Endogenous β-d-galactoside binding lectin during the expansion of the yolk sac in the developing chick embryo

Summary A lectin with an affinity for -d-galactoside-containing saccharides is present in the developing yolk sac from the chick embryo at stages from 2 to 7 days of incubation. This activity is present in the area vitellina (less differentiated) and the area vasculosa (more differentiated). In both areas, lectin activity increases significantly during the spreading of the yolk sac up to 5 days of incubation. At all of the stages studied lectin activity was significantly higher in the area vasculosa, as compared to the area vitellina.Lectins were purified by affinity chromatography and examined by SDS-PAGE. Under reducing conditions two components are evident. A more prominent band of subunit molecular weight of 14,200±100 for the area vitellina and 13,700±300 for the area vasculosa and a second band with molecular weight of about 68,000±700 and 68,000±1,200 for the area vitellina and area vasculosa respectively, were observed. The -d-galactoside-binding lectin appears to be similar if not identical to that of the early chick blastoderm.     

Immunochemical evidence for the involvement of adrenal ferredoxin in the side-chain cleavage of 20α-hydroxycholesterol

A goat antibody produced against homogeneous bovine adrenal ferrodoxin has been employed to study the involvement of this iron-sulfur protein in the side-chain cleavage of 20α-hydroxycholesterol catalyzed by a soluble fraction, supernatant S₁, prepared from sonicated bovine adrenocortical mitochondria. When added to this supernatant, the antibody inhibited the side-chain cleavage of 20α-hydroxycholesterol as well as the side-chain cleavage of cholesterol, the 11β-hydroxylation of deoxycorticosterone, and the NADPH-dependent reduction of cytochrome $\text{c}$. These results demonstrate that, similar to the NADPH-cytochrome $\text{c}$ reductase and both the cholesterol side-chain cleavage and steroid 11β-hydroxylase reactions, adrenal ferredoxin is also required for the side-chain cleavage of 20α-hydroxycholesterol.     

High-resolution structural insights on the sugar-recognition and fusion tag properties of a versatile β-trefoil lectin domain from the mushroom Laetiporus sulphureus

In this work, we analyzed at high resolution the sugar-binding mode of the recombinant N-terminal ricin-B domain of the hemolytic protein LSLa (LSL(150)) from the mushroom Laetiporus sulphureus and also provide functional in vitro evidence suggesting that, together with its putative receptor-binding role, this module may also increase the solubility of its membrane pore-forming partner. We first demonstrate that recombinant LSL(150) behaves as an autonomous folding unit and an active lectin. We have determined its crystal structure at 1.47?? resolution and also that of the [LSL(150):(lactose)β, γ)] binary complex at 1.67?? resolution. This complex reveals two lactose molecules bound to the β and γ sites of LSL(150), respectively. Isothermal titration calorimetry indicates that LSL(150) binds two lactoses in solution with highly different affinities. Also, we test the working hypothesis that LSL(150) exhibits in vivo properties typical of solubility tags. With this aim, we have fused an engineered version of LSL(150) (LSL(t)) to the N-terminal end of various recombinant proteins. All the designed LSL(150)-tagged fusion proteins were successfully produced at high yield, and furthermore, the target proteins were purified by a straightforward affinity procedure on agarose-based matrices due to the excellent properties of LSL(150) as an affinity tag. An optimized protocol for target protein purification was devised, which involved removal of the LSL(150) tag through in-column cleavage of the fusion proteins with His(6)-tagged TEV endoprotease. These results permitted to set up a novel, lectin-based system for production and purification of recombinant proteins in E. coli cells with attractive biotechnological applications.     

Further evidence of the involvement of the Wnt signaling pathway in Dupuytren’s disease

Genetic background plays an important role in the development of Dupuytren’s disease. A genome-wide association study (GWAS) showed that nine loci are associated with the disease, six of which contain genes that are involved in Wnt signaling (WNT2, WNT4, WNT7B, RSPO2, SFRP4, SULF1). To obtain insight in the role of these genes, we performed expression studies on affected and unaffected patient’s tissues. Surgically obtained nodules and cords from eight Dupuytren’s patients were compared to patient-matched control tissue (unaffected transverse palmar fascia). The Wnt-related genes found in the GWAS, the classical Wnt-downstream protein β-catenin, as well as (myo)fibroblast markers were analyzed using real-time qPCR and immunohistochemical stainings for mRNA levels and protein levels, respectively. The collagen-coding genes COL1A1 and COL3A1 were highly upregulated on mRNA level, both in cords and nodules. Three Wnt-related genes were found to be differently regulated compared to control tissue: WNT2 was downregulated in nodules, WNT7B was upregulated in nodules, and SFRP4 was upregulated in nodules and cords. Immunohistochemistry revealed significantly less staining of Wnt2 in cords, but significantly more staining for Wnt7b in nodules. There was significantly more staining of α-SMA in nodules and cord and β-catenin in nodules than in control tissue. We found differences in expression, both at mRNA and protein level, in several Wnt-related genes found earlier to be associated with Dupuytren’s disease. Of these, Wnt7b was upregulated and found in close association with both α-SMA and β-catenin expressing cells, making it a candidate pro-fibrotic mediator in Dupuytren’s disease.     

Is there a role for mannan-binding lectin in the diagnosis of inflammatory bowel disease?

Mannan-binding lectin (MBL) activates the lectin-complement pathway as part of the innate immune defence by binding to the surface of microorganisms. Therefore, MBL2 presents an interesting candidate gene for the inflammatory bowel diseases, ulcerative colitis (UC) and Crohn's disease (CD). In our study, we evaluated the MBL serum concentrations and genotypes for diagnostic and classification purposes of patients with CD and UC. The MBL serum concentration was analysed in 98 CD patients and in 83 UC patients. In total, 82 patients with inflammatory rheumatic disorders and 189 healthy individuals served as controls. All study subjects were genotyped for the MBL2 polymorphisms G54D, G57E and R52C and the NOD2 (CARD15) mutations R702W, G908R and L1007fsinsC. Neither the median MBL serum concentration nor the MBL2 genotype distribution differed significantly between cohorts. Measurement of MBL serum concentrations offers no benefit for the diagnosis of CD or UC.     

Muscle injury-induced thymosin β4 acts as a chemoattractant for myoblasts

Thymosin β4 (Tβ4) is a major intracellular G-actin-sequestering peptide. There is increasing evidence to support important extracellular functions of Tβ4 related to angiogenesis, wound healing and cardiovascular regeneration. We investigated the expression of 'Tβ4' and 'thymosin β10', a closely related peptide, during skeletal muscle regeneration in mice and chemotactic responses of myoblasts to these peptides. The mRNA levels of 'Tβ4' and 'thymosin β10' were up-regulated in the early stage of regenerating muscle fibres and inflammatory haematopoietic cells in the injured skeletal muscles of mice. We found that both Tβ4 and its sulphoxized form significantly accelerated wound closure and increased the chemotaxis of C2C12 myoblastic cells. Furthermore, we showed that primary myoblasts and myocytes derived from muscle satellite cells of adult mice were chemoattracted to sulphoxized form of Tβ4. These data indicate that muscle injury enhances the local production of Tβ4, thereby promoting the migration of myoblasts to facilitate skeletal muscle regeneration.     

Nucleotide sequence evidence of the unicentric origin of the βC mutation in Africa

Summary The origin of the ^C mutation was studied by characterizing nucleotide sequence polymorphisms on ^C chromosomes of patients from various African countries. In the majority of cases, the ^C mutation was found in linkage disequilibrium with a single chromosomal structure as defined by classical RFLP haplotypes, intergenic nucleotide sequence polymorphisms immediately upstream of the -globin gene, and intragenic -globin gene polymorphisms (frameworks). In addition, three atypical variant chromosomes carrying the ^C mutation were observed, and are most probably explained either by a meiotic recombination (two cases) or by one nucleotide substitution occurring in an unstable array of tandemly repeated sequences (one case). These data demonstrate the unicentric origin of the ^C mutation in central West Africa, with subsequent mutational modification in a small number of instances. The data also supports gene flow of the ^C chromosome from subsaharan Africa to North Africa.     

Immunohistochemical and in situ hybridization analysis of galectin-3, a β-galactoside binding lectin, in the urinary system of adult mice

Galectin is an animal lectin that has high affinity to β-galactoside of glycoconjugates. In the present study, cellular expression of galectin subtypes in the urinary system of adult mice was examined by in situ hybridization and immunohistochemistry. The major subtype expressed in the murine urinary system was galectin-3, which was expressed continuously from the kidney to the distal end of the urethra. The renal cortex expressed galectin-3 more intensely than the medulla. Renal galectin-3 immunoreactivity was strongest in the cortical collecting ducts, where principal cells were the sole cellular source. All cell layers of the transitional epithelium from the renal pelvis to the urethra strongly expressed galectin-3 at the mRNA and protein levels. An electron microscopic study demonstrated diffuse cytoplasmic localization of galectin-3 in principal cells of the collecting ducts and in the bladder epithelial cells. Urethral galectin-3 expression at the pars spongiosa decreased in intensity near the external urethral orifice, where the predominant subtype of galectin was substituted by galectin-7. The muscular layer of the ureter and urinary bladder contained significant signals for galectin-1. Taken together, the observations indicate that the adult urinary system shows intense and selective expression of galectin-3 in epithelia of the uretic bud- and cloaca-derivatives.     

Analysis of the β-oxidation of trans-unsaturated fatty acid in recombinant Saccharomyces cerevisiae expressing a peroxisomal PHA synthase reveals the involvement of a reductase-dependent pathway

The degradation of fatty acids having cis- or trans-unsaturated bond at an even carbon was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanaote is synthesized by the polymerization of the β-oxidation intermediates 3-hydroxy-acyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The synthesis of polyhydroxyalkanoate in cells grown in media containing 10-cis-heptadecenoic acid was dependent on the presence of 2,4-dienoyl-CoA reductase activity as well as on Δ³,Δ²-enoyl-CoA isomerase activity. The synthesis of polyhydroxyalkanoate from 10-trans-heptadecenoic acid in mutants devoid of 2,4-dienoyl-CoA reductase revealed degradation of the trans fatty acid directly via the enoyl-CoA hydratase II activity of the multifunctional enzyme (MFE), although the level of polyhydroxyalkanoate was 10–25% to that of wild type cells. Polyhydroxyalkanoate produced from 10-trans-heptadecenoic acid in wild type cells showed substantial carbon flux through both a reductase-dependent and a direct MFE-dependent pathway. Flux through β-oxidation was more severely reduced in mutants devoid of Δ³,Δ²-enoyl-CoA isomerase compared to mutants devoid of 2,4-dienoyl-CoA reductase. It is concluded that the intermediate 2-trans,4-trans-dienoyl-CoA is metabolized in vivo in yeast by both the enoyl-CoA hydratase II activity of the multifunctional protein and the 2,4-dienoyl-CoA reductase, and that the synthesis of the intermediate 3-trans-enoyl-CoA in the absence of the Δ³,Δ²-enoyl-CoA isomerase leads to the blockage of the direct MFE-dependent pathway in vivo.     

Crystal structure of a β-prism II lectin from Remusatia vivipara

The crystal structure of a β-prism II (BP2) fold lectin from Remusatia vivipara, a plant of traditional medicinal value, has been determined at a resolution of 2.4??. This lectin (RVL, Remusatia vivipara lectin) is a dimer with each protomer having two distinct BP2 domains without a linker between them. It belongs to the "monocot mannose-binding" lectin family, which consists of proteins of high sequence and structural similarity. Though the overall tertiary structure is similar to that of lectins from snowdrop bulbs and garlic, crucial differences in the mannose-binding regions and oligomerization were observed. Unlike most of the other structurally known proteins in this family, only one of the three carbohydrate recognition sites (CRSs) per BP2 domain is found to be conserved. RVL does not recognize simple mannose moieties. RVL binds to only N-linked complex glycans like those present on the gp120 envelope glycoprotein of HIV and mannosylated blood proteins like fetuin, but not to simple mannose moieties. The molecular basis for these features and their possible functional implications to understand the different levels of carbohydrate affinities in this structural family have been investigated through structure analysis, modeling and binding studies. Apart from being the first structure of a lectin to be reported from the Araceae/Arum family, this protein also displays a novel mode of oligomerization among BP2 lectins.     

Deletion of the N-terminal dirigent domain in maize β-glucosidase aggregating factor and its homolog sorghum lectin dramatically alters the sugar-specificities of their lectin domains

Maize β-glucosidase aggregating factor (BGAF) and its homolog Sorghum Lectin (SL) are modular proteins consisting of an N-terminal dirigent domain and a C-terminal jacalin-related lectin (JRL) domain. BGAF is a polyspecific lectin with a monosaccharide preference for galactose, whereas SL displays preference for GalNAc. Here, we report that deletion of the N-terminal dirigent domain in the above lectins dramatically changes their sugar-specificities. Deletions in the N-terminal region of the dirigent domain of BGAF abolished binding to galactose/lactose, but binding to mannose was unaffected. Glucose, which was a poor inhibitor of hemagglutinating activity of BGAF, displayed higher inhibitory effect on the hemagglutinating activity of deletion mutants. Deletion of the dirigent domain in SL abolished binding to GalNAc, but binding to mannose was not affected. Surprisingly, fructose, an extremely poor inhibitor (minimum inhibitory concentration (MIC) = 125 mM) of SL hemagglutinating activity, was found to be a very potent inhibitor (MIC = 1 mM) of hemagglutinating activity of its JRL domain. These results indicate that the dirigent domain in this class of modular lectins, at least in the case of maize BGAF and SL, influences sugar specificity.     

Expression of a library of fungal β-glucosidases in Saccharomyces cerevisiae for the development of a biomass fermenting strain

Converting cellulosic biomass to ethanol involves the enzymatic hydrolysis of cellulose and the fermentation of the resulting glucose. The yeast Saccharomyces cerevisiae is naturally ethanologenic, but lacks the enzymes necessary to degrade cellulose to glucose. Towards the goal of engineering S. cerevisiae for hydrolysis of and ethanol production from cellulose, 35 fungal β-glucosidases (BGL) from the BGL1 and BGL5 families were screened for their ability to be functionally expressed and displayed on the cell surface. Activity assays revealed that the BGL families had different substrate specificities, with only the BGL1s displaying activity on their natural substrate, cellobiose. However, growth on cellobiose showed no correlation between the specific growth rates, the final cell titer, and the level of BGL1 activity that was expressed. One of the BGLs that expressed the highest levels of cellobiase activity, Aspergillus niger BGL1 (Anig-Bgl101), was then used for further studies directed at developing an efficient cellobiose-fermenting strain. Expressing Anig-Bgl101 from a plasmid yielded higher ethanol levels when secreted into the medium rather than anchored to the cell surface. In contrast, ethanol yields from anchored and secreted Anig-Bgl101 were comparable when integrated on the chromosome. Flow cytometry analysis revealed that chromosomal integration of Anig-Bgl101 resulted in a higher percentage of the cell population that displayed the enzyme but with overall lower expression levels.     

Do synovial biopsies help to support evidence for involvement of innate immunity in the immunopathology of Beh?et's disease?

Behçet's disease is a complex vasculitis of unknown etiology. Abundant neutrophils suggest the involvement of innate immunity. Cytokines are skewed to the T-helper-1 pattern. Few sterile organs are easily accessible for analysis in Behçet's disease. Cañete and coworkers identify inflamed joints as a feasible model and suggest the involvement of innate immunity in Behçet's disease.     

Ferritin acts as a target site for the snowdrop lectin （GNA） in the midgut of the cotton leafworm Spodoptera littoralis

The snowdrop lectin GNA （Galanthus nivalis agglutinin） has been shown to possess insecticidal activity to a range of economically important insect pests. However, the precise mechanism of insecticidal action of GNA against insects remains unknown. In this investigation, we attempted to purify and identify receptor（s） responsible for binding of GNA in the larval midgut of a major lepidopteran pest （the cotton leafworm, Spodoptera littoralis） to better understand its mode of action. Therefore, cytoplasmic as well as membrane proteins from 800 larval midguts were chromatographed on a column with immobilized GNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the proteins eluted from the GNA column followed by sequencing of the GNA-binding proteins and BLAST analyses revealed that the N-terminal sequences of a 24 kDa polypeptide purified from the cytoplasmic and membrane protein fraction revealed sequence similarity to sequences encoding heavy chain homologs of ferritin from Manduca sexta （76% sequence identity）, Calpodes ethlius （80% sequence identity） and Bombyx mori （61% sequence identity）. Furthermore, the N-terminal sequence of a 31 kDa polypeptide from the membrane protein fraction showed sequence similarity to a light chain homolog of ferritin from Manduca sexta （88% sequence identity）.     

Immunohistochemical localization of 14 kDa β-galactoside-binding lectin in various organs of rat

Summary Immunohistochemical localization of 14 kDa -galactoside-binding lectin in various organs of adult rat was achieved using a monospecific antibody raised against lectin purified from rat lung. The antibody-stained cells were formed into small aggregates, thin fascicles, or thick bundles in the walls of blood vessels, gastrointestinal tracts and urogenital organs. From the patterns of distribution, as well as their organization, these immunoreactive cells were regarded as smooth muscle cells. This was confirmed by a double immunofluorescence study using a mixture of anti 14 kDa lectin and anti -smooth muscle-specific actin antibodies. Strong 14 kDa lectin immunoreactivity was seen in the pericellular matrix of smooth muscle cells in intact organs as well as in detergent-treated organs from which all cellular components were extracted. From these findings, it is suggested that the 14 kDa lectin may be externalized by smooth muscle cells into their pericellular matrix and participate in the crosslinking of the complementary glycoconjugate(s) localized at that site. The macromolecular complex of glycoconjugates thus formed around smooth muscle cells may play a role in anchoring smooth muscle cells to the pericellular connective tissue thereby permitting the force of muscle contraction to be efficiently transmitted to the surrounding connective tissue proper.