Identification and Functional Characterization of Plant Toxins.

The evolutionary arms race between plants and herbivores has led, over millions of years, to the production of many substances that prevent plants from being over-eaten by plant-feeding animals [...].

Extracts Prepared from a Canadian Toxic Plant Induce Light-Dependent Perinuclear Vacuoles in Human Cells.

We are investigating plant species from the Canadian prairie ecological zone by phenotypic cell assays to discover toxins of biological interest. We provide the first report of the effects of extracts prepared from the shrub Symphoricarpos occidentalis in several human cell lines. S. occidentalis (Caprifoliaceae) extracts are cytotoxic, and, strikingly, treated cells undergo light-dependent vacuolation near the nucleus. The range of irradiation is present in standard ambient light and lies in the visible range (400-700 nm). Vacuolization in treated cells can be induced with specific wavelengths of 408 or 660 nm at 1 J/cm2 energies. Vacuolated cells show a striking phenotype of a large perinuclear vacuole (nuclear associated vacuole, NAV) that is distinct from vesicles observed by treatment with an autophagy-inducing agent. Treatment with S. occidentalis extracts and light induces an intense lamin A/C signal at the junction of a nuclear vacuole and the nucleus. Further study of S. occidentalis extracts and vacuolation provide chemical tools that may contribute to the understanding of nuclear envelope organization and human cell biology.

Antimalarials and Phytotoxins from Botryosphaeria dothidea Identified from a Seed of Diseased Torreya taxifolia.

The metabolic pathways in the apicoplast organelle of Plasmodium parasites are similar to those in plastids in plant cells and are suitable targets for malaria drug discovery. Some phytotoxins released by plant pathogenic fungi have been known to target metabolic pathways of the plastid; thus, they may also serve as potential antimalarial drug leads. An EtOAc extract of the broth of the endophyte Botryosphaeria dothidea isolated from a seed collected from a Torreya taxifolia plant with disease symptoms, showed in vitro antimalarial and phytotoxic activities. Bioactivity-guided fractionation of the extract afforded a mixture of two known isomeric phytotoxins, FRT-A and flavipucine (or their enantiomers, sapinopyridione and (-)-flavipucine), and two new unstable γ-lactam alkaloids dothilactaenes A and B. The isomeric mixture of phytotoxins displayed strong phytotoxicity against both a dicot and a monocot and moderate cytotoxicity against a panel of cell lines. Dothilactaene A showed no activity. Dothilactaene B was isolated from the active fraction, which showed moderate in vitro antiplasmodial activity with high selectivity index. In spite of this activity, its instability and various other biological activities shown by related compounds would preclude it from being a viable antimalarial lead.

Antiproliferative potential from aqueous Viscum album L. preparations and their main constituents in comparison with ricin and purothionin on human cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Mistletoe has been used since ancient times in Europe mostly for medicinal purposes. Since 1917, mistletoe preparations have been applied in cancer therapy and today are the most frequently used complementary medicine in tumor treatment. The main cytotoxic constituents of Viscum album are lectins and viscotoxins. AIM OF THE STUDY: The aim of this in vitro study was to investigate the antiproliferative potential of Viscum album preparations from different host trees and to assess the impact of mistletoe lectin 1 (ML-1) and viscotoxin A (VT-A) in comparison to a structurally similar lectin and thionin. MATERIALS AND METHODS: By means of widely accepted 2D Alamar Blue Assay, based on population counting of living cells using a fluorescent cell viability dye, the potential impact to inhibit tumor cell of the mistletoe preparations (Iscucin®) and their single compounds (ML-1 and VT-A) on the cell growth of six human cancer cell lines were evaluated. Also the mixture of ML-1 and VT-A corresponding to the contents in the specific mistletoe preparations were monitored. Ricin and purothionin were used as reference lectin and reference thionin, respectively. RESULTS: The lung carcinoma cell line HCC827 was very sensitive to the Iscucin® preparations. Very strong antiproliferative effects were found with Iscucin®Salicis and Tiliae and a strong with Iscucin®Crataegi, Mali and Populi. The IC50 concentrations of the Iscucin® preparations correlated with their respective ML-1 contents, but the ML-1 levels were much lower than the IC50 concentration of isolated ML-1 (1 ng/ml - 56 ng/ml). ML-1 was much more effective than ricin. Iscucin® preparations, ML-1 and ricin showed antiproliferative activity on human tumor cells. VT-A and purothionin had no effect on cell viability in the concentration ranges tested. CONCLUSION: The complete mistletoe extract is more potent to inhibit tumor cell proliferation than isolated ML-1 at an equivalent concentration level. Phenolic compounds found in all Iscucin® preparations might contribute to uphold the cytotoxic activity of ML-1 by antioxidative action. However, further studies are necessary to evaluate the role of VT-A and possible synergistic actions to the antiproliferative effect of aqueous mistletoe extracts.

Improved dialysis removal of protein-bound uremic toxins by salvianolic acids.

BACKGROUND: Indoxyl sulfate (IS) and p-cresyl sulfate (pCS) are two key protein-bound uremic toxins that accumulate in patients with end-stage renal disease. IS and pCS cannot be efficiently removed by conventional hemodialysis because they are highly bound to proteins. One promising means to optimize the removal of protein-bound uremic toxins involves using binding competitors to liberate uremic toxins from protein-binding partners. PURPOSE: In this study, we try to identify potential binding competitors that can enhance the dialysis removal of IS and pCS in natural compounds of phytomedicine. METHODS: We employed microdialysis to evaluate whether Danhong injection (DHI) and its salvianolic acids can increase the free fractions of IS and pCS and thus improve their dialysis efficiency in vitro. Furthermore, we confirmed the positive effects of DHI and salvianolic acids in vivo on chronic kidney disease model rats in which IS and pCS had heavily accumulated. RESULTS: DHI significantly increased the dialysis efficiency of IS and pCS by 99.13% and 142.00% in vitro (10-fold dilution), respectively, and by 135.61% and 272.13% in vivo (4.16 ml/kg). Salvianolic acids including lithospermic acid (LA), salvianolic acid A (SaA), tanshinol (DSS), caffeic acid (CA), salvianolic acid B (SaB), protocatechuic aldehyde (PA) and rosmarinic acid (RA) significantly enhanced the dialysis removal of IS and pCS in a concentration-dependent manner. LA, the best competitor of the tested salvianolic acids, increased dialysis efficiency levels of IS and pCS by 197.23% and 198.31% in vitro (400 µM), respectively, and by 119.55% and 127.56% in vivo (24.69 mg/kg). CONCLUSION: The removal of protein-bound uremic toxins IS and pCS using DHI or salvianolic acids as protein-bound competitors is superior to previously reported strategies and drugs and may contribute to clinical hemodialysis therapeutic practice.

Potential Uses of Isolated Toxin Peptides in Neuropathic Pain Relief: A Literature Review.

Neuropathic pain is a subset of chronic pain that is caused by neurons that are damaged or firing aberrantly in the peripheral or central nervous systems. The treatment guidelines for neuropathic pain include antidepressants, calcium channel α2 delta ligands, topical therapy, and opioids as a second-line option. Pharmacotherapy has not been effective in the treatment of neuropathic pain except in the treatment of trigeminal neuralgia with carbamazepine. The inability to properly treat neuropathic pain causes frustration in both the patients and their treating physicians. Venoms, which are classically believed to be causes of pain and death, have peptide components that have been implicated in pain relief. Although some venoms are efficacious and have shown benefits in patients, their side-effect profile precludes their more widespread use. This review identifies and explores the use of venoms in neuropathic pain relief. This treatment can open doors to potential therapeutic targets. We believe that further research into the mechanisms of action of these receptors as well as their functions in nature will provide alternative therapies as well as a window into how they affect neuropathic pain.

Toxins and pharmacologically active compounds from species of the family Bufonidae (Amphibia, Anura).

ETHNOPHARMACOLOGICAL RELEVANCE: Among amphibians, 15 of the 47 species reported to be used in traditional medicines belong to the family Bufonidae, which demonstrates their potential in pharmacological and natural products research. For example, Asian and American tribes use the skin and the parotoid gland secretions of some common toads in the treatment of hemorrhages, bites and stings from venomous animals, skin and stomach disorders, as well as several types of cancers. OVERARCHING OBJECTIVE: In addition to reviewing the occurrence of chemical constituents present in the family Bufonidae, the cytotoxic and biomedical potential of the active compounds produced by different taxa are presented. METHODOLOGY: Available information on bioactive compounds isolated from species of the family Bufonidae was obtained from ACS Publications, Google, Google Scholar, Pubmed, Sciendirect and Springer. Papers written in Chinese, English, German and Spanish were considered. RESULTS: Recent reports show more than 30% of amphibians are in decline and some of bufonid species are considered to be extinct. For centuries, bufonids have been used as traditional folk remedies to treat allergies, inflammation, cancer, infections and other ailments, highlighting their importance as a prolific source for novel drugs and therapies. Toxins and bioactive chemical constituents from skin and parotid gland secretions of bufonid species can be grouped in five families, the guanidine alkaloids isolated and characterized from Atelopus, the lipophilic alkaloids isolated from Melanophryniscus, the indole alkaloids and bufadienolides known to be synthesized by species of bufonids, and peptides and proteins isolated from the skin and gastrointestinal extracts of some common toads. Overall, the bioactive secretions of this family of anurans may have antimicrobial, protease inhibitor and anticancer properties, as well as being active at the neuromuscular level. CONCLUSION: In this article, the traditional uses, toxicity and pharmacological potential of chemical compounds from bufonids have been summarized. In spite of being reported to be used to treat several diseases, neither extracts nor metabolites from bufonids have been tested in such illness like acne, osteoporosis, arthritis and other illnesses. However, the cytotoxicity of these metabolites needs to be evaluated on adequate animal models due to the limited conditions of in vitro assays. Novel qualitative and quantitative tools based on MS spectrometry and Nuclear Magnetic Resonance spectroscopy is now available to study the complex secretions of bufonids.

Detection of aristolochic acids I and II in "Chiniy-trèf", a traditional medicinal preparation containing caterpillars feeding on Aristolochia trilobata L. in Martinique, French West Indies.

"Chiniy-trèf" is a traditional medicinal preparation used in Martinique, French West Indies, for the prevention of all kinds of attempted poisoning and hex. It is produced by the maceration in alcohol (mostly rum) of larvae (caterpillars) of the butterfly Battus polydamas ssp. cebriones, feeding on the leaves of Aristolochia trilobata. Aristolochic acids I and II that are well-known nephrotoxic and carcinogenic substances were identified on two samples of "chiniy-trèfl" by chromatographic methods.

The relative toxicity of Delphinium stachydeum in mice and cattle.

Larkspurs (Delphinium spp.) are poisonous plants on rangelands throughout the Western United States and Canada. Larkspur-induced poisoning in cattle is due to norditerpene alkaloids that are represented by two main structural groups of norditerpene alkaloids, the N-(methylsuccinimido) anthranoyllycoctonine type (MSAL-type) and the non-MSAL type. Information on the alkaloid composition and resulting toxicity in mice and cattle is lacking for a number of Delphinium species, including Delphinium stachydeum. The objective of this study was to determine the alkaloid composition of D. stachydeum and to characterize its relative toxicity in mice and cattle compared to two reference species Delphinium barbeyi and Delphinium occidentale. D. stachydeum contains the non-MSAL-type alkaloids but not the MSAL-type alkaloids. D. stachydeum was less toxic than D. barbeyi and D. occidentale in the mouse model. D. stachydeum was less toxic than the MSAL-containing D. barbeyi but much more toxic than the non-MSAL-containing D. occidentale in cattle as measured by heart rate and time of exercise. These results indicate that predictions of Delphinium toxicity can't be accurately made based solely on results from the mouse model or the absence of the MSAL-type alkaloids in the plant.

Identification of RIP-II toxins by affinity enrichment, enzymatic digestion and LC-MS.

Type 2 ribosome-inactivating protein toxins (RIP-II toxins) were enriched and purified prior to enzymatic digestion and LC-MS analysis. The enrichment of the RIP-II family of plant proteins, such as ricin, abrin, viscumin, and volkensin was based on their affinity for galactosyl moieties. A macroporous chromatographic material was modified with a galactose-terminated substituent and packed into miniaturized columns that were used in a chromatographic system to achieve up to 1000-fold toxin enrichment. The galactose affinity of the RIP-II proteins enabled their selective enrichment from water, beverages, and extracts of powder and wipe samples. The enriched fractions were digested with trypsin and RIP-II peptides were identified based on accurate mass LC-MS data. Their identities were unambiguously confirmed by LC-MS/MS product ion scans of peptides unique to each of the toxins. The LC-MS detection limit achieved for ricin target peptides was 10 amol and the corresponding detection limit for the full method was 10 fmol/mL (0.6 ng/mL). The affinity enrichment method was applied to samples from a forensic investigation into a case involving the illegal production of ricin and abrin toxins.

Suaveolic acid: a potent phytotoxic substance of Hyptis suaveolens.

Hyptis suaveolens (Lamiaceae) is an exotic invasive plant in many countries. Earlier studies reported that the aqueous, methanol, and aqueous methanol extract of H. suaveolens and its residues have phytotoxic properties. However, to date, the phytotoxic substances of this plant have not been reported. Therefore, the objectives of this study were isolation and identification of phytotoxic substances of H. suaveolens. Aqueous methanol extract of this plant was purified by several chromatographic runs through bioassay guided fractionation using garden cress (Lepidium sativum) as a test plant. Final purification of a phytotoxic substance was achieved by reverse phase HPLC and characterized as 14α-hydroxy-13ß-abiet-8-en-18-oic acid (suaveolic acid) by high-resolution ESI-MS, (1)H-,(13)C-NMR, CD, and specific rotation. Suaveolic acid inhibited the shoot growth of garden cress, lettuce (Lactuca sativa), Italian ryegrass (Lolium multiflorum), and barnyard grass (Echinochloa crus-galli) at concentrations greater than 30 µM. Root growth of all but lettuce was also inhibited at concentrations greater than 30 µM. The inhibitory activities were concentration dependent. Concentrations required for 50% growth inhibition of suaveolic acid for those test plant species were ranged from 76 to 1155 µM. Therefore, suaveolic acid is phytotoxic and may be responsible for the phytotoxicity of H. suaveolens plant extracts.

Novel linear and step-gradient counter-current chromatography for bio-guided isolation and purification of cytotoxic podophyllotoxins from Dysosma versipellis (Hance).

Dysosma versipellis (Hance) is a famous traditional Chinese medicine for the treatment of snakebite, weakness, condyloma accuminata, lymphadenopathy, and tumors for thousands of years. In this work, four podophyllotoxin-like lignans including 4'-demethylpodophyllotoxin (1), α-peltatin (2), podophyllotoxin (3), ß-peltatin (4) as major cytotoxic principles of D. versipellis were successfully isolated and purified by several novel linear and step gradient counter-current chromatography methods using the systems of hexane/ethyl acetate/methanol/water (4:6:3:7 and 4:6:4:6, v/v/v/v). Compared with isocratic elution, linear and step-gradient elution can provide better resolution and save more time for the separation of photophyllotoxin and its congeners. Their cytotoxicities were further evaluated and their structures were validated by high-resolution electrospray TOF MS and nuclear magnetic resonance spectra. All components showed potent anticancer activity against human hepatoma cells HepG2.

Larvicidal activity of a toxin from the seeds of Jatropha curcas Linn. against Aedes aegypti Linn. and Culex quinquefasciatus Say.

The larvicidal effects of the crude protein extract and purified toxin, Jc-SCRIP, from the seed coat of Jatropha curcas Linn. against the third instar larvae of mosquitoes, Aedes aegypti Linn. and Culex quinquefasciatus Say, were investigated. This test compared the effects of the purified toxin with crude protein extracts from seed kernels of J. curcas and Ricinus communis. At various concentrations of purified toxin and crude protein extract, the larval mortality of both Ae. aegypti and Cx. quinquefasciatus were positively correlated with increased exposure time. The larvae of Cx. quinquefasciatus were more susceptible to the toxin and both extracts than the larvae of Ae. aegypti. After 24 hours of exposure, the extract showed larvicidal activity against Ae. aegypti and Cx. quinquefasciatus with (LC50) values of 3.89 mg/ml and 0.0575 mg/ml, respectively. The toxin, Jc-SCRIP, showed larvicidal activity against Ae. aegypti and Cx. quinquefasciatus with (LC50) values of 1.44 mg/ml and 0.0303 mg/ ml, respectively. These results indicated that the crude protein extract and Jc-SCRIP were more toxic to the third instar larvae of Cx. quinquefasciatus than that of Ae. aegypti. The potent larvicidal activities of the seed coat extract and the Jc-SCRIP toxin from J. curcas suggest that they may be used as bioactive agents to control the mosquito population.

The antimutagenic effect of mistletoe lectin (Viscum album L. var. coloratum agglutinin).

A galactose- and N-acetyl-D-galactosamine-specific lectin (Viscum album L. var. coloratum agglutinin, VCA), which is known for its anticancer activity, was isolated from mistletoe. In this study, we investigated the antimutagenic potentials of VCA by using the pre-incubation method of the Ames test (Salmonella typhimurium TA98 and TA100) in the presence or absence of S9 mixture. Viscum album L. var. coloratum agglutinin was assessed for its antimutagenic properties against the mutagens 2-aminoanthracene (2AA) and furylfuramide (AF-2) for strain TA98, and sodium azide (NaN(3) ) and 2-aminoanthracene (2AA) for strain TA100. The concentrations used for this test compound were 100, 200 and 400 µg per plate. Viscum album L. var. coloratum agglutinin showed moderate, but not negligible, protective effects regarding the antimutagenic properties against the direct-acting mutagens NaN(3) and AF-2. Furthermore, VCA was more effective in preventing the mutagenicity of the indirect-acting mutagen 2-AA (in the presence of S9) when tested with both TA98 and TA100. In conclusion, this report has shown broad ranging antimutagenic effects of VCA to numerous mutagens in TA98 and TA100 Salmonella typhimurium strains. Although the data presented here cannot be applied in vivo, they can support other antimutagenic and anticarcinogenic findings for VCA.

Soil biota reduce allelopathic effects of the invasive Eupatorium adenophorum.

Allelopathy has been hypothesized to play a role in exotic plant invasions, and study of this process can improve our understanding of how direct and indirect plant interactions influence plant community organization and ecosystem functioning. However, allelopathic effects can be highly conditional. For example allelopathic effects demonstrated in vivo can be difficult to demonstrate in field soils. Here we tested phytotoxicity of Eupatorium adenophorum (croftonweed), one of the most destructive exotic species in China, to a native plant species Brassica rapa both in sand and in native soil. Our results suggested that natural soils from different invaded habitats alleviated or eliminated the efficacy of potential allelochemicals relative to sand cultures. When that soil is sterilized, the allelopathic effects returned; suggesting that soil biota were responsible for the reduced phytotoxicity in natural soils. Neither of the two allelopathic compounds (9-Oxo-10,11-dehydroageraphorone and 9b-Hydroxyageraphorone) of E. adenophorum could be found in natural soils infested by the invader, and when those compounds were added to the soils as leachates, they showed substantial degradation after 24 hours in natural soils but not in sand. Our findings emphasize that soil biota can reduce the allelopathic effects of invaders on other plants, and therefore can reduce community invasibility. These results also suggest that soil biota may have stronger or weaker effects on allelopathic interactions depending on how allelochemicals are delivered.

A cytotoxic type-2 ribosome inactivating protein (from leafless mistletoe) lacking sugar binding activity.

Articulatin-D, a 66 kDa ribosome inactivating protein (RIP) comprised of 29 kDa A-chain linked to 35 kDa B-chain, is purified from leafless mistletoe (Viscum articulatum) parasitic on Dalbergia sp. from Western Ghats (India). N-terminal sequence and LC-MS/MS analyses of A- and B-chain confirmed that articulatin-D is a type-2 RIP having high homology with other mistletoe lectins. Translation inhibition and diagnostic N-glycosidase activity of articulatin-D illustrate the presence of catalytically active A-chain. Its inability to: (i) bind to acid treated Sepharose CL-6B column, (ii) agglutinate trypsin-treated and untreated RBCs of human (A, B, O, AB), mice, rat, rabbit, buffalo, porcine, pigeon, cock, fish, sheep and goat even with 10mg/ml of purified articulatin-D, (iii) show change in circular dichroism spectra after addition of sugar to the native protein, (iv) bind to different sugars (galactose, lactose, gal-NAc, rhamnose, arabinose, fucose and mannose) immobilized on Sepharose 4B matrix, and (v) show change in enthalpy during titration with galactose confirm that the B-chain of articulatin-D lacks sugar binding activity. Despite this, articulatin-D is highly toxic as characterized with low IC(50) against different cancer cell lines (Jurkat: 0.31 ± 0.02 nM, MOLT-4: 0.51 ± 0.03 nM, U-937: 0.64 ± 0.07 nM, HL-60: 0.79 ± 0.11 nM, Raji: 1.45 ± 0.09 nM). Toxicity of RIPs has been ascribed to the absence/presence of B-chain with sugar binding activity. Identification of articulatin-D, the first cytotoxic RIP with B-chain lacking sugar binding activity opens new vistas in understanding cytotoxic action of RIPs.

Determination of total retronecine esters-type hepatotoxic pyrrolizidine alkaloids in plant materials by pre-column derivatization high-performance liquid chromatography.

A pre-column derivatization high-performance liquid chromatography method with diode array detection was developed and validated to determine the total retronecine esters-type hepatotoxic pyrrolizidine alkaloids (RET-HPAs) in herbs. The RET-HPAs reacted with o-chloranil in methanolic solution heated for 3 h, and an oxidative derivative was produced that could be detected at a maximal absorption of 223 nm. The analysis was performed using a C18 column with an isocratic elution of methanol and aqueous 0.01% triethylamine (adjusted to pH 4 with formic acid), and the detection was carried out with DAD at 223 nm. The validation of the method included linearity, sensitivity, recovery and stability. It showed a good linear regression (r(2) > 0.9900) in the range of 2.5-250 microM with a limit of detection (S/N = 3) of 0.5 microM. The method provided desirable repeatability with overall intra- and inter-day variations of less than 4.6%. The obtained recoveries for both of the extraction and derivatization process were between 94.6 and 100.7% (n = 3).

Quinoid radio-toxin (QRT) induced metabolic changes in mice: an ex vivo and in vivo EPR investigation.

Radio-toxins are toxic metabolites produced by ionizing irradiation and have toxic effects similar to those caused by direct irradiation. We have investigated the effect of a quinoid radio-toxin (QRT) obtained from gamma-irradiated potato tuber on various organs in mice using ex vivo and in vivo EPR spectroscopy. Results indicate a decrease in the activity of ribonucleotide reductase enzyme in spleen of mice treated with 0.2mg QRT. A dose of 2mg QRT was fatal to mice within 45-60 min of treatment. Nitrosyl hemoglobin complexes alpha-(Fe(2+)-NO)alpha-(Fe(2+))beta-(Fe(2+))(2) were detected from spleen, blood, liver, kidney, heart, and lung tissue samples of mice treated with lethal doses of QRT. A significant decrease of pO(2) in liver and brain was observed after administration of QRT at the lethal dose. The time of the appearance of the nitrosyl hemoglobin complex and its intensity varied with the dose of QRT and the type of tissue. These results indicate that the effect of the QRT is more prominent in spleen and to a lesser extent in liver and blood. The QRT action at the lethal doses resulted in an increased hypoxia over time with disruption of compensatory adaptive response. The results indicate similar outcome of QRT as observed with gamma-irradiation.

Isolation and characterization of two Korean mistletoe lectins.

Two isolectins (KML-IIU and the KML-IIL) were individually isolated from the previously reported Korean mistletoe lectin, KML-C, by using an immunoaffinity column. Molecular weights of the KML-IIU and the KML-IIL were 64 kDa and 60 kDa respectively. Both of the lectins were composed of heterogeneous A and B subunits linked with a disulfide bond, and showed the same carbohydrate-binding specificities for Gal and GalNAc. However, they are different not only in biophysical properties (glycosylation and amino acid compositions) but also bioactivities (cell killing and cytokine induction). The KML-IIL showed 17-145 times stronger in cytotoxicities to various human and mouse cancer cell lines than the KML-IIU. The KML-IIL also induced TNF-alpha secretion from mouse peritoneal macrophages 4.5 times better than the KML-IIU. The results demonstrated isolectins in Korean mistletoe were varied in bioactivities and the KML-IIL may be developed as an anti-cancer agent.

Complete structure determination of N-acetyl-D-galactosamine-binding mistletoe lectin-3 from Viscum album L. album.

The primary structure of the B chain of the N-acetyl-D-galactosamine-recognizing mistletoe lectin-3 (ML-3B) has been deduced from proteolytic digest peptides of the purified glycoprotein, their HPLC-separation and Edman degradation and confirmation of the peptide sequences by MALDI-MS. ML-3B consists of 262 amino acid residues including 10 cysteine moieties. The structure and linkage of the carbohydrate side chains, connected to two N-glycosylation sites at positions Asn(95) and Asn(135) of the lectin, were determined by a combination of glycosidase treatment and MALDI-MS of corresponding glycopeptide fragments. The sequence alignment reveals a high homology with other B chains of type-II RIPs, although there are remarkable differences in the D-galactose-specific mistletoe lectin-1B chain. The recently published primary structure of the mistletoe lectin-3A chain1 and the now available primary sequence of the 3B chain allowed the construction of a preliminary homology model of ML-3. The model demonstrates, unequivocally, that ML-3 is a member of the type-II RIP family with rigid conservation of the enzymatic active site of the A chain and an identical overall protein fold. Specific amino acid residue exchanges and the different glycosylation pattern in comparison with ML-1 are discussed and related to the properties of the two glycoproteins. The knowledge of the complete primary structure of mistletoe lectin-3 is a major contribution towards more insight into the mechanism of the biological activity of commercial mistletoe preparations.