Electrophoretic separation of gill proteins of the clam Codakia orbicularis

Marine organisms inhabiting the coastal environment of the Caribbean islands have attracted the attention of a number of research scientists. These organisms generally live in areas of high environmental stress and may, therefore, contain specialized proteins and enzymes which exhibit valuable biotechnological applications. Among them, a small number of clams have been studied. Our work relates to the clam Codakia orbicularis. This bivalve lives in areas of high hydrogen sulfide concentrations on the Caribbean island of Guadeloupe. Its enzymatic system must, therefore, have evolved to allow its adaptation to this high-stress environment. C. orbicularis also contains endosymbiotic bacteria which are housed in the bacteriocytes of the gills. Its protein content can, therefore, be expected to have an impact on this symbiotic relationship. We have analysed gill protein extracts of this clam by various biochemical techniques: SDS-PAGE, IEF, PAS, and Western blotting using a panel of lectins, in order to establish its protein and glycoprotein profiles. This biochemical analysis, the first of its kind, constitutes an important step in separating and characterizing the proteins involved in the biochemical pathways of this organism whose stock is in decline in Guadeloupe. Our results show the presence of three major proteins whose molecular weights vary between 14,000 and 24,000 Daltons, and some of which are glycoproteins with predominantly alpha-mannose and N-acetylgalactosamine moieties. Their pI values are in the range between 4.5 and 5.6. These protein profiles are different from those observed for Lucina pectinata, a clam which has been the subject of earlier studies in the literature.     

Two-domain hemoglobin from the blood clam,Barbatia lima. The cDNA-derived amino acid sequence

The blood clam,Barbatia lima, from Kochi, Japan, expresses a tetrameric (α ₂ β ₂) and a polymeric hemoglobin in erythrocytes. The latter hemoglobin is composed of unusual 34-kDa hemoglobin with a two-domain structure, and its molecular mass (about 430 kDa) is exceptionally large for an intracellular hemoglobin. The 3′ and 5′ parts of the cDNA ofB. lima two-domain globin have been amplified separately by polymerase chain reaction and the complete nucleotide sequence of 1147 bp was determined. The open reading frame is 930 nucleotides in length and encodes a protein with 309 amino acid residues, of which 73 amino acids were identified directly by protein sequencing. The mature protein begins with the acetylated Ser, and thus the N-terminus Met is cleaved. The molecular mass for the protein was calculated to be 35,244 Da. The cDNA-derived amino acid sequence ofB. lima two-domain globin shows 89% homology with that of two-domain globin fromB. reeveana, a North American species. The sequence homology between the two domains is 75%, suggesting that the two-domain globin resulted from the gene duplication of an ancestral 17-kDa globin.     

Environmental transmission of a sulfur-oxidizing bacterial gill endosymbiont in the tropical lucinid bivalve Codakia orbicularis.

Codakia orbicularis is a large tropical member of the bivalve mollusk family Lucinidae which inhabits shallow-water sea-grass beds (Thalassia testudinum environment) and harbors sulfur-oxidixing endosymbiotic bacteria within bacteriocytes of its gill filaments. When a C. orbicularis-specific 16S rDNA (DNA encoding rRNA) primer is used with a bacterium-specific 16S rDNA reverse primer in amplifications by PCR, the primer set was unsuccessful in amplifying symbiont DNA targets from ovaries, eggs, veligers, and metamorphosed juveniles (600 microns to 1 mm in shell length) cultivated in sterile sand, whereas successful amplifications were obtained from gill tissue of adult specimens and from metamorphosed juveniles (600 microns to 1 mm in shell length) cultivated in unsterilized sea-grass bed sand. To ascertain the presence of the symbiont target in juveniles, restriction fragment length polymorphism analysis, Southern blotting, and transmission electron microscopy were used. Specific hybridizations and observation of endosymbiotic bacteria in the gills of numerous juveniles cultivated in unsterilized sea-grass bed sand showed that the sulfur-oxidizing endosymbionts of C. orbicularis are environmentally transmitted to the new generation after larval metamorphosis.     

Expression and purification of a novel mannose-binding lectin from Pinellia ternata

Pinellia ternata agglutinin (PTA) from the tubers of P. ternata is a monocot mannose-binding lectin that catalytically agglutinated rabbit erythrocytes. The potential effect of PTA has gained considerable interest in recent years owing to clinical use of native PTA as the preparation against cancer and for plant protection against insect pests. Here we report a successful strategy to allow high-level expression of PTA as inclusion bodies in Escherichia coli M15. Purification of refolded recombinant protein from solubilized inclusion bodies by Ni-NTA agarose affinity chromatography yielded biological activity recombinant PTA (final yield of about 10 mg/L). The recombinant PTA agglutinated rabbit erythrocytes to a dilution similar to that determined for “native” lectin purified from P. ternata. The expression and purification system makes it possible to obtain sufficient quantities of biologically active and homogenous recombinant PTA sufficient to carry out advanced clinical trials. This is the first report on the large-scale expression and purification of biologically active recombinant PTA from E. coli.     

Expression and purification of a novel mannose-binding lectin from Pinellia ternata

Pinellia ternata agglutinin (PTA) from the tubers of P. ternata is a monocot mannose-binding lectin that catalytically agglutinated rabbit erythrocytes. The potential effect of PTA has gained considerable interest in recent years owing to clinical use of native PTA as the preparation against cancer and for plant protection against insect pests. Here we report a successful strategy to allow high-level expression of PTA as inclusion bodies in Escherichia coli M15. Purification of refolded recombinant protein from solubilized inclusion bodies by Ni-NTA agarose affinity chromatography yielded biological activity recombinant PTA (final yield of about 10 mg/L). The recombinant PTA agglutinated rabbit erythrocytes to a dilution similar to that determined for "native" lectin purified from P. ternata. The expression and purification system makes it possible to obtain sufficient quantities of biologically active and homogenous recombinant PTA sufficient to carry out advanced clinical trials. This is the first report on the large-scale expression and purification of biologically active recombinant PTA from E. coli.     

Two-domain hemoglobin from the blood clam,Barbatia lima. The cDNA-derived amino acid sequence

The blood clam,Barbatia lima, from Kochi, Japan, expresses a tetrameric ( _{2 2}) and a polymeric hemoglobin in erythrocytes. The latter hemoglobin is composed of unusual 34-kDa hemoglobin with a two-domain structure, and its molecular mass (about 430 kDa) is exceptionally large for an intracellular hemoglobin. The 3 and 5 parts of the cDNA ofB. lima two-domain globin have been amplified separately by polymerase chain reaction and the complete nucleotide sequence of 1147 bp was determined. The open reading frame is 930 nucleotides in length and encodes a protein with 309 amino acid residues, of which 73 amino acids were identified directly by protein sequencing. The mature protein begins with the acetylated Ser, and thus the N-terminus Met is cleaved. The molecular mass for the protein was calculated to be 35,244 Da. The cDNA-derived amino acid sequence ofB. lima two-domain globin shows 89% homology with that of two-domain globin fromB. reeveana, a North American species. The sequence homology between the two domains is 75%, suggesting that the two-domain globin resulted from the gene duplication of an ancestral 17-kDa globin.     

Molecular cloning and characterization of a novel mannose-binding lectin gene from Amorphophallus konjac

A new lectin gene was cloned from Amorphophallus konjac. The full-length cDNA of Amorphophallus konjac agglutinin (aka) was 736 bp and contained a 474 bp open reading frame encoding a 158 amino acid protein. Homology analysis revealed that the lectin from this Araceae species belonged to the superfamily of monocot mannose-binding proteins. Molecular modeling of AKA indicated that the three-dimensional structure of AKA strongly resembles that of the snowdrop lectin. Southern blot analysis of the genomic DNA revealed that aka belonged to a low-copy gene family. Northern blot analysis demonstrated that aka expression was tissue-specific with the strongest expression being found in root.     

Isolation, characterization, and cDNA-derived amino acid sequence of glycocyamine kinase from the tropical marine worm Namalycastis sp

We isolated cytoplasmic glycocyamine kinase (GK) and creatine kinase (CK) from the tropical marine worm Namalycastis sp. by ammonium sulfate fractionation, gel filtration on Sephacryl S-200, and DEAE-5PW chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the isolated GK is highly purified and appears to be a heterodimer of two distinct subunits, alpha and beta, with molecular masses of approximately 40 kDa. The complete nucleotide sequences of the cDNAs for Namalycastis GKalpha and GKbeta were 1527 (encoding 374 amino acids) and 1579 bp (encoding 390 amino acids), respectively. The predicted amino acid sequences differ only in the N-terminal 50 residues. This is consistent with the characteristics of Neanthes GKalpha and GKbeta chains, which we have previously shown to be generated by alternative splicing. The recombinant enzymes GKalpha, GKbeta, and CK from Namalycastis were successfully expressed in Escherichia coli as maltose-binding protein fusion proteins. In contrast to the stable GKbeta enzyme, GKalpha was quite unstable, and its activity decreased remarkably with time. Thus, the N-terminal 50 residues appear to play a key role in enzyme stability. The kinetic parameters for the native GK heterodimer were similar to GKbeta, suggesting that GKalpha would have an activity similar to GKbeta if part of a heterodimer. This is the first report of precise kinetic parameters for GK. Finally, based on our results, we present a model for pluriphosphagen function in Namalycastis wherein cytoplasmic GK and CK and mitochondrial CK function together with phosphocreatine and phosphoglycocyamine to enable cells to respond quickly to a sudden large energy requirement.     

Purification and antibacterial characterization of a novel isoform of the Manila clam lectin (MCL-4) from the plasma of the Manila clam, Ruditapes philippinarum

In many bivalve molluscs, lectins are present in the hemolymph and are thought to be important for internal host defense mechanisms. For this study, we purified a novel isoform of the Manila clam lectin (designated MCL-4) from the plasma of the Manila clam, Ruditapes philippinarum, using affinity chromatography and gel filtration. Native PAGE results showed that the MCL-4 consisted of 70 kDa protein. MCL-4 was found to be composed of 58-kDa and 43-kDa bands when examined using SDS-PAGE under reducing and non-reducing conditions. The native MCL-4 was revealed as a 147 kDa molecular mass protein by gel filtration. The purified MCL-4 agglutinates calcium-dependently in the erythrocytes of sheep and rabbit, but not in cells of the three species of marine bacteria tested. However, the phagocytic ability of the R. philippinarum hemocytes for the MCL-4-opsonized Vibrio tubiashii cells was significantly greater than that for the BSS-treated bacterial cells. Addition of purified MCL-4 markedly suppressed Alteromonas haloplanktis growth. These results suggest that MCL-4, because of its opsonizing and bacteriostatic properties, might contribute to the host defense mechanisms against invading microorganisms in R. philippinarum.     

Crystallization and preliminary x-ray diffraction analysis of a novel mannose-binding lectin with antiretroviral properties from Polygonatum cyrtonema hua

A novel antiretroviral protein Polygonatum cyrtonema lectin (PCL) belonging to the monocot mannose-binding lectin (MMBL) superfamily has been crystallized using hanging-drop vapor-diffusion method. The crystals diffract to 2.0 A resolution and belong to space group P2(1), with unit-cell parameters of a=39.308 A, b=48.317 A, c=112.221 A, and beta=90.12 degrees . Preliminary analysis indicates that the asymmetric unit contains four PCL molecules with a solvent content of about 45%. A set of X-ray data has been collected for the crystal structure determination.     

Isolation of mannose-binding C-type lectin from Heliothis virescens pupae

A mannose-binding C-type lectin (MBL) was isolated by affinity chromatography from Heliothis virescens immune pupal hemolymph. The immune pupal hemolymph was obtained after bacterial injection of live Enterobacter cloacae bacteria. MBL in mammals acts as an opsonin for phagocytosis and activates the lectin complement pathway of the innate immune response, which leads to killing of gram-negative bacteria and enveloped viruses. The affinity-purified and reduced pupal MBL showed a single band of 36 kDa by SDS-PAGE (12% gel). A dot-immunoblot ELISA (using guinea pig anti-MBL IgG as primary antibody) demonstrated specificity of the antibody for the affinity-purified pupal MBL. The immune pupal hemolymph contained 21 microg of MBL per ml of hemolymph. The amino acid composition of the purified pupal MBL was determined with high amounts of arginine and histidine detected. The presence of MBL in insect pupae has not before been reported and could be important in pupal innate immunity to bacterial infection.     

KM+, a mannose-binding lectin from Artocarpus integrifolia: amino acid sequence, predicted tertiary structure, carbohydrate recognition, and analysis of the beta-prism fold

The complete amino acid sequence of the lectin KM+ from Artocarpus integrifolia (jackfruit), which contains 149 residues/mol, is reported and compared to those of other members of the Moraceae family, particularly that of jacalin, also from jackfruit, with which it shares 52% sequence identity. KM+ presents an acetyl-blocked N-terminus and is not posttranslationally modified by proteolytic cleavage as is the case for jacalin. Rather, it possesses a short, glycine-rich linker that unites the regions homologous to the alpha- and beta-chains of jacalin. The results of homology modeling implicate the linker sequence in sterically impeding rotation of the side chain of Asp141 within the binding site pocket. As a consequence, the aspartic acid is locked into a conformation adequate only for the recognition of equatorial hydroxyl groups on the C4 epimeric center (alpha-D-mannose, alpha-D-glucose, and their derivatives). In contrast, the internal cleavage of the jacalin chain permits free rotation of the homologous aspartic acid, rendering it capable of accepting hydrogen bonds from both possible hydroxyl configurations on C4. We suggest that, together with direct recognition of epimeric hydroxyls and the steric exclusion of disfavored ligands, conformational restriction of the lectin should be considered to be a new mechanism by which selectivity may be built into carbohydrate binding sites. Jacalin and KM+ adopt the beta-prism fold already observed in two unrelated protein families. Despite presenting little or no sequence similarity, an analysis of the beta-prism reveals a canonical feature repeatedly present in all such structures, which is based on six largely hydrophobic residues within a beta-hairpin containing two classic-type beta-bulges. We suggest the term beta-prism motif to describe this feature.     

Molecular cloning and characterization of a mannose-binding lectin gene from Crinum asiaticum

Full-length cDNA of a mannose-binding lectin or agglutinin gene was cloned from a traditional Chinese medicinal herb Crinum asiaticum var. sinicum through RACE-PCR cloning. The full-length cDNA of C. asiaticum agglutinin (caa) was 820 bp and contained a 528 bp open reading frame encoding a lectin precursor (preproprotein) of 175 amino acid residues with a 22 aa signal peptide. The coding region of the caa gene was high in G/C content. The first 20 bp of the 5' UTR had a dC content of 50%, which was a typical feature of the leader sequence. By cutting away the signal peptide, the CAA proprotein was 15.79 kDa with a pl of 9.27 and contained 3 mannose-binding sites (QDNY). Random coil and extended strand constituted interlaced domination of the main part of the secondary structure. B-lectin conserved domain existed within N24 to G130. Predicted three-dimensional structure of CAA proprotein was very similar to that of GNA (Galanthus nivalis agglutinin). It is significant that besides certain homologies to known monocot mannose-binding lectins from Amaryllidaceae, Orchidaceae, Alliaceae and Liliaceae, caa also showed high similarity to gastrodianin type antifungal proteins. No intron was detected within the region of genomic sequence corresponding to the caa full-length cDNA. Southern blot analysis indicated that the caa gene belonged to a low-copy gene family. Northern blot analysis demonstrated that caa mRNA was constitutively expressed in all the tested tissue types including the root, bulb, leaf, rachise, flower and fruit tissues.     

Effects of Long-Term Starvation on a Host Bivalve (Codakia orbicularis,Lucinidae) and Its Symbiont Population

The bivalve Codakia orbicularis, hosting sulfur-oxidizing gill endosymbionts, was starved (in artificial seawater filtered through a 0.22-μm-pore-size membrane) for a long-term experiment (4 months). The effects of starvation were observed using transmission electron microscopy, fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH), and flow cytometry to monitor the anatomical and physiological modifications in the gill organization of the host and in the symbiotic population housed in bacteriocytes. The abundance of the symbiotic population decreased through starvation, with a loss of one-third of the bacterial population each month, as shown by CARD-FISH. At the same time, flow cytometry revealed significant changes in the physiology of symbiotic cells, with a decrease in cell size and modifications to the nucleic acid content, while most of the symbionts maintained a high respiratory activity (measured using the 5-cyano-2,3-ditolyl tetrazolium chloride method). Progressively, the number of symbiont subpopulations was reduced, and the subsequent multigenomic state, characteristic of this symbiont in freshly collected clams, turned into one and five equivalent genome copies for the two remaining subpopulations after 3 months. Concomitant structural modifications appeared in the gill organization. Lysosymes became visible in the bacteriocytes, while large symbionts disappeared, and bacteriocytes were gradually replaced by granule cells throughout the entire lateral zone. Those data suggested that host survival under these starvation conditions was linked to symbiont digestion as the main nutritional source.The entire marine Lucinidae family, found in a wide range of sulfidic habitats, lives in association with chemoautotrophic sulfide-oxidizing bacterial symbionts, generally hosted in the gills of the bivalve. Lucinids are usually found in shallow water, such as intertidal mud or seagrasses (4, 53), in deeper water, e.g., Bathyaustriella thionipta (30), and in deep oceans at a 2,000-m depth, i.e., Lucinoma kazani (21, 55). The chemoautotrophic endosymbionts involved in such relationships are always localized inside specialized cells called bacteriocytes, and they have been found in several genera of the Lucinidae family, such as Codakia (4, 28), Loripes (39, 43), Lucina, and Lucinoma (17). Sulfur granules inside the symbiont cytoplasm have been demonstrated in most of the investigated species. The intracellular symbionts take energy from the oxidation of reduced sulfur compounds (27, 56, 59) and synthesize organic molecules by CO₂ fixation in a Calvin-Benson cycle, translocated to the host (18). This relationship between the host and its symbionts represents the autotrophic pathway for host nutrition (27). It has also been suggested that in symbiotic bivalves, intracellular digestion of the symbionts may be a nutrient source for the host, based on studies of hydrothermal vent and shallow water bivalves (6, 26, 39).The relative importance of the autotrophic versus heterotrophic nutritional pathway can be estimated by measuring the carbon isotope (δ-¹³C) ratios in the host tissue. Measuring this δ-¹³C ratio on a wide range of invertebrates suggested that bivalves, including members of the Lucinidae, that live in reduced sediment may obtain a significant proportion of their organic carbon from chemoautotrophic endosymbionts (4, 10, 51, 52, 57). This suggestion was in agreement with the reduced functional digestive system previously described for the Lucinidae family (2, 53). Structural and morphological studies of gills of a few lucinids (belonging to the genera Lucina and Lucinoma) strongly suggested that symbionts play an important role in host nutrition since they occupy about 30% of the gill tissue and produce most of the host energy (17). Nevertheless, an alternative pathway for feeding, i.e., heterotrophic particulate feeding, could occur in some of the lucinid bivalves, since diatoms were found in the stomach of some lucinids (57). Duplessis et al. (22) showed that particulate feeding could be an important part of the nutritional strategy in symbiont-bearing Lucinoma, as opposed to the anatomical features that gave the impression that this bivalve relied only on symbiont nutrition.In natural habitats, chemoautotrophic bivalves live at the interface between an anoxic sulfide-generating zone and water column oxygenated sediment. However, even if they are not close to a vent, these symbiotic organisms often have to deal with environments that are periodically depleted of oxygen (5, 12) and with extremely low sulfide concentration (13, 15, 16). These natural environmental variations lead to annual and seasonal changes in the δ-¹³C ratio, as observed for some thyasirid species (13, 14). This δ-¹³C ratio variation may be assumed to correspond to the capability of the host to rely on both autotrophic and heterotrophic pathways, and the preponderance of one pathway versus the other in the mixotrophic diet has been considered to be the way in which these organisms deal with changes in the chemical composition of their environment.Apart from the decrease in symbiont abundance suggested by transmission electron microscopy (TEM) analysis and a decrease in sulfur and protein content in the gill tissue of thyasirids (20, 38, 40), little is known about the physiological status of these symbionts and the changes undergone by the symbiotic population of starved bivalves. A previous study of the population was carried out under natural conditions with Codakia orbicularis, a chemoautotrophic bivalve. This tropical bivalve lives in shallow-water sediment among the roots of seagrasses (Thalassia testudinum) (1). Like all lucinids studied so far, it is associated with sulfur-oxidizing symbionts (4, 27, 28) containing elemental sulfur in their cytoplasm (42). The bacterial symbiont of C. orbicularis, environmentally transmitted to the host (31), belongs to a single taxonomic group (Gammaproteobacteria) (25) and is shared by several other tropical lucinids (24, 25, 34, 35). Only a few data are available on the physiology of this symbiont. It was characterized by the presence of Rubisco and ATP sulfurylase enzymes and a δ-¹³C ratio typical of chemoautotrophic bivalves (4). Unlike other related thyasirids tested for nitrate respiration even under oxygenated conditions (37), the symbionts of C. orbicularis use oxygen as the primary electron acceptor (23). Initial investigations of the population of Codakia orbicularis'' symbiont revealed that the symbiotic population hosted by freshly collected individuals contained a high proportion of large bacterial cells containing multiple copies of their genome, typical of actively growing cells, despite the absence of dividing cells (11). It was assumed that the host could maintain a pure culture of the symbiont inside the bacteriocytes by regulating the entry and growth of newly recruited symbionts from sediment and probably regulating symbiont densities by host digestion (11).This study was undertaken to investigate the dynamics of the symbiotic population hosted by C. orbicularis under experimental conditions based on long-term starvation of bivalves, i.e., incubated without planktonic food. We set out the ultrastructural, structural, and physiological changes that occurred in the symbiont population by examining the host gill sections using TEM and fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH). A purified fraction of gill endosymbionts was analyzed by flow cytometry (FCM) to investigate the nucleic acid content and cell size of symbionts and by using the 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) method and epifluorescence microscopy to detect the respiratory activity of symbionts. The modifications induced by host starvation in the symbiotic population are described for the period of long-term starvation.     

Curculin,a sweet-tasting and taste-modifying protein,is a non-functional mannose-binding lectin

A three-dimensional model of curculin, a sweet-tasting and taste-modifying protein from the fruits of Curculigo latifolia, was built from the X-ray coordinates of GNA, a mannose-binding lectin from snowdrop (Galanthus nivalis). The three mannose-binding sites present in GNA were found in curculin but are devoid of mannose-binding activity as shown by docking experiments performed with mannose. Some regions well exposed on the surface of the three-dimensional model of curculin could act as epitopes responsible for the sweet-tasting properties of this protein.     

Respiration Strategies Utilized by the Gill Endosymbiont from the Host Lucinid Codakia orbicularis (Bivalvia: Lucinidae)

The large tropical lucinid clam Codakia orbicularis has a symbiotic relationship with intracellular, sulfide-oxidizing chemoautotrophic bacteria. The respiration strategies utilized by the symbiont were explored using integrative techniques on mechanically purified symbionts and intact clam-symbiont associations along with habitat analysis. Previous work on a related symbiont species found in the host lucinid Lucinoma aequizonata showed that the symbionts obligately used nitrate as an electron acceptor, even under oxygenated conditions. In contrast, the symbionts of C. orbicularis use oxygen as the primary electron acceptor while evidence for nitrate respiration was lacking. Direct measurements obtained by using microelectrodes in purified symbiont suspensions showed that the symbionts consumed oxygen; this intracellular respiration was confirmed by using the redox dye CTC (5-cyano-2,3-ditolyl tetrazolium chloride). In the few intact chemosymbioses tested in previous studies, hydrogen sulfide production was shown to occur when the animal-symbiont association was exposed to anoxia and elemental sulfur stored in the thioautotrophic symbionts was proposed to serve as an electron sink in the absence of oxygen and nitrate. However, this is the first study to show by direct measurements using sulfide microelectrodes in enriched symbiont suspensions that the symbionts are the actual source of sulfide under anoxic conditions.     

Structural mechanism governing the quaternary organization of monocot mannose-binding lectin revealed by the novel monomeric structure of an orchid lectin

Two isoforms of an antifungal protein, gastrodianin, were isolated from two subspecies of the orchid Gastrodia elata, belonging to the protein superfamily of monocot mannose-specific lectins. In the context that all available structures in this superfamily are oligomers so far, the crystal structures of the orchid lectins, both at 2.0 A, revealed a novel monomeric structure. It resulted from the rearrangement of the C-terminal peptide inclusive of the 12th beta-strand, which changes from the "C-terminal exchange" into a "C-terminal self-assembly" mode. Thus, the overall tertiary scaffold is stabilized with an intramolecular beta-sheet instead of the hybrid observed on subunit/subunit interface in all known homologous dimeric or tetrameric lectins. In contrast to the constrained extended conformation with a cis peptide bond between residues 98 and 99 commonly occurring in oligomers, a beta-hairpin forms from position 97 to 101 with a normal trans peptide bond at the corresponding site in gastrodianin, which determines the topology of the C-terminal peptide and thereby its unique fold pattern. Sequence and structure comparison shows that residue replacement and insertion at the position where the beta-hairpin occurs in association with cis-trans inter-conversion of the specific peptide bond (97-98) are possibly responsible for such a radical structure switch between monomers and oligomers. Moreover, this seems to be a common melody controlling the quaternary states among bulb lectins through studies on sequence alignment. The observations revealed a structural mechanism by which the quaternary organization of monocot mannose binding lectins could be governed. The mutation experiment performed on maltose-binding protein-gastrodianin fusion protein followed by a few biochemical detections provides direct evidence to support this conclusion. Potential carbohydrate recognition sites and biological implications of the orchid lectin based on its monomeric state are also discussed in this paper.     

Respiration strategies utilized by the gill endosymbiont from the host lucinid Codakia orbicularis (Bivalvia: Lucinidae)

The large tropical lucinid clam Codakia orbicularis has a symbiotic relationship with intracellular, sulfide-oxidizing chemoautotrophic bacteria. The respiration strategies utilized by the symbiont were explored using integrative techniques on mechanically purified symbionts and intact clam-symbiont associations along with habitat analysis. Previous work on a related symbiont species found in the host lucinid Lucinoma aequizonata showed that the symbionts obligately used nitrate as an electron acceptor, even under oxygenated conditions. In contrast, the symbionts of C. orbicularis use oxygen as the primary electron acceptor while evidence for nitrate respiration was lacking. Direct measurements obtained by using microelectrodes in purified symbiont suspensions showed that the symbionts consumed oxygen; this intracellular respiration was confirmed by using the redox dye CTC (5-cyano-2,3-ditolyl tetrazolium chloride). In the few intact chemosymbioses tested in previous studies, hydrogen sulfide production was shown to occur when the animal-symbiont association was exposed to anoxia and elemental sulfur stored in the thioautotrophic symbionts was proposed to serve as an electron sink in the absence of oxygen and nitrate. However, this is the first study to show by direct measurements using sulfide microelectrodes in enriched symbiont suspensions that the symbionts are the actual source of sulfide under anoxic conditions.     

Purification and cDNA-derived sequence of adenylosuccinate synthetase from Dictyostelium discoideum

Adenylosuccinate synthetase (IMP:L-aspartate ligase (GDP), EC 6.3.4.4) plays an important role in purine biosynthesis catalyzing the GTP-dependent conversion of IMP to AMP. The enzyme was purified from the cytosol of Dictyostelium discoideum using GTP-agarose chromatography as the critical step. It has an apparent molecular mass of 44 kDa. Monoclonal antibodies identified several forms of the enzyme with pI values between 8.1 and 9.0. Michaelis-Menten constants (Km) were low for the nucleotide substrates IMP (Km = 30 microM) and GTP (Km = 35 microM) as compared with the value for aspartic acid (Km = 440 microM). These values are in good agreement with constants reported from other organisms. Immunological studies indicated that the protein is predominantly localized in the cytosol and only partially associated with particulate fractions. The enzyme is present throughout the developmental cycle of D. discoideum. Using monoclonal antibodies, the gene was cloned from a lambda gt11 expression library. The complete sequence represents the first reported primary structure of an eucaryotic adenylosuccinate synthetase. Southern blots hybridized with a cDNA probe demonstrate that adenylosuccinate synthetase is encoded by a single gene and contains at least one intron. The deduced amino acid sequence shows 43% identity to adenylosuccinate synthetase from Escherichia coli. Homologous regions include short sequence motifs, such as the glycine-rich loop which is typical for GTP-binding proteins.