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R C Hughes

Publications and source records attributed to R C Hughes.

At least 19 recordsLinked to original sources
PubMed2000

Interaction of a novel cysteine and histidine-rich cytoplasmic protein with galectin-3 in a carbohydrate-independent manner.

We have used the yeast two-hybrid system to search for cytoplasmic proteins that might assist in the intracellular trafficking of the soluble beta-galactoside-binding protein, galectin-3. We utilised as bait murine full-length galectin-3 to screen a murine 3T3 cDNA library. Several interacting clones were found to encode a partial open reading frame and a full-length clone was obtained by rapid amplification of cDNA ends methodology. In various assays in vitro the novel protein was shown to bind galectin-3 in a carbohydrate-independent manner. The novel protein contains an unusually high content of cysteine and histidine residues and shows significant sequence homologies with several metal ion-binding motifs present in known proteins. Confocal immunofluorescence microscopy of permeabilised 3T3 cells shows a prominent perinuclear, as well as cytoplasmic, localisation of the novel protein.

3T3 Cells↗
PubMed1999

Secretion of the galectin family of mammalian carbohydrate-binding proteins.

Galectins are cytosolic proteins that lack any signal sequence for transport into the endoplasmic reticulum and are not glycosylated, although several galectins contain consensus sites for N-glycosylation, indicating that these proteins do not traverse the ER-Golgi network. However, there is abundant evidence for the extracellular localisation of some galectins at cell surfaces, in the extracellular matrix and in cell secretions consistent with other evidence for extracellular roles of galectins as modulators of cell adhesion and signalling. How then are galectins secreted if not through the classical secretory pathway? Do all galectins share the same secretory pathway? Can a particular galectin utilise more than one secretory pathway? If galectins play important extracellular roles how is their secretion regulated in relation to function? These are still largely unanswered questions but recent studies are beginning to give glimpses into some novel aspects of the secretion of these intriguing proteins.

ATP-Binding Cassette Transporters↗
PubMed1999

Galectin-3 modulates rat mesangial cell proliferation and matrix synthesis during experimental glomerulonephritis induced by anti-Thy1.1 antibodies.

Galectin-3 is a beta-galactoside-binding protein synthesized by macrophages and other inflammatory cells and expressed in various branching epithelia, including the developing kidney. The expression of galectin-3 has been studied in a rat model of acute mesangial proliferative glomerulonephritis in which a single injection of anti-Thy1.1 antibodies leads to destruction of mesangial cells expressing a Thy1.1 epitope on their surface. The glomerular lesion is characterized by expansion of the mesangial matrix, especially laminin and collagen type IV, and mesangial hypercellularity. Galectin-3 expression, which is sparse in mature rat kidney and confined to the apical face of some distal tubules, is increased within 1-3 days following antibody administration, with the recruitment of glomerular macrophages and pronounced neo-expression in the cytoplasm and at the basal face of distal tubules. At later times, galectin-3 is detected immunohistochemically in the repopulating mesangial cell mass, preceding the extensive mesangial deposition of laminin and collagen type IV. Mesangial cells in culture do not produce appreciable amounts of galectin-3 but do bind and endocytose exogenously added lectin. Addition of galectin-3 to primary cultures of mesangial cells prepared from normal rats induces a 1.5-fold increase in the synthesis of collagen type IV and it also acts in synergy with a quantitatively similar stimulatory effect of transforming growth factor beta (TGF-beta) on matrix synthesis. Exogenous galectin-3 prolongs the survival of mesangial cells in serum-free cultures and also protects these cells against cytotoxic effects of TGF-beta. The data support the notion that the increased expression and secretion of galectin-3 in infiltrating macrophages and in distal tubular epithelia, together with up-regulation of IL-1beta and TGF-beta genes, play a role in mesangial hypercellularity in the progression of one model of inflammatory renal disease.

Animals↗
PubMed1999

Kinetic measurements of binding of galectin 3 to a laminin substratum.

Galectin 3, a beta-galactoside binding protein, contains a C-terminal carbohydrate recognition domain (CRD) and an N-terminal segment including multiple repeats of a proline/tyrosine/glycine-rich motif. Previous work has shown that galectin 3 but not the isolated CRD binds to laminin, a multivalent ligand, with positive cooperativety indicating the formation of multiple interactions although the lectin in solution is monomeric. Using surface plasmon resonance, we find that hamster galectin 3 at sub-micromolar concentrations or its isolated CRD at all concentrations binds to a laminin substratum with similar association (k(ass); 10-30,000 M(-1) S(-1)) and dissociation (k(diss); 0.2-0.3 S1(-1)) rates and weak affinity (Ka; 1-3 x 10(5) M(-1)). At higher concentrations of galectin 3 the off rate decreases ten fold leading to increased affinity. Ligation of an N-terminal epitope of galectin 3 with a monoclonal Fab fragment increases association and dissociation rates ten fold. A recombinant protein obtained by deletion of the first 93 N-terminal residues binds to laminin with positive cooperativity and a slowly dissociating fraction (K(diss); 0.002 S(-1)) accumulates on the substratum. The data suggest that homophilic interactions between CRD as well as N terminal domains are implicated in galectin 3 aggregation on the substratum leading to positive binding cooperativity.

Amino Acid Sequence↗
PubMed1999

Determinants in the N-terminal domains of galectin-3 for secretion by a novel pathway circumventing the endoplasmic reticulum-Golgi complex.

Galectin-3 is a beta-galactoside-binding protein that is secreted from many cells although the protein lacks a signal sequence for transfer into the endoplasmic reticulum and Golgi compartments and entry into classical secretory pathways. Previously it was shown that attachment of the first 120 amino acid residues of the N-terminal sequence of hamster galectin-3 to the cytoplasmic protein chloramphenicol acetyltransferase (CAT) supported the rapid secretion of the fusion protein from transiently transfected Cos cells under conditions in which CAT protein was not secreted. Here we report that progressive N-terminal truncation gradually reduced secretion of the fusion proteins, eventually to very low levels compared with the starting product, but did not totally eliminate secretion until a significant majority of the sequence was removed. Mutant CAT fusion proteins containing internal deletions in residues 97-120 of the galectin-3 N-terminal sequence were also secreted to a similar extent to the starting product, but further deletion of residues 89-96 abolished detectable secretion. Proline to alanine mutagenesis of the sequence YP(90)SAP(93)GAY in two secretion-competent CAT fusion proteins greatly reduced or abolished their secretion, whereas similar mutagenesis of proline pairings present elsewhere in the galectin-3 N-terminal segments of these proteins had no effect. The results indicate that this sequence is one essential determinant for secretion of galectin-3-CAT fusion proteins and by inference galectin-3, at least from transfected Cos cells. However, the short sequence of residues 89-96 by itself is insufficient to direct secretion of CAT fusion proteins and appears to be active only in the context of a larger portion of the galectin-3 N-terminal sequence.

Amino Acid Sequence↗
PubMed1999

Galectin-3 and polarized growth within collagen gels of wild-type and ricin-resistant MDCK renal epithelial cells.

Previous studies (Q. Bao and R. C. Hughes (1995) J. Cell Sci., 108, 2791-2800) showed that the beta-galactoside-binding protein, galectin-3, is secreted onto the basolateral surface domains of Madin-Darby canine kidney MDCK cells growing as polarized cysts within a collagen gel. The growth and enlargement of such cysts were shown to be increased significantly when cultured in the presence of antibodies directed against the lectin and were slowed down by addition of exogenous galectin-3. These results suggested a role for galectin-3, interacting with appropriately glycosylated surface receptors, as a negative growth regulator in the development of MDCK cysts, a well-known model for renal epithelial morphogenesis. In the present report we have tested this proposal by use of a ricin-resistant mutant of MDCK cells that is unable to transfer galactose residues during biosynthesis of cellular glycoconjugates and hence lacks extracellular receptors for galectin-3. We find that when grown within collagen gels, the mutant cell cysts grow significantly faster than wild-type cell cysts. Furthermore, they form nonspherical and tubular cysts that are induced in wild-type cell cysts only under the influence of the morphogen, hepatocyte growth factor (HGF).

Animals↗
PubMed1998

The tryptophan fluorescence of Tetracarbidium conophorum agglutinin II and a solution-based assay for the binding of a biantennary glycopeptide.

The plant lectin Tetracarbidium conophorum agglutinin II binds to glycoproteins and glycopeptides in a structurally specific manner [Animashaun et al., (1994) Glycoconjugate J. 11, 299-303]. We have characterized the steady-state and time-resolved fluorescence of the tryptophan residues of this lectin. The fluorescence (lambda[ex] = 295 nm, lambda[em] = 350 nm) decay is complex and can be described by four decay times with the following values: tau1 = 7.4 nsec, alpha1 = 0.22; tau2 = 2.9 nsec, alpha2 = 0.25: tau3 = 1.0 nsec, alpha3 = 0.34, tau4 = 0.2 nsec, alpha4 = 0.18. The addition of a biantennary glycopeptide (carbohydrate sequence [see text]) to the lectin results in a quench and an 8 nm blue shift of the emission spectrum. The effect is saturable, and is described by an association constant of 1.8 x 10(5) M(-1). The tryptophan fluorescence of Tetracarbidium conophorum agglutinin II may therefore be utilized to characterize thermodynamically the binding interactions between this lectin and complex glycoprotein.

Carbohydrate Sequence↗
PubMed1998

Evidence for subsites in the galectins involved in sugar binding at the nonreducing end of the central galactose of oligosaccharide ligands: sequence analysis, homology modeling and mutagenesis studies of hamster galectin-3.

A model of the carbohydrate recognition domain CRD, residues 111-245, of hamster galectin-3 has been made using homology modeling and dynamics minimization methods. The model is based on the known x-ray structures of bovine galectin-1 and human galectin-2. The oligosaccharides NeuNAc-alpha2,3-Gal-beta1,4-Glc and GalNAc-alpha1, 3-[Fuc-alpha1,2]-Gal-beta1,4-Glc, known to be specific high-affinity ligands for galectin-3, as well as lactose recognized by all galectins were docked in the galectin-3 CRD model structure and a minimized binding conformation found in each case. These studies indicate a putative extended carbohydrate-binding subsite in the hamster galectin-3 involving Arg139, Glu230, and Ser232 for NeuNAc-alpha2,3-; Arg139 and Glu160 for fucose-alpha1,2-; and Arg139 and Ile141 for GalNAc-alpha1,3- substituents on the primary galactose. Each of these positions is variable within the whole galectin family. Two of these residues, Arg139 and Ser232, were selected for mutagenesis to probe their importance in this newly identified putative subsite. Residue 139 adopts main-chain dihedral angles characteristic of an isolated bridge structural feature, while residue 232 is the C-terminal residue of beta-strand-11, and is followed immediately by an inverse gamma-turn. A systematic series of mutant proteins have been prepared to represent the residue variation present in the aligned sequences of galectins-1, -2, and -3. Minimized docked models were generated for each mutant in complex with NeuNAc-alpha2,3-Gal-beta1,4-Glc, GalNAc-alpha1, 3-[Fuc-alpha1,2]-Gal-beta1,4- Glc, and Gal-beta1,4-Glc. Correlation of the computed protein-carbohydrate interaction energies for each lectin-oligosaccharide pair with the experimentally determined binding affinities for fetuin and asialofetuin or the relative potencies of lactose and sialyllactose in inhibiting binding to asiolofetuin is consistent with the postulated key importance of Arg139 in recognition of the extended sialylated ligand.

Amino Acid Sequence↗
PubMed1997

Macrophage surface glycoproteins binding to galectin-3 (Mac-2-antigen).

Galectin-3 (formerly called Mac-2 antigen) is a approximately 30 kDa carbohydrate-binding protein expressed on the surface of inflammatory macrophages and several macrophage cell lines. We have purified from lysates of the murine macrophage cell line WEHI-3 glycoproteins that bind to a galectin-3 affinity column. Several of these receptors are labelled after biotinylation of intact cells showing their location at the cell surface. N-terminal aminoacid sequencing of intact galectin-3-binding glycoproteins isolated from preparative SDS-gels or of chemically derived fragments showed several homologies with known proteins and identification was confirmed by immunoprecipitation with specific antibodies. The glycoproteins were shown to be: the alpha-subunit(CD11b) of the CD11b/CD18 integrin(Mac-1 antigen); the lysosomal membrane glycoproteins LAMPs 1 and 2 which are known in part to be expressed at cell surfaces; the Mac-3 antigen, a mouse macrophage differentiation antigen defined by the M3/84 monoclonal antibody and related immunochemically to LAMP-2; the heavy chain of CD98, a 125 kDa heterodimeric glycoprotein identified by the 4F2/RL388 monoclonal antibodies respectively on human and mouse monocytes/macrophages and on activated T cells. Further studies showed that CD11b/CD18, CD98 and Mac-3 are major surface receptors for galectin-3 on murine peritoneal macrophages elicited by thioglycollate.

Amino Acid Sequence↗
PubMed1997

The complex-type oligosaccharide binding lectin Datura stramonium agglutinin detects type II A muscle fibres in the branchial biceps from man and cat.

Complex-type oligosaccharides were detected in the sarcoplasm of muscle fibres from cat and human biceps using lectins and anticarbohydrate antibodies. The lectin Datura stramonium agglutinin strongly stained type II A fibres as identified by myosin ATPase activity after alkaline and acid preincubation. In contrast, all muscle fibres showed a moderate coarse granular staining after incubation with Tetracarpidum conophorum agglutinin and Telfairia occidentalis agglutinin which recognize tri-antennary complex glycans poorly bound by D. stramonium agglutinin. Strong sarcoplasmic staining in all muscle fibres was obtained after incubation with an antibody against branched N-acetyllactosamine structure while an antibody against binary 2 --> 3 sialyllactosamine glycans failed to detect the muscle fibres. Treatment of the muscle sections with sialidase prior to incubation with D. stramonium agglutinin did not influence the lectin staining pattern. Staining of blots from electrophoretically separated muscle proteins obtained by homogenization, solubilization and centrifugation of small muscle pieces showed D. stramonium agglutinin binding to a number of bands ranging from 200 kDa to 30 kDa. No D. stramonium agglutinin positive bands were observed in blots from separated mitochondrial proteins while blots from sarcoplasmic reticulum separated by electrophoresis stained many bands in the range from 200 kDa to 30 kDa. It may be concluded that all muscle fibres in human and cat biceps hold intracellular non-sialylated complex-type oligosaccharides and further, that a specific tri-antennary complex-type glycoform is strongly expressed in type II A fibres as recognized by D. stramonium agglutinin. These results indicate a different glycosylation of certain myofibrillar-associated proteins in muscle fibre types.

Animals↗
PubMed1997

Detection and distribution of the carbohydrate binding protein galectin-3 in human notochord, intervertebral disc and chordoma.

Galectin-3 is an endogenous carbohydrate-binding protein which plays a role in cell differentiation, morphogenesis and cancer biology. We investigated the occurrence and distribution of galectin-3 in the embryonic and fetal human notochord, the developing human vertebral column, adult intervertebral discs and in six chordomas, which are tumors thought to originate from notochordal remnants. By means of Western blots, the expression of galectin-3 was confirmed in tissue probes from the vertebral column region beginning with the 8th gestational week. These results were supported by immunohistochemical data which revealed the presence of galectin-3 in the cytoplasm of cells of the notochord also from the 8th gestational week onwards. Notochordal immunostaining became stronger with increasing gestational age. A persisting notochordal remnant in an adult intervertebral disc and various cells of the nucleus pulposus also contained galectin-3. All chordomas showed moderate or strong immunoreactivity irrespective of their cellular composition. Subcellularly, galectin-3 was localized mostly in the cytoplasm, while a subset of tumor cells also showed nuclear distribution. Differences in staining patterns of chordoma cells could not, in general, be correlated to any histological features of these tumors.

Adolescent↗
PubMed1997

Plasma membrane targetting, vesicular budding and release of galectin 3 from the cytoplasm of mammalian cells during secretion.

Galectin 3, a 30 kDa galactoside-binding protein distributed widely in epithelial and immune cells, contains no signal sequence and is externalized by a mechanism independent of the endoplasmic reticulum (ER)-Golgi complex. We show here that hamster galectin 3 overexpressed in transfected cos-7 cells is secreted at a very low rate. A chimaera of galectin 3 fused to the N-terminal acylation sequence of protein tyrosine kinase p56(lck), Nt-p56(lck)-galectin 3, which is myristoylated and palmitoylated and rapidly transported to plasma membrane domains, is efficiently released from transfected cells indicating that movement of cytoplasmic galectin 3 to plasma membrane domains is a rate limiting step in lectin secretion. N-terminal acylation is not sufficient for protein secretion since p56(lck) and the chimaera Nt-p56(lck)-CAT are not secreted from transfected cells. The amino-terminal half of galectin 3 is sufficient to direct export of a chimaeric CAT protein indicating that part of the signal for plasma membrane translocation lies in the N-terminal domains of the lectin. Immunofluorescence studies show that Nt-p56(lck)-galectin 3 aggregates underneath the plasma membrane and is released by membrane blebbing. Vesicles of low buoyant density isolated from conditioned medium are enriched in galectin 3. The lectin is initially protected from exogenous collagenase but is later released in soluble protease-sensitive form from the lectin-loaded vesicles. Using murine macrophages, which secrete their endogenous galectin 3 at a moderate rate especially in the presence of Ca2+-ionophores, we were also able to trap a galectin 3-loaded vesicular fraction which was released into the culture supernatant.

Acylation↗
PubMed1997

Epithelial galectin-3 during human nephrogenesis and childhood cystic diseases.

Galectin-3 is a beta-galactoside-binding protein with putative roles in development, oncogenesis, and inflammation. Its expression in human nephrogenesis has not been previously reported. This study examines galectin-3 expression in early human embryos by Western blot and immunohistochemistry. This 33-kD protein was detected in the apical domain of distal tubules of the mesonephros and also in the mesonephric duct. In the metanephros, the adult kidney precursor, galectin-3 was detected in the apical domains of ureteric bud branches, and there was intense expression in fetal medullary and papillary collecting ducts in both the cytoplasm and plasma membranes. Low levels of galectin-3 were detected in the cytoplasm of a subset of cells in adult collecting ducts; these were alpha-intercalated cells because they expressed basal band 3 protein. In human multicystic dysplastic kidneys, all diseased epithelia had an embryonic apical expression pattern of galectin-3 and, in addition, all cystic epithelia in autosomal recessive polycystic kidneys expressed this molecule. It is concluded that galectin-3 is expressed by cells of the mesonephric duct/ureteric bud lineage, and it is speculated that the different subcellular locations may be implicated in both the regulation of normal growth and differentiation of this lineage, as well as in the pathogenesis of cystic epithelia.

Adult↗
PubMed1996

Galectin-3 stimulates uptake of extracellular Ca2+ in human Jurkat T-cells.

Galectin-3, a mammalian galactoside-binding protein, is not expressed in the Jurkat T-lymphoblastoid cell line. However, Jurkat cells express surface glycoprotein receptors for galectin-3, one of which is shown to be the glycosylated heavy chain of CD98 (4F2 antigen), a T-cell activation marker. Addition of galectin-3 to Jurkat cells triggers a sustained influx of extracellular Ca2+ in a concentration dependent manner. The induced increase in cytosolic [Ca2+]i is blocked by sugar hapten inhibitors of galectin-3. The galectin-3-induced effect is insensitive to voltage-gated Ca2+ channel antagonists such as prenylamine, nifedipine and diltiazem and to pertussis toxin but is inhibited by cholera toxin. The results suggest that galectin-3 released by accessory cells such as macrophages may bind in vivo to T-cell activation antigens and also participate in Ca2+ signalling.

Animals↗
PubMed1996

Immunocytochemical evidence for a modulation of galectin 3 (Mac-2), a carbohydrate binding protein, in pulmonary fibrosis.

Galectin 3 is endogenous mammalian carbohydrate-binding protein with affinity for terminal beta-galactose residues, polylactosamine glycans, and ABH-blood group carbohydrate epitopes. To determine the distribution and regulation of galectin 3 during pulmonary injury, which is known to be accompanied by profound changes in the carbohydrate moieties of cell surface glycoproteins of alveolar cells, a rat model of irradiation-induced lung inflammation and repair was used. Immunocytochemistry showed that in normal rat lungs, galectin 3 was localized to alveolar macrophages, with weaker staining of bronchial epithelial cells. Shortly after irradiation-induced lung injury, when there is active proliferation of type II alveolar epithelial cells and re-epithelialization of alveolar basement membranes by type I cells, the total galectin concentration in the lung increased dramatically. This increase was due in part to an increased population of galectin 3-positive interstitial and alveolar macrophages. In addition, galectin 3 was expressed prominently at the surface of the newly formed type I alveolar epithelium and to lesser extent at the apical surface of type II cells. These findings suggest that the increased synthesis and secretion of galectin 3 during irradiation-induced lung injury, together with ligation of secreted lectin at the surface of alveolar epithelial cells, may play roles in pulmonary alveolar epithelial expansion and differentiation during injury and repair.

Animals↗
PubMed1996

Effects of the carbohydrate-binding protein galectin-3 on the invasiveness of human breast carcinoma cells.

Galectin-3 is a Mr 30,000 protein with carbohydrate-binding specificity for type I and II ABH blood group epitopes and polylactosamine glycans expressed on cell surface and extracellular matrix glycoproteins such as laminin. Cell lines propagated from human normal mammary epithelia and from benign or infiltrating components of primary breast tumours express low levels of galectin-3 in the cytoplasm. However, galectin-3 when added exogenously in solution or when bound within a three-dimensional matrix markedly enhanced the migration of the primary tumour cell lines through a Matrigel barrier. Galectin-3 expression in the cytoplasm and intercellularly on surface membranes was greatly increased in cell lines propagated from malignant ascites and pleural effusions of late stage breast cancer. These cell lines were non-invasive in the Matrigel assay and exogenous galectin-3 had no enhancing effect on invasiveness. These results suggest that galectin-3 could play multiple roles in cell metastasis at an early invasive stage by acting in a paracrine manner to stimulate cell migration through an extracellular matrix, and in later stage cancers in synergy with other mediators of cell-cell aggregation. However, endogenous galectin-3 expression in human breast cancers is not correlated directly with their invasive potential in vitro.

Antigens, Differentiation↗
PubMed1995

Cross-linking of galectin 3, a galactose-binding protein of mammalian cells, by tissue-type transglutaminase.

The 30 kDa beta-galactoside-binding protein of baby hamster kidney (BHK) cells [Mehul et al. (1994), J. Biol. Chem. 269, 18250-18258] homologous to galectin 3, a widely distributed mammalian lectin, has been found to be a substrate for tissue type transglutaminase, as shown by the incorporation in a calcium- and time-dependent manner of 5-(biotinamido) pentylamine in the presence of guinea pig liver transglutaminase. The amino-terminal domain of hamster galectin 3, which is a repetitive sequence rich in glutamine, tyrosine, glycine and proline, is also an excellent substrate. A single lysine residue in the N-terminal domain is an essential requirement for transglutaminase-mediated oligomerization, and two equivalent glutamine residues present in identical sequence repeats within this domain appear to be involved as amine acceptors in cross-linking reactions. Transglutaminase-mediated cross-linking of galectin 3 to itself or to matrix components may be one mechanism for stabilisation of a multivalent binding form of the lectin in cell secretions or in extracellular matrices.

Amino Acid Sequence↗