Antimicrobial susceptibility of urinary bacterial isolates of pregnant women attending antenatal clinics of selected hospitals in Ilorin, Nigeria

Background: Fragmented service provision and a lack of efficient cooperation between health and welfare sectors serving children and families remain ongoing challenges in South Africa. The coronavirus disease 2019 (COVID-19) pandemic escalated this fragmentation. A community of practice (CoP) was established by the Centre for Social Development in Africa to promote collaboration between the sectors and to assist communities in their environments. Aim: To explore and describe collaboration on child health promotion between professional nurses and social workers, who formed part of the CoP during the COVID-19 pandemic. Setting: The study was conducted in five public schools from four of the seven district regions of the City of Johannesburg, Gauteng province. Methods: A qualitative, exploratory, descriptive research design was employed to conduct psychosocial and health screenings of children and their families. Focus group interviews were conducted, and field notes were used to collect and confirm data from the team. Results: Four themes emerged. Participants shared their positive and negative experiences faced during the fieldwork, their realisation of the value of collaboration between various sectors and their desire and capacity to do more. Conclusion: Participants indicated that collaboration between the health and welfare sectors is vital to support and promote the health of children and their families. The COVID-19 pandemic highlighted the need for collaboration between these sectors in the children and their families' ongoing struggles. Contribution: The importance of these sectors being engaged as a team highlighted the multisectoral influence shaping child development outcomes, supporting children's human rights and advancing social and economic justice.

Non-telecentric two-photon microscopy for 3D random access mesoscale imaging.

Diffraction-limited two-photon microscopy permits minimally invasive optical monitoring of neuronal activity. However, most conventional two-photon microscopes impose significant constraints on the size of the imaging field-of-view and the specific shape of the effective excitation volume, thus limiting the scope of biological questions that can be addressed and the information obtainable. Here, employing a non-telecentric optical design, we present a low-cost, easily implemented and flexible solution to address these limitations, offering a several-fold expanded three-dimensional field of view. Moreover, rapid laser-focus control via an electrically tunable lens allows near-simultaneous imaging of remote regions separated in three dimensions and permits the bending of imaging planes to follow natural curvatures in biological structures. Crucially, our core design is readily implemented (and reversed) within a matter of hours, making it highly suitable as a base platform for further development. We demonstrate the application of our system for imaging neuronal activity in a variety of examples in zebrafish, mice and fruit flies.

Rhizoctonia bataticola lectin induces apoptosis and inhibits metastasis in ovarian cancer cells by interacting with CA 125 antigen differentially expressed on ovarian cells.

A N-glycan specific lectin from Rhizoctonia bataticola [RBL] was shown to induce growth inhibitory and apoptotic effect in human ovarian, colon and leukemic cells but mitogenic effect on normal PBMCs as reported earlier, revealing its clinical potential. RBL has unique specificity for high mannose tri and tetra antennary N-glycans, expressed in ovarian cancer and also recognizes glycans which are part of CA 125 antigen, a well known ovarian cancer marker. Hence, in the present study diagnostic and therapeutic potential of RBL was investigated using human ovarian epithelial cancer SKOV3 and OVCAR3 cells known for differentially expressing CA 125. RBL binds differentially to human ovarian normal, cyst and cancer tissues. Flow cytometry, western blot analysis of membrane proteins showed the competitive binding of RBL and CA 125 antibody for the same binding sites on SKOV3 and OVCAR3 cells. RBL has strong binding to both SKOV3 and OVCAR3 cells with MFI of 173 and 155 respectively. RBL shows dose and time dependent growth inhibitory effect with IC50 of 2.5 and 8 µg/mL respectively for SKOV3 and OVCAR3 cells. RBL induces reproductive cell death, morphological changes, nuclear degradation and increased release of ROS in SKOV3 and OVCAR3 cells leading to cell death. This is also supported by increase in hypodiploid population, altered MMP leading to apoptosis possibly involving intrinsic pathway. Adhesion, wound healing, invasion and migration assays demonstrated anti-metastasis effect of RBL apart from its growth inhibitory effect. These results show the promising potential of RBL both as a diagnostic and therapeutic agent.

A core fucose specific lectin from Cephalosporium curvulum induces cellular apoptosis in hepatocellular and pancreatic cancer cells and effective in detecting AFP.

Cephalosporium curvulum lectin (CSL), a lectin from pathogenic fungus has exquisite specificity towards α1-6 linkage of core fucosylated glycans, expressed in hepatocellular and pancreatic cancer. Interaction and effect of CSL and other fucose specific lectins LCA and AOL on HepG2 and PANC-1 cells was investigated. CSL, LCA and AOL exhibited strong binding to PANC-1 cells which could be effectively blocked by competing glycoprotein mucin. Effect of CSL, LCA and AOL on PANC-1 and HepG2 cells was determined by MTT assay and all the three lectins inhibited the cell growth which could be blocked by mucin, cell cycle analysis revealed that CSL increased hypodiploid HepG2 cell population indicating cellular apoptosis. CSL induced apoptosis in HepG2 cells was confirmed by Annexin V/PI assay. CSL induced increase in early apoptotic HepG2 cell population, a time dependent increase in the expression of caspases-3, 9 and cytochrome-c was observed by western blotting suggesting the possible involvement of intrinsic caspase dependent apoptosis. Increase in ROS and decrease in MMP demonstrated involvement of intrinsic caspase dependent apoptosis. Quantification of AFP in HCC patients using CSL lectin-antibody sandwich ELISA, supports diagnostic potential of CSL.

Sclerotium rolfsii lectin induces opposite effects on normal PBMCs and leukemic Molt-4 cells by recognising TF antigen and its variants as receptors.

Sclerotium rolfsii lectin (SRL) exerts apoptotic effect against various cancer cells and an antitumor activity on mice with colon and breast cancer xenografts. The current study aimed to explore its exquisite carbohydrate specificity on human peripheral blood mononuclear cells (PBMCs) and leukemic T-cells. SRL, showed strong binding (>98%) to resting/activated PBMCs, leukemic Molt-4 and Jurkat cell lines. The glycans mediated binding to these cells was effectively blocked by mucin and fetuin, exhibiting 97% and 94% inhibition respectively. SRL showed mitogenic stimulation of PBMCs at 10 µg/ml as determined by thymidine incorporation assay. In contrast, lectin induced a dose dependent growth inhibition of Molt-4 cells with 58% inhibition at 25 µg/ml. Many common membrane receptors in activated PBMCs, Molt 4 and Jurkat cells were identified by lectin blotting. However, membrane receptors that are recognized by SRL in normal resting PBMCs were totally different and are high molecular weight glycoproteins. Treatment of membrane receptors with glycosidases prior to lectin probing, revealed that fucosylated Thomsen-Friedenreich(TF) antigen glycans are increasingly expressed on transformed Molt-4 leukemic cells compared to other cells. The findings highlight the opposite effects of SRL on transformed and normal hematopoietic cells by recognizing different glycan-receptors. SRL has promising potential for diagnostics and therapeutic applications in leukaemia.

No evidence of ongoing HIV replication or compartmentalization in tissues during combination antiretroviral therapy: Implications for HIV eradication.

HIV persistence during combination antiretroviral therapy (cART) is the principal obstacle to cure. Mechanisms responsible for persistence remain uncertain; infections may be maintained by persistence and clonal expansion of infected cells or by ongoing replication in anatomic locations with poor antiretroviral penetration. These mechanisms require different strategies for eradication, and determining their contributions to HIV persistence is essential. We used phylogenetic approaches to investigate, at the DNA level, HIV populations in blood, lymphoid, and other infected tissues obtained at colonoscopy or autopsy in individuals who were on cART for 8 to 16 years. We found no evidence of ongoing replication or compartmentalization of HIV; we did detect clonal expansion of infected cells that were present before cART. Long-term persistence, and not ongoing replication, is primarily responsible for maintaining HIV. HIV-infected cells present when cART is initiated represent the only identifiable source of persistence and is the appropriate focus for eradication.

An L-fucose specific lectin from Aspergillus niger isolated from mycotic keratitis patient and its interaction with human pancreatic adenocarcinoma PANC-1 cells.

An L-fucose specific lectin from pathogenic fungus Aspergillus niger isolated from the corneal smears of keratitis patient was purified in a single step using mucin coupled sepharose-4B column by 58-fold. The purified lectin, ANL has molecular mass of 30 kDa by SDS-PAGE and 31.6 kDa by ESI-MS. ANL is a glycoprotein with 2.59% carbohydrate. ANL is blood group nonspecific and also agglutinates rabbit erythrocytes. ANL is heat stable up to 50 °C and over a pH range of 7-10. Hapten inhibition studies revealed that ANL is specific to L-fucose, galactose, lactose and glycoproteins, showing highest MIC of 3.125 µg for L-fucose, mucin and fetuin. ANL has potent antibacterial activity against Klebsiella pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli and also it inhibits the biofilm formation by them. ANL showed strong binding to human pancreatic adenocarcinoma PANC-1 cells which was effectively blocked by L-fucose and mucin respectively by 76.2% and 84.2%. ANL showed dose and time dependent growth inhibitory effect on PANC-1 cells with IC50 of 1.25 µg/ml at 48 h. Effect of ANL was compared with another fucose specific lectin AOL, from Aspergillus oryzae showing an IC50 of 1.85 µg/ml at 48 h revealing promising clinical potential of ANL.

Investigation of TF-binding lectins from dietary sources and SRL on proliferation and cell cycle progression in human colon HT29 and SW620 cells.

TF antigen binding lectins from dietary sources PNA, ACA, ABL, JAC, and SRL from Sclerotium rolfsii have been reported to induce diverse effects on cancer cell proliferation by different mechanisms. This study aimed to compare effects of these lectins on growth and cell cycle progression in colon cancer HT29 and SW620 cells. As reported SRL, ABL, and JAC inhibited while PNA and ACA increased cell proliferation. ABL and JAC treated HT29 cells showed increased cell population in G0/G1 phase. PNA, ACA, ABL, and JAC increased SW620 cell population in S and decreased in G2/M phase. In contrast, SRL and JAC increased hypodiploid population in both the cells. PNA and ACA reduced whereas SRL and ABL diminished cell cyclin D1 expression. SRL, PNA, and ACA also reduced cellular cyclin D3 level while SRL, ABL, and JAC reduced cyclin E levels. ABL decreased CDK5 levels while SRL and ACA completely abolished CDK5 expression. All the lectins completely abolished cyclin D2 expression. These results not only confirms growth regulatory effects of TF-binding lectins but also indicates different effects of these lectins on cell growth is associated with regulation on expression of cell cycle associated proteins in G1-S phase and on cell cycle progression.

Purification, characterization and fine sugar specificity of a N-Acetylgalactosamine specific lectin from Adenia hondala.

Plant lectins are gaining interest because of their interesting biological properties. Several Adenia species, that are being used in traditional medicine to treat many health ailments have shown presence of lectins or carbohydrate binding proteins. Here, we report the purification, characterization and biological significance of N-Acetyl galactosamine specific lectin from Adenia hondala (AHL) from Passifloraceae family. AHL was purified in a single step by affinity chromatography on asialofetuin Sepharose 4B column, characterized and its fine sugar specificity determined by glycan array analysis. AHL is human blood group non specific and also agglutinates rabbit erythrocytes. AHL is a glycoprotein with 12.5% of the carbohydrate, SDS-PAGE, MALDI-TOF-MS and ESI-MS analysis showed that AHL is a monomer of 31.6 kDa. AHL is devoid of DNase activity unlike other Ribosome inactivating proteins (RIPs). Glycan array analysis of AHL revealed its highest affinity for terminal lactosamine or polylactosamine of N- glycans, known to be over expressed in hepatocellular carcinoma and colon cancer. AHL showed strong binding to human hepatocellular carcinoma HepG2 cells with MFI of 59.1 expressing these glycans which was effectively blocked by 93.1% by asialofetuin. AHL showed dose and time dependent growth inhibitory effects on HepG2 cells with IC50 of 4.8 µg/ml. AHL can be explored for its clinical potential.

Potent insect gut binding lectin from Sclerotium rolfsii impart resistance to sucking and chewing type insects in cotton.

Cotton is an important crop that is continuously cultivated around the world. However, its production has decreased in recent times due to wide ranging insects and also current practices of using synthetic insecticides that are not precise and their residues impairing the biodiversity. Hence, the search for newer classes of efficient entomotoxic proteins continues. Genetically modified cotton crops with cry genes from Bacillus thuringiensis, have been cultivated across the world, which overcome the chewing type insect menace. In the present study, we assess the development of transgenic cotton plants by Agrobacterium, wherein the confirmed kanamycin resistant T0 plants were advanced to T1 generation and the gene integration was studied by molecular analysis. Western blot and ELISA assays demonstrated the expression of 0.46% lectin of the total soluble leaf proteins. In planta bioassay showed 69% of aphid, Aphis gossypii population reduction with T1 generation plants. Whereas 100% insect mortality is occurred in Spodoptera litura larvae by 96 h. Present findings shows the potent insecticidal effect of Sclerotium rolfsii lectin on sucking (homopteran) and chewing (lepidopteron) insects, underlining its significance and strengthening genetic resources in cotton breeding against different order insect pests.

Efficacy studies of Sclerotium rolfsii lectin on breast cancer using NOD SCID mouse model.

Expression of altered glycans such as TF, Tn, and sTn antigens has been observed in a number of carcinomas which are targeted in cancer therapy. Sclerotium rolfsii lectin (SRL) is known to recognize TF and its substituted forms. Clinical potential of SRL has been demonstrated by studying its interaction with different types of cancer cells. Here we report, in vitro studies of SRL on breast cancer MDA-MB-468 cells and in vivo studies with MCF-7 xenografts. In vitro growth inhibitory studies of SRL on metastatic triple negative breast cancer MDA-MB-468 cells were performed by MTT assay, flow cytometry, adhesion, and CAM assay. In vivo efficacy studies of SRL were performed using NOD SCID mice bearing MCF-7 xenografts. SRL has strong binding to MDA-MB-468 cells with MFI of 85.5 and has growth inhibitory effect with IC50 of 32 µg/ml at 48 hr. SRL has antiangiogenesis effect and also anti adhesive effect with fibronectin and collagen at 20 µg/ml by 36% and 42%, respectively. In vivo efficacy studies of SRL on NOD SCID mice bearing MCF-7 xenogratfs revealed 61.77% and 75.71% tumor regressing effect, respectively, at 20 and 30 mg/kg body weight without any toxicity. All these results substantiate clinical potential of SRL on breast cancer.

A mitogenic lectin from Rhizoctonia bataticola arrests growth, inhibits metastasis, and induces apoptosis in human colon epithelial cancer cells.

The correlation between colorectal cancer (CRC) progression and altered expression of N-glycans can be considered in search for new biomarkers and anticancer agents to control CRC. Earlier N-glycan specific mitogenic lectin from Rhizoctonia bataticola (RBL) has been reported which has growth inhibitory and apoptotic effect on human ovarian and leukemic cells, but mitogenic effect on normal PBMCs revealing its clinical potential. Here, we report the effect of RBL on human colon cancer HT 29, SW480, and SW620 cell growth and its differential binding to human normal colon and cancer tissues. RBL has strong binding to both primary and metastatic colon cancer cells with MFI of 403, 404, and 192, respectively for HT 29, SW480, and SW620 cells. RBL shows dose and time dependent growth inhibitory effect with IC50 of 5, 6.4, and 6.8 µg/mL, respectively for HT 29, SW480, and SW620 cells. RBL inhibited the clonogenicity of colon carcinoma cells. RBL arrests metastatic SW620 cell growth at S phase, increased hypodiploid population by 6.1%, 14.3%, and 23.2%, respectively at 12, 24, and 36 h. Further, RBL induces SW620 cell apoptosis in time dependent manner, showed increased release of ROS and nuclear degradation compared to lectin untreated control. Adhesion, wound healing, invasion, and migration assays demonstrated anti-metastasis effect of RBL in SW620 cells apart from its growth inhibitory effect. Anti angiogenic effect of RBL was demonstrated by CAM assay. All these results show the promising potential of RBL both as diagnostic and therapeutic agent.

High mannose N-glycan binding lectin from Remusatia vivipara (RVL) limits cell growth, motility and invasiveness of human breast cancer cells.

Breast cancer known for its high metastatic potential is responsible for large mortality rate amongst women; hence it is imperative to search for effective anti-metastatic molecules despite anticancer drugs. The current study describes the potential of Remusatia vivipara lectin (RVL), inducing apoptosis in breast cancer cells there by limiting motility and invasiveness. RVL binds to the cell surface glycans of MDA-MB-468 and MCF-7 cells, exhibiting strong glycan mediated cytotoxic effect, but show marginal effect on non-tumorigenic MCF-10A cells. RVL elicits increased cellular stress, apoptotic vacuoles and nuclear disintegration in both MDA-MB-468 and MCF-7 cells accompanied by depletion of G0/G1, S and G2/M phases. Lectin interaction induced production of reactive oxygen species through altering mitochondrial membrane potential progressing to apoptosis. Further, RVL strongly elicited reproductive cell death in MDA-MB-468 cells and showed strong inhibitory effect on neovascularization demonstrated in chorioallantoic membrane assay. Treatment of MDA-MB-468 cells with RVL, suppress the motility and invasive property as shown by scratch wound heal and Boyden chamber transwell assays respectively. These results provide an insight into significance of interaction of RVL with specific cell surface high mannose N-glycans resulting in curtailing the metastatic ability of cancer cells.

Purification, characterization and biological significance of mannose binding lectin from Dioscorea bulbifera bulbils.

Dioscorea bulbifera or air potato has been used as a folk remedy to treat cancer. A mannose binding lectin from bulbils of D. bulbifera was purified in a single step by affinity chromatography on mucin coupled Sepharose 4B column, determined by its fine sugar specificity by glycan array analysis and studied for its clinical potential in cancer and HIV research. SDS-PAGE showed that lectin is a monomer of Mr 24kDa. DBL agglutinated only rabbit erythrocytes and was inhibited by mucin, asialomucin, fetuin, asialofetuin and transferrin but not by any monosaccharides. Glycan array analysis of DBL revealed its affinity toward high mannose N-linked glycans with enhanced affinity for terminal mannose including N-linked glycans of HIV envelope glycoprotein gp120 and has strong anti-reverse transcriptase activity. DBL showed strong binding to non-metastatic human colon epithelial cancer HT 29, metastatic SW 620 and hepatocellular HepG2 cell lines. DBL showed dose and time dependent growth inhibitory effects on all the three cell lines HT 29, SW 620 and HepG2 with IC50 of 110µg, 9.8µg, 40µg respectively at 72h. Inhibitory effect of DBL was effectively blocked in presence of competing glycans like mucin. DBL has promising clinical potential both in cancer and HIV research.

Pharmacokinetics, biodistribution and antitumour effects of Sclerotium rolfsii lectin in mice.

Sclerotium rolfsii lectin (SRL) is a lectin isolated from the fungus Sclerotium rolfsii and has exquisite binding specificity towards the oncofetal Thomsen-Friedenreich antigen (TF-Ag; Galß1-3GalNAcα-O-Ser/Thr) and its derivatives. Previous studies have shown that SRL inhibits the proliferation of human colon, breast and ovarian cancer cells in vitro and suppresses tumour growth in mice when introduced intratumourally. The present study assessed the effect of SRL on tumour growth when introduced intraperitoneally in BALB/c nude mice and investigated the pharmacokinetics and biodistribution of SRL in Swiss albino mice. When 9 doses of SRL (30 mg/kg body weight/mice) was administered to BALB/c nude mice bearing human colon cancer HT-29 xenografts, a substantial reduction in tumour size was observed. A 35.8% reduction in tumour size was noted in the treated animals after 17 days. SRL treatment also inhibited angiogenesis, and the tumours from the treated animals were observed to carry fewer blood vessels and express less angiogenesis marker protein CD31, than that from the control animals. Pharmacokinetics and biodistribution analysis revealed that SRL was detected in the serum after 1 h and its level peaked after 24 h. SRL was not detected in any of the organs apart from the kidney where a trace amount was detected after 24 h of SRL injection. No significant changes were observed in any of the biochemical parameters tested including SGOT, SGPT, LDH, CREAT and BUN in the SRL-treated mice compared to these levels in the controls. This suggests that SRL has good potential to be developed as a therapeutic agent for cancer treatment and warrant further investigations in vivo and subsequent clinical trials.

Apparent seroprevalence, isolation and identification of risk factors for brucellosis among dairy cattle in Goa, India.

Brucellosis is a highly contagious zoonotic infection affecting livestock and human beings. The disease has been reported worldwide except in few countries where it has been eradicated. The prevalence of brucellosis among cattle from 11 farms having a history of abortions was studied. A total of 481 samples comprising of blood, milk, vaginal swabs, vaginal discharges, placental tissues and fetal tissues were collected from 296 animals. Clinical samples were processed for the isolation of Brucella. Serum samples (n=296) were tested by Rose Bengal Plate Test (RBPT) and indirect ELISA. A total of 90 (30.40%) and 123 (41.55%) samples were positive by RBPT and indirect ELISA, respectively. Also 27.02% samples were positive by both the tests. Brucella isolates (n= 8) were recovered from clinical samples using Brucella selective media. All the isolates demonstrated PCR amplification for the bcsp31 and IS711 genes. Amplification of Brucella abortus specific primer was demonstrated by all the isolates in AMOS PCR indicating isolates to be of either B. abortus biotype 1, 2 or 4. Risk factors for transmission of brucellosis among cattle population were studied by field surveys. It was observed that lack of awareness about brucellosis (OR=8.739, P=0.138) and inadequate floor space (OR=0.278, P=0.128) were crucial risk factors for transmission of bovine brucellosis.

Exquisite specificity of mitogenic lectin from Cephalosporium curvulum to core fucosylated N-glycans.

Lectins are carbohydrate binding proteins that are gaining attention as important tools for the identification of specific glycan markers expressed during different stages of the cancer. We earlier reported the purification of a mitogenic lectin from human pathogenic fungus Cephalosporium curvulum (CSL) that has complex sugar specificity when analysed by hapten inhibition assay. In the present study, we report the fine sugar specificity of CSL as determined by glycan array analysis. The results revealed that CSL has exquisite specificity towards core fucosylated N-glycans. Fucosylated trimannosyl core is the basic structure required for the binding of CSL. The presence of fucose in the side chain further enhances the avidity of CSL towards such glycans. The affinity of CSL is drastically reduced towards the non-core fucosylated glycans, in spite of their side chain fucosylation. CSL showed no binding to the tested O-glycans and monosaccharides. These observations suggest the unique specificity of CSL towards core fucosylated N-glycans, which was further validated by binding of CSL to human colon cancer epithelial and hepatocarcinoma cell lines namely HT29 and HepG2, respectively, that are known to express core fucosylated N-glycans, using AOL and LCA as positive controls. LCA and AOL are fucose specific lectins that are currently being used clinically for the diagnosis of hepatocellular carcinomas. Most of the gastrointestinal markers express core fucosylated N-glycans. The high affinity and exclusive specificity of CSL towards α1-6 linkage of core fucosylated glycans compared to other fucose specific lectins, makes it a promising molecule that needs to be further explored for its application in the diagnosis of gastrointestinal cancer.

Molecular mechanism of anticancer effect of Sclerotium rolfsii lectin in HT29 cells involves differential expression of genes associated with multiple signaling pathways: A microarray analysis.

Sclerotium rolfsii lectin (SRL) is a lectin isolated from fungus S. rolfsii and has high binding specificity toward the oncofetal Thomsen-Friedenreich carbohydrate antigen (Galß1-3GalNAc-α-O-Ser/Thr, T or TF), which is expressed in more than 90% of human cancers. Our previous studies have shown that binding of SRL to human colon, breast and ovarian cancer cells induces cell apoptosis in vitro and suppresses tumor growth in vivo. This study investigated the SRL-mediated cell signaling in human colon cancer HT29 cells by mRNA and miRNA microarrays. It was found that SRL treatment results in altered expression of several hundred molecules including mitogen-activated protein kinase (MAPK) and c-JUN-associated, apoptosis-associated and cell cycle and DNA replication-associated signaling molecules. Pathway analysis using GeneSpring 12.6.1 revealed that SRL treatment induces changes of MAPK and c-JUN-associated signaling pathways as early as 2 h while changes of cell cycle, DNA replication and apoptosis pathways were significantly affected only after 24 h. A significant change of cell miRNA expression was also observed after 12 h treatment of the cells with SRL. These changes were further validated by quantitative real time polymerase chain reaction and immunoblotting. This study thus suggests that the presence of SRL affects multiple signaling pathways in cancer cells with early effects on cell proliferation pathways associated with MAPK and c-JUN, followed by miRNA-associated cell activity and apoptosis. This provides insight information into the molecular mechanism of the anticancer activity of this fungal lectin.

Molecular Cloning, Carbohydrate Specificity and the Crystal Structure of Two Sclerotium rolfsii Lectin Variants.

SRL is a cell wall associated developmental-stage specific lectin secreted by Sclerotium rolfsii, a soil-born pathogenic fungus. SRL displays specificity for TF antigen (Galß1→3GalNAc-α-Ser//Thr) expressed in all cancer types and has tumour suppressing effects in vivo. Considering the immense potential of SRL in cancer research, we have generated two variant gene constructs of SRL and expressed in E. coli to refine the sugar specificity and solubility by altering the surface charge. SSR1 and SSR2 are two different recombinant variants of SRL, both of which recognize TF antigen but only SSR1 binds to Tn antigen (GalNAcα-Ser/Thr). The glycan array analysis of the variants demonstrated that SSR1 recognizes TF antigen and their derivative with high affinity similar to SRL but showed highest affinity towards the sialylated Tn antigen, unlike SRL. The carbohydrate binding property of SSR2 remains unaltered compared to SRL. The crystal structures of the two variants were determined in free form and in complex with N-acetylglucosamine at 1.7 Å and 1.6 Å resolution, respectively. Structural analysis highlighted the structural basis of the fine carbohydrate specificity of the two SRL variants and results are in agreement with glycan array analysis.