A Cell-Free Protein Expression System Derived from Human Primary Peripheral Blood Mononuclear Cells.

Historically, some of the first cell-free protein expression systems studied in vitro translation in various human blood cells. However, because of limited knowledge of eukaryotic translation and the advancement of cell line development, interest in these systems decreased. Eukaryotic translation is a complex system of factors that contribute to the overall translation of mRNA to produce proteins. The intracellular translateome of a cell can be modified by various factors and disease states, but it is impossible to individually measure all factors involved when there is no comprehensive understanding of eukaryotic translation. The present work outlines the use of a coupled transcription and translation cell-free protein expression system to produce recombinant proteins derived from human donor peripheral blood mononuclear cells (PBMCs) activated with phytohemagglutinin-M (PHA-M). The methods outlined here could result in tools to aid immunology, gene therapy, cell therapy, and synthetic biology research and provide a convenient and holistic method to study and assess the intracellular translation environment of primary immune cells.

N-glycan profiles in H9N2 avian influenza viruses from chicken eggs and human embryonic lung fibroblast cells.

N-glycosylation can affect the host specificity, virulence and infectivity of influenza A viruses (IAVs). In this study, the distribution and evolution of N-glycosylation sites in the hemagglutinin (HA) and neuraminidase (NA) of H9N2 virus were explored using phylogenetic analysis. Then, one strain of the H9N2 subtypes was proliferated in the embryonated chicken eggs (ECE) and human embryonic lung fibroblast cells (MRC-5) system. The proliferated viral N-glycan profiles were analyzed by a glycomic method that combined the lectin microarray and MALDI-TOF/TOF-MS. As a result, HA and NA of H9N2 viruses prossess six and five highly conserved N-glycosylation sites, respectively. Sixteen lectins (e.g., MAL-II, SNA and UEA-I) had increased expression levels of the glycan structures in the MRC-5 compared with the ECE system; however, 6 lectins (e.g., PHA-E, PSA and DSA) had contrasting results. Eleven glycans from the ECE system and 13 glycans from the MRC-5 system were identified. Our results showed that the Fucα-1,6GlcNAc(core fucose) structure was increased, and pentaantennary N-glycans were only observed in the ECE system. The SAα2-3/6Gal structures were highly expressed and Fucα1-2Galß1-4GlcNAc structures were only observed in the MRC-5 system. We conclude that the existing SAα2-3/6Gal sialoglycans make the offspring of the H9N2 virus prefer entially attach to each other, which decreases the virulence. Alterations in the glycosylation sites for the evolution and role of IAVs have been widely described; however, little is known about the exact glycan structures for the same influenza strain from different hosts. Our findings may provide a novel way for further discussing the molecular mechanism of the viral transmission and virulence associated with viral glycosylation in avian and human hosts as well as vital information for designing a vaccine against influenza and other human viruses.

Phytohemagglutinin from Phaseolus vulgaris (PHA-E) displays a novel glycan recognition mode using a common legume lectin fold.

Phytohemagglutinin from Phaseolus vulgaris (PHA-E), a legume lectin, has an unusual specificity toward biantennary galactosylated N-glycan with bisecting N-acetylglucosamine (GlcNAc). To investigate the interaction in detail, we have solved the crystal structures of PHA-E without ligand and in complex with biantennary N-glycan derivatives. PHA-E interacts with the trisaccharide unit (Galß1-4GlcNAcß1-2Man) in a manner completely different from that of mannose/glucose-specific legume lectins. The inner mannose residue binds to a novel site on the protein, and its rotation is opposite to that occurring in the monosaccharide-binding site of other lectins around the sugar O3 axis. Saturation-transfer difference NMR using biantennary di-galactosylated and bisected glycans reveals that PHA-E interacts with both antennas almost equally. The unique carbohydrate interaction explains the glycan-binding specificity and high affinity.

Multivariate heredity of melanin-based coloration, body mass and immunity.

The genetic covariation among different traits may cause the appearance of correlated response to selection on multivariate phenotypes. Genes responsible for the expression of melanin-based color traits are also involved in other important physiological functions such as immunity and metabolism by pleiotropy, suggesting the possibility of multivariate evolution. However, little is known about the relationship between melanin coloration and these functions at the additive genetic level in wild vertebrates. From a multivariate perspective, we simultaneously explored inheritance and selection of melanin coloration, body mass and phytohemagglutinin (PHA)-mediated immune response by using long-term data over an 18-year period collected in a wild population of the common kestrel Falco tinnunculus. Pedigree-based quantitative genetic analyses showed negative genetic covariance between melanin-based coloration and body mass in male adults and positive genetic covariance between body mass and PHA-mediated immune response in fledglings as predicted by pleiotropic effects of melanocortin receptor activity. Multiple selection analyses showed an increased fitness in male adults with intermediate phenotypic values for melanin color and body mass. In male fledglings, there was evidence for a disruptive selection on rump gray color, but a stabilizing selection on PHA-mediated immune response. Our results provide an insight into the evolution of multivariate traits genetically related with melanin-based coloration. The differences in multivariate inheritance and selection between male and female kestrels might have resulted in sexual dimorphism in size and color. When pleiotropic effects are present, coloration can evolve through a complex pathway involving correlated response to selection on multivariate traits.

QUES, a new Phaseolus vulgaris genotype resistant to common bean weevils, contains the Arcelin-8 allele coding for new lectin-related variants.

In common bean (Phaseolus vulgaris L.), the most abundant seed proteins are the storage protein phaseolin and the family of closely related APA proteins (arcelin, phytohemagglutinin and α-amylase inhibitor). High variation in APA protein composition has been described and the presence of arcelin (Arc) has been associated with bean resistance against two bruchid beetles, the bean weevil (Acanthoscelides obtectus Say) and the Mexican bean weevil (Zabrotes subfasciatus Bohemian). So far, seven Arc variants have been identified, all in wild accessions, however, only those containing Arc-4 were reported to be resistant to both species. Although many efforts have been made, a successful breeding of this genetic trait into cultivated genotypes has not yet been achieved. Here, we describe a newly collected wild accession (named QUES) and demonstrate its resistance to both A. obtectus and Z. subfasciatus. Immunological and proteomic analyses of QUES seed protein composition indicated the presence of new Arc and arcelin-like (ARL) polypeptides of about 30 and 27 kDa, respectively. Sequencing of cDNAs coding for QUES APA proteins confirmed that this accession contains new APA variants, here referred to as Arc-8 and ARL-8. Moreover, bioinformatic analysis showed the two proteins are closely related to APA components present in the G12949 wild bean accession, which contains the Arc-4 variant. The presence of these new APA components, combined with the observations that they are poorly digested and remain very abundant in A. obtectus feces, so-called frass, suggest that the QUES APA locus is involved in the bruchid resistance. Moreover, molecular analysis indicated a lower complexity of the locus compared to that of G12949, suggesting that QUES should be considered a valuable source of resistance for further breeding purposes.

Comparative safety testing of genetically modified foods in a 90-day rat feeding study design allowing the distinction between primary and secondary effects of the new genetic event.

This article discusses the wider experiences regarding the usefulness of the 90-day rat feeding study for the testing of whole foods from genetically modified (GM) plant based on data from a recent EU-project [Poulsen, M., Schrøder, M., Wilcks, A., Kroghsbo, S., Lindecrona, R.H., Miller, A., Frenzel, T., Danier, J., Rychlik, M., Shu, Q., Emami, K., Taylor, M., Gatehouse, A., Engel, K.-H., Knudsen, I., 2007a. Safety testing of GM-rice expressing PHA-E lectin using a new animal test design. Food Chem. Toxicol. 45, 364-377; Poulsen, M., Kroghsbo, S., Schrøder, M., Wilcks, A., Jacobsen, H., Miller, A., Frenzel, T., Danier, J., Rychlik, M., Shu, Q., Emami, K., Sudhakar, D., Gatehouse, A., Engel, K.-H., Knudsen, I., 2007b. A 90-day safety in Wistar rats fed genetically modified rice expressing snowdrop lectin Galanthus nivalis (GNA). Food Chem. Toxicol. 45, 350-363; Schrøder, M., Poulsen, M., Wilcks, A., Kroghsbo, S., Miller, A., Frenzel, T., Danier, J., Rychlik, M., Emami, K., Gatehouse, A., Shu, Q., Engel, K.-H., Knudsen, I., 2007. A 90-day safety study of genetically modified rice expressing Cry1Ab protein (Bacillus thuringiensis toxin) in Wistar rats. Food Chem. Toxicol. 45, 339-349]. The overall objective of the project has been to develop and validate the scientific methodology necessary for assessing the safety of foods from genetically modified plants in accordance with the present EU regulation. The safety assessment in the project is combining the results of the 90-day rat feeding study on the GM food with and without spiking with the pure novel gene product, with the knowledge about the identity of the genetic change, the compositional data of the GM food, the results from in-vitro/ex-vivo studies as well as the results from the preceding 28-day toxicity study with the novel gene product, before the hazard characterisation is concluded. The results demonstrated the ability of the 90-day rat feeding study to detect the biological/toxicological effects of the new gene product in the GM food. The authors consider on this basis that the 90-day, rodent feeding study with one high dose level and a dietary design based upon compositional data on the GM food and toxicity data on the gene product is sensitive and specific enough to verify the presence/absence of the biological/nutritional/toxicological effects of the novel gene insert and further by the use of spiking able to separate potentially unintended effects of the novel gene product from other unintended effects at the level of intake defined in the test and within the remit of the test. Recommendations for further work necessary in the field are given.

Evolutionary analysis of the APA genes in the Phaseolus genus: wild and cultivated bean species as sources of lectin-related resistance factors?

The APA (Arcelin/Phytohemagglutinin/alpha-Amylase inhibitor) gene family is composed of various members, present in Phaseolus species and coding for lectin and lectin-related seed proteins having the double role of storage and defense proteins. Here members of the APA family have been identified by immunological, functional, and molecular analyses and representative genes were sequenced in nine wild species of Phaseolus. All taxa possessed at least one member of the true lectin gene. No arcelin type sequences have been isolated from the species examined. Among the wild species studied, only P. costaricensis contained an alpha-amylase inhibitor (alpha-AI). In addition P. augusti, P. maculatus, P. microcarpus, and P. oligospermus showed the presence of the lectin-related alpha-amylase inhibitor-like (AIL) genes and alpha-AI activity. Data from Southern blot analysis indicated the presence of only one lectin gene in P. parvulus and P. filiformis, while an extensive gene duplication of the APA locus was found in the other Phaseolus species. Phylogenetic analysis carried out on the nucleotide sequences showed the existence of two main clusters and clearly indicated that lectin-related genes originated from a paralogous duplication event preceding the development of the ancestor to the Phaseolus genus. The finding of detectable alpha-AI activity in species containing AIL genes suggests that exploiting APA genes variability in the Phaseolus genus may represent a valuable tool to find new members that may have acquired insecticidal activities.

Safety testing of GM-rice expressing PHA-E lectin using a new animal test design.

The 90-day animal study is the core study for the safety assessment of genetically modified foods in the SAFOTEST project. The model compound tested in the 90-day study was a rice variety expressing the kidney bean Phaseolus vulgaris lectin agglutinin E-form (PHA-E lectin). Female Wistar rats were given a nutritionally balanced purified diet with 60% parental rice, 60% PHA-E rice or 60% PHA-E rice spiked with 0.1% recombinant PHA-E lectin for 90 days. This corresponded to a mean daily PHA-E lectin intake of approximately 0, 30 and 100mg/kg body weight for each group, respectively. The spiking was used to increase the specificity and to demonstrate the sensitivity of the study. A range of biological, biochemical, microbiological and pathological parameters were examined and significant differences in weight of small intestine, stomach and pancreas and plasma biochemistry were seen between groups. Included in this paper are also data from the molecular characterisation and chemical analysis of the PHA-E rice, from the construction and production of the PHA-E lectin, and from the preceding 28-day in vivo study where the toxicity of the pure PHA-E lectin was determined. In conclusion, the combined use of information from the compositional analysis, the 28-day study and the characterisation of the PHA-E rice and the PHA-E lectin has improved the design of the 90-day study. The spiking procedure has facilitated the interpretation of the results of the study and transferred it into a valuable tool for the future safety testing of genetically modified foods.

Agrobacterium-mediated transformation of chickpea with alpha-amylase inhibitor gene for insect resistance.

Chickpea is the world's third most important pulse crop and India produces 75% of the world's supply. Chickpea seeds are attacked by Callosobruchus maculatus and C. chinensis which cause extensive damage. The alpha-amylase inhibitor gene isolated from Phaseolus vulgaris seeds was introduced into chickpea cultivar K850 through Agrobacterium-mediated transformation. A total of 288 kanamycin resistant plants were regenerated. Only 0.3% of these were true transformants. Polymerase chain reaction (PCR) analysis and Southern hybridization confirmed the presence of 4.9 kb alpha-amylase inhibitor gene in the transformed plants. Western blot confirmed the presence of alpha-amylase inhibitor protein. The results of bioassay study revealed a significant reduction in the survival rate of bruchid weevil C. maculatus reared on transgenic chickpea seeds. All the transgenic plants exhibited a segregation ratio of 3:1.

Development of four phylogenetically-arrayed BAC libraries and sequence of the APA locus in Phaseolus vulgaris.

The APA family of seed proteins consists of three subfamilies, in evolutionary order of hypothesized appearance: phytohaemagglutinins (PHA), alpha-amylase inhibitors (alphaAI), and arcelins (ARL). The APA family plays a defensive role against mammalian and insect seed predation in common bean (Phaseolus vulgaris L.). The main locus (APA) for this gene family is situated on linkage group B4. In order to elucidate the pattern of duplication and diversification at this locus, we developed a BAC library in each of four different Phaseolus genotypes that represent presumptive steps in the evolutionary diversification of the APA family. Specifically, BAC libraries were established in one P. lunatus (cv. 'Henderson: PHA+ alphaAI- ARL-) and three P. vulgaris accessions (presumed ancestral wild G21245 from northern Peru: PHA+ alphaAI+ ARL-; Mesoamerican wild G02771: PHA+ alphaAI+ ARL+; and Mesoamerican breeding line BAT93: PHA+ alphaAI+ ARL-). The libraries were constructed after HindIII digestion of high molecular weight DNA, obtained with a novel nuclei isolation procedure. The frequency of empty or cpDNA-sequence-containing clones in all libraries is low (generally <1%). The Henderson, G21245, and G02771 libraries have a 10x genome coverage, whereas the BAT93 library has a 20x coverage to allow further, more detailed genomic analysis of the bean genome. The complete sequence of a 155 kbp-insert clone of the G02771 library revealed six sequences belonging to the APA gene family, including members of the three subfamilies, as hypothesized. The different subfamilies were interspersed with retrotransposon sequences. In addition, other sequences were identified with similarity to chloroplast DNA, a dehydrin gene, and the Arabidopsis flowering D locus. Linkage between the dehydrin gene and the D1711 RFLP marker identifies a potential syntenic region between parts of common bean linkage group B4 and cowpea linkage group 2.

Identification of a cDNA clone encoding for a galactose-binding lectin from peanut (Arachis hypogaea) seedling roots.

A cDNA clone obtained from developing peanut (Arachis hypogaea) seedling roots, when expressed in Escherichia coli and insect cells (Sf9) gave a 29 kDa subunit protein. The native recombinant protein agglutinates neuraminidase treated human erythrocytes and the agglutination is inhibited by galactose. Nucleotide sequence and predicted amino acid sequence analyses indicate that it is different from peanut seed (PNA and SGL) and nodule (NGLa and NGLb) galactose-binding lectins.

Mutated plant lectin library useful to identify different cells.

The 24 nucleotides comprising the carbohydrate-recognition domain of Maackia amurensis hemagglutinin (MAH) cDNA were randomly mutated. The mutant lectins were expressed as glutathione-S-transferase fusion proteins in Escherichia coli and 16 clones were randomly chosen. Although all of 16 recombinant lectins reacted strongly with anti-MAH polyclonal antibody, the carbohydrate-recognition domain of each was unique. As shown by agglutination studies, each mutant MAH lectin was able to bind to erythrocytes from one or more of five animal species in very distinct patterns. Thus, novel plant lectin libraries can be used to discriminate in a highly specific manner among a variety of cell types. This technology may prove to be very useful in a number of different applications requiring a high level of specificity in cell identification.

Functional phytohemagglutinin (PHA) and Galanthus nivalis agglutinin (GNA) expressed in Pichia pastoris correct N-terminal processing and secretion of heterologous proteins expressed using the PHA-E signal peptide.

Phytohemagglutinin (Phaseolus vulgaris agglutinin; PHA; E- and L-forms) and snowdrop lectin (Galanthus nivalis agglutinin; GNA) were expressed in Pichia pastoris using native signal peptides, or the Saccharomyces alpha-factor preprosequence, to direct proteins into the secretory pathway. PHA and GNA were present as soluble, functional proteins in culture supernatants when expressed from constructs containing the alpha-factor preprosequence. The recombinant lectins, purified by affinity chromatography, agglutinated rabbit erythrocytes at concentrations similar to the respective native lectins. However, incomplete processing of the signal sequence resulted in PHA-E, PHA-L and GNA with heterogenous N-termini, with the majority of the protein containing N-terminal extensions derived from the alpha-factor prosequence. Polypeptides in which most of the alpha-factor prosequence was present were also glycosylated. Inclusion of Glu-Ala repeats at the C-terminal end of the alpha-factor preprosequence led to efficient processing N-terminal to the Glu-Ala sequence, but inefficient removal of the repeats themselves, resulting in polypeptides with heterogenous N-termini still containing N-terminal extensions. In contrast, PHA expressed with the native signal peptide was secreted, correctly processed, and also fully functional. No expression of GNA from a construct containing the native GNA signal peptide was observed. The PHA-E signal peptide directed correct processing and secretion of both GNA and green fluorescent protein (GFP) when used in expression constructs, and is suggested to have general utility for synthesis of correctly processed proteins in Pichia.

CENP-B autoantigen is a conserved protein from humans to higher plants: identification of the aminoterminal domain in Phaseolus vulgaris.

Centromeres are critical structures in cell division, and CENP-B is the most important protein of the centromeric complex recognized by autoantibodies from patients with scleroderma. Our major aim was to demonstrate whether CENP-B is a conserved protein along the phylogenic scale including the higher plants. Vegetal and human cell proteins were extracted from Phaseolus vulgaris and HEp-2 cells and were characterized by PAGE, Western blot, and human autoimmune sera containing anti-CENP-B autoantibodies. The aminoterminus of the gene encoding for CENP-B from HEp-2 cells and Phaseolus vulgaris was isolated by reverse transcriptase-PCR using complementary oligonucleotides to the human CENP-B gene. Also, in situ hybridization was performed on vegetal tissues and HEp-2 cells using human CENP-B box probes. Our main results were as follows: 1) Autoimmune sera were reactive to a vegetal protein of 80 kDa. 2) Affinity-purified anti-CENP-B antibodies recognized a protein from Phaseolus vulgaris with molecular mass similar to that found in human cells. Vegetal and HEp-2 cells CENP-B proteins were immunologically identical. 3) Using RT-PCR, we were able to amplify a cDNA encoding for the aminoterminus domain of CENP-B from Phaseolus vulgaris that had the same molecular behaviour as the cDNA from HEp-2 cells. 4) Complementary oligonucleotides for human CENP-B box hybridized a DNA sequence from Phaseolus vulgaris. In conclusion, CENP-B protein is a conserved protein along the phylogenic scale from humans to higher plants.

Sialic acid-binding motif of Maackia amurensis lectins.

Maackia amurensis hemagglutinin (MAH) and leukoagglutinin (MAL) are leguminous lectins which recognize carbohydrate chains containing sialic acid residues linked alpha2,3 to penultimate galactose residues. In the present investigation, cDNA clones encoding MAL were isolated from a cDNA library constructed from germinated Maackia amurensis seeds and sequenced. From the reading frame of the cloned cDNAs, MAL was predicted to be composed of 287 amino acid residues, and showed strong similarity to MAH (86.2% identity). In leguminous lectins, most amino acid residues involved in sugar-binding were previously shown to be conserved. However, in both MAL and MAH lectins, the conserved glycine and asparagine were shown to be substituted by lysine and aspartic acid, respectively. Substitutions were made at position 105 and/or 135 of MAH to examine the roles of amino acid residues postulated to be important in binding to sialic acids. Recombinant MAH bound to the sialic acid-containing CB-II glycopeptide of human glycophorin A. By contrast, mutant lectins with lysine-105 substituted with glycine and/or aspartic acid-135 with asparagine did not bind to sialic acid residues. This indicates that these characteristic substitutions are important in sialic acid binding.

Conserved RY-repeats mediate transactivation of seed-specific promoters by the developmental regulator PvALF.

Transcription of genes DLEC2 and PHSbeta is specifically and coordinately regulated during maturation of Phaseolus embryos. Over-expression of the seed- specific factor PvALF in cotyledon cells results in transactivation of either promoter. PvALF is related to the VP1 protein of maize, which transactivates gene expression via G-boxes, Sph elements and AT-rich sequences. We used deletions and base substitutions in the DLEC2 and PHSbeta promoters to demonstrate that several conserved RY-repeats were necessary for PvALF induction of both genes. A comprehensive mutational and transactivation analysis was used to define functionally the sequence of the DLEC2 repeat RY3 asG/CCATGCxxG/C. We also found that an interaction between RY3 and the 3'-flanking tetranucleotide CCAC increased PvALF transactivation. A preferred spacing and phasing requirement for the RY3 and CCAC motifs suggested the possibility of interactions between cellular factors that recognize either element. The high conservation of Sph-RY motifs in seed-specific promoters from monocots and dicots indicates that organ and temporal specification by factors similar to VP1 and PvALF is common among seed plants.

Expression of a synthetic antifreeze protein in potato reduces electrolyte release at freezing temperatures.

A synthetic antifreeze protein gene was expressed in plants and reduced electrolyte leakage from the leaves at freezing temperatures. The synthetic AFP was expressed as a fusion to a signal peptide, directing it to the extracytoplasmic space where ice crystallization first occurs. The gene was introduced to Solanum tuberosum L. cv. Russet Burbank by Agrobacterium-mediated transformation. Transformants were identified by PCR screening and expression of the introduced protein was verified by immunoblot. Electrolyte-release analysis of transgenic plant leaves established a correlation between the level of transgenic protein expression and degree of tolerance to freezing. This is the first identification of a phenotype associated with antifreeze protein expression in plant tissue.

Rare codons are not sufficient to destabilize a reporter gene transcript in tobacco.

In plants, as in other eukaryotes, most synonymous codons of the genetic-code are not used with equal frequency, but instead some codons are preferred, whereas others are rare. Circumstantial evidence led to the suggestion that rare codons have a negative influence on mRNA stability. To address this question experimentally, rare codons encoded by a Bacillus thuringiensis (B.t.) toxin gene (cryIA(c)) or a synthetic sequence were introduced into a phytohemagglutinin (PHA) reporter gene. In neither case was the mRNA stability appreciably diminished in stably transformed tobacco cell cultures nor was the accumulation of mRNA in transgenic plants affected. Thus rare codons do not appear to be sufficient to cause rapid degradation of the PHA mRNA and potentially other mRNAs in plants.

Premature nonsense codons decrease the stability of phytohemagglutinin mRNA in a position-dependent manner.

Premature termination of translation has often been associated with decreased mRNA accumulation in plants, but the affected step in gene expression has not been identified. To investigate this problem, the expression of wild-type and mutant alleles of the bean phytohemagglutinin (PHA) gene has been examined in tobacco cells and transgenic plants. Measurement of mRNA decay rates in stably transformed cell lines demonstrated that premature nonsense codons markedly destabilized the mRNA. This decreased stability was also reflected by decreased accumulation of transcripts containing premature nonsense codons in transgenic plants. The positional dependence of the nonsense codon effect was evaluated by introducing premature nonsense codons at different distances from the PHA AUG start codon. Transcripts with nonsense codons about 20, 40 or 60% of the way through the normal PHA coding region yielded highly unstable mRNAs, whereas a transcript with a nonsense codon at 80% was as stable as wild-type. The ability to recognize and rapidly degrade certain transcripts with early nonsense codons could provide plant cells with a means to minimize the production of wasteful and possible deleterious truncated proteins.

Molecular cloning of two different mannose-binding lectins from tulip bulbs.

Two lectins were isolated from the bulbs of Tulipa cv. Apeldoorn and their corresponding cDNA clones analyzed. The first, called TxLMII (second mannose-binding Tulipa hybrid lectin), is a novel mannose-binding tulip lectin. Based on its molecular structure, carbohydrate-binding specificity and amino acid sequence, TxLMII belongs to the superfamily of mannose-binding monocot lectins which are also found in representatives of the plant families Amaryllidaceae, Alliaceae, Orchidaceae and Araceae. Molecular cloning of the second lectin, called TxLCI (first Tulipa hybrid lectin with complex specificity), allowed determination unambiguously of the molecular structure of this previously described protein. In addition, evidence is presented that each TxLCI subunit possesses a mannose-binding site and an N-acetylgalactosamine-binding site, which act independently of each other. Both binding sites are located in a separate domain of the lectin polypeptide. Since the first domain of TxLCI shows sequence similarity to TxLMII, it is suggested that their genes evolved from a common ancestor.