Crystal structure of a lectin from Canavalia maritima (ConM) in complex with trehalose and maltose reveals relevant mutation in ConA-like lectins

The crystal structure of Canavalia maritima lectin (ConM) complexed with trehalose and maltose revealed relevant point mutations in ConA-like lectins. ConM with the disaccharides and other ConA-like lectins complexed with carbohydrates demonstrated significant differences in the position of H-bonds. The main difference in the ConM structure is the replacement of Pro202 by Ser202, a residue that promotes the approximation of Tyr12 to the carbohydrate-binding site. The O-6' of the second glucose ring in maltose interacts with Tyr12, while in trehalose the interaction is established by the O-2' and Tyr12, explaining the higher affinity of ConM for disaccharides compared to monosaccharides.     

cDNA cloning and 1.75 A crystal structure determination of PPL2, an endochitinase and N-acetylglucosamine-binding hemagglutinin from Parkia platycephala seeds

Parkia platycephala lectin 2 was purified from Parkia platycephala (Leguminosae, Mimosoideae) seeds by affinity chromatography and RP-HPLC. Equilibrium sedimentation and MS showed that Parkia platycephala lectin 2 is a nonglycosylated monomeric protein of molecular mass 29 407+/-15 Da, which contains six cysteine residues engaged in the formation of three intramolecular disulfide bonds. Parkia platycephala lectin 2 agglutinated rabbit erythrocytes, and this activity was specifically inhibited by N-acetylglucosamine. In addition, Parkia platycephala lectin 2 hydrolyzed beta(1-4) glycosidic bonds linking 2-acetoamido-2-deoxy-beta-D-glucopyranose units in chitin. The full-length amino acid sequence of Parkia platycephala lectin 2, determined by N-terminal sequencing and cDNA cloning, and its three-dimensional structure, established by X-ray crystallography at 1.75 A resolution, showed that Parkia platycephala lectin 2 is homologous to endochitinases of the glycosyl hydrolase family 18, which share the (betaalpha)8 barrel topology harboring the catalytic residues Asp125, Glu127, and Tyr182.     

Lack of tyrosine 320 impairs spontaneous endocytosis and enhances release of HLA-B27 molecules

Several lines of evidence suggest that endocytosis of MHC class I molecules requires conserved motifs within the cytoplasmic domain. In this study, we show, in the C58 rat thymoma cell line transfected with HLA-B27 molecules, that replacement of the highly conserved tyrosine (Tyr320) in the cytoplasmic domain of HLA-B27 does not hamper cell surface expression of beta2-microglobulin H chain heterodimers or formation of misfolded molecules. However, Tyr320 replacement markedly impairs spontaneous endocytosis of HLA-B27. Although wild-type molecules are mostly internalized via endosomal compartments, Tyr320-mutated molecules remain at the plasma membrane in which partial colocalization with endogenous transferrin receptors can be observed, also impairing their endocytosis. Finally, we show that Tyr320 substitution enhances release of cleaved forms of HLA-B27 from the cell surface. These studies show for the first time that Tyr320 is most likely part of a cytoplasmic sorting motif involved in spontaneous endocytosis and shedding of MHC class I molecules.     

Crystal structure of the lectin of Camptosema pedicellatum: implications of a conservative substitution at the hydrophobic subsite

Lectins have been used as models for studies of the molecular basis of protein-carbohydrate interaction and specificity by deciphering codes present in the glycan structures. The purpose of the present study was to purify and solve the complete primary and crystal structure of the lectin of Camptosema pedicellatum (CPL) complexed with 5-bromo-4-chloro-3-indolyl-α-d-mannose (X-Man) using tandem mass spectrometry. CPL was purified by single-step affinity chromatography. Mass spectrometry findings revealed that purified CPL features a combination of chains weighing 25,298 ± 2 (α-chain), 12,835 ± 2 (β-chain) and 12,481 ± 2 Da (γ-chain). The solved crystal structure of CPL features a conservative mutation in the hydrophobic subsite, a constituent of the carbohydrate recognition domain (CRD), indicating the relevance of hydrophobic interactions in the establishment of interactions with carbohydrates. The substitution and the analysis of the interactions with X-Man also revealed that the hydrophobic effect caused by a minor change in the hydrophobic subsite interferes in the formation of H-bonds due to the reorientation of the indolyl group in the CRD.     

Characterisation of the osteoclastogenic potential of human osteoblastic and fibroblastic conditioned media

Although M‐CSF and RANKL are sufficient to promote in vitro osteoclastogenesis, in vivo this is a complex process which requires the action of many signalling molecules and cellular crosstalks. In this work, isolated or combined conditioned media, obtained from human adult skin fibroblast and bone marrow cells, were tested for their osteoclastogenic potential, through an indirect co‐culture system, in the absence of recombinant M‐CSF and RANKL. Osteoclastogenesis was assessed on human peripheral blood mononuclear cells (PBMC) and CD14+ cell cultures by quantification of total protein content, tartrate‐resistant acid phosphatase (TRAP) activity, presence of multinucleated cells positive for TRAP, RT‐PCR of TRAP, CATK, CA2, c‐myc and c‐src and presence of multinucleated cells displaying actin rings, vitronectin and calcitonin receptors. Cultures supplemented with M‐CSF and RANKL were used as positive controls. It was observed that the conditioned medium from dexamethasone osteogenic‐induced bone marrow cell cultures displayed the highest osteoclastogenic potential, with similar behaviour to that observed in the presence of both M‐CSF and RANKL. Comparatively, fibroblastic conditioned medium elicited a slightly lower osteoclastogenic response. Combination of both conditioned media resulted in a significant increase of TRAP activity. On the other hand, conditioned medium from non‐osteogenic‐induced bone marrow cell cultures presented the lowest osteoclastogenic potential. These results were observed for both PBMC and CD14+ cell cultures, suggesting that fibroblast and osteoblast cells are able to modulate osteoclastogenesis in the absence of physical cell–cell interactions. In addition, osteoclastogenic potential of bone marrow cells increases with their osteoblastic differentiation. J. Cell. Biochem. 109: 205–216, 2010. © 2009 Wiley‐Liss, Inc.     

GANT-61 Induces Autophagy and Apoptosis in Glioblastoma Cells despite their heterogeneity

Cellular and Molecular Neurobiology - Glioblastoma (GBM) is the most common adult primary tumor of the CNS characterized by rapid growth and diffuse invasiveness into the brain parenchyma. The GBM...     

Effects of nandrolone and resistance training on the blood pressure,cardiac electrophysiology,and expression of atrial β-adrenergic receptors

AimsThis study was performed to assess isolated and combined effects of nandrolone and resistance training on the blood pressure, cardiac electrophysiology, and expression of the β₁- and β₂-adrenergic receptors in the heart of rats.Main methodsWistar rats were randomly divided into four groups and submitted to a 6-week treatment with nandrolone and/or resistance training. Cardiac hypertrophy was accessed by the ratio of heart weight to the final body weight. Blood pressure was determined by a computerized tail-cuff system. Electrocardiography analyses were performed. Western blotting was used to access the protein levels of the β₁- and β₂-adrenergic receptors in the right atrium and left ventricle.Key findingsBoth resistance training and nandrolone induced cardiac hypertrophy. Nandrolone increased systolic blood pressure depending on the treatment time. Resistance training decreased systolic, diastolic and mean arterial blood pressure, as well as induced resting bradycardia. Nandrolone prolonged the QTc interval for both trained and non-trained groups when they were compared to their respective vehicle-treated one. Nandrolone increased the expression of β₁- and β₂-adrenergic receptors in the right atrium for both trained and non-trained groups when they were compared to their respective vehicle-treated one.SignificanceThis study indicated that nandrolone, associated or not with resistance training increases blood pressure depending on the treatment time, induces prolongation of the QTc interval, and increases the expression of β₁- and β₂-adrenergic receptors in the cardiac right atrium, but not in the left ventricle.     

Interactions between indole-3-acetic acid (IAA) with a lectin from Canavalia maritima seeds reveal a new function for lectins in plant physiology

Indole-3-acetic acid (IAA) bound is considered a storage molecule and is inactive. However, some studies have proposed an additional possible regulatory mechanism based on the ability of lectins to form complexes with IAA. We report the first crystal structure of ConM in complex with IAA at 2.15 Å resolution. Based on a tetrameric model of the complex, we hypothesize how the lectin controls the availability of IAA during the early seedling stages, indicating a possible new physiological role for these proteins. A free indole group is also bound to the protein. The ConM interaction with different forms of IAA is a strategy to render the phytohormone unavailable to the cell. Thus, this new physiological role proposed for legume lectins might be a novel mechanism by which IAA levels are decreased in addition to the destruction and formation of new complexes in the later stages of seed germination.