Secreted and transmembrane mucins inhibit gene transfer with AAV4 more efficiently than AAV5

Adeno-associated virus (AAV) is a promising vector for gene transfer in cystic fibrosis. AAV4 and AAV5 both bind to the apical surface of differentiated human airway epithelia, but only AAV5 infects. Both AAV4 and AAV5 require 2,3-linked sialic acid for binding. However, AAV5 interacts with sialic acid on N-linked carbohydrates, whereas AAV4 interacts with sialic acid on O-linked carbohydrates. Because mucin is decorated with O-linked carbohydrates, we hypothesized that mucin binds AAV4 and inhibits gene transfer. To evaluate the effect of secreted mucin, we studied mucin binding and gene transfer to COS cells and the basolateral membrane of well differentiated human airway epithelia. AAV4 bound mucin more efficiently than AAV5, and mucin inhibited gene transfer with AAV4. Moreover, O-glycosidase-pretreated mucin did not block gene transfer with AAV4. Similar to secreted mucin, the transmembrane mucin MUC1 inhibited gene transfer with AAV4 but not AAV5. MUC1 inhibited AAV4 by blocking internalization of the virus. Thus, O-linked carbohydrates of mucin are potent inhibitors of AAV4. Furthermore, whereas mucin plays an important role in innate host defense, its activity is specific; some vectors or pathogens are more resistant to its effects.     

Sialic acid-specific lectin-mediated adhesion of Tritrichomonas foetus and Tritrichomonas mobilensis

Tritrichomonas foetus is an obligate parasite of the bovine urogenital tract producing infection associated with inflammatory changes, abortion, and infertility, Tritrichomonas mobilensis was isolated from squirrel monkey colon, and symptoms involve diarrheal complications. Both tritrichomonads produced hemagglutinins with the properties of sialic acid-specific lectins. Assays on the adherence of these protozoans to Chinese hamster ovary (CHO) cells and to bovine cervical and monkey colon mucus were performed to assess the function of the lectins in adhesion. Sialic acid at concentration as low as 2 mM inhibited the adhesion to CHO cells, less effectively to the mucus. Predigestion with Clostridium perfringens sialidase prevented the adhesion to both epithelial cells and the mucus. Inhibition of endogenous sialidases with 2,3-dehydro-2-deoxy-NeuAc increased the adhesion of T. mobilensis to CHO cells. Specific anti-T. foetus lectin (TFL) and anti-T. mobilensis lectin (TML) antibodies inhibited adhesion of the trichomonads to the epithelial cells and to the mucus. TFL histochemistry disclosed the presence of lectin ligands on keratinized vaginal epithelia, cervical mucosa, and mucin and on endometrial glands and their secretions. TML histochemistry showed reactivity with the luminal membranes of colonic glandular epithelium and less with the colonic mucin. Both lectins bound to the surface membrane of CHO cells. Anti-lectin antibodies showed granular cytoplasmic and strong membrane localization of the lectins in both tritrichomonads. Although the 2 tritrichomonads have different habitats, the results indicate that both these protozoa use lectins with sialic acid specificity for adhesion to mucosal surfaces.     

Inhibition of Helicobacter pylori colonization by sulfated gastric mucin.

The role of human gastric mucin in mucosal protection against Helicobacter pylori colonization was investigated. H. pylori cells were incubated with purified intact mucin or its acidic fractions and then examined for their inhibitory capacity of H. pylori attachment to erythrocytes. Titration data established that the inhibitory activity of mucin was associated with its acidic component as the fraction enriched in sialic acid and sulfate showed 16-fold higher inhibitory titer than that of the intact mucin. While the inhibitory titer of acidic mucin fraction was not affected by the removal of sialic acid, the desulfation led to a complete loss of its inhibitory activity, thus pointing towards the importance of sulfate ester groups in this process. The results for the first time point towards the involvement of sulfomucins in the protection of gastric mucosa against colonization by H. pylori.     

Specificity of the sialic acid-binding lectin from the snail Cepaea hortensis.

The specificity of the sialic acid-binding lectin from the snail Cepaea hortensis, purified by affinity chromatography on fetuin-Sepharose, was studied by hemagglutination inhibition assay applying 32 sialic acid derivatives and 14 glycoproteins. 2-alpha-Methyl-9-O-acetyl-NeuAc was the most potent inhibitor, followed closely by 2-alpha-methyl-NeuAc and 2-alpha-benzyl-NeuAc. An axially orientated carboxyl group is a prerequisite for maximal lectin-sugar binding. Neither size nor polarity of the alpha-anomeric substituent significantly influenced inhibition potency. An intact sialic acid N-acetyl group is essential for optimal lectin-sugar interaction. The trihydroxypropyl side chain also is of great importance. However, a bulky hydrophobic substituent at the side chain like a 9-O-tosyl residue did not decrease binding to the lectin. The lectin did not distinguish between NeuAc alpha 2----3Gal beta 1----4Glc and NeuAc alpha 2----6Gal beta 1----4Glc. Among other sugars tested, only N-acetylglucosamine showed inhibition, although 50-fold less. The most potent glycoprotein inhibitors were those carrying O-chains only or preferentially, as ovine submaxillary mucin, bovine submaxillary mucin, and glycophorin A. Tamm-Horsfall protein was an exception being a strong inhibitor, although carrying only N-chains. Asialoglycoproteins were inactive. Glycoproteins containing the NeuAc alpha 2----3Gal sequence inhibited the lectin as well as those with NeuAc alpha 2----6GalNAc. From the results a model of the lectin's binding site for sialic acid is suggested.     

Fibrinogen induces adhesion, spreading, and microfilament organization of human endothelial cells in vitro

Human umbilical vein endothelial cells (ECs) have been shown to attach to a substratum of fibrinogen (fg). Later, ECs undergo spreading, organization of thick microfilament bundles of the stress fiber type, and formation of focal contacts (adhesion plaques) that correspond to accumulation of vinculin at the cytoplasmic aspect of the ventral membrane. The rate of attachment to fg and the type of spreading is virtually identical to that obtained on substrata coated with fibronectin (FN). Antibodies to fg, but not to FN, prevent EC adhesion to fg; conversely, antibodies to FN, but not to fg, prevent adhesion of ECs to a FN-coated substratum. The removal of residual FN contamination from fg preparations by means of DEAE-cellulose chromatography does not result in any difference in EC adhesion on fg. Moreover, pretreatment of cells with inhibitors of synthesis and release of proteins does not impair their adhesion capacity on an fg-coated substratum. In contrast, human arterial smooth muscle cells do not adhere and spread on fg substrata but do so on FN. The synthetic peptides (Gly-Arg-Gly-Asp[GRGD] and Gly-Arg-Gly-Asp-Ser-Pro[GRGDSP]) containing the tripeptide Arg-Gly-Asp (RGD), originally found to be responsible for the cell binding activity of FN, have been found to inhibit EC spreading and the redistribution of their cytoskeleton, including the formation of stress fibers and the localization of vinculin either on fg or on FN. Conversely, the synthetic peptide Arg-Gly-Gly (RGG) was completely uneffective in inhibiting the adhesion and the sequence of events leading to spreading and cytoskeletal organization. These results indicate that ECs, but not smooth muscle cells, specifically adhere and spread on an fg substratum and this occurs by recognition mechanisms similar to those reported for FN.     

Endothelial alpha 2,6-linked sialic acid inhibits VCAM-1-dependent adhesion under flow conditions.

We have previously shown that costimulation of endothelial cells with IL-1 + IL-4 markedly inhibits VCAM-1-dependent adhesion under flow conditions. We hypothesized that sialic acids on the costimulated cell surfaces may contribute to the inhibition. Northern blot analyses showed that Gal beta 1-4GlcNAc alpha 2, 6-sialyltransferase (ST6N) mRNA was up-regulated in cultured HUVEC by IL-1 or IL-4 alone, but that the expression was enhanced by costimulation, whereas the level of Gal beta 1-4GlcNAc/Gal beta 1-3GalNAc alpha2,3-sialyltransferase (ST3ON) mRNA was unchanged. Removing both alpha 2,6- and alpha 2,3-linked sialic acids from IL-1 + IL-4-costimulated HUVEC by sialidase significantly increased VCAM-1-dependent adhesion, whereas removing alpha 2,3-linked sialic acid alone had no effect; adenovirus-mediated overexpression of ST6N with costimulation almost abolished the adhesion, which was reversible by sialidase. The same treatments of IL-1-stimulated HUVEC had no effect. Lectin blotting showed that VCAM-1 is decorated with alpha 2,6- but not alpha 2,3-linked sialic acids. However, overexpression of alpha 2,6-sialyltransferase did not increase alpha 2,6-linked sialic acid on VCAM-1 but did increase alpha 2,6-linked sialic acids on other proteins that remain to be identified. These results suggest that alpha 2,6-linked sialic acids on a molecule(s) inducible by costimulation with IL-1 + IL-4 but not IL-1 alone down-regulates VCAM-1-dependent adhesion under flow conditions.     

Purification and properties of a Ca2+-independent sialic acid-binding lectin from human placenta with preferential affinity to O-acetylsialic acids

A Ca2+-independent sialic acid-specific lectin from two developmental stages of human placenta was similarly purified to apparent homogeneity by DEAE-cellulose chromatography, affinity chromatography on bovine submaxillary mucin, and gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration disclosed a molecular mass of 53 kDa. The specificity of the lectin for O-acetylsialic acids was substantiated by the dependence of hemagglutination on the presence of acetylated sialic acids on the surface of mammalian erythrocytes of various sources, by hapten inhibition in hemagglutination assays with protease-treated rabbit erythrocytes and by hapten inhibition of binding of labeled N-acetylneuraminic acid-bovine serum albumin to the lectin in a solid-phase assay. Bovine and equine submaxillary mucins that contain 9(7,8)-O-acetyl and 4-O-acetylsialic acids were potent inhibitors in contrast to the non-acetylated sialic acids of ovine submaxillary mucin. Absence of inhibitory efficiency of other negatively charged substances like phosphorylated sugars, glucuronic acid, heparin, or oligodeoxynucleotides emphasized the importance of structural features instead of simple ionic interaction. In the presence of acetylation, the pattern of inhibition by gangliosides in the solid-phase assay indicated a preference to alpha-2,8- or alpha-2,6-linked sialic acids in comparison to alpha-2,3-linked moieties. Chemical modification of the lectin by group-specific reagents allowed to emphasize the role of primarily lysine residues, but also, although less pronounced, arginine, tryptophan, and carboxyl groups for ligand binding and/or maintenance of the active conformational state. Application of reagents, specific for histidine or tyrosine residues, failed to affect lectin activity.     

Mouse large can modify complex N- and mucin O-glycans on alpha-dystroglycan to induce laminin binding

The human LARGE gene encodes a protein with two putative glycosyltransferase domains and is required for the generation of functional alpha-dystroglycan (alpha-DG). Monoclonal antibodies IIH6 and VIA4-1 recognize the functional glycan epitopes of alpha-DG that are necessary for binding to laminin and other ligands. Overexpression of full-length mouse Large generated functionally glycosylated alpha-DG in Pro(-5) Chinese hamster ovary (CHO) cells, and the amount was increased by co-expression of protein:O-mannosyl N-acetylglucosaminyltransferase 1. However, functional alpha-DG represented only a small fraction of the alpha-DG synthesized by CHO cells or expressed from an alpha-DG construct. To identify features of the glycan epitopes induced by Large, the production of functionally glycosylated alpha-DG was investigated in several CHO glycosylation mutants. Mutants with defective transfer of sialic acid (Lec2), galactose (Lec8), or fucose (Lec13) to glycoconjugates, and the Lec15 mutant that cannot synthesize O-mannose glycans, all produced functionally glycosylated alpha-DG upon overexpression of Large. Laminin binding and the alpha-DG glycan epitopes were enhanced in Lec2 and Lec8 cells. In Lec15 cells, functional alpha-DG was increased by co-expression of core 2 N-acetylglucosaminyltransferase 1 with Large. Treatment with N-glycanase markedly reduced functionally glycosylated alpha-DG in Lec2 and Lec8 cells. The combined data provide evidence that Large does not transfer to Gal, Fuc, or sialic acid on alpha-DG nor induce the transfer of these sugars to alpha-DG. In addition, the data suggest that human LARGE may restore functional alpha-DG to muscle cells from patients with defective synthesis of O-mannose glycans via the modification of N-glycans and/or mucin O-glycans on alpha-DG.     

A sialic acid-specific lectin from the slug Limax flavus

The slug, Limax flavus, contains a lectin that appears to be highly specific for sialic acid residues of glycoproteins. The carbohydrates which inhibited the hemagglutinating activity of the slug lectin and the concentration of the carbohydrate which gave a 50% inhibition are as follows: N-acetylneuraminic acid, 0.13 mm; N-glycolylneuraminic acid, 0.90 mm; d-glucosamine, 4.9 mm; d-galactosamine, 7.6 mm; N-acetyl-d-glucosamine, 23 mm; and N-acetyl-d-galactosamine, 24 mm. d-Galactose, d-glucose, d-mannose, α-methyl-d-glucoside, α-methyl-d-mannoside, l-arabinose, d-xylose, l-fucose, d-glucuronic acid, lactose, and sucrose were found to be ineffective as inhibitors of the hemagglutinating activity of the slug lectin. Hemagglutination by slug lectin was strongly inhibited by bovine submaxillary mucin and fetuin but not by sialic acid-free bovine submaxillary mucin or fetuin.     

Subsets of sialylated, sulfated mucins of diverse origins are recognized by L-selectin. Lack of evidence for unique oligosaccharide sequences mediating binding

Previous studies have shown that the mucin-type polypeptidesGlyCAM-1, CD34, and MAdCAM-1 can function as ligands for L-selectinonly when they are synthesized by the specialized high-endothelialvenules (HEV) of lymph nodes. Since sialylation, sulfation,and possibly fucosylation are required for generating recognition,we reasoned that other mucins known to have such componentsmight also bind L-selectin. We show here that soluble mucinssecreted by human colon carcinoma cells, as well as those derivedfrom human bronchial mucus can bind to human L-selectin in acalcium-dependent manner. As with GlyCAM-1 synthesized by lymphnode HEY, 2–3 linked sialic acids and sulfation seem toplay a critical role in generating this L-selectin binding.In each case, only a subset of the mucin molecules is recognizedby L-selectin. Binding is not destroyed by boiling, suggestingthat recognition may be based primarily upon carbohydrate structures.Despite this, O-linked oligosaccharide chains released fromthese ligands by beta-elimination do not show any detectablebinding to L-selectin. Following protease treatment of the ligands,binding persists in a subset of the resulting fragments, indicatingthat specific recognition is determined by certain regions ofthe original mucins. How ever, O-linked oligosaccharides releasedfrom the subset of non-binding mucin fragments do not show verydifferent size and charge profiles compared to those that dobind. Furthermore, studies with polylactosamine-degrading endoglycosidasessuggest that the core structures involved in generating bindingcan vary among the different ligands. Taken together, thesedata indicate that a single unique oligosaccharide structuremay not be responsible for high-affinity binding. Rather, diversemucins with sialylated, sulfated, fucosylated lactosamine-typeO-linked oligosaccharides can generate high-affinity L-selectinligands, but only when they present these chains in unique spacingand/or clustered combinations, presumably dictated by the polypeptidebackbone. L-selectin mucins sialic sialic acid sulfate adhesion     

Lack of evidence for sialidase activity in Helicobacter pylori

The role of sialic acid for the adhesion of Helicobacter pylori to gastric mucosa cells and/or to the mucin layer is still under debate. Several but not all H. pylori strains express a sialic acid-binding adhesin, specific for terminal α-2,3-sialic acid residues. Recently, the production of sialidase by H. pylori was reported [Dwarakanath, A.D. et al. (1995) FEMS Immunol. Med. Microbiol. 12, 213–216]. We analysed several strains isolated from gastric biopsies cultivated both in liquid media and on agar plates for sialidase. Activity of this enzyme was first assayed using the fluorigenic substrate 4-methylumbelliferyl-α-d-N-acetylneuraminic acid. Since the fluorimetric assay can give false-positive results caused by non-specific interactions with umbelliferyl-tagged substances, we used also the more sensitive and specific assay with sialyl-[³H]lactitol as a substrate. No evidence for sialidase activity of H. pylori strains, cultivated under both inducible and non-inducible conditions, was obtained.     

Interaction between cells and elastin fibers: An ultrastructural and immunocytochemical study

Mesenchymal cells (fibroblasts, smooth muscle cells) and endothelial cells were shown to interact with elastin fibers. The strong adhesion of elastin fibers to these cells is mediated by a cell membrane complex with a major glycoprotein component of 120 kDa designated as elastonectin. This interaction was studied by transmission electron microscopy (TEM) and immunocytochemical techniques using antibodies raised against the elastin adhesive proteins. When fibroblasts and smooth muscle cells were cultured in presence of elastin fibers, TEM showed an adhesion mechanism that takes place over several sites along the plasma membrane of these cells. Endothelial cells showed a very close association with elastin, emitting “pseudopodia” that embody the fibers. TEM, indirect immunofluorescence, immunoperoxidase, and confocal microscopy showed the presence and localization of cell membrane components synthesized in large quantities when cells were incubated in presence of elastin. Cells without elastin fibers barely revealed the adhesive membrane complex. These results confirm and extend previous findings concerning the presence of an inducible cell membrane complex that mediates the adhesion of elastin fibers to these cell types. © 1994 Wiley-Liss, Inc.     

Heterogeneity of glycoconjugates in the secretory cells of the chinchilla middle ear and eustachian tubal epithelia: a lectin-gold cytochemical study

The present study was conducted to characterize and localize the glycoconjugates in the tubotympanum (auditory or eustachian tube and middle ear cavity) of chinchilla on an ultrastructural level, using lectin-gold complexes with six different lectins: BPA, ConA, RCA-1, WGA, LFA, and SNA. A comparison of the affinity of these lectins demonstrated the heterogeneity of secretory cells. The glandular serous cells and epithelial dark granulated cells produced "serum"-type glycoprotein. The glandular mucous cells and goblet cells produced dominantly "mucin"-type glycoprotein in the light granules, but "serum"-type glycoprotein in the dark cores. The labeling of LFA and SNA showed that sialic acids existed mainly in the mucinous granules of secretory cells and ciliated epithelium glycocalyx, and in the mucous blanket. The results also suggested that the dominant linkage of sialic acids of mucin is a Neu5Ac(alpha 2-6)Gal/GalNAc sequence. Furthermore, the data obtained from ConA and BPA suggested that initial O-glycosylation of mucin took place in the cis side of the Golgi apparatus and that initial N-glycosylation of the serum occurred in the rough endoplasmic reticulum.     

链球菌神经氨酸酶的作用机制及酶活性的测定技术

神经氨酸酶不仅存在于流感病毒,在细菌中也有分布。细菌的神经氨酸酶可裂解宿主体内糖结合物末端的神经氨酸残基,有助于细菌实现在宿主体内的定殖、穿透和扩散,是细菌重要的毒力因子之一。链球菌是自然界广泛存在的人畜共患的病原菌,在多种链球菌中均可检测出神经氨酸酶。肺炎链球菌的神经氨酸酶研究最为透彻,该菌可产生3种神经氨酸酶(NanA,NanB,NanC),NanA不但可以发挥酶的催化作用,分解唾液酸残基,暴露细菌的黏附受体,还能不依赖酶活基团,辅助细菌感染宿主细胞;NanB催化后产物可作为细菌的碳源;NanC可辅助细菌入侵脑部。在无乳链球菌和猪链球菌中,神经氨酸酶的活性一直未得到确切的验证,可能是由于它们的荚膜均含有神经氨酸,所以其神经氨酸酶的活性逐渐在进化中丧失。另外一些链球菌,例如化脓链球菌和C、G、L群链球菌,其神经氨酸酶的底物偏好相近,均对唾液类黏蛋白的催化活性较强,利于链球菌在含唾液类黏蛋白的组织中扩散。在口腔链球菌和血链球菌中,神经氨酸酶破坏血液成分中的神经氨酸链。由此可见,神经氨酸酶的特异性催化作用与链球菌在宿主体内的定植部位密切相关。此外,随着科技的发展,对神经氨酸酶的活性检测,也由早期的硫代巴比妥法,转为现在的荧光值和吸光度的测定,更为便捷和敏感。本文旨在对链球菌的神经氨酸酶的作用机制、与毒力关系及酶活测定方法等研究进展作一综述,为从事相关研究的科学工作者提供参考。     

Development of an enzyme-linked glycosorbent method to monitor the inhibition of sialic acid-dependent Helicobacter pylori adhesion

Horseradish peroxidase conjugation with fetuin, which expresses sialic acid-dependent binding specificity to Helicobacter pylori, was used to develop an enzyme-linked glycosorbent method. This method yielded results that were consistent with those from a hemagglutination assay using a microscope and allowed the quantitative analysis of inhibitors of sialic acid-dependent Helicobacter pylori adhesion to host cells. The results of inhibitor screening with carbohydrates, including commercially available polysaccharides and extracted from various sources, displayed not only the relative inhibition potencies among carbohydrates, but also their respective concentration-dependencies.     

A 23 kDa membrane glycoprotein bearing NeuNAc{alpha}2-3Gal{beta}1-3GalNAc O-linked carbohydrate chains acts as a receptor for Streptococcus sanguis OMZ 9 on human buccal epithelial cells

Streptococcus sanguis colonizes several human oral surfaces,including both hard and soft tissues. Large salivary mucin likeglycoproteins bearing sialic acid residues are known to bindvarious S.sanguis strains. However, the molecular basis forthe adhesion of S.sanguis to human buccal epithelial cells (HBEC)has not been established. The present study shows that S.sanguisOMZ 9 binds to exfoliated HBEC in a sialic acid-sensitive manner.The desialylation of such cells invariably abolhhes adhesionof S.sanguis OMZ 9 to the cell surface. A soluble glycopeptidebearing short sialylated O-linked carbohydrate chains behavesas a potent inhibitor of the attachment of S.sanguis OMZ 9 toexfoliated HBEC. The resialylation of desialylated HBEC withCMP-sialic acid and Galß1,3GalNAc     

New aspects of siglec binding specificities, including the significance of fucosylation and of the sialyl-Tn epitope. Sialic acid-binding immunoglobulin superfamily lectins

The siglecs (sialic acid-binding immunoglobulin superfamily lectins) are immunoglobulin superfamily members recognizing sialylated ligands. Most prior studies of siglec specificities focused on alpha2-3- and alpha2-6-sialyllactos(amin)es and on one or two of the siglecs at a time. Here, we explore several new aspects of specificities of the first six reported siglecs, using sialylated glycans presented in multivalent form, on synthetic polyacrylamide backbones, or on mucin polypeptides. First, we report that binding of siglec-1 (sialoadhesin), siglec-3 (CD33), siglec-4a (myelin-associated glycoprotein), and siglec-5 to alpha2-3 sialyllactosamine is affected markedly by the presence of an alpha1-3-linked fucose. Thus, while siglecs may not interfere with selectin-mediated recognition, fucosylation could negatively regulate siglec binding. Second, in contrast to earlier studies, we find that siglec-3 prefers alpha2-6-sialyllactose. Third, siglec-5 binds alpha2-8-linked sialic acid, making it the siglec least specific for linkage recognition. Fourth, siglecs-2 (CD22), -3, -5, and -6 (obesity-binding protein 1) showed significant binding to sialyl-Tn (Neu5Acalpha2-6-GalNAc), a tumor marker associated with poor prognosis. Fifth, siglec-6 is an exception among siglecs in not requiring the glycerol side chain of sialic acid for recognition. Sixth, all siglecs require the carboxyl group of sialic acid for binding. Finally, the presentation of the sialyl-Tn epitope and/or more extended structures that include this motif may be important for optimal recognition by the siglecs. This was concluded from studies using ovine, bovine, and porcine submaxillary mucins and Chinese hamster ovary cells transfected with ST6GalNAc-I and/or the mucin polypeptide MUC1.     

NAD+ Biosynthesis Ameliorates a Zebrafish Model of Muscular Dystrophy

Muscular dystrophies are common, currently incurable diseases. A subset of dystrophies result from genetic disruptions in complexes that attach muscle fibers to their surrounding extracellular matrix microenvironment. Cell-matrix adhesions are exquisite sensors of physiological conditions and mediate responses that allow cells to adapt to changing conditions. Thus, one approach towards finding targets for future therapeutic applications is to identify cell adhesion pathways that mediate these dynamic, adaptive responses in vivo. We find that nicotinamide riboside kinase 2b-mediated NAD+ biosynthesis, which functions as a small molecule agonist of muscle fiber-extracellular matrix adhesion, corrects dystrophic phenotypes in zebrafish lacking either a primary component of the dystrophin-glycoprotein complex or integrin alpha7. Exogenous NAD+ or a vitamin precursor to NAD+ reduces muscle fiber degeneration and results in significantly faster escape responses in dystrophic embryos. Overexpression of paxillin, a cell adhesion protein downstream of NAD+ in this novel cell adhesion pathway, reduces muscle degeneration in zebrafish with intact integrin receptors but does not improve motility. Activation of this pathway significantly increases organization of laminin, a major component of the extracellular matrix basement membrane. Our results indicate that the primary protective effects of NAD+ result from changes to the basement membrane, as a wild-type basement membrane is sufficient to increase resilience of dystrophic muscle fibers to damage. The surprising result that NAD+ supplementation ameliorates dystrophy in dystrophin-glycoprotein complex– or integrin alpha7–deficient zebrafish suggests the existence of an additional laminin receptor complex that anchors muscle fibers to the basement membrane. We find that integrin alpha6 participates in this pathway, but either integrin alpha7 or the dystrophin-glycoprotein complex is required in conjunction with integrin alpha6 to reduce muscle degeneration. Taken together, these results define a novel cell adhesion pathway that may have future therapeutic relevance for a broad spectrum of muscular dystrophies.     

Ultrastructure and function of the fractalkine mucin domain in CX(3)C chemokine domain presentation

Fractalkine (FKN), a CX(3)C chemokine/mucin hybrid molecule on endothelium, functions as an adhesion molecule to capture and induce firm adhesion of a subset of leukocytes in a selectin- and integrin-independent manner. We hypothesized that the FKN mucin domain may be important for its function in adhesion, and tested the ability of secreted alkaline phosphatase (SEAP) fusion proteins containing the entire extracellular region (FKN-SEAP), the chemokine domain (CX3C-SEAP), or the mucin domain (mucin-SEAP) to support firm adhesion under flow. CX3C-SEAP induced suboptimal firm adhesion of resting peripheral blood mononuclear cells, compared with FKN-SEAP, and mucin-SEAP induced no firm adhesion. CX3C-SEAP and FKN-SEAP bound to CX(3)CR1 with similar affinities. By electron microscopy, fractalkine was 29 nm in length with a long stalk (mucin domain), and a globular head (CX(3)C). To test the function of the mucin domain, a chimeric protein replacing the mucin domain with a rod-like segment of E-selectin was constructed. This chimeric protein gave the same adhesion of peripheral blood mononuclear cells as intact FKN, both when immobilized on glass and when expressed on the cell surface. This implies that the function of the mucin domain is to provide a stalk, extending the chemokine domain away from the endothelial cell surface to present it to flowing leukocytes.