lectin against several viruses has already been demonstrated. The aim of this work was therefore to test the antiviral and virucidal activities of BanLec against BVDV-1 and BoHV-1. Its antiviral activity was assessed by measuring the viral titer and viability of susceptible Madin-Darby Bovine Kidney cells (MDBK) treated with BanLec before and after viral infection. The virucidal properties of BanLec were determined by preincubation of the lectin with the viruses, followed by measurement of the viral load in exposed cells. Treatment with 25 μg/mL BanLec resulted in high levels of inhibition against BVDV-1 (99.98%) and BoHV-1 (99.68%) without affecting cell viability, demonstrating promising potential as an antiviral agent.
Lectins are proteins or glycoproteins of non-immune origin which have at least one non-catalytic domain that bind reversibly to specific mono or oligosaccharides. Traditional Chinese Medicine (TCM) involves a broad range of medicinal practices sharing common concepts which have been developed in China and are based on a tradition of more than thousands of years. Plant materials which are commonly used in TCM as a complementary or alternative for Western medical treatments contain a considerable number of important lectins. These lectins have been reported to have various applications and uses such as cancer treatment, glycoconjugate research, biomarker development, and others. Here, we summarize the available literature related to lectins from TCM and recent trends in their potential biomedical applications.
Banana is one of the oldest cultivated plant known for its dietary and medicinal properties. Banana is grown in the tropical and subtropical regions of the world and constitutes the staple food of the people. They are classified as dessert or sweet bananas and cooking bananas or plantains depending on whether they can be eaten raw or not. Banana plant parts such as the roots, pseudostem, fruits, and inflorescence is used in some or the other way and therefore it is rightly called as the “Kalpatharu” in India. Banana and plantains contain important bioactive compounds such as phenolics, flavonoids, carotenoids, biogenic amines, sterols, and antimicrobial compounds which make bananas a perfect functional food for health improvement. Presently, research is focused on exploring and identifying compounds, refining the techniques of isolation and purification, and using it in modern medicines. Moreover, bananas are also being used as a platform to produce and accumulate important nutrients like vitamins and minerals by biofortification strategies.
Glycan-lectin interactions are commonly observed in nature. Analytical methods, which are used to detect lectins that rely on the use of glycan ligand-modified nanoprobes as affinity probes, have been developed. Most of the existing methods are focused on the use of synthetic glycan ligands. Nevertheless, naturally available glycoproteins, such as ovalbumin in chicken egg whites, are good sources for fabricating glycan-immobilized nanoprobes. In this study, we generated functionalized gold nanoparticles (Au NPs) from a one-pot reaction by reacting chicken egg white (cew) proteins with aqueous tetrachloroaurate. The generated Au@cew NPs are mainly encapsulated by ovalbumin, in which the surface is decorated by abundant hybrid mannose and Galβ(1→4)GlcNAc-terminated glycan ligands. Thus, the generated Au@cew NPs containing hybrid mannose and Galβ(1→4)GlcNAc have the capability to selectively bind with their corresponding lectins. Lectins including concanavalin A, banana lectin, and ricin B that have binding moieties toward specific sugars were used as the model samples. Our results showed that the generated AuNPs can be used as multiplex affinity probes for these model lectins. Lectins can be selectively released from the Au@cew NP-lectin conjugates by using specific sugars, such as mannose, glucose, and β-lactose, as the releasing agents to release specific lectins from the conjugates. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used as the tool to characterize the released species from the nanoprobes. The limit of detection of these model lectins using the current approach was low (in nM). The feasibility of using the Au@cew NP-based affinity MALDI-MS to selectively detect specific lectins from complex samples was also demonstrated.
The banana is a herbaceous perennial of the MUSACEAE family that grows to 5–9 m in height. It has a tuberous subterranean rhizome, from which the leaves emerge. The lower part of the leaves is folded within each other producing a ‘false stem’ from which the long, narrow blades protrude and spread out. In the center of the folded leaf-sheats, a growing point forms from the top of the rhizome, grows up and emerges as an overhanging inflorescence with a succession of reddish brown bracts. The bracts unfold from the base to the tip and fall off. Within the lower 1–12 bracts arise 14–18 female flowers in double rows. These develop into fruits without having to be fertilized, a process known as parthenocarpy. The next few bracts contained bisexual flowers that are rich in nectar but do not develop any further. In the upper bracts only male flowers are formed.
Bunch of Williams Cavendish, Kluai Hom Kiau bananas on sale in a Thai market, Hand of pisang masak hijau, Ripe ‘fingers’ of Williams banana, Musa acuminata Colla (AAA Group) ‘Dwarf Cavendish’.
In this article enzymes and other biologically active proteins of the banana are reviewed. The bulk of the literature is on the banana fruit. Polyphenol oxidase isolated from roots may be associated with resistance against the parasitic nematode Radolpholus similis. Lipoxygenase purified from leaves may be involved in leaf aroma production. Key enzymes of carbohydrate metabolism including ATP-dependent and pyrophosphate-dependent phosphofructokinase have been isolated from banana fruits. Acid phosphatase, pectate lyase and β-1, 3-glucanase from fruits are associated with fruit ripening. Starch phosphorlases, polygalacturonase and pectin methylesterase have been reported from fruits. The allergen present in bananas have been indentified to be a class I chitinase. Banana fruit lectin displays unique carbohydrate binding properties. It stimulates nitric oxide production by macrophages, [methyl-3H] thymidine uptake and cytokine production by splenocytes, inhibits tumor cell proliferation, and reduces the activity of HIV-1 reverse transcriptase. Thaumatin-like protein from banana fruits exhibits antifungal and HIV-1 reverse transcriptase inhibitory activities but is devoid of mitogenic activity toward splenocytes and inhibitory activity toward tumor cells. Banana fruit chitinases demonstrate antifungal activity. Other than the proteins mentioned above, fructooligosaccharides and small molecules with biological activity have also been reported.
Recombinant banana lectin isoform (rBanLec) attaches specifically to the mucosal surface, crosses the epithelial barrier and then directly affects the immune response in mouse colon. Structural characteristics, specificity and physiological impacts of rBanLec reported until now highly resemble those of its natural counterpart. Here, we demonstrated that a dose-dependent stimulation of the colon with rBanLec skewed the immune response towards Th1/Th17 direction and this effect was counterbalanced by the rise in IL-10 production. Qualitative and quantitative characteristics of the established cytokine network were dependent on the applied rBanLec concentration. In addition, rBanLec enhanced local NO production and myeloperoxidase activity and promoted an increase in local IgA and IgG production. Stimulation with rBanLec can be beneficial in prevention of pathologies raised due to inappropriate cell-mediated immune response as well as in prevention of the pathogen invasion via the colon.
Abstract OrysaEULD1A is one of the five EUL genes in rice (Oryza sativa) encoding a putative carbohydrate-binding protein belonging to the family of Euonymus related lectins (EUL). The OrysaEULD1A sequence comprises two highly similar EUL domains (91 % sequence similarity and 72 % sequence identity) separated by a 23 amino acid linker sequence and preceded by a 19 amino acid N-terminal sequence. In the present study, the full-length protein OrysaEULD1A as well as its individual domains OrysaEULD1A domain 1 and 2 were expressed in Pichia pastoris. After purification of the recombinant proteins, their carbohydrate-binding specificity was analyzed and compared. Interestingly, all recombinant lectins showed clear specificity towards galactosylated structures. Furthermore, all recombinant proteins agglutinated red blood cells indicating that the full-length protein OrysaEULD1A and its domains are true lectins. These results taken together with data previously reported for single domain EUL proteins indicate that although the amino acids, responsible for the formation of the carbohydrate-binding site, are identical for all EUL proteins in rice, these lectins show different carbohydrate specificities. This promiscuity of the carbohydrate-binding site can be attributed to gene divergence.
To explore the potential usage of recombinant banana lectin as an immunostimulator it was of interest to analyze the effect it exerted in vivo in mice, upon two different routes of exposure. By Western blotting analysis of the gastric and intestinal luminal content it was found that recombinant banana lectin is stable in vivo in the mouse digestive tract, where it specifically interacts with the mucosal surfaces which can be inhibited by the addition of glucose. Its attachment to mice jejunum has been confirmed by immunofluorescence. In addition, it was able to induce systemic immunity, evidenced by specific antibody production, by oral application, a property that might be exploited for the induction of systemic immune responses.
Banana lectin (BanLec) was isolated from slightly overripe bananas (PCI 6-7) by homogenation in NaCl solution, followed by extraction in the presence of glucose, ammonium sulfate precipitation, and affinity chromatography. Yields were approximately 10-fold greater that those of previously published methods using acidic extraction from very overripe fruit (Peel Color Index [PCI] 7+). By dilution of added isotopically labeled recombinant lectin, the content of total exchangeable BalLec was shown to be constant or to slightly decrease with increasing stage of ripeness, even though extractable BanLec increased, followed by rapid decrease in overripened fruit. In the course of this study we observed that recombinant BanLec expressed in Escherichia coli, although chemically and functionally identical to native BanLec, differed slightly in its apparent molecular size on gel filtration, probably due to differences in its native folding.
The plant kingdom is an underexplored source of valuable proteins which, like plant lectins, display unique interacting specificities. Furthermore, plant protein diversity remains under-exploited due to the low availability and heterogeneity of native sources. All these hurdles could be overcome with recombinant production. A narrow phylogenetic gap between the native source and the recombinant platform is likely to facilitate proper protein processing and stability; therefore, the plant cell chassis should be specially suited for the recombinant production of many plant native proteins. This is illustrated herein with the recombinant production of two representatives of the plant jacalin-related lectin (JRLs) protein family in Nicotiana benthamiana using state-of-the-art magnICON technology. Mannose-specific Banlec JRL was produced at very high levels in leaves, reaching 1.0mg of purified protein per gram of fresh weight and showing strong agglutination activity. Galactose-specific jacalin JRL, with its complicated processing requirements, was also successfully produced in N. benthamiana at levels of 0.25mg of purified protein per gram of fresh weight. Recombinant Jacalin (rJacalin) proved efficient in the purification of human IgA1, and was able to discriminate between plant-made and native IgA1 due to their differential glycosylation status. Together, these results show that the plant cell factory should be considered a primary option in the recombinant production of valuable plant proteins.
Hydrolytically stable non-glycosidically linked tail-to-tail pseudodisaccharides are linked by a single bridging atom remote from the anomeric centre of the constituent monosaccharides. Some such pseudodisaccharides with sulfur or oxygen bridges were found to act as disaccharide mimetics in their binding to the Banana Lectin and to Concanavalin A. A versatile synthetic route to a small library of such compounds is described.
A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.
Two mAb were used to develop new techniques for the purification and quantitation of the major feline salivary allergen, Felis domesticus allergen I (Fel d I). The allergen was purified from aqueous house dust extract with a high Fel d I content by affinity chromatography over a monoclonal immunosorbent and elution with 4 mM HCl, pH 2.5. This single step procedure gave 40 to 50% recovery of 90% pure allergen which, following final purification by size exclusion HPLC, showed a single line on immunodiffusion and crossed immunoelectrophoresis against monospecific anti-Fel d I and polyclonal anti-cat dander antibodies. The m.w. of native Fel d I was 39,000 on size exclusion HPLC, and 17,000 under nonreducing conditions on gel electrophoresis. The N-terminal amino acid sequence (33 residues) showed no homology with other known protein sequences. The combination of the SDS-PAGE and N-terminal sequence data suggests that Fel d I is a non-covalently linked homodimer. A two-site RIA was developed using mAb directed against different epitopes on Fel d I. This assay was species-specific, highly sensitive (0.0004 U/ml), and showed an excellent correlation with a polyclonal inhibition RIA (n = 27, r = 0.93, p less than 0.001). Cat allergen extracts used for immediate skin tests showed marked differences in Fel d I content (from 0.1 to 30 U/ml). Consistently high Fel d I levels were found at monthly intervals in six dust samples from four houses with cats (10 to 100 U/g of dust). Comparisons of Fel d I and mite and pollen allergen levels showed that house dust can contain greater than 100 micrograms/g of either of these allergens and is a potent source of foreign environmental antigens. Monoclonal affinity chromatography provides a major breakthrough in the purification of Fel d I, from a source material that would otherwise have been considered impossible (house dust). The mAb assay for Fel d I is both more sensitive and more easily standardized than existing techniques. These techniques will allow full structural and antigenic analysis of Fel d I and more detailed studies on the relationship between cat antigen exposure and the development of asthma.
A purified allergen was obtained from D. pteronyssinus culture by gel filtration, block electrophoresis, and preparative isoelectricfocusing (IEF). The antigenic activity on IEF was predominantly distributed in two peaks (pI 4.7 to 5.4 and pI 6.6 to 7.1), which were completely cross-reactive on immunodiffusion and by cross-inhibition of radioimmunoassay. These fractions had the same m.w. (24,000) and a very similar amino acid composition. The common antigen in each fraction was designated Dermatophagoides pteronyssinus antigen P1. In seven, symptomatic mite allergic subjects skin test reactivity to antigen P1 and crude mite extract was closely correlated. There was also a very good correlation between IgE-binding activity (BA) for P1 measured by antigen-binding radioimmunoassay and IgE antibody (ab) to D. pteronyssinus measured by using the radioallergosorbent test (RAST) (r = 0.82, p < 0.001). RAST experiments showed that up to three-quarters of the IgE ab to D. pteronyssinus were directed against P1, and that IgE ab to P1 accounted for on average 12% of the total serum IgE. These data strongly suggest that P1 is the major allergen of D. pteronyssius. By immunodiffusion, SDS-PAGE, and radioimmunoassay antigen P1 appeared to be present as a large proportion (10 to 20%) of the protein in crude mite extract. Our results support the view that the importance of an inhalant allergen is largely determined by its physical properties, and by the quantitites inhaled during natural exposure.
It is shown that apple allergens are probably proteins and that they can be extracted in an active form only if reactions with phenolic compounds present in apple are inhibited. This is accomplished by incorporating chelators and solid polyvinylpolypyrrolidone in the extraction medium. Phenol, commonly used as a preservative, should not be added. With the RAST, serum-IgE antibodies capable of reacting with apple allergens were detected in 90% of patients with clinical apple allergy, in 44% of patients with clinical birch-pollen allergy and in 5—10% of patients with other atopic allergies. RAST inhibition indicated that apple and birch-pollen allergens cross-react.
The complete amino acid sequence of phospholipase A2 from the venom of the common European honey-bee (Apis mellifica) has been determined. The sequence of amino acid residues at the N-terminus was obtained by direct application of the Edman degradation technique and that at the C-terminus by digestion with carboxypeptidases A and B. Digestion of the reduced and carboxymethylated enzyme with trypsin yielded a completely soluble peptide mixture, all the components of which were isolated and their structures determined. Overlaps of the tryptic peptides were deduced from the structures of peptides isolated after digestion of the reduced and carboxymethylated enzyme with chymotrypsin and of the reduced and aminoethylated enzyme with a protease specific for cleavage at lysine residues. Two remaining ambiguities were resolved by peptides obtained from a digest of the reduced, carboxymethylated and maleylated enzyme with trypsin. The phospholipase A2 consists of a single chain of 128 amino acid residues and contains attached carbohydrate residues. The composition and point of attachment of the carbohydrate moiety have been established.
The hemagglutinating properties of a large proportion of anti-M and anti-N reagents, and sera containing antibodies to MN-related antigens, have been shown to be unaffected by treatment of red blood cells with neuraminidase. These antibodies, which define NANA-independent MN-system structures, provide further evidence that MN blood group specificity may also be determined by moieties other than N-acetylneuraminic acid.
Honeybee venom was separated into seven fractions by gel filtration on Sephadex G-75. Allergenic activities of these fractions were assessed by the paper disc radioallergosorbent test (RAST) with a panel of sera from 24 individuals who had systemic reactions to bee stings, 7 who had large local reactions, and 10 control subjects who had reactions of 5 cm or less following bee stings. Three fractions were identified by enzyme or direct hemolytic activity. Twenty-nine of 31 sera from patients having either systemic or large local reactions to bee stings were positive when radioallergosorbent tested with whole bee venom; 22 were positive to phospholipase A, and 28 were positive to both fractions 1 and 2. Thirteen sera combined most strongly with fraction 1, 12 sera most strongly with fraction 2, hyaluronidase, and three sera about equally with fractions 1, 2, and 3. Reactions with other fractions were much weaker. Fractions 1 and 2 were potent inhibitors of RAST with whole venom in the sera reacting most strongly with fractions 1 or 2, respectively. Fraction 3, phospholipase A, and commercial bee venom phospholipase A were significantly less potent inhibitors with the sera tested. In the cases in which IgE antibody bindings to fractions 1, 2, and 3 were of similar magnitude, inhibitions of RAST using the various fractions both on the discs and as inhibitors demonstrated substantial cross-reactivity between the fractions. These results strongly indicate that by using RAST with human sera from bee sting-sensitive individuals, fraction 1, the high molecular materials, and fraction 2, hyaluronidase, are the major allergens in honeybee venom. Phospholipase A appears to be of secondary importance.
Three fractions of rye-grass (Lolium perenne) pollen extract have been isolated by preparative isoelectric focusing (i.e.f.) and characterized in terms of physicochemical and immunochemical properties. The purified components were designated 'R7' and 'R14' on the basis of their positions in relation to other rye-grass pollen extract components on SDS/polyacrylamide-gel electrophoresis and their apparent molecular masses were assessed as 31 and 11 kDa respectively. On i.e.f., R14 split into two components, one acidic (pI 5.0) and one basic (pI 9.0), termed 'R14a' and 'R14b' respectively, and R7 focused at pI 5.8. R7 and R14a were shown to be allergenic by skin-prick test and all three components were recognized by rye-grass-pollen-specific human IgE. On SDS/polyacrylamide-gel electrophoresis and i.e.f., R7 behaved in a manner identical with that shown by an authentic sample of Rye I and gave an amino acid analysis similar to published data [Johnson & Marsh (1966) Immunochemistry 3, 91-100] for Rye group-I isoallergens; the amino acid sequence of the first 27 N-terminal amino acids was also determined. Physicochemical analysis revealed that R14a was equivalent to Rye II and 14b to Rye III. Preparative i.e.f. followed by gel-permeation chromatography proved to be a rapid and efficient method for purifying the allergenic components of Rye I (R7), Rye II (R14a) and Rye III (R14b) from rye-grass pollen extract.
Electroblotting of crude ryegrass pollen extracts and purified group I, II and III allergens identified 14 IgE-binding components, 8 of which were previously unrecognized. In addition to allergen groups I, II and III, which are already regarded as clinically important, and on the basis of the frequencies and intensities of IgE binding with sera from 42 ryegrass pollen-allergic patients, proteins with molecular weights (MWs) of 60, 32, 30 and 28 kD were identified as allergens of possible major clinical importance. Six other pollen components with MWs ranging from 23 to 80 kD and which reacted with IgE antibodies in the sera of 33-50% of patients, should also be viewed as proteins with potential clinical relevance for at least a proportion of the patients. The electrophoretic separation patterns of ryegrass pollen extracts in both alkaline and acid gels and IgE-probed membrane transfers produced in this study should serve as useful reference patterns for standardization purposes. In addition, the identification of the complete allergen recognition pattern by individual patients will permit safer and more effective diagnosis and therapy of ryegrass pollen sensitivities.
We have investigated the requirements for lectin-induced proliferation of highly purified human T cells. To study activation, independent of growth factor production, we cultured the cells in the presence of an excess of interleukin 2 (IL2), which was a product of cDNA cloned in E. coli. In the presence of IL2, the same cooperative effect of lectin and accessory cells was found that we have previously described for IL2 production. Thus, analogous to induction of IL2 production, the acquisition of responsiveness to IL2 can be completely monocyte dependent, but a 10-fold increase in lectin concentration completely abolishes the requirement for accessory cells. Furthermore, two stimuli (IL1 and phorbol myristate acetate), which are able to replace monocytes at the level of IL2 production, also induce responsiveness to IL2 under accessory cell-dependent conditions. Thus, very similar conditions are required for proliferation and for the induction of IL2 production. There is only a quantitative difference: proliferation of cells in the presence of exogenous IL2 occurs already at low lectin concentrations, whereas IL2 production and consequently proliferation in the absence of exogenous IL2 requires higher lectin concentrations.
At high lectin concentrations, when IL2 production has become the only limiting factor, purified T cells cannot be induced to proliferate in the absence of exogenous IL2 because the lectin concentration that induces IL2 production independent of accessory cells inhibits mitogenesis. However, after addition of thiols to the medium, which enhances the IL2 production, a very narrow range of lectin concentration can be found which is just below toxic values and still high enough to induce IL 2 production in the absence of accessory cells. Under these conditions, accessory cells are no longer a prerequisite for lectin-induced T cell proliferation.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
The fusion glycoprotein (F0) was isolated from Newcastle disease virus (NDV) particles metabolically labelled with [2-3H]mannose; it was successively digested with protease and with endo-beta-N-acetylglucosaminidase from Streptomyces griseus. In this manner, the majority of the oligosaccharides in NDV F0 could be liberated. After reduction with NaBH4, they were separated by high-performance liquid chromatography, and were subjected to structural analysis. Using micromethylation/capillary gas chromatography/mass fragmentography, alpha-mannosidase digestion, and acetolysis, it was found that the enzymatically released NDV F0 oligosaccharides are common oligomannosidic glycoprotein glycans of size classes (Man)8GlcNAc, Man)7GlcNAc, (Man)6GlcNAc, (Man)9GlcNAc, and (Man)5GlcNAc (in order of prevalence). The major structural isomers present in the NDV F0 (Man)8GlcNAc to (Man)5GlcNAc fractions were shown to lack mannose residues D2, D1D2 or D2D3, D1D2D3, and CD1D2D3, respectively, of (Man)9GlcNAc.
IgE in some human sera reacted with an antigen present in a large number of unrelated foods: potato, spinach, wheat, buckwheat, peanut, honey and others. The antigen, which was periodate-sensitive and heat-stable, was also found in pollen. Even more surprisingly, these antibodies often reacted in vitro with bee and vespid venom and were sometimes apparently induced by Hymenoptera stings.
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