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H Leffler

Publications and source records attributed to H Leffler.

At least 19 recordsLinked to original sources
PubMed2001

Sd(a)-antigen-like structures carried on core 3 are prominent features of glycans from the mucin of normal human descending colon.

This paper describes structural characterization by NMR, MS and degradative studies of mucin glycans from normal human descending colon obtained freshly at autopsy. The saccharides were mainly based on core 3 (GlcNAcbeta1-3GalNAc). Among the terminal saccharide determinants Sd(a)/Cad-antigen-like structures were prominent, and Lewis x, sialyl Lewis x and sulphated Lewis x were found as minor components, whereas blood group H and A antigenic determinants were absent. The saccharides were markedly different from those of mucins from colon cancers or colon cancer cell lines analysed so far, in which cores 1 and 2 are prominent features, and in which various other terminal determinants have been found, but not Sd(a)/Cad.

Autopsy↗
PubMed2001

Galectins structure and function--a synopsis.

The 20 or so galectins expected to be found in man, and their many possible functional effects promise a rich and fruitful research field in the future. At present, the biomedically most promising areas for use of galectins or their ligands are in inflammation, immunity, and cancer. Many good stories can be formulated, but the field lacks the cohesion of knowing basic galectin function. The only basic common denominators among galectins are beta-galactoside binding, and the unusual combination of intra- and extracellular expression with non-classical secretion in between. Maybe that is all there is, and nature has used these properties for multiple, otherwise unrelated functions. Then again, maybe there is some deeper common function that has so far been overlooked. If it exists, this probably lies somewhere in the detailed integration of galectin activity in the complexities of cell physiology.

Animals↗
PubMed2001

Protein identification by in-gel digestion, high-performance liquid chromatography, and mass spectrometry: peptide analysis by complementary ionization techniques.

A biologically active protein fraction was isolated from rabbit intestine, purified by one-dimensional SDS-PAGE and stained with Coomassie Brilliant Blue. A predominant band of approximately 110-130 kDa was excised and digested in-gel with trypsin. The resulting peptides were extracted then separated by microbore reversed-phase high-performance liquid chromatography (HPLC). Mass spectrometric data from one HPLC fraction obtained by two different ionization techniques proved to be complementary. Matrix-assisted laser desorption/ionization (MALDI) showed nine peptide masses, which by post source decay analysis and database searching were attributed to two proteins. Nanoflow electrospray analysis performed on a hybrid tandem mass spectrometer of quadrupole-quadrupole-orthogonal acceleration time-of-flight (QqTOF) geometry detected six additional peptide components. On the basis of the additional peptides and superior quality collision-induced dissociation spectra typical of this instrument type, two further proteins were identified. The resolution afforded by the QqTOF instrument permitted charge state determination for the fragment ions while preserving the high detection sensitivity that was essential in obtaining the composition of this mixture of proteins.

Animals↗
PubMed2001

Distance mapping of protein-binding sites using spin-labeled oligosaccharide ligands.

The binding of a nitroxide spin-labeled analog of N-acetyllactosamine to galectin-3, a mammalian lectin of 26 kD size, is studied to map the binding sites of this small oligosaccharide on the protein surface. Perturbation of intensities of cross-peaks in the (15)N heteronuclear single quantum coherence (HSQC) spectrum of full-length galectin-3 owing to the bound spin label is used qualitatively to identify protein residues proximate to the binding site for N-acetyllactosamine. A protocol for converting intensity measurements to a more quantitative determination of distances between discrete protein amide protons and the bound spin label is then described. This protocol is discussed as part of a drug design strategy in which subsequent perturbation of chemical shifts of distance mapped amide cross-peaks can be used effectively to screen a library of compounds for other ligands that bind to the target protein at distances suitable for chemical linkage to the primary ligand. This approach is novel in that it bypasses the need for structure determination and resonance assignment of the target protein.

Amino Sugars↗
PubMed2001

Lipopolysaccharide-induced gelatinase granule mobilization primes neutrophils for activation by galectin-3 and formylmethionyl-Leu-Phe.

We have earlier shown that galectin-3, a lactose-binding mammalian lectin that is secreted from activated macrophages, basophils, and mast cells, induces activation of the NADPH oxidase in exudated but not in peripheral blood neutrophils (A. Karlsson, P. Follin, H. Leffler, and C. Dahlgren, Blood 91:3430-3438, 1998). The alteration in responsiveness occurring during extravasation correlated with mobilization of the gelatinase and/or specific granules to the cell surface, indicating a role for mobilizable galectin-3 receptors. In this study we have investigated galectin-3-induced NADPH oxidase activation, measured as superoxide production, in lipopolysaccharide (LPS)-primed neutrophils. Upon galectin-3 challenge, the LPS-primed cells produced superoxide, both extracellularly and intracellularly. A primed extracellular response to formylmethionyl-Leu-Phe (fMLF) was also achieved. The exposure of complement receptors 1 and 3 as well as the formyl peptide receptor on the cell surface was markedly increased after LPS treatment, indicating that granule fusion with the plasma membrane had occurred. Further assessment of specific markers for neutrophil granules showed that the LPS treatment had mobilized the gelatinase granules but only a minor fraction of the specific granules. We thus suggest that the mechanism behind LPS priming lies at the level of granule (receptor) mobilization for galectin-3 as well as for fMLF.

Antigens, CD↗
PubMed2000

Salivary agglutinin, which binds Streptococcus mutans and Helicobacter pylori, is the lung scavenger receptor cysteine-rich protein gp-340.

Salivary agglutinin is a high molecular mass component of human saliva that binds Streptococcus mutans, an oral bacterium implicated in dental caries. To study its protein sequence, we isolated the agglutinin from human parotid saliva. After trypsin digestion, a portion was analyzed by matrix-assisted laser/desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), which gave the molecular mass of 14 unique peptides. The remainder of the digest was subjected to high performance liquid chromatography, and the separated peptides were analyzed by MALDI-TOF/post-source decay; the spectra gave the sequences of five peptides. The molecular mass and peptide sequence information showed that salivary agglutinin peptides were identical to sequences in lung (lavage) gp-340, a member of the scavenger receptor cysteine-rich protein family. Immunoblotting with antibodies that specifically recognized either lung gp-340 or the agglutinin confirmed that the salivary agglutinin was gp-340. Immunoblotting with an antibody specific to the sialyl Le(x) carbohydrate epitope detected expression on the salivary but not the lung glycoprotein, possible evidence of different glycoforms. The salivary agglutinin also interacted with Helicobacter pylori, implicated in gastritis and peptic ulcer disease, Streptococcus agalactiae, implicated in neonatal meningitis, and several oral commensal streptococci. These results identify the salivary agglutinin as gp-340 and suggest it binds bacteria that are important determinants of either the oral ecology or systemic diseases.

Agglutinins↗
PubMed1999

Identification of CD66a and CD66b as the major galectin-3 receptor candidates in human neutrophils.

The mammalian lectin galectin-3 is a potent stimulus of human neutrophils, provided that the receptor(s) for the lectin has been mobilized to the cell surface before activation. We have recently shown that the receptors for galectin-3 are stored in intracellular mobilizable granules. Here we show supportive evidence for this in that DMSO-differentiated (neutrophil-like) HL-60 cells, which lack gelatinase and specific granules, are nonresponsive when exposed to galectin-3. Neutrophil granules were subsequently used for isolation of galectin-3 receptors by affinity chromatography. Proteins eluted from a galectin-3-Sepharose column by lactose were analyzed on SDS-polyacrylamide gels and showed two major bands of 100 and 160 kDa and a minor band of 120 kDa. By immunoblotting, these proteins were shown to correspond to CD66a (160 kDa), CD66b (100 kDa), and lysosome-associated membrane glycoprotein-1 and -2 (Lamp-1 and -2; 120 kDa). The unresponsive HL-60 cells lacked the CD66 Ags but contained the Lamps, implying that neutrophil CD66a and/or CD66b may be the functional galectin-3 receptors. This conclusion was supported by the subcellular localization of the CD66 proteins to the gelatinase and specific granules in resting neutrophils.

Antigens, CD↗
PubMed1999

Separate oligosaccharide determinants mediate interactions of the low-molecular-weight salivary mucin with neutrophils and bacteria.

The low-molecular-weight human salivary mucin (MG2) coats oral surfaces, where it is in a prime location for governing cell adhesion. Since oligosaccharides form many of the interactive facets on mucin molecules, we examined MG2 glycosylation as it relates to the molecule's adhesive functions. Our previous study of MG2 oligosaccharide structures showed that the termini predominantly carry T, sialyl-T, Lewisx (Lex), sialyl Lex (sLex), lactosamine, and sialyl lactosamine determinants [Prakobphol, A., et al. (1998) Biochemistry 37, 4916-4927]. In addition, we showed that sLex determinants confer L-selectin ligand activity to this molecule. Here we studied adhesive interactions between MG2 and cells that traffic in the oral cavity: neutrophils and bacteria. Under flow conditions, neutrophils tethered to MG2-coated surfaces at forces between 1.25 and 2 dyn/cm2, i.e., comparable to the shear stress generated at the tooth surface by salivary flow ( approximately 0.8 dyn/cm2). MG2 was also found in association with neutrophils isolated from the oral cavity, evidence that the cells interact with this mucin in vivo. Since MG2 serves as an adhesion receptor for bacteria, the MG2 saccharides that serve this function were also identified. Seven of 18 oral bacteria strains that were tested adhered to MG2. Importantly, six of these seven strains adhered via T antigen, sialyl-T antigen, and/or lactosamine sequences. No adherence to Lex and sLex epitopes was detected in all the strains that were tested. Together, these results suggest that distinct subsets of MG2 saccharides function as ligands for neutrophil L-selectin and receptors for bacterial adhesion, a finding with interesting implications for both oral health and mucin function.

Bacterial Adhesion↗
PubMed1999

Molecular characterization of alpha-lactalbumin folding variants that induce apoptosis in tumor cells.

This study characterized a protein complex in human milk that induces apoptosis in tumor cells but spares healthy cells. The active fraction was purified from casein by anion exchange chromatography. Unlike other casein components the active fraction was retained by the ion exchanger and eluted after a high salt gradient. The active fraction showed N-terminal amino acid sequence identity with human milk alpha-lactalbumin and mass spectrometry ruled out post-translational modifications. Size exclusion chromatography resolved monomers and oligomers of alpha-lactalbumin that were characterized using UV absorbance, fluorescence, and circular dichroism spectroscopy. The high molecular weight oligomers were kinetically stable against dissociation into monomers and were found to have an essentially retained secondary structure but a less well organized tertiary structure. Comparison with native monomeric and molten globule alpha-lactalbumin showed that the active fraction contains oligomers of alpha-lactalbumin that have undergone a conformational switch toward a molten globule-like state. Oligomerization appears to conserve alpha-lactalbumin in a state with molten globule-like properties at physiological conditions. The results suggest differences in biological properties between folding variants of alpha-lactalbumin.

Amino Acid Sequence↗
PubMed1999

Macrophage inflammatory protein-2 is required for neutrophil passage across the epithelial barrier of the infected urinary tract.

IL-8 is a major human neutrophil chemoattractant at mucosal infection sites. This study examined the C-X-C chemokine response to mucosal infection, and, specifically, the role of macrophage inflammatory protein (MIP)-2, one of the mouse IL-8 equivalents, for neutrophil-epithelial interactions. Following intravesical Escherichia coli infection, several C-X-C chemokines were secreted into the urine, but only MIP-2 concentrations correlated to neutrophil numbers. Tissue quantitation demonstrated that kidney MIP-2 production was triggered by infection, and immunohistochemistry identified the kidney epithelium as a main source of MIP-2. Treatment with anti-MIP-2 Ab reduced the urine neutrophil numbers, but the mice had normal tissue neutrophil levels. By immunohistochemistry, the neutrophils were found in aggregates under the pelvic epithelium, but in control mice the neutrophils crossed the urothelium into the urine. The results demonstrate that different chemokines direct neutrophil migration from the bloodstream to the lamina propria and across the epithelium and that MIP-2 serves the latter function. These findings suggest that neutrophils cross epithelial cell barriers in a highly regulated manner in response to chemokines elaborated at this site. This is yet another mechanism that defines the mucosal compartment and differentiates the local from the systemic host response.

Animals↗
PubMed1998

X-ray crystal structure of the human galectin-3 carbohydrate recognition domain at 2.1-A resolution.

Galectins are a family of lectins which share similar carbohydrate recognition domains (CRDs) and affinity for small beta-galactosides, but which show significant differences in binding specificity for more complex glycoconjugates. We report here the x-ray crystal structure of the human galectin-3 CRD, in complex with lactose and N-acetyllactosamine, at 2.1-A resolution. This structure represents the first example of a CRD determined from a galectin which does not show the canonical 2-fold symmetric dimer organization. Comparison with the published structures of galectins-1 and -2 provides an explanation for the differences in carbohydrate-binding specificity shown by galectin-3, and for the fact that it fails to form dimers by analogous CRD-CRD interactions.

Animals↗
PubMed1998

Galectin-3 activates the NADPH-oxidase in exudated but not peripheral blood neutrophils.

Galectin-3, a lactose-binding mammalian lectin that is secreted from activated macrophages, basophils, and mast cells, was investigated with respect to its ability to activate the human neutrophil NADPH-oxidase. The galectin-3-induced activity was determined with in vivo exudated cells (obtained from a skin chamber) and compared with that of peripheral blood neutrophils. Galectin-3 was found to be a potent activator of the NADPH-oxidase only in exudated neutrophils and the binding of galectin-3 to the surface of these cells was increased compared with peripheral blood cells. Different in vitro priming protocols resulting in degranulation were used to mimic the exudation process in terms of increasing the receptor exposure on the cell surface. Galectin-3 could induce an oxidative response similar to that in exudated cells only after a significant amount of the intracellular organelles had been mobilized. This increase in oxidative response was paralleled by an increased binding of galectin-3 to the surface of the cells. The major conclusion of the study is that galectin-3 is a potent stimulus of the neutrophil respiratory burst, provided that the cells have first experienced an extravasation process. The results also imply that the neutrophil response to galectin-3 could be mediated through receptors mobilized from intracellular granules, and we report the presence of galectin-3-binding proteins in such organelles.

Alkaline Phosphatase↗
PubMed1998

Human low-molecular-weight salivary mucin expresses the sialyl lewisx determinant and has L-selectin ligand activity.

Previously we showed that the low-molecular-weight mucin (MG2, encoded by MUC7), a major component of human submandibular/sublingual saliva, is a bacterial receptor that coats the tooth surface. Here we tested the hypothesis that the structure of its carbohydrate residues contains important information about its function. Purified MG2 (Mr 120 000) was digested with trypsin, and the resulting Mr 90 000 fragment, which carried primarily O-linked oligosaccharides, was subjected to reductive beta-elimination. The released oligosaccharides were characterized by using nuclear magnetic resonance spectroscopy and mass spectrometry. Of the 41 different structures we detected, the most prominent included NeuAcalpha2-->3Galbeta1-->3GalNAc-ol (sialyl-T antigen), Galbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-->6(Galbeta1 -->3)GalNAc-ol [type 2 core with Lewisx (Lex) determinant], and NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->6(Galbeta1--> 3) GalNAc-ol [type 2 core with sialyl Lex (sLex) determinant]. We also detected di-, tri-, and pentasaccharides with one sulfate group. Lex, sLex, and related sulfated structures are ligands for selectins, adhesion molecules that mediate leukocyte trafficking. Therefore, we investigated whether MG2 was a selectin ligand. In an enzyme-linked immunosorbent assay, L-selectin chimeras interacted with immobilized MG2 in a Ca2+-dependent manner. L-Selectin chimeras also bound to MG2 immobilized on nitrocellulose. Together, these results suggest that the saccharides that MG2 carries could specify some of its important functions, which may include mediating leukocyte interactions in the oral cavity.

Carbohydrate Sequence↗
PubMed1998

Galectin-4 and galectin-6 are two closely related lectins expressed in mouse gastrointestinal tract.

Galectins are a family of carbohydrate-binding proteins that share a conserved sequence and affinity for beta-galactosides. Some, such as galectin-1, are isolated as dimers and have a single carbohydrate recognition domain (CRD) in each monomer, whereas others, such as galectin-4, are isolated as monomers and have two CRDs in a single polypeptide chain. In the course of studying mouse colon mRNA for galectin-4, we detected a related mRNA that encodes a new galectin that also has two CRDs in a single peptide chain. The new galectin, galectin-6, lacks a 24-amino acid stretch in the link region between the two CRDs that is present in galectin-4. Otherwise, these two galectins have 83% amino acid identity. Expression of both galectin-4 and galectin-6 is confined to the epithelial cells of the embryonic and adult gastrointestinal tract. Galectin-4 is expressed at about equal levels in colon and small intestine but much less in stomach, whereas galectin-6 is expressed at about equal levels throughout the gastrointestinal tract.

Animals↗
PubMed1998

Sequence, structure, and chromosomal mapping of the mouse Lgals6 gene, encoding galectin-6.

In the accompanying paper (Gitt, M. A., Colnot, C., Poirier, F., and Barondes, S. H., and Leffler, H. (1998) J. Biol. Chem. 273, 2954-2960), we reported that mouse gastrointestinal tract specifically expresses two closely related galectins, galectins-4 and -6, each with two carbohydrate recognition domains in the same peptide. Here, we report the isolation, characterization, and chromosomal mapping of the complete mouse Lgals6 gene, which encodes galectin-6, and of a fragment of a distinct gene, Lgals4, which encodes galectin-4. The coding sequence of galectin-6 is specified by eight exons. The upstream region contains two putative promoters. Both Lgals6 and the closely related Lgals4 are clustered together about 3.2 centimorgans proximal to the apoE gene on mouse chromosome 7. The syntenic human region is 19q13.1-13.3.

Animals↗
PubMed1997

Sulfated lewis X determinants as a major structural motif in glycans from LS174T-HM7 human colon carcinoma mucin.

This article describes oligosaccharide structures of mucin isolated from nude mouse xenograft tumors produced by LS174T-HM7 cells, a subline of the human colon carcinoma LS174T with higher metastatic tendency and higher mucin production. A striking feature of the oligosaccharides of the LS174T-HM7 xenograft tumor mucin was a predominance of sulfated Lewis X determinants: HSO3-Galbeta1-4(Fucalpha1-3)GlcNAc. In addition to one previously known saccharide with one sulfated Lewis X determinant, the HM7 xenograft tumor mucin contained multiple novel structures containing one, two, or three sulfated Lewis X determinants. This determinant, known to act as a selectin ligand, has been found previously in minor saccharide components of human milk as well as mucins, but never before as a predominant structure in one mucin source.

Animals↗
PubMed1997

Palmitoyl carnitine, a lysophospholipase-transacylase inhibitor, prevents Candida adherence in vitro.

Candida adherence is poorly understood. The results of this study indicate that interactions of Candida with the lyso-forms of phospholipids may be one important attachment mechanism. C. tropicalis and C. albicans adhered to purified lysophospholipids immobilized on microtiter wells, as well as to a human laryngeal epidermoid carcinoma (HEp-2) cell line. Adherence to both lysophospholipids and HEp-2 cells was significantly reduced by palmitoyl carnitine, a lysophospholipase-transacylase inhibitor. Over time there was a positive correlation between Candida adherence and its transacylase activity. The data suggest that palmitoyl carnitine interferes with Candida adherence to lysophospholipids and the HEp-2 cell line by blocking the interaction between the Candida-associated transacylase enzyme receptor site and its lysophospholipid substrate ligand.

Acyltransferases↗