Buck (Capra hircus) genes encode new members of the spermadhesin family

Spermadhesins are the major proteins of boar seminal plasma and form a group of polypeptides probably involved in reproduction. In previous work, a member of the spermadhesin family from buck seminal plasma, called BSFP, was characterized by mass spectrometry and N-terminal sequencing. The present study aimed to clone and characterize the BSFP gene and investigate its expression along the genital tract using real-time polymerase chain reaction (PCR). The cDNAs of the seminal vesicle, testis, epididymis, bulbourethral gland, and ductus deferens were prepared from a buck. Following 3'- and 5'-end amplifications using seminal vesicle cDNA, we cloned and sequenced four highly similar (97-98%) nucleotide sequences encoding spermadhesins, which were named Bodhesin-1(Bdh-1), Bdh-2, Bdh-3, and Bdh-4. All deduced amino acid sequences contained the CUB domain signature and were 49-52% similar to boar AWN. Among the four Bdh amino acid sequences, Bdh-2 was the most similar to the BSFP N-terminal fragment. By using real-time PCR, it was verified specific amplifications for all Bdh in the seminal vesicle, testis, epididymis, and bulbourethral gland, with the exception of Bdh-2 in epididymis. The amplicons had a melting temperature and size of approximately 78 degrees C and 130 bp, respectively. Bdh expression was higher in the seminal vesicle when compared to the other tissues. The present work confirms that goat is the fifth mammalian species, after pig, cattle, horse, and sheep, in which spermadhesin molecules are found. To the best of our knowledge, this is the first report on buck spermadhesin genes using molecular cloning and expression profile.     

Pro-inflammatory effect of Arum maculatum lectin and role of resident cells

Arum maculatum agglutinin (AMA) is a monocot lectin isolated from tubers of Arum maculatum L. (Araceae) which exhibits different specificity towards oligo-mannosidic-type and N-acetyllactosaminic-type glycans. We have investigated the effect of this lectin on the cells of the immune system. Models of neutrophil migration in vivo, neutrophil chemotaxis in vitro and macrophage cultures were used to study the lectin inflammatory activity. When administered into rat peritoneal cavities, AMA (80, 200 and 500 microg/mL/cavity) induced significant and dose-dependent neutrophil migration. This effect was inhibited by incubation with alpha-methyl-d-mannoside. A 83% depletion in the number of resident cells following peritoneal lavage did not reduce the AMA-induced neutrophil migration, as compared to sham animals (not washed). However, pre-treatment with 3% thioglycolate which increases the peritoneal macrophage population by 236%, enhanced the neutrophil migration induced by AMA (200 microg/mL/cavity) (119%, p < 0.05). Reduction of peritoneal mast cell population by chronic treatment of cavities with compound 48/80 did not modify AMA-induced neutrophil migration. The neutrophil chemotaxy assay in vitro shows that the lectin (300 microg/mL) induces neutrophil chemotaxy (368% p < 0.05) compared to RPMI. Finally, injection into peritoneal cavities of supernatants from macrophage cultures obtained after stimulation with AMA (300 microg/mL) enhanced neutrophil migration (110% p < 0.05). Summarizing, our data suggest that A. maculatum agglutinin presents pro-inflammatory activity, inducing neutrophil migration by two ways, one which is independent on resident cells and another one dependent on the presence of these cells.     

Kinetic sedimentation of Rhizobium-aggregates produced by leguminous lectins

Lectins from Canavalia brasiliensis (CnBr), Cratylia floribunda (CFL), Vatairea macrocarpa (VML) and Phaseolus vulgaris (PHA) aggregate Rhizobiumbacteria. The relationship between specific sedimentation rate, (based on bacterial dry biomass) of bacterial aggregates and lectin concentrations was hyperbolic and showed bacterial surface affinity by lectins. R. tropici (Rt), R. leguminosarum bv. phaseoli (Rlp) and R. etli (Re) surfaces showed predominantly receptors of galactosidic nature. The Rt surfaces showed very high affinities (k _s = ±8.6 × 10^–8 ag lectin protein ml^–1) by Gal-specific lectins (PHA and VML), and very low affinities (k_s=± 4.9 × 10^–6) by Glc-specific lectins (CnBr and CFL). The Rlp surface had intermediate affinities by lectins. The Re surface showed high affinities by PHA (k_s= ±1.26 × 10^–8) and intermediate affinities by VML, CnBr and CFL. The relationship between sedimentation specific (based on lectin weight) and bacterial density was a sigmoid and showed lectin affinity by Rt surfaces. The bacterial sedimentation showed positive cooperative binding of lectins. The V_max induced by Glc-specific lectins was ±20 of that produced by Gal-specific lectins. The PHA affinity (k_s= 1.19 mg dry biomass ml^–1) was larger than VML (k_s = 1.23). The Glc-specific lectin affinities were smaller than those of Gal-specific. The apparent binding site number of lectins (n_app) was: 2.7-PHA; 2.2-VML; 3.2-CFL and 3.2-CnBr. The dissociation constant, k_s, of lectin-binding kinetics decreased with sugar-hapten treatment (10 M). The n_app decreased in PHA and CFL, increasing in VML + sugar-hapten treatment. This study showed that there is a difference in Rhizobium surfaces for lectin binding.     

HCA and HML isolated from the red marine algae Hypnea cervicornis and Hypnea musciformis define a novel lectin family

HCA and HML represent lectins isolated from the red marine algae Hypnea cervicornis and Hypnea musciformis, respectively. Hemagglutination inhibition assays suggest that HML binds GalNAc/Gal substituted with a neutral sugar through 1-3, 1-4, or 1-2 linkages in O-linked mucin-type glycans, and Fuc(alpha1-6)GlcNAc of N-linked glycoproteins. The specificity of HCA includes the epitopes recognized by HML, although the glycoproteins inhibited distinctly HML and HCA. The agglutinating activity of HCA was inhibited by GalNAc, highlighting the different fine sugar epitope-recognizing specificity of each algal lectin. The primary structures of HCA (9193+/-3 Da) and HML (9357+/-1 Da) were determined by Edman degradation and tandem mass spectrometry of the N-terminally blocked fragments. Both lectins consist of a mixture of a 90-residue polypeptide containing seven intrachain disulfide bonds and two disulfide-bonded subunits generated by cleavage at the bond T50-E51 (HCA) and R50-E51 (HML). The amino acid sequences of HCA and HML display 55% sequence identity (80% similarity) between themselves, but do not show discernible sequence and cysteine spacing pattern similarities with any other known protein structure, indicating that HCA and HML belong to a novel lectin family. Alignment of the amino acid sequence of the two lectins revealed the existence of internal domain duplication, with residues 1-47 and 48-90 corresponding to the N- and C-terminal domains, respectively. The six conserved cysteines in each domain may form three intrachain cysteine linkages, and the unique cysteine residues of the N-terminal (Cys46) and the C-terminal (Cys71) domains may form an intersubunit disulfide bond.     

Interaction of Diocleinae lectins with glycoproteins based in surface plasmon resonance

Interaction of glucose/mannose-binding lectins in solution with immobilized glycoproteins was followed in real time using surface plasmon resonance technology. The lectins which share many biochemical and structural features could be clearly differentiated in terms of their specificity for complex glycoconjugates. The most prominent interaction of the lectins with PHA-E comparing with soybean agglutinin, both glycoproteins exhibiting high mannose oligosaccharides, suggests that the whole structure of the glycoproteins themselves, may interfere in affinity. These findings also support the hypothesis that minor amino acid replacements in the primary sequence of the lectins might be responsible for their divergence in fine specificity and biological activities. This is the first report using surface plasmon resonance technology that evidences differences of Diocleinae lectins in respect their fine glycan-specificity.     

Porcine spermadhesin PSP-I/PSP-II stimulates macrophages to release a neutrophil chemotactic substance: modulation by mast cells

The complex of porcine seminal plasma heterodimers I and II (PSP-I/PSP-II), which are heterodimers of glycosylated spermadhesins, is the major component of porcine seminal fluid. The proinflammatory and immunostimulatory activities of this spermadhesin complex suggest its participation in modulation of the uterine immune activity that may ensure reproductive success. Spermadhesin PSP-I/PSP-II induced the migration of neutrophils into the peritoneal cavity of rats via activation of resident cells. In the present study, we have investigated the involvement of macrophages and mast cells in the neutrophil chemotactic activity of PSP-I/PSP-II and the underlying mechanism. Macrophages and mast cells were isolated, cultured, and stimulated with purified PSP-I/PSP-II. Pharmacological modulation was performed using the glucocorticoid dexamethasone, indomethacin (cyclooxygenase inhibitor), MK886 (leukotriene inhibitor), and the supernatant of spermadhesin-stimulated mast cells. Macrophages stimulated with PSP-I/PSP-II released into the culture supernatant a neutrophil chemotactic substance. This activity was partly inhibited by both dexamethasone (85%) and the supernatant of spermadhesin-stimulated mast cells (74%) but not by indomethacin and MK886. An anti-tumor necrosis factor (TNF) alpha antibody neutralized (by 68%) the neutrophil chemotactic activity of PSP-I/PSP-II-stimulated macrophages. An anti-interleukin (IL)-4 antibody blocked the inhibitory activity of spermadhesin-stimulated mast cells on release of a neutrophil chemotactic substance by PSP-I/PSP-II-stimulated macrophages. As a whole, these data indicate that the neutrophil migration-inducing ability of spermadhesin PSP-I/PSP-II involves the release of the inflammatory cytokine TNFalpha by stimulated macrophages and that this activity is modulated by the lymphokine IL-4 liberated by mast cells. The balance between these two cytokines may control onset of the local inflammatory reaction, avoiding excessive neutrophil recruitment that would lead to tissue damage.     

Determination of the Amino Acid Sequence of a New Phospholipase A<Subscript>2</Subscript> (MIDCA1) Isolated from <Emphasis Type="Italic">Micrurus dumerilii carinicauda</Emphasis> Venom

A new Phospholipase A₂ (PLA₂) from Micrurus dumerilii carinicauda venom was isolated and its primary structure determined. This new PLA₂ showed a low enzymatic activity when compared with other PLA₂s and it is moderately basic with an isoelectric point of 8.0. Its amino acid sequence showed the presence of 120 amino acid residues and its sequence was: NLIQFLNMIQCTTPGREPLVAFANYGCYCGRGGSGTPVDELDRCCQVHDNCYDTAKKVFGCSPYFTMYSYDCSEGKLTCKDNNTKCKAAVCNCDRTAALCFAKAPYNDKNYKIDLTKRCQ. The structural model of MIDCA1, when compared with other strong neurotoxic PLA₂s, such as Naja naja, showed significant differences in the β-wing and neurotoxic sites, despite the high level of amino acid sequence similarity. These observations indicate a dissociation between the biological and catalytic activity of this new PLA₂, supporting the view that other regions of the protein are involved in the biological effects.     

Lectin from Canavalia brasiliensis (ConBr) protects hippocampal slices against glutamate neurotoxicity in a manner dependent of PI3K/Akt pathway

The excitotoxicity induced by excessive activation of the glutamatergic neurotransmission pathway is involved in several neuropathologies. In this sense, molecules that prevent the release of glutamate or the excessive activation of its receptors can be useful in preventing the neuronal cell death observed in these diseases. Lectins are proteins capable of reversible binding to the carbohydrates in glycoconjugates, and some have been used in the study and purification of glutamate receptors. ConBr is a mannose/glucose-binding lectin purified from Canavalia brasiliensis seeds. In the present study, we aimed to evaluate the neuroprotective activity of ConBr against glutamate-induced excitotoxicity. Hippocampal slices were isolated from adult male mice and incubated for 6 h in Krebs saline/DMEM buffer alone (control), in the presence of glutamate or glutamate plus ConBr. The phosphorylation of Akt and mitogen activated protein kinases (MAPKs) such as ERK1/2, p38^MAPK and JNK1/2/3 was evaluated with western blotting. The results indicate that glutamate provoked a reduction in the hippocampal slice viability (−25%), diminished the phosphorylation of Akt and augmented p38^MAPK and ERK1 phosphorylation. No changes were observed in the phosphorylation of JNK1/2/3 or ERK2. Notably, ConBr, through a mechanism dependent on carbohydrate interaction, prevented the reduction of cell viability and Akt phosphorylation induced by glutamate. Furthermore, in the presence of the PI3K inhibitor LY294002, ConBr was unable to reverse glutamate neurotoxicity. Taken together, our data suggest that the neuroprotective effect of ConBr against glutamate neurotoxicity requires oligosaccharide interaction and is dependent on the PI3K/Akt pathway.     

Halilectin 1 (H‐1) and Halilectin 2 (H‐2): two new lectins isolated from the marine sponge Haliclona caerulea

Two new lectins named Halilectin 1 (H‐1) and Halilectin 2 (H‐2) were isolated from the marine sponge Haliclona caerulea using a combination of affinity chromatography on stroma fixed onto Sephadex G‐25 and cation and anion exchange chromatography. H‐1 is a monomeric protein with a molecular mass of 40 kDa estimated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and 15 kDa estimated using a TSK gel. Conversely, H‐2 is a homodimeric protein with 15 kDa monomers linked via weak interactions. H‐1 more effectively agglutinates trypsinized rabbit erythrocytes, whereas H‐2 more effectively agglutinates native rabbit erythrocytes. The hemagglutinating activity of H‐1 could be not inhibited by any tested sugars, but H‐2 was inhibited by orosomucoid and porcine stomach mucin. Neither lectin was dependent on divalent ions. H‐1 was stable at basic pH range and temperatures up to 50 °C, whereas H‐2 was stable at acid pH range and temperatures up to 80 °C. The H. caerulea lectins exhibited dose‐dependent toxicity against Artemia nauplii. Additionally, 76% of the primary structure of H‐2 was determined using tandem mass spectrometry to contain a unique amino acid sequence with no similarity to any members of the animal lectin family. Copyright © 2012 John Wiley & Sons, Ltd.     

Purification and primary structure of a mannose/glucose‐binding lectin from Parkia biglobosa Jacq. seeds with antinociceptive and anti‐inflammatory properties

Parkia biglobosa (subfamily Mimosoideae), a typical tree from African savannas, possess a seed lectin that was purified by combination of ammonium sulfate precipitation and affinity chromatography on a Sephadex G‐100 column. The P. biglobosa lectin (PBL) strongly agglutinated rabbit erythrocytes, an effect that was inhibited by d ‐mannose and d ‐glucose‐derived sugars, especially α‐methyl‐d ‐mannopyranoside and N‐acetyl‐d ‐glucosamine. The hemagglutinating activity of PBL was maintained after incubation at a wide range of temperature and pH and also was independent of divalent cations. By sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis, PBL exhibited an electrophoretic profile consisting of a single band with apparent molecular mass of 45 kDa. An analysis using electrospray ionization–mass spectrometry indicated that purified lectin possesses a molecular average mass of 47 562 ± 4 Da, and the analysis by gel filtration showed that PBL is a dimer in solution. The complete amino acid sequence of PBL, as determined using tandem mass spectrometry, consists of 443 amino acid residues. PBL is composed of a single non‐glycosylated polypeptide chain of three tandemly arranged jacalin‐related domains. Sequence heterogeneity was found in six positions, indicating that the PBL preparations contain highly homologous isolectins. PBL showed important antinociceptive activity associated to the inhibition of inflammatory process. Copyright © 2013 John Wiley & Sons, Ltd.