Differential analysis of site-specific glycans on plasma and cellular fibronectins: application of a hydrophilic affinity method for glycopeptide enrichment

Isolation of glycopeptides utilizing hydrogen bonding between glycopeptide glycans and a carbohydrate-gel matrix in the organic phase is useful for site-specific characterization of oligosaccharides of glycoproteins, when combined with mass spectrometry. In this study, recovery of glycopeptides was improved by including divalent cations or increasing the organic solvent in the binding solution, without losing specificity, whereas it was still less effective for those with a long peptide backbone exceeding 50 amino acid residues. The method was then applied to the analysis of glycan heterogeneities at seven N-glycosylation sites in each of the plasma and cellular fibronectins (FNs). There was a remarkable site-specific difference in fucosylation between these isoforms; Asn1244 selectively escaped the global fucosylation of cellular FN, whereas only Asn1007 and Asn2108 of the plasma isoform underwent modification. In addition, a new O-glycosylation site was identified at Thr279 in the connecting segment between the fibrin- and heparin-binding domain and the collagen-binding domain, and the glycopeptide was reactive to a peanut agglutinin lectin. Considering that another mucin-type O-glycosylation site lies within a different connecting segment, the O-glycosylation of FN was suggested to play a significant role in segregating the neighboring domains and thus maintaining the topology of FN and the domain functions. In addition, the method was applied to apolipoprotein B-100 (apoB100) whose N-glycan structures at 17 of 19 potential sites have been reported, and characterized the remaining sites. The results also demonstrated that the enriched glycopeptide provides resources for site-specific analysis of oligosaccharides in glycoproteomics.     

Land use legacy effects on woody vegetation in agricultural landscapes of south‐western Ethiopia

Aim

Past land use legacy effects—extinction debts and immigration credits—might be particularly pronounced in regions characterized by complex and dynamic landscape change. The aim of this study was to evaluate how current woody plant species distribution, composition and richness related to historical and present land uses.

Location

A smallholder farming landscape in south‐western Ethiopia.

Methods

We surveyed woody plants in 72 randomly selected 1‐ha sites in farmland and grouped them into forest specialist, generalist and pioneer species. First, we investigated woody plant composition and distribution using non‐metric multidimensional scaling. Second, we modelled species richness in response to historical and current distance from the forest edge. Third, we examined diameter class distributions of trees in recently converted vs. permanent farmland.

Results

Historical distance was a primary driver of woody plant composition and distribution. Generalist and pioneer species richness increased with historical distance. Forest specialists, however, did not respond to historical distance. Only few old individuals of forest specialist species remained in both recently converted and permanent farmlands.

Main conclusions

Our findings suggest that any possible extinction debt for forest specialist species in farmland at the landscape scale was rapidly paid off, possibly because farmers cleared large remnant trees. In contrast, we found substantial evidence of immigration credits in farmland for generalist and pioneer species. This suggests that long‐established farmland may have unrecognized conservation values, although apparently not for forest specialist species. We suggest that conservation policies in south‐western Ethiopia should recognize not only forests, but also the complementary value of the agricultural mosaic—similar to the case of European cultural landscapes. A possible future priority could be to better reintegrate forest species in the farmland mosaic.

     

An N-Glycosylation Site on the��-Propeller Domain of the Integrin ��5 Subunit Plays Key Roles in Both Its Function and Site-specific Modification by��1,4-N-Acetylglucosaminyltransferase III

Recently we reported that N-glycans on the β-propeller domain of the integrin α5 subunit (S-3,4,5) are essential for α5β1 heterodimerization, expression, and cell adhesion. Herein to further investigate which N-glycosylation site is the most important for the biological function and regulation, we characterized the S-3,4,5 mutants in detail. We found that site-4 is a key site that can be specifically modified by N-acetylglucosaminyltransferase III (GnT-III). The introduction of bisecting GlcNAc into the S-3,4,5 mutant catalyzed by GnT-III decreased cell adhesion and migration on fibronectin, whereas overexpression of N-acetylglucosaminyltransferase V (GnT-V) promoted cell migration. The phenomenon is similar to previous observations that the functions of the wild-type α5 subunit were positively and negatively regulated by GnT-V and GnT-III, respectively, suggesting that the α5 subunit could be duplicated by the S-3,4,5 mutant. Interestingly GnT-III specifically modified the S-4,5 mutant but not the S-3,5 mutant. This result was confirmed by erythroagglutinating phytohemagglutinin lectin blot analysis. The reduction in cell adhesion was consistently observed in the S-4,5 mutant but not in the S-3,5 mutant cells. Furthermore mutation of site-4 alone resulted in a substantial decrease in erythroagglutinating phytohemagglutinin lectin staining and suppression of cell spread induced by GnT-III compared with that of either the site-3 single mutant or wild-type α5. These results, taken together, strongly suggest that N-glycosylation of site-4 on the α5 subunit is the most important site for its biological functions. To our knowledge, this is the first demonstration that site-specific modification of N-glycans by a glycosyltransferase results in functional regulation.Glycosylation is a crucial post-translational modification of most secreted and cell surface proteins (1). Glycosylation is involved in a variety of physiological and pathological events, including cell growth, migration, differentiation, and tumor invasion. It is well known that glycans play important roles in cell-cell communication, intracellular signal transduction, protein folding, and stability (2, 3).Integrins comprise a family of receptors that are important for cell adhesion. The major function of integrins is to connect cells to the extracellular matrix, activate intracellular signaling pathways, and regulate cytoskeletal formation (4). Integrin α5β1 is well known as a fibronectin (FN)³ receptor. The interaction between integrin α5 and FN is essential for cell migration, cell survival, and development (5–8). In addition, integrins are N-glycan carrier proteins. For example, α5β1 integrin contains 14 and 12 putative N-glycosylation sites on the α5 and β1 subunits, respectively. Several studies suggest that N-glycosylation is essential for functional integrin α5β1. When human fibroblasts were cultured in the presence of 1-deoxymannojirimycin, which prevents N-linked oligosaccharide processing, immature α5β1 integrin appeared on the cell surface, and FN-dependent adhesion was greatly reduced (9). Treatment of purified integrin α5β1 with N-glycosidase F, which cleaves between the innermost N-acetylglucosamine (GlcNAc) and asparagine N-glycan residues of N-linked glycoproteins, prevented the inherent association between subunits and blocked α5β1 binding to FN (10).A growing body of evidence indicates that the presence of the appropriate oligosaccharide can modulate integrin activation. N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the addition of GlcNAc to mannose that is β1,4-linked to an underlying N-acetylglucosamine, producing what is known as a “bisecting” GlcNAc linkage as shown in Fig. 1B. GnT-III is generally regarded as a key glycosyltransferase in N-glycan biosynthetic pathways and contributes to inhibition of metastasis. The introduction of a bisecting GlcNAc catalyzed by GnT-III suppresses additional processing and elongation of N-glycans. These reactions, which are catalyzed in vitro by other glycosyltransferases, such as N-acetylglucosaminyltransferase V (GnT-V), which catalyzes the formation of β1,6 GlcNAc branching structures (Fig. 1B) and plays important roles in tumor metastasis, do not proceed because the enzymes cannot utilize the bisected N-glycans as a substrate. Introduction of the bisecting GlcNAc to integrin α5 by overexpression of GnT-III resulted in decreased in ligand binding and down-regulation of cell adhesion and migration (11–13). Contrary to the functions of GnT-III, overexpression of GnT-V promoted integrin α5β1-mediated cell migration on FN (14). These observations clearly demonstrate that the alteration of N-glycan structure affected the biological functions of integrin α5β1. Similarly characterization of the carbohydrate moieties in integrin α3β1 from non-metastatic and metastatic human melanoma cell lines showed that expression of β1,6 GlcNAc branched structures was higher in metastatic cells compared with non-metastatic cells, confirming the notion that the β1,6 GlcNAc branched structure confers invasive and metastatic properties to cancer cells. In fact, Partridge et al. (15) reported that GnT-V-modified N-glycans containing poly-N-acetyllactosamine, the preferred ligand for galectin-3, on surface receptors oppose their constitutive endocytosis, promoting intracellular signaling and consequently cell migration and tumor metastasis.Open in a separate windowFIGURE 1.Potential N-glycosylation sites on the α5 subunit and its modification by GnT-III and GnT-V. A, schematic diagram of potential N-glycosylation sites on the α5 subunit. Putative N-glycosylation sites are indicated by triangles, and point mutations are indicated by crosses (N84Q, N182Q, N297Q, N307Q, N316Q, N524Q, N530Q, N593Q, N609Q, N675Q, N712Q, N724Q, N773Q, and N868Q). B, illustration of the reaction catalyzed by GnT-III and GnT-V. Square, GlcNAc; circle, mannose. TM, transmembrane domain.In addition, sialylation on the non-reducing terminus of N-glycans of α5β1 integrin plays an important role in cell adhesion. Colon adenocarcinomas express elevated levels of α2,6 sialylation and increased activity of ST6GalI sialyltransferase. Elevated ST6GalI positively correlated with metastasis and poor survival. Therefore, ST6GalI-mediated hypersialylation likely plays a role in colorectal tumor invasion (16, 17). In fact, oncogenic ras up-regulated ST6GalI and, in turn, increased sialylation of β1 integrin adhesion receptors in colon epithelial cells (18). However, this is not always the case. The expression of hyposialylated integrin α5β1 was induced by phorbol esterstimulated differentiation in myeloid cells in which the expression of the ST6GalI was down-regulated by the treatment, increasing FN binding (19). A similar phenomenon was also observed in hematopoietic or other epithelial cells. In these cells, the increased sialylation of the β1 integrin subunit was correlated with reduced adhesiveness and metastatic potential (20–22). In contrast, the enzymatic removal of α2,8-linked oligosialic acids from the α5 integrin subunit inhibited cell adhesion to FN (23). Collectively these findings suggest that the interaction of integrin α5β1 with FN is dependent on its N-glycosylation and the processing status of N-glycans.Because integrin α5β1 contains multipotential N-glycosylation sites, it is important to determine the sites that are crucial for its biological function and regulation. Recently we found that N-glycans on the β-propeller domain (sites 3, 4, and 5) of the integrin α5 subunit are essential for α5β1 heterodimerization, cell surface expression, and biological function (24). In this study, to further investigate the underlying molecular mechanism of GnT-III-regulated biological functions, we characterized the N-glycans on the α5 subunit in detail using genetic and biochemical approaches and found that site-4 is a key site that can be specifically modified by GnT-III.     

Transfer of maternal cholesterol to embryo and fetus in pregnant mice

Cholesterol is essential for antenatal development. However, the transport of maternal cholesterol to the embryo has not been sufficiently studied, and that to the fetus is still controversial. To this end, a 1 mg dose of [3,4-(13)C(2)]cholesterol was injected daily into pregnant mice and the labeled cholesterol was measured by gas chromatography-mass spectrometry. After venous injections from days 10 to 17 of gestation, [(13)C]cholesterol levels in total ((12)C and (13)C) cholesterol were increased to 5.1% and 2.8% in maternal and fetal plasma, respectively. Labeled cholesterol was identified in the liver, kidneys, and intestines, but not in the brain, of the fetus. After injections from days 1 to 8, [(13)C]cholesterol levels were increased to 12.4% and 8.0% of total cholesterol in maternal plasma and the embryo, respectively. The level of 11.5% in the yolk sac was higher than that in the embryo. Intrauterine transfer of maternal cholesterol to the embryo as well as the fetus was evident in mice, and both the placenta and the yolk sac appear to be sites of intermediate passage in murine pregnancy.     

Activin A regulates activities of peripheral blood natural killer cells of mouse in an autocrine and paracrine manner

Activin A, a multifunctional cytokine of transforming growth factor-β (TGF-β) superfamily, can be produced by the diverse immune cells. NK cells in peripheral blood are one of the major immune cells applied to cancer therapy in recent years. However, whether activin A can be produced by natural killer (NK) cells and be involved in regulation of peripheral blood NK cells activities of mouse are not well characterized. Here, we found that activin type IIA and IIB receptors and signaling molecules Smad2, 3 were expressed in peripheral blood NK cells of mouse by flow cytometry and RT-PCR. The cultured blood NK cells of mouse not only produced activin βA chain protein by intracellular cytokine staining, but also secreted mature activin A protein by enzyme-linked immunosorbent assay (ELISA), and the production was promoted by IL-2. In addition, IL-2 as a positive control obviously promoted IFNγ production of mouse blood NK cells in vitro. However, activin A suppressed IFNγ production, but enhanced IL-2 synthesis and did not alter IL-10 production. Moreover, we found that activin A significantly suppressed the ability of NK cells to lyse target cells. These data revealed that blood NK cells of mouse were not only the target cells in response to activin A, but also the source of activin A, suggesting that activin A may play an important role in regulation of NK cells activities of mouse in an autocrine / paracrine manner.     

Woody plant diversity,composition and structure in relation to environmental variables and land-cover types in Lake Wanchi watershed,central highlands of Ethiopia

Knowledge of vegetation dynamics associated with human land-use change and environmental variables is crucial for sustainable watershed management. The objective of this study was to analyse woody plant diversity in response to the effects of anthropogenic disturbances, and topographic variables in different land-cover types. Woody plants ≥5 cm diameter at breast height (DBH) were recorded from 20-by-20 m plots, those <5 cm DBH were recorded from 10-by-10 m plots, and tree seedlings were recorded in 3-by-3 m plots. Nonmetric multidimensional scaling with Bray–Curtis distance was employed to analyse woody species composition. Woody species richness was analysed using generalised linear modelling. Tree diameter-class distributions were examined for population structure. The study identified 104 woody plants belonging to 52 families, where 74.5% were indigenous and 16.7% were endemic to Ethiopia. Asteraceae, Fabaceae and Rosaceae were the most species-rich families. There was evidence for the effect of anthropogenic disturbance and elevation on woody species composition. Wetlands contained the highest woody species richness, while woody species richness declined with increasing elevation and disturbance. Evidence of good regeneration was observed in wetlands. To sustain the ecological functions and services provided by the watershed, rehabilitation of the forest and shrub-cover should be a high priority for local management authorities.     

Efficient DNA Extraction from Nail Clippings Using the Protease Solution from <Emphasis Type="Italic">Cucumis melo</Emphasis>

Owing to the increasing importance of genomic information, obtaining genomic DNA easily from biological specimens has become more and more important. This article proposes an efficient method for obtaining genomic DNA from nail clippings. Nail clippings can be easily obtained, are thermostable and easy to transport, and have low infectivity. The drawback of their use, however, has been the difficulty of extracting genomic material from them. We have overcome this obstacle using the protease solution obtained from Cucumis melo. The keratinolytic activity of the protease solution was 1.78-fold higher than that of proteinase K, which is commonly used to degrade keratin. With the protease solution, three times more DNA was extracted than when proteinase K was used. In order to verify the integrity of the extracted DNA, genotype analysis on 170 subjects was performed by both PCR–RFLP and Real Time PCR. The results of the genotyping showed that the extracted DNA was suitable for genotyping analysis. In conclusion, we have developed an efficient extraction method for using nail clippings as a genome source and a research tool in molecular epidemiology, medical diagnostics, and forensic science.