Spitzenkorper localization and intracellular traffic of green fluorescent protein-labeled CHS-3 and CHS-6 chitin synthases in living hyphae of Neurospora crassa

The subcellular location and traffic of two selected chitin synthases (CHS) from Neurospora crassa, CHS-3 and CHS-6, labeled with green fluorescent protein (GFP), were studied by high-resolution confocal laser scanning microscopy. While we found some differences in the overall distribution patterns and appearances of CHS-3-GFP and CHS-6-GFP, most features were similar and were observed consistently. At the hyphal apex, fluorescence congregated into a conspicuous single body corresponding to the location of the Spitzenkörper (Spk). In distal regions (beyond 40 μm from the apex), CHS-GFP revealed a network of large endomembranous compartments that was predominantly comprised of irregular tubular shapes, while some compartments were distinctly spherical. In the distal subapex (20 to 40 μm from the apex), fluorescence was observed in globular bodies that appeared to disintegrate into vesicles as they advanced forward until reaching the proximal subapex (5 to 20 μm from the apex). CHS-GFP was also conspicuously found delineating developing septa. Analysis of fluorescence recovery after photobleaching suggested that the fluorescence of the Spk originated from the advancing population of microvesicles (chitosomes) in the subapex. The inability of brefeldin A to interfere with the traffic of CHS-containing microvesicles and the lack of colocalization of CHS-GFP with the endoplasmic reticulum (ER)-Golgi body fluorescent dyes lend support to the idea that CHS proteins are delivered to the cell surface via an alternative route distinct from the classical ER-Golgi body secretory pathway.Fungal hyphae elongate and branch by a complex process based on polarized secretion. Many studies have investigated the cellular and molecular components involved in shaping fungal cells, but no detailed understanding of the mechanisms that govern and regulate polarized fungal growth has been achieved (4, 25). In the yeast Saccharomyces cerevisiae, many of the main components of the secretory pathway, including some of the enzymes involved in cell wall formation, have been extensively characterized (32). Filamentous fungi encode homologues of some key components known from the yeast secretory pathway, but despite their apparent orthology, relatively little is known about how this pathway is organized to accomplish the highly polarized growth typical of hyphae. There are some differences in cell wall synthesis between filamentous fungi and S. cerevisiae. In hyphae of septate fungi, vesicles and other components accumulate at the apex, as part of the Spitzenkörper (Spk) (14, 22-24, 28). The composition and mode of action of this pleomorphic and dynamic structure have intrigued fungal biologists for many decades.Fungal cells have at least two types of well-defined secretory vesicles (5). It has been suggested that macrovesicles, or conventional secretory vesicles, carry the components of the amorphous phase of the cell wall, in addition to the load of extracellular enzymes (5, 27). There is a large body of evidence characterizing the chitin synthase (CHS)-carrying microvesicles as chitosomes (3, 8, 13, 30). CHS are β-glycosyltransferases that catalyze the polymerization of N-acetylglucosamine from UDP N-acetylglucosamine into chitin (47), a major structural polymer of the fungal cell wall (2). Chitin synthesis occurs in highly localized fashion both at the hyphal apices (7) and at nascent septa (29). Chitosomes are the smallest vesicles with the ability to form chitin microfibrils in vitro and have been suggested to carry and transport CHS to the cell surface at the apex of hyphae for cell wall synthesis (13, 37, 48, 55, 56). In recent years, studies on fungal CHS have concentrated mainly on gene identification. Given this wealth of information, we chose CHS as candidate markers to investigate vesicle traffic in fungal hyphae.Fungi have multiple chs genes grouped into two divisions, with seven classes, primarily on the basis of similarities in the primary sequence of the predicted proteins (12, 16, 37, 50). Division I includes classes I, II, and III, which share a catalytic domain surrounded by a hydrophilic N-terminal region and a hydrophobic C-terminal region (12). Division II includes classes IV, V, and VII, all with a catalytic domain preceded by a cytochrome b₅-like domain. In addition, classes V and VII contain an N-terminal myosin motor-like domain, suggesting a direct interaction with the actin cytoskeleton (15, 20, 58). Class VI has not been assigned to either division and includes recently identified CHS of unknown function (16). Earlier studies suggest that the various CHS have specific roles in chitin cell wall synthesis that are time or space dependent (60). In contrast to most filamentous fungi, S. cerevisiae (46) and Candida albicans (40) have only three or four CHS isozymes, respectively. S. cerevisiae Chs1p, C. albicans Chs2p, and C. albicans Chs8p belong to class I; S. cerevisiae Chs2p and C. albicans Chs1p belong to class II; and S. cerevisiae Chs3p and C. albicans Chs3p belong to class IV (46). While potential roles in hyphal growth have been suggested for some of the seven CHS classes described in filamentous fungi (9, 64, 65), we lack specific information on the cellular localization and trafficking to their sites of action in regions of active cell wall growth for most of these proteins.The goal of this study was to elucidate the traffic of CHS-containing vesicles en route from their site of genesis to their site of exocytosis in living hyphae of Neurospora crassa. The availability of an almost-complete genome sequence for this fungus allowed the identification of seven open reading frames with high homology to previously described chs genes (10). We chose to trace the intracellular location and secretory paths of CHS-3 and CHS-6. Neurospora CHS-3 belongs to the previously reported class I CHS with known homologues in all fungi tested, including S. cerevisiae Chs1p. In contrast, CHS-6 is a newly identified CHS assigned to class VI, homologous to Aspergillus fumigatus ChsD (39) and Coccidioides posadasii CHS-6 (34) but with no apparent homologues in S. cerevisiae or C. albicans. To trace both proteins, we fused green fluorescent protein (GFP) to the carboxyl terminus of the CHS coding regions and analyzed the fate of the resulting CHS-3-GFP and CHS-6-GFP fusion proteins by high-resolution confocal laser scanning microscopy (CLSM) in living hyphae of N. crassa.     

PER-TIM interactions with the photoreceptor cryptochrome mediate circadian temperature responses in Drosophila

Drosophila cryptochrome (CRY) is a key circadian photoreceptor that interacts with the period and timeless proteins (PER and TIM) in a light-dependent manner. We show here that a heat pulse also mediates this interaction, and heat-induced phase shifts are severely reduced in the cryptochrome loss-of-function mutant cry^b. The period mutant per^L manifests a comparable CRY dependence and dramatically enhanced temperature sensitivity of biochemical interactions and behavioral phase shifting. Remarkably, CRY is also critical for most of the abnormal temperature compensation of per^L flies, because a per^L; cry^b strain manifests nearly normal temperature compensation. Finally, light and temperature act together to affect rhythms in wild-type flies. The results indicate a role for CRY in circadian temperature as well as light regulation and suggest that these two features of the external 24-h cycle normally act together to dictate circadian phase.     

Aqueous two-phase recovery of bio-nanoparticles: a miniaturization study for the recovery of bacteriophage T4

Biotechnology industry has recently been demanding nanoparticulate products (20-200 nm) such as viruses, plasmids, virus-like particles and drug delivery assemblies. These products are mainly used as gene delivery systems in gene therapy protocols. During the process development for the manufacture of these products, it is crucial to optimize the recovery and purification steps. Unfortunately, the high value of some bio-nanoparticles complicates the optimization studies. The solvent extraction method with aqueous two-phase systems (ATPS) has been used to successfully recover bioproducts on a large scale. In this study, the potential miniaturization of ATPS is presented. The partition behavior of pure bovine serum albumin (BSA) in PEG-800-phosphate and bacteriophage T4 in PEG 8000-phosphate and PEG 600-sulphate systems were studied at three different scales (10 g, 2 g and 300 microl). The results obtained showed that the volume ratio (V(R)) for BSA (V(R)=1.0) was comparable to the blank systems at the scales studied. Additionally, the partition coefficient (K) was also similar (K=0.05) with more than 82% of BSA concentrated in the bottom phase. Same system was challenged with bacteriophage T4 showing a V(R)=1.0 and K greater than 5 with the infective particles concentrated in the top phase. The bacteriophage T4 was concentrated in opposite phase in the PEG-600-sulfate system with a consistent V(R)=0.8 and K<0.2 for the scales analyzed. The partition behavior the bacteriophage T4 was comparable to that reported previously for adenoviral vectors in same system at 15 ml scale. The results obtained demonstrated that the miniaturization of ATPS is feasible and reproducible for the two models selected. This provides significant information about the miniaturization process of such ATPS for their potential generic applications in the recovery of different bio-nanoparticle products.     

Unraveling multistate unfolding of pig kidney fructose-1,6-bisphosphatase using single tryptophan mutants

Pig kidney fructose-1,6-bisphosphatase is a homotetrameric enzyme which does not contain tryptophan. In a previous report the guanidine hydrochloride-induced unfolding of the enzyme has been described as a multistate process [Reyes, A. M., Ludwig, H. C., Ya?ez, A. J., Rodriguez, P. H and Slebe, J. C. (2003) Biochemistry 42, 6956-6964]. To monitor spectroscopically the unfolding transitions, four mutants were constructed containing a single tryptophan residue either near the C1-C2 or the C1-C4 intersubunit interface of the tetramer. The mutants were shown to retain essentially all of the structural and kinetic properties of the enzyme isolated from pig kidney. The enzymatic activity, intrinsic fluorescence, size-exclusion chromatographic profiles and 1-anilinonaphthalene-8-sulfonate binding by the mutants were studied under unfolding equilibrium conditions. The unfolding profiles were multisteps, and formation of hydrophobic structures was detected. The enzymatic activity of wild-type and mutant FBPases as a function of guanidine hydrochloride concentration showed an initial enhancement (maximum approximately 30%) followed by a biphasic decay. The activity and fluorescence results indicate that these transitions involve conformational changes in the fructose-1,6-bisphosphate and AMP domains. The representation of intrinsic fluorescence data as a 'phase diagram' reveals the existence of five intermediates, including two catalytically active intermediates that have not been previously described, and provides the first spectroscopic evidence for the formation of dimers. The intrinsic fluorescence unfolding profiles indicate that the dimers are formed by selective disruption of the C1-C2 interface.     

Study of the role of CCR5 in a mouse model of intranasal challenge with Yersinia pestis

CCR5 is a chemokine receptor used by HIV-1 to enter cells and has recently been found to act as a pathogen associated molecule pattern receptor. Current positive selection for the high frequency of a CCR5-Delta32 allele in humans has been attributed to resistance to HIV, smallpox, and plague infections. Using an intranasal mouse model of Y. pestis infection, we have found that lack of CCR5 does not enhance host resistance to Y. pestis infection and that CCR5-mediated responses might have a protective role. CCR5-/- mice exhibited higher levels of circulating RANTES and MIP-1alpha than those exhibited by wild-type mice at the baseline and throughout the course of Y. pestis infection. High levels of RANTES and MIP-1alpha, which are CCR5 ligands that mediate Natural Killer cell migration, may reflect compensation for the absence of CCR5 signaling.     

The ITGAV rs3738919-C allele is associated with rheumatoid arthritis in the European Caucasian population: a family-based study

The integrin alpha(v)beta3, whose alpha(v) subunit is encoded by the ITGAV gene, plays a key role in angiogenesis. Hyperangiogenesis is involved in rheumatoid arthritis (RA) and the ITGAV gene is located in 2q31, one of the suggested RA susceptibility loci. Our aim was to test the ITGAV gene for association and linkage to RA in a family-based study from the European Caucasian population. Two single nucleotide polymorphisms were genotyped by PCR-restriction fragment length polymorphism in 100 French Caucasian RA trio families (one RA patient and both parents), 100 other French families and 265 European families available for replication. The genetic analyses for association and linkage were performed using the comparison of allelic frequencies (affected family-based controls), the transmission disequilibrium test, and the genotype relative risk.We observed a significant RA association for the C allele of rs3738919 in the first sample (affected family-based controls, RA index cases 66.5% versus controls 56.7%; P = 0.04). The second sample showed the same trend, and the third sample again showed a significant RA association. When all sets were combined, the association was confirmed (affected family-based controls, RA index cases 64.6% versus controls 58.1%; P = 0.005). The rs3738919-C allele was also linked to RA (transmission disequilibrium test, 56.5% versus 50% of transmission; P = 0.009) and the C-allele-containing genotype was more frequent in RA index cases than in controls (RA index cases 372 versus controls 339; P = 0.002, odds ratio = 1.94, 95% confidence interval = 1.3-2.9). The rs3738919-C allele of the ITGAV gene is associated with RA in the European Caucasian population, suggesting ITGAV as a new minor RA susceptibility gene.     

Improved BLAST searches using longer words for protein seeding

MOTIVATION: The blastp and tblastn modules of BLAST are widely used methods for searching protein queries against protein and nucleotide databases, respectively. One heuristic used in BLAST is to consider only database sequences that contain a high-scoring match of length at most 5 to the query. We implemented the capability to use words of length 6 or 7. We demonstrate an improved trade-off between running time and retrieval accuracy, controlled by the score threshold used for short word matches. For example, the running time can be reduced by 20-30% while achieving ROC (receiver operator characteristic) scores similar to those obtained with current default parameters. AVAILABILITY: The option to use long words is in the NCBI C and C++ toolkit code for BLAST, starting with version 2.2.16 of blastall. A Linux executable used to produce the results herein is available at: ftp://ftp.ncbi.nlm.nih.gov/pub/agarwala/protein_longwords     

Comparative genomic analysis of the odorant-binding protein family in 12 <Emphasis Type="Italic">Drosophila</Emphasis> genomes: purifying selection and birth-and-death evolution

Background  

Chemoreception is a widespread mechanism that is involved in critical biologic processes, including individual and social behavior. The insect peripheral olfactory system comprises three major multigene families: the olfactory receptor (Or), the gustatory receptor (Gr), and the odorant-binding protein (OBP) families. Members of the latter family establish the first contact with the odorants, and thus constitute the first step in the chemosensory transduction pathway.     

Positive Mode LC-MS/MS Analysis of Chondroitin Sulfate Modified Glycopeptides Derived from Light and Heavy Chains of The Human Inter-α-Trypsin Inhibitor Complex*

The inter-α-trypsin inhibitor complex is a macromolecular arrangement of structurally related heavy chain proteins covalently cross-linked to the chondroitin sulfate (CS) chain of the proteoglycan bikunin. The inter-α-trypsin inhibitor complex is abundant in plasma and associated with inflammation, kidney diseases, cancer and diabetes. Bikunin is modified at Ser-10 by a single low-sulfated CS chain of 23–55 monosaccharides with 4–9 sulfate groups. The innermost four monosaccharides (GlcAβ3Galβ3Galβ4Xylβ-O-) compose the linkage region, believed to be uniform with a 4-O-sulfation to the outer Gal. The cross-linkage region of the bikunin CS chain is located in the nonsulfated nonreducing end, (GalNAcβ4GlcAβ3)_n, to which heavy chains (H1-H3) may be bound in GalNAc to Asp ester linkages. In this study we employed a glycoproteomics protocol to enrich and analyze light and heavy chain linkage and cross-linkage region CS glycopeptides derived from the IαI complex of human plasma, urine and cerebrospinal fluid samples. The samples were trypsinized, enriched by strong anion exchange chromatography, partially depolymerized with chondroitinase ABC and analyzed by LC-MS/MS using higher-energy collisional dissociation. The analyses demonstrated that the CS linkage region of bikunin is highly heterogeneous. In addition to sulfation of the Gal residue, Xyl phosphorylation was observed although exclusively in urinary samples. We also identified novel Neu5Ac and Fuc modifications of the linkage region as well as the presence of mono- and disialylated core 1 O-linked glycans on Thr-17. Heavy chains H1 and H2 were identified cross-linked to GalNAc residues one or two GlcA residues apart and H1 was found linked to either the terminal or subterminal GalNAc residues. The fragmentation behavior of CS glycopeptides under variable higher-energy collisional dissociation conditions displays an energy dependence that may be used to obtain complementary structural details. Finally, we show that the analysis of sodium adducts provides confirmatory information about the positions of glycan substituents.Glycosylation is the most complex protein post-translational modification known today but unfortunately glycomics characterization is often kept apart from the proteomic characterization of proteins of biological tissues and samples. Such dual approaches may be complementary but also limiting because they will give neither the whole picture of all protein iso/glycoforms in a tissue nor the detailed structure of any of the single proteins included. Glycoproteomics approaches are now becoming available bridging these two fields by keeping the glycan and the peptide parts together in giving simultaneous and specific information on the glycan structures, their attachment sites and the identities of the core proteins (1). Here we report on new liquid chromatography-tandem MS (LC-MS/MS) protocols that can be used to decipher the structural heterogeneity of proteoglycans and even proteoglycan complexes, i.e. glycoproteins known to be inherently demanding to structurally characterize either alone or in mixtures. Because of the theoretically immense but biologically limited number of glycan structures appearing in nature, and given the limitations of MS analysis of glycopeptides (defining types, numbers and sequences of monosaccharides rather than monosaccharide identities, linkage positions and configurations), it is important to build on earlier experience from analyses of single isolated proteins and to use nomenclature, abbreviations and symbols that are well accepted and understood. Throughout this report we have, used the CFG nomenclature of glycan structures (http://www.functionalglycomics.org/static/consortium/Nomenclature.shtml) to increase the intelligibility of our findings (Fig. 1).Open in a separate windowFig. 1.Schematic depiction of the inter-α-trypsin inhibitor complex and its degradation by chondroitinase ABC. Both the linkage and cross-linkage regions are highlighted as well as the molecular details of the GalNAc to aspartic acid ester bond that cross-links the heavy chains to the CS chain of bikunin. In addition to CS-glycopeptides the endolytic activity of the chondroitinase ABC digestion is also expected to generate free disaccharides of the CS chain (faded region). The formation of unsaturated GlcA by chondrotinase ABC is shown below the complex and the monosaccharide symbols are explained in the upper left quadrant.Proteoglycans constitute a group of O-glycosylated proteins all carrying one or more complex glycan (glycosaminoglycan or GAG)¹ chains attached to Ser residues of the core proteins through a common linkage tetrasaccharide, called the GAG linkage region, which is composed of the innermost four monosaccharides (GlcAβ3Galβ3Galβ4Xylβ-O-) at the reducing end of the GAG chain (GlcA is glucuronic acid; Gal is galactose; and Xyl is xylose) (2).The structural difference between various subclasses of proteoglycans emanates from the repeated extension of the linkage tetrasaccharide by two monosaccharides; e.g. GlcAβ3GalNAcβ4 in chondroitin sulfate proteoglycans (CSPGs), GlcAβ4GlcNAcα4 in heparan sulfate proteoglycans (HSPGs) and from the extent and positions of O- and N-sulfations, GlcA epimerization and from the actual chain length of the glycosaminoglycan. The GAG chains may also be covalently attached, or cross-linked, to other proteins at the nonreducing end of the chain through a C-terminal aspartic acid ester linkage. In this report we concentrate on the structural characterization of the human inter-α-trypsin inhibitor CSPG complex.Chondroitin sulfate proteoglycans play a significant role in maintaining the structural integrity of most extracellular matrices. In addition to their structural role as matrix components, eukaryote CSPGs are known to be involved in more specialized functions such as signal transduction, morphogenesis and regulation of stem cell behavior and differentiation (3–9). In many cases, their biological activity is mediated by selective binding of protein ligands to distinct glycan structural variants (10, 11). Previous studies have made possible the analysis of the average fine structure of chondroitin sulfate (CS) chains facilitating the identification of discrete glycan domains likely involved in ligand interaction (12–16). The CSPG saccharide linkage region differs significantly from the structure of the rest of the glycan chain and its assembly has been shown to be essential for the regulation of the GAG biosynthesis (17–19).The glycomics approach for CSPGs structural analysis involves the release of the GAG chains from the core proteins, digestion of the released chain into disaccharides followed by their subsequent analysis by e.g. ion exchange chromatography, nuclear magnetic resonance spectroscopy or mass spectrometry. However, in this work-flow the identities of the core proteins as well as the attachment sites are lost hindering the assignment of specific structures to particular proteoglycan isoforms. In order to obtain integrated glycan-protein information, we recently developed a glycoproteomics approach allowing site-specific analysis of CSPG linkage region glycopeptides (20). Samples from human urine, plasma and cerebrospinal fluid were trypsinized and subjected to strong anion exchange (SAX) chromatography in order to enrich for anionic GAG-substituted glycopeptides. The fractions were then treated with chondroitinase ABC to depolymerize CS-chains into free disaccharides and residual hexasaccharide substituted peptides comprising the tetrasaccharide linkage region (21). The resulting CS-glycopeptides were finally characterized by reversed phase nLC-MS/MS run in positive mode using higher-energy collisional dissociation (HCD). In that study, 13 novel CSPGs were successfully identified and site-specific information regarding 13 already established CSPGs was obtained. However, the fine-structure analysis of the CS glycopeptides and their substitution patterns was hampered by the relatively weak intensities of sulfated and/or phosphorylated fragment ions obtained under the HCD conditions used. This problem became especially obvious for bikunin, the major component of the inter-α-trypsin inhibitor (IαI) complex and the most abundant and heterogeneous CSPG of those samples.Bikunin is a small acidic glycoprotein of about 40 kDa that is modified at Ser-10 by the attachment of one single CS chain. The linkage region of this GAG chain has been the target for several studies concluding a uniform galactose-4-sulfate tetrasaccharide structure (GlcAβ3Galβ3(4-O-SO₃)Galβ4Xylβ-O-) in both human plasma and urinary samples (22, 23). The IαI complex is a unique macromolecular arrangement of structurally related proteins, i.e. heavy chains, covalently linked to the CS chain of bikunin, also called the light chain of the IαI complex (Fig. 1) (24–27). In humans, one bikunin molecule is typically cross-linked to both heavy chain 1 (H1) and heavy chain 2 (H2) in the IαI-complex, but only to heavy chain 3 (H3) in the pre-α-trypsin inhibitor complex (28). Free bikunin is mainly found in urine and also known as the urinary trypsin inhibitor (UTI) (29). The plasma and urinary levels of bikunin are often raised during pathological processes such as chronic and acute inflammations, infections, cancer, renal diseases and diabetes (30–36). During inflammation, the CS chain may undergo changes in size and sulfation that correlate with the severity of the inflammatory response (37, 38).In the present work, we further developed our glycoproteomics approach to enable a detailed characterization and relative quantification of the IαI linkage and cross-linkage region glycopeptides derived from human plasma, urine and CSF samples. The use of alternating collisional energies proved to have a major impact on the fragmentation of CS glycopeptides and added valuable complementary structural information. The addition of sodium ions readily increased the stability of labile sulfate substituents upon HCD conditions allowing a more precise glycan characterization.The CS linkage region of bikunin was found to display a much larger heterogeneity than previously realized. The linkage region modifications were found to include sulfate, phosphate, fucose and sialic acid substitutions with notable differences across body fluids. We were also able to specifically identify glycopeptides derived from the cross-linking regions of H1 and H2. The heavy chains were often simultaneously attached to the same CS-chain, in close proximity to each other and sometimes even to adjacent GalNAc residues. Taken together, this study demonstrates that the linkage region of human CSPGs displays a vast greater structural complexity than previously perceived. Given that specific modifications of the linkage region may function as a molecular regulator of CS biosynthesis, these findings could also assist in further elucidating the mechanisms of CS chain elongation and substitution.