Plant lectins and their usage in preparing targeted nanovaccines for cancer immunotherapy.

Plant lectins, a natural source of glycans with a therapeutic potential may lead to the discovery of new targeted therapies. Glycans extracted from plant lectins are known to act as ligands for C-type lectin receptors (CLRs) that are primarily present on immune cells. Plant-derived glycosylated lectins offer diversity in their N-linked oligosaccharide structures that can serve as a unique source of homogenous and heterogenous glycans. Among the plant lectins-derived glycan motifs, Man9GlcNAc2Asn exhibits high-affinity interactions with CLRs that may resemble glycan motifs of pathogens. Thus, such glycan domains when presented along with antigens complexed with a nanocarrier of choice may bewilder the immune cells and direct antigen cross-presentation - a cytotoxic T lymphocyte immune response mediated by CD8+ T cells. Glycan structure analysis has attracted considerable interest as glycans are looked upon as better therapeutic alternatives than monoclonal antibodies due to their cost-effectiveness, reduced toxicity and side effects, and high specificity. Furthermore, this approach will be useful to understand whether the multivalent glycan presentation on the surface of nanocarriers can overcome the low-affinity lectin-ligand interaction and thereby modulation of CLR-dependent immune response. Besides this, understanding how the heterogeneity of glycan structure impacts the antigen cross-presentation is pivotal to develop alternative targeted therapies. In the present review, we discuss the findings on structural analysis of glycans from natural lectins performed using GlycanBuilder2 - a software tool based on a thorough literature review of natural lectins. Additionally, we discuss how multiple parameters like the orientation of glycan ligands, ligand density, simultaneous targeting of multiple CLRs and design of antigen delivery nanocarriers may influence the CLR targeting efficacy. Integrating this information will eventually set the ground for new generation immunotherapeutic vaccine design for the treatment of various human malignancies.

Site-Specific N-Glycosylation Characterization of Windmill Palm Tree Peroxidase Using Novel Tools for Analysis of Plant Glycopeptide Mass Spectrometry Data.

Plant secretory (Class III) peroxidases are redox enzymes that rely on N-glycosylation for full enzyme activity and stability. Peroxidases from palm tree leaves comprise the most stable and active plant peroxidases characterized to date. Herein, site-specific glycosylation and microheterogeneity of windmill palm tree (Trachycarpus fortunei) peroxidase are reported. The workflow developed in this study includes novel tools, written in R, to aid plant glycan identification, pGlycoFilter, for annotation of glycopeptide fragmentation spectra, gPSMvalidator, and for relative quantitation of glycoforms, glycoRQ. Mass spectrometry analysis provided a detailed glycosylation profile at the 13 sites of N-linked glycosylation on windmill palm tree peroxidase. Glycan microheterogeneity was observed at each site. Site Asn211 was the most heterogeneous and contained 30 different glycans. Relative quantitation revealed 90% of each glycosylation site was occupied by three or fewer glycans, and two of the 13 sites were partially unoccupied. Although complex and hybrid glycans were identified, the majority of glycans were paucimannosidic, characteristic of plant vacuolar glycoproteins. Further studies pertaining to the glycan structure-activity relationships in plant peroxidases can benefit from the work outlined here.

Unidentified N-glycans by N-glycosidase A were Identified by Nglycosidase F under Denaturing Conditions in Plant Glycoprotein.

BACKGROUND: The identification of N-glycans in plant glycoproteins or plant-made pharmaceuticals is essential for understanding their structure, function, properties, immunogenicity, and allergenicity (induced by plant-specific core-fucosylation or xylosylation) in the applications of plant food, agriculture, and plant biotechnology. N-glycosidase A is widely used to release the Nglycans of plant glycoproteins because the core-fucosylated N-glycans of plant glycoproteins are hydrolyzed by N-glycosidase A but not by N-glycosidase F. However, the efficiency of Nglycosidase A activity in plant glycoproteins remains unclear. OBJECTIVE: The aim of the study was to elucidate the efficient use of N-glycosidases to identify and quantify the N-glycans of plant glycoproteins; it aimed at identification of released N-glycans by Nglycosidase F and assessment of their relative quantities with a focus on unidentified N-glycans by N-glycosidase A in plant glycoproteins, Phaseolus vulgaris lectin (PHA) and horseradish peroxidase (HRP). METHODS: Liquid chromatography-tandem mass spectrometry was used to analyze and compare the N-glycans of PHA and HRP treated with either N-glycosidase A or F under denaturing conditions. The relative quantities (%) of each N-glycan (>0.1%) to the total N-glycans (100%) were determined. RESULTS: N-glycosidase A and F released 9 identical N-glycans of PHA, but two additional corefucosylated N-glycans were released by only N-glycosidase A, as expected. By contrast, in HRP, 8 N-glycans comprising 6 core-fucosylated N-glycans, 1 xylosylated N-glycan, and 1 mannosylated N-glycan were released by N-glycosidase A. Moreover, 8 unexpected N-glycans comprising 1 corefucosylated N-glycan, 4 xylosylated N-glycans, and 3 mannosylated N-glycans were released by Nglycosidase F. Of these, 3 xylosylated and 2 mannosylated N-glycans were released by only Nglycansodase F. CONCLUSION: These results demonstrate that N-glycosidase A alone is insufficient to release the Nglycans of all plant glycoproteins, suggesting that to identify and quantify the released N-glycans of the plant glycoprotein HRP, both N-glycosidase A and F treatments are required.

Molecular characterization of tomato α1,3/4-fucosidase, a member of glycosyl hydrolase family 29 involved in the degradation of plant complex type N-glycans.

In this study, we identified a gene in tomato that encodes an acidic α-fucosidase (LOC101254568 or Solyc03g006980, α-Fuc'ase S1-1), which may be involved in the turnover of plant complex-type N-glycans. Recombinant α-Fuc'ase S1-1 (rFuc'ase S1-1) was expressed using a baculovirus-insect cell expression system. rFuc'ase Sl-1 is 55 kDa in size and has an optimum pH around 4.5. It substantially hydrolyzed the non-reducing terminal α1,3-fucose residue on LNFP III and α1,4-fucose residues of Lea epitopes on plant complex-type N-glycans, but not the α1,2-fucose residue on LNFP I or the α1,3-fucose residue on pyridylaminated Fucα1-3GlcNAc. Furthermore, we found that this tomato α-Fuc'ase S1-1 was inactive toward the core penta-oligosaccharide unit [Manß1-4(Xylß1-2)GlcNAcß1-4(Fucα1-3)GlcNAc-PA] of plant complex-type N-glycans. Molecular 3D modelling of α-Fuc'ase Sl-1 and structure/sequence interpretation based on comparison with a homologous α-fucosidase from Bifidobacterium longum subsp. infantis (Blon_2336) indicated that residues Asp193 and Glu237 might be important for substrate binding.

A glycoprotein from plant(ZPP):study on tumor common antigen / 中国免疫学杂志

Objective:Tumor common antigen of an extract of plant and applying in early stage diagnosis for tumor were studied.Methods:Tumor cells and lymphocytes proliferate responses were determined by 3H-TdR intervening.Fresh tumor tissue from human transplant to mice,the survival rate of transplanted tumor was observed.DNA content of tumor cells was measured by flowcytometery.The chemical nature was analyzed by chromatography and measured by ultraviolet-spectrometer.Results:The extract of plant markedly stimulated the lymphocytes from tumor patients and atypical hyperplasia patient to proliferate,but failed to health persons(P