Hyaluronic acid by atomic force microscopy.

Hyaluronic acid (HA) of different molecular weights has been examined by atomic force microscopy (AFM) in air. This technique allows 3-D surface images of soft samples without any pretreatment, such as shadowing or staining. In the present study we examined the supermolecular organization of HA chains when deposited on mica and graphite, to better understand the interchain and intrachain interactions of HA molecules in solution. The concentration of the solution deposited varied from 0.001 to 1 mg/ml. On both substrates, and independent of the concentration, high-molecular-mass HA formed networks in which molecules ran parallel for hundreds of nanometers, giving rise to flat sheets and tubular structures that separate and rejoin into similar neighboring aggregates. Accurate measurements of the thickness of the thinnest sheets were consistent with a monolayer of HA molecules, 0.3 nm thick, strongly indicating lateral aggregation forces between chains as well as rather strong hydrophilic interactions between mica and HA. The results agree with an existing model of HA tertiary structure in solution in which the network is stabilized by both hydrophilic and hydrophobic interactions. Our images support this model and indicate that hydrophobic interactions between chains may exert a pivotal role in aqueous solution.     

Neutrophil extracellular traps: casting the NET over pathogenesis

Neutrophil extracellular traps (NETs) are considered to be part of the human innate immunity because they trap and kill pathogens. NETs are formed by activated neutrophils and consist of a DNA backbone with embedded antimicrobial peptides and enzymes. They are involved in host defense during pneumococcal pneumonia, streptococcal necrotizing fasciitis, appendicitis and insemination. Recently, bacterial virulence factors that counteract NETs have been identified. These include the degradation of the NET-backbone by DNases enabling the liberation of bacteria from NETs, as well as capsule formation, which reduces bacterial trapping. Furthermore, pathogens can resist NET-mediated killing by adding positive charge to their cell surface.     

Molecular modelling of secondary and tertiary structures of hyaluronan,compared with electron microscopy and NMR data. Possible sheets and tubular structures in aqueous solution

Electron microscopy shows that hyaluronan (HA) forms sheets and tube-like structures in solution. Molecular modelling by Tartu plastic space-filling atomic models revealed that hydroxymethyl and carboxylate groups of HA anti-parallel chains can be joined by H-bonds. Using these bonds, HA molecules can be modelled as sheets and tubules. These tertiary structures have three kinds of lateral contact: (1) antiparallel chains stacked by hydrophobic patches; (2) parallel chains joined by both stacking interactions and H-bonds; and (3) crossing chains joined by H-bonds and stacking interactions. Sheet and tubular structures may explain some viscoelastic and biological properties of HA.     

FGF-2 release from the lens capsule by MMP-2 maintains lens epithelial cell viability

The lens is an avascular tissue, separated from the aqueous and vitreous humors by its own extracellular matrix, the lens capsule. Here we demonstrate that the lens capsule is a source of essential survival factors for lens epithelial cells. Primary and immortalized lens epithelial cells survive in low levels of serum and are resistant to staurosporine-induced apoptosis when they remain in contact with the lens capsule. Physical contact with the capsule is required for maximal resistance to stress. The lens capsule is also a source of soluble factors including fibroblast growth factor 2 (FGF-2) and perlecan, an extracellular matrix component that enhances FGF-2 activity. Matrix metalloproteinase 2 (MMP-2) inhibition as well as MMP-2 pretreatment of lens capsules greatly reduced the protective effect of the lens capsule, although this could be largely reversed by the addition of either conditioned medium or recombinant FGF-2. These data suggest that FGF-2 release from the lens capsule by MMP-2 is essential to lens epithelial cell viability and survival.     

Functional molecular mass of a vertebrate hyaluronan synthase as determined by radiation inactivation analysis.

Hyaluronan (HA), a linear polysaccharide composed of N-acetylglucosamine-glucuronic acid repeats, is found in the extracellular matrix of vertebrate tissues as well as the capsule of several pathogenic bacteria. The HA synthases (HASs) are dual-action glycosyltransferases that catalyze the addition of two different sugars from UDP-linked precursors to the growing HA chain. The prototypical vertebrate hyaluronan synthase, xlHAS1 (or DG42) from Xenopus laevis, is a 588-residue membrane protein. Recently, the streptococcal enzyme was found to function as a monomer of protein with approximately 16 lipid molecules. The vertebrate enzymes are larger than the streptococcal enzymes; based on the vertebrate HAS deduced amino acid sequence, two additional membrane-associated regions at the carboxyl terminus are predicted. We have utilized radiation inactivation to measure the target size of yeast-derived recombinant xlHAS1. The target size of HAS activity was confirmed using two internal standards. First, samples were spiked with glucose-6-phosphate dehydrogenase, an enzyme of known molecular weight. Second, parallel samples of native xlHAS1 and a xlHAS1-green fluorescent protein fusion (833 residues) were compared; substantial confidence was gained by using this novel internal standard. Our test also corroborated the basic tenets of radiation inactivation theory. We found that the vertebrate HAS protein functions catalytically as a monomer.     

Recognition of a single transmembrane degron by sequential quality control checkpoints

To understand the relationship between conformational maturation and quality control-mediated proteolysis in the secretory pathway, we engineered the well-characterized degron from the alpha-subunit of the T-cell antigen receptor (TCRalpha) into the alpha-helical transmembrane domain of homotrimeric type I integral membrane protein, influenza hemagglutinin (HA). Although the membrane degron does not appear to interfere with acquisition of native secondary structure, as assessed by the formation of native intrachain disulfide bonds, only approximately 50% of nascent mutant HA chains (HA(++)) become membrane-integrated and acquire complex N-linked glycans indicative of transit to a post-ER compartment. The remaining approximately 50% of nascent HA(++) chains fail to integrate into the lipid bilayer and are subject to proteasome-dependent degradation. Site-specific cleavage by extracellular trypsin and reactivity with conformation-specific monoclonal antibodies indicate that membrane-integrated HA(++) molecules are able to mature to the plasma membrane with a conformation indistinguishable from that of HA(wt). These apparently native HA(++) molecules are, nevertheless, rapidly degraded by a process that is insensitive to proteasome inhibitors but blocked by lysosomotropic amines. These data suggest the existence in the secretory pathway of at least two sequential quality control checkpoints that recognize the same transmembrane degron, thereby ensuring the fidelity of protein deployment to the plasma membrane.     

Alternate structural conformations of Streptococcus pneumoniae hyaluronan lyase: insights into enzyme flexibility and underlying molecular mechanism of action

Streptococcus pneumoniae hyaluronan lyase is a surface enzyme of this Gram-positive bacterium. The enzyme degrades several biologically important, information-rich linear polymeric glycans: hyaluronan, unsulfated chondroitin, and some chondroitin sulfates. This degradation facilitates spreading of bacteria throughout the host tissues and presumably provides energy and a carbon source for pneumococcal cells. Its beta-elimination catalytic mechanism is an acid/base process termed proton acceptance and donation leading to cleavage of beta-1,4 linkages of the substrates. The degradation of hyaluronan occurs in two stages, initial endolytic cuts are followed by processive exolytic cleavage of one disaccharide at a time. In contrast, the degradation of chondroitins is purely endolytic. Structural studies together with flexibility analyses of two streptococcal enzymes, from S.pneumoniae and Streptococcus agalactiae, allowed for insights into this enzyme's molecular mechanism. Here, two new X-ray crystal structures of the pneumococcal enzyme in novel conformations are reported. These new conformations, complemented by molecular dynamics simulation results, directly confirm the predicted domain motions presumed to facilitate the processive degradative process. One of these new structures resembles the S.agalactiae enzyme conformation, and provides evidence of a uniform mechanistic/dynamic behavior of this protein across different bacteria.     

Covalent transfer of heavy chains of inter-alpha-trypsin inhibitor family proteins to hyaluronan in in vivo and in vitro expanded porcine oocyte-cumulus complexes

Previous studies have shown that the heavy chains (HCs) of serum-derived inter-alpha-trypsin inhibitor (IalphaI) molecules become covalently linked to hyaluronan (HA) during in vivo mouse cumulus expansion and significantly contribute to cumulus matrix organization. Experiments with mice suggest that the incorporation of such proteins in cumulus matrix appears to be rather complex, involving LH/hCG-induced changes in blood-follicle barrier and functional cooperation between cumulus cells, granulosa cells, and oocyte within the follicle. We demonstrate here that HC-HA covalent complexes are formed during in vivo porcine cumulus expansion as well. Western blot analysis with IalphaI antibody revealed that follicular fluids from medium-sized follicles and those from large follicles unstimulated with hCG contain high levels of all forms of IalphaI family members present in pig serum. The same amount of HCs were covalently transferred from IalphaI molecules to HA when pig oocyte-cumulus complexes (OCCs) were stimulated in vitro with FSH in the presence of pig serum or follicular fluid from unstimulated or hCG-stimulated follicles. In addition, HC-HA coupling activity was stimulated in cumulus cells by FSH treatment also in the absence of oocyte. Collectively, these results indicate that IalphaI molecules can freely cross the blood follicle barrier and that follicular fluid collected at any stage of folliculogenesis can be successfully used instead of serum for improving OCC maturation. Finally, pig cumulus cells show an autonomous ability to promote the incorporation of IalphaI HCs in the cumulus matrix.     

Golgi linked protein glycosylation and associated diseases

One of the Golgi's main functions is the glycosylation of secreted proteins. A large variety of glycan chains can be synthesized in the Golgi, and it is increasingly clear that these are critical in basic cellular functions as well as the development of multicellular organisms. The structurally best-documented glycans are N-glycans, yet these are also the most enigmatic in their function. In contrast, O-glycan function is far better understood, but here the structures and biosynthetic pathways are very incomplete. The critical importance of glycans is highlighted by the broad spectrum of diseases they are associated with, such as a number of inherited diseases, but also cancers or diabetes. The molecular clues to these, however, are only just being elucidated. Although some glycan structures are known to be involved in signaling or adhesion to the extracellular matrix, for most the functions are not yet known. This review aims at summarizing current knowledge as much as to point out critical areas key for future progress.     

Terminal sialic acids on CD44 N‐glycans can block hyaluronan binding by forming competing intramolecular contacts with arginine sidechains

Specific sugar residues and their linkages form the basis of molecular recognition for interactions of glycoproteins with other biomolecules. Seemingly small changes, like the addition of a single monosaccharide in the covalently attached glycan component of glycoproteins, can greatly affect these interactions. For instance, the sialic acid capping of glycans affects protein‐ligand binding involved in cell–cell and cell–matrix interactions. CD44 is a single‐pass transmembrane glycoprotein whose binding with its carbohydrate ligand hyaluronan (HA), an extracellular matrix component, mediates processes such as leukocyte homing, cell adhesion, and tumor metastasis. This binding is highly regulated by glycosylation of the N‐terminal extracellular hyaluronan‐binding domain (HABD); specifically, sialic acid capped N‐glycans of HABD inhibit ligand binding. However, the molecular mechanism behind this sialic acid mediated regulation has remained unknown. Two of the five N‐glycosyation sites of HABD have been previously identified as having the greatest inhibitory effect on HA binding, but only if the glycans contain terminal sialic acid residues. These two sites, Asn25 and Asn120, were chosen for in silico glycosylation in this study. Here, from extensive standard molecular dynamics simulations and biased simulations, we propose a molecular mechanism for this behavior based on spontaneously‐formed charge‐paired hydrogen bonding interactions between the negatively‐charged sialic acid residues and positively‐charged Arg sidechains known to be critically important for binding to HA, which itself is negatively charged. Such intramolecular hydrogen bonds would preclude associations critical to hyaluronan binding. This observation suggests how CD44 and related glycoprotein binding is regulated by sialylation as cellular environments fluctuate. Proteins 2014; 82:3079–3089. © 2014 Wiley Periodicals, Inc.     

PTX3 interacts with inter-alpha-trypsin inhibitor: implications for hyaluronan organization and cumulus oophorus expansion

Pentraxin 3 (PTX3) and heavy chains (HCs) of inter-alpha-trypsin inhibitor (IalphaI) are essential for hyaluronan (HA) organization within the extracellular matrix of the cumulus oophorus, which is critical for in vivo oocyte fertilization and female fertility. In this study, we examined the possibility that these molecules interact and cooperate in this function. We show that HCs and PTX3 colocalize in the cumulus matrix and coimmunoprecipitate from cumulus matrix extracts. Coimmunoprecipitation experiments and solid-phase binding assays performed with purified human IalphaI and recombinant PTX3 demonstrate that their interaction is direct and not mediated by other matrix components. PTX3 does not bind to IalphaI subcomponent bikunin and, accordingly, bikunin does not compete for the binding of PTX3 to IalphaI, indicating that PTX3 interacts with IalphaI subcomponent HC only. Recombinant PTX3-specific N-terminal region, but not the PTX3-pentraxin C-terminal domain, showed the same ability as full-length protein to bind to HCs and to enable HA organization and matrix formation by Ptx3(-/-) cumulus cell oocyte complexes cultured in vitro. Furthermore, a monoclonal antibody raised against PTX3 N terminus, which inhibits PTX3/IalphaI interaction, also prevents recombinant full-length PTX3 from restoring a normal phenotype to in vitro-cultured Ptx3(-/-) cumuli. These results indicate that PTX3 directly interacts with HCs of IalphaI and that such interaction is essential for organizing HA in the viscoelastic matrix of cumulus oophorus, highlighting a direct functional link between the two molecules.     

Cytological studies of the nematocysts of Hydra. II. The stenoteles

Entire hydras or tentacles were prepared for electron microscopy as described in the preceding paper. The stenotele capsule has been observed to be composed of an external membrane, a thick chitinous or keratin layer, and an inner membrane. A sac-like extension of the capsular wall into the capsule bears spines and stylets on its inner surface and evagination of this structure occurs on discharge. Profiles of tubular or membranous structures often are seen within the capsules of resting stenoteles. These structures are presumably related to the external filament. The spines often reveal a flattened aspect which suggests that at least some of them might more accurately be called "vanes." A cnidocil has been found to accompany each stenotele. This study revealed several aspects of the developmental stages of stenoteles: A vacuole is formed which is nearly surrounded by the nematocyte nucleus. The vacuole content changes in density and a capsular wall is formed at the periphery of the vacuole. Tubules differentiate from the capsular matrix, and spines and stylets develop somewhat later. An operculum is formed from the nematocyte cytoplasm.     

Structure and interactions of proteoglycans in the extracellular matrix produced by cultured human fibroblasts.

Subconfluent cultures of human embryonic skin fibroblasts were labelled with [35S]sulphate for 3 days, after which cell-free extracellular matrix was isolated. A chondroitin sulphate proteoglycan (CSPG) and a heparan sulphate proteoglycan (HSPG) were purified from the matrix. Chromatography on Sepharose CL-2B gave peak Kav. values of 0.35 and 0.38 respectively for the CSPG and the HSPG. The polysaccharide chains released from the two PGs were of similar size (Kav. 0.50 on Sepharose CL-4B). Approx. 50% of the CSPG showed affinity for hyaluronic acid (HA). However, it differed immunologically from the HA-aggregating CSPG of human articular cartilage, and had a larger core protein (apparent molecular mass 290 kDa) than had the cartilage PG. Neither metabolically [35S]sulphate-labelled PGs, isolated from the medium of fibroblast cultures, nor chemically 3H-labelled polysaccharides (HA, CS, HS and heparin) were incorporated into the extracellular matrix when added to unlabelled cell cultures. These results indicate that the matrix PGs are not derived from the PGs present in the medium and that an interation between polysaccharide chains and matrix components is not sufficient for incorporation of PGs into the matrix. Incubation of cell-free 35S-labelled matrix with unlabelled polysaccharides did not lead to the release of any 35S-labelled material, supporting this conclusion. Furthermore, so-called 'link proteins' were not present in the fibroblast cultures, indicating that the CSPGs were anchored in the matrix in a manner different from the link-stabilized association of CSPG with HA in chondrocyte matrix. The identification of a proteinase, secreted by fibroblasts in culture, that after activation with heparin has the ability to release 35S-labelled PGs from the matrix may also indicate that the core proteins are important for the association of the PGs to the matrix.     

Glycan-Dependent Immunogenicity of Recombinant Soluble Trimeric Hemagglutinin

Recombinant soluble trimeric influenza A virus (IAV) hemagglutinin (sHA₃) has proven an effective vaccine antigen against IAV. Here, we investigate to what extent the glycosylation status of the sHA₃ glycoprotein affects its immunogenicity. Different glycosylation forms of subtype H5 trimeric HA protein (sH5₃) were produced by expression in insect cells and different mammalian cells in the absence and presence of inhibitors of N-glycan-modifying enzymes or by enzymatic removal of the oligosaccharides. The following sH5₃ preparations were evaluated: (i) HA proteins carrying complex glycans produced in HEK293T cells; (ii) HA proteins carrying Man₉GlcNAc₂ moieties, expressed in HEK293T cells treated with kifunensine; (iii) HA proteins containing Man₅GlcNAc₂ moieties derived from HEK293S GnTI(−) cells; (iv) insect cell-produced HA proteins carrying paucimannosidic N-glycans; and (v) HEK293S GnTI(−) cell-produced HA proteins treated with endoglycosidase H, thus carrying side chains composed of only a single N-acetylglucosamine each. The different HA glycosylation states were confirmed by comparative electrophoretic analysis and by mass spectrometric analysis of released glycans. The immunogenicity of the HA preparations was studied in chickens and mice. The results demonstrate that HA proteins carrying terminal mannose moieties induce significantly lower hemagglutination inhibition antibody titers than HA proteins carrying complex glycans or single N-acetylglucosamine side chains. However, the glycosylation state of the HA proteins did not affect the breadth of the antibody response as measured by an HA1 antigen microarray. We conclude that the glycosylation state of recombinant antigens is a factor of significant importance when developing glycoprotein-based vaccines, such as recombinant HA proteins.     

Cytological Studies of the Nematocysts of Hydra : II. The Stenoteles

Entire hydras or tentacles were prepared for electron microscopy as described in the preceding paper. The stenotele capsule has been observed to be composed of an external membrane, a thick chitinous or keratin layer, and an inner membrane. A sac-like extension of the capsular wall into the capsule bears spines and stylets on its inner surface and evagination of this structure occurs on discharge. Profiles of tubular or membranous structures often are seen within the capsules of resting stenoteles. These structures are presumably related to the external filament. The spines often reveal a flattened aspect which suggests that at least some of them might more accurately be called "vanes." A cnidocil has been found to accompany each stenotele. This study revealed several aspects of the developmental stages of stenoteles: A vacuole is formed which is nearly surrounded by the nematocyte nucleus. The vacuole content changes in density and a capsular wall is formed at the periphery of the vacuole. Tubules differentiate from the capsular matrix, and spines and stylets develop somewhat later. An operculum is formed from the nematocyte cytoplasm.     

Organization of intestinal epithelial cells into multicellular structures requires laminin and functional actin microfilaments

Epithelial cell organization into multicellular structures is a critical biological process required for both organogenesis and repair following injury. The basement membrane and the cytoskeleton have important roles in this process; however, the functions of individual components of basement membrane and cytoskeleton are poorly understood. We used IEC-6 cells, a rat intestinal crypt cell line, grown on a three-dimensional gel of reconstituted basement membrane as a model system to determine which extracellular matrix and cytoskeletal components mediate intestinal epithelial cell organization. The cells entered the gel and formed hollow, tubular structures that resembled intestinal crypts. These structures were characterized by a single layer of polarized cells with apical tight junctions and microvilli on the luminal surface. Antiserum to laminin and the pentapeptide Tyr-Ile-Gly-Ser-Arg (which prevents cell attachment to laminin) inhibited this organization, but a control pentapeptide (Tyr-Tyr-Gly-Asp-Ala) and antiserum to collagen IV did not. Cytochalasin B, which interferes with actin microfilament polymerization, also inhibited organization of cells into multicellular structures, but vinblastine and Colcemid, which disrupt microtubules, and cycloheximide, which inhibits protein synthesis, did not. We conclude that organization of intestinal epithelial cells on a basement membrane into multicellular structures results from specific interactions between cells and laminin and requires intact actin microfilaments.     

Isolation and characterization of enterotoxigenic Escherichia coli mutants that produce abnormal heat-labile enterotoxins

Abstract Bacteroides fragilis populations were separated according to the size of surface structure. Subculture of the separated populations produced cultures enriched for 3 different structures; a large capsule, a small capsule and an electron-dense layer (EDL). The ability of these subpopulations to haemagglutinate (HA) erythrocytes from a number of species was examined. Populations which produced either a large or s small capsule did not have HA activity, whereas those with an extracellular EDL did. By mixing populations with EDL and those with either the large or small capsule, the degree of HA could be altered. HA was dependent on the proportion of EDL-bearing bacteria present. Fimbriae were not observed on electron microscopy.     

Ultrastructural characteristics of the external surfaces of Pasteurella pneumotropica from mice and Pasteurella multocida from rabbits

The surface structures of the cells of Pasteurella pneumotropica from mice and Pasteurella multocida from rabbits were examined by transmission electron microscopy after ruthenium red staining and polycationic ferritin labelling. P. pneumotropica strains ATCC 35149 and K 79114 had slight extracellular fibrous materials associated with cell walls with ruthenium red staining. Ferritin labelling method revealed thick strands or sparsely ferritin-labelled materials on the cell surface of the strains. P. multocida strains Pm-78 and P-2440 had ferritin-labelled capsules surrounded with the cell wall. Strain Pm-78, which was serotyped as A:12, had a thick capsule, whereas serotype -:3 strain P-2440 had a thin and irregular capsule.     

Integrin-β5 and zyxin mediate formation of ventral stress fibers in response to transforming growth factor β

Cell adhesion to the extracellular matrix is an essential element of various biological processes. TGF-β cytokines regulate the matrix components and cell–matrix adhesions. The present study investigates the molecular organization of TGF-β-induced matrix adhesions. The study demonstrates that in various mouse and human epithelial cells TGF-β induces cellular structures containing 2 matrix adhesions bridged by a stretch of actin fibers. These structures are similar to ventral stress fibers (VSFs). Suppression of integrin-β5 by RNA interference reduces VSFs in majority of cells (>75%), while overexpression of integrin-β5 fragments revealed a critical role of a distinct sequence in the cytoplasmic domain of integrin-β5 in the VSF structures. In addition, the integrity of actin fibers and Src kinase activity contribute to integrin-β5-mediated signaling and VSF formation. TGF-β-Smad signaling upregulates actin-regulatory proteins, such as caldesmon, zyxin, and zyxin-binding protein Csrp1 in mouse and human epithelial cells. Suppression of zyxin markedly inhibits formation of VSFs in response to TGF-β and integrin-β5. Zyxin is localized at actin fibers and matrix adhesions of VSFs and might bridge integrin-β5-mediated adhesions to actin fibers. These findings provide a platform for defining the molecular mechanism regulating the organization and activities of VSFs in response to TGF-β.