Structural study of the N-glycans of intercellular adhesion molecule-5 (telencephalin)

Intercellular adhesion molecule-5 (ICAM-5, telencephalin) is a dendritically polarized membrane glycoprotein expressed in tissues distinct from those expressing other ICAMs. Here, we determined the N-glycan structure of ICAM-5 purified from adult rat brain and compared it with that of other ICAMs. N-glycans were released by N-glycosidase F digestion and labeled with p-amino benzoic octylester (ABOE). ABOE-labeled glycans were analyzed by high performance liquid chromatography (HPLC) and mass spectrometry. The N-glycans obtained from rat brain ICAM-5 consisted of approximately 85% neutral, 10.2% sialylated-only, 2.8% sulfated-only, and 1.2% sialylated and sulfated glycans. Compared with the N-glycan structures of human ICAM-1 expressed in CHO cells, HEK cells, or mouse myeloma cells and ICAM-3 isolated from human T-cells, rat brain ICAM-5 had less highly branched glycans, sialylated glycans, and N-acetyllactosamine structures. In contrast, high-mannose-type N-glycans and Lewis X were more commonly found in rat brain ICAM-5 than in human ICAM-1 expressed in CHO cells, HEK cells, or mouse myeloma cells and ICAM-3 isolated from human T-cells. In addition, sulfated glycans contained GlcNAc 6-O-sulfate on the non-reducing terminal side. Our data will be important for the elucidation of the roles of the N-glycans expressed in neural cells, including those present on ICAM-5.     

Monosaccharide Sequence of Protein-Bound Glycans of Uukuniemi Virus

Uukuniemi virus, a member of the Bunyaviridae family, was grown in BHK-21 cells in the presence of [³H]mannose. The purified virions were disrupted with sodium dodecyl sulfate and digested with pronase. The [³H]mannose-labeled glycopeptides of the mixture of the two envelope glycoproteins G1 and G2 were characterized by degrading the glycans with specific exo-and endoglycosidases, by chemical methods, and by analyzing the products with lectin affinity and gel chromatography. The glycopeptides of Uukuniemi virus fell into three categories: complex, high-mannose type, and intermediate. The complex glycopeptides probably contained mainly two NeuNAc-Gal-GlcNAc branches attached to a core (Man)₃(GlcNAc)₂ peptide. The high-mannose-type glycans were estimated to contain at least five mannose units attached to two N-acetylglucosamine residues. Both glycan species appeared to be similar to the asparagine-linked oligosaccharides found in many soluble and membrane glycoproteins. The results suggested that the intermediate glycopeptides contained a mannosyl core. In about half of the molecules, one branch appeared to be terminated in mannose, and one appeared to be terminated in N-acetylglucosamine. Such glycans are a novel finding in viral membrane proteins. They may represent intermediate species in the biosynthetic pathway from high-mannose-type to complex glycans. Their accumulation could be connected with the site of maturation of the members of the Bunyaviridae family. Electron microscopic data suggest that the virions bud into smooth-surfaced cisternae in the Golgi region. The relative amounts of [³H]mannose in the complex, high-mannose-type, and intermediate glycans were 25, 62, and 13%, respectively, which corresponded to the approximate relative number of oligosaccharide chains of 2:2.8:1, respectively, in the roughly equimolar mixture of G1 and G2. Endoglycosidase H digestion of isolated [³⁵S]methionine-labeled G1 and G2 proteins suggested that most of the complex and intermediate chains were attached to G1 and that most of the high-mannose-type chains were attached to G2.     

Simultaneous expression of keratan sulphate epitope (a sulphated poly-N-acetyllactosamine) and blood group ABH antigens in papillary carcinomas of the human thyroid gland

Summary The monoclonal antibody 5-D-4 recognizes heavily sulphated forms of keratan sulphate epitope. It reacted strongly with the cell surfaces of most thyroid papillary carcinomas from all the individuals examined, independently of the blood group of the patients. Cells of follicular variants of papillary carcinomas were also labelled by 5-D-4. In contrast, no labelling with this antibody was observed in other types of thyroid neoplasms, or in normal tissues. The reactivity of 5-D-4 with papillary carcinomas was markedly reduced or abolished by prior digestion with endo-β-galactosidase keratanase II, or N-glycosidase F. Although keratanase digestion had no effect on 5-D-4 labelling, it revealed the binding sites ofGriffonia simplicifolia agglutinin II (GSA-II), which recognizes terminalN-acetylglucosamine in a limited number of carcinoma cells from some individuals. Blood group ABH antigens, which are simultaneously expressed together with keratan sulphate epitope in cancer cells, were eliminated by digestion with endo-β-galactosidase and N-glycosidase F, but were resistant to keratanase and keratanase II treatment. These results indicate that keratan sulphate oligosaccharides are cancer-associated and are probably oncofoetal antigens, as are the blood group antigens in human thyroid glands. The results suggests that poly-N-acetyllactosamine, which is ubiquitously and consistently produced in papillary carcinomas, is modified in two different ways: sulphation on the 6-position of at least some units of either galactose, orN-acetylglucosamine or both, and decoration of non-reducing termini with the blood group antigens. Along with the endo-β-galactosidase-GSA-II labelling procedure, labelling with 5-D-4 may be a useful diagnostic means for distinguishing papillary carcinoma from other types of thyroid neoplasms.     

Histochemical demonstration of different types of poly-N-acetyllactosamine structures in human thyroid neoplasms using lectins and endo-β-galactosidase digestion

Summary Blood-group-related antigens expressed in papillary carcinomas and other types of neoplasm of the human thyroid glands have been shown to be carried by poly-N-acetyllactosamines containing a linear domain susceptible to endo-β-galactosidase digestion. To make clear more precisely the backbone poly-N-acetyllactosamine structures, labelled lectins specific to different types of these structures and specific to core structures with β1-6GlcNAc branching of N- and O-linked glycoproteins were employed in conjunction with prior endo-β-galactosidase digestion on formalin-fixed, paraffin-embedded neoplasms of the human thyroid glands. In papillary carcinomas,Datura stramonium agglutinin (DSA) and succinyl wheat germ agglutinin (Suc-WGA) reacted most consistently and frequently with papillary carcinomas from all the individuals examined. Pokeweed mitogen (PWM) likewise stained the cells of papillary carcinomas from all the individuals examined, but in some individuals the number of lectin-reactive cells were very small.Lycoperscion esculentum aggultinin (LEA),Solanum tuberosum agglutinin (STA),Phaseolus vulgaris agglutinin L (PHA-L) andArtocarpus integrifolia agglutinin (jacalin) similarly bound to the cancer cells from most of the individuals, and in these cases the number of reactive cells was usually much more restricted than was the case with DSA or PWM. In adenoma and other types of carcinoma, such as follicular carcinomas, these lectins specific to poly-N-acetyllactosamine exhibited slight or no reactivity with the cells, whereas PHA-L and jacalin similarly bound to the cells of adenomas and carcinomas from most of the individuals examined. Prior digestion with endo-β-galactosidase completely eliminated or markedly reduced the reactivity with PWM and LEA in papillary carcinomas. Reactivity with DSA, Suc-WGA, STA, PHA-L and jacalin was slightly reduced or not at all affected by enzyme digestion. These results confirmed that poly-N-acetyllactosamine species found in papillary carcinomas are quite different from those in other types of thyroid neoplasm, suggesting that at least three different types of poly-N-acetyllactosamine, that is, linear unbranched short and long sequences and highly branched ones are produced in these cells.     

O-linked glycans mediate apical sorting of human intestinal sucrase-isomaltase through association with lipid rafts.

The plasma membrane of polarised epithelial cells is characterised by two structurally and functionally different domains, the apical and basolateral domains. These domains contain distinct protein and lipid constituents that are sorted by specific signals to the correct surface domain [1]. The best characterised apical sorting signal is that of glycophosphatidylinositol (GPI) membrane anchors [2], although N-linked glycans on some secreted proteins [3] and O-linked glycans [4] also function as apical sorting signals. In the latter cases, however, the underlying sorting mechanisms remain obscure. Here, we have analysed the role of O-glycosylation in the apical sorting of sucrase-isomaltase (SI), a highly polarised N- and O-glycosylated intestinal enzyme, and the mechanisms underlying this process. Inhibition of O-glycosylation by benzyl-N-acetyl-alpha-D-galactosaminide (benzyl-GalNAc) was accompanied by a dramatic shift in the sorting of SI from the apical membrane to both membranes. The sorting mechanism of SI involves its association with sphingolipid- and cholesterol-rich membrane rafts because this association was eliminated when O-glycosylation was inhibited by benzyl-GaINAc. The results demonstrate for the first time that O-linked glycans mediate apical sorting through association with lipid rafts.     

Fractionation of sulfated galactan from the red alga Botryocladia occidentalis separates its anticoagulant and anti-SARS-CoV-2 properties

Sulfation pattern and molecular weight (MW) play a key role in the biological actions of sulfated glycans. Besides anticoagulant effects, certain sulfated glycans can also exhibit anti-SARS-CoV-2 properties. To develop a more selective antiviral carbohydrate, an efficient strategy to separate these two actions is required. In this work, low MW fractions derived from the red alga Botryocladia occidentalis sulfated galactan (BoSG) were generated, structurally characterized, and tested for activity against SARS-CoV-2 and blood coagulation. The lowest MW fraction was found to be primarily composed of octasaccharides of monosulfated monosaccharides. Unlike heparin or native BoSG, we found that hydrolyzed BoSG products had weak anticoagulant activities as seen by aPTT and inhibitory assays using purified cofactors. In contrast, lower MW BoSG-derivatives retained anti-SARS-CoV-2 activity using SARS-CoV-2 spike (S)-protein pseudotyped lentivirus vector in HEK-293T-hACE2 cells monitored by GFP. Surface plasmon resonance confirmed that longer chains are necessary for BoSG to interact with coagulation cofactors but is not required for interactions with certain S-protein variants. We observed distinct affinities of BoSG derivatives for the S-proteins of different SARS-CoV-2 strains, including WT, N501Y (Alpha), K417T/E484K/N501Y (Gamma), and L542R (Delta) mutants, and stronger affinity for the N501Y-containing variants. Docking of the four possible monosulfated BoSG disaccharides in interactions with the N501Y mutant S-protein predicted potential binding poses of the BoSG constructs and favorable binding in close proximity to the 501Y residue. Our results demonstrate that depolymerization and fractionation of BoSG are an effective strategy to segregate its anticoagulant property from its anti-SARS-CoV-2 action.     

Carbohydrate Recognition Properties of Human Ficolins: GLYCAN ARRAY SCREENING REVEALS THE SIALIC ACID BINDING SPECIFICITY OF M-FICOLIN*

Ficolins are oligomeric innate immune recognition proteins consisting of a collagen-like region and a fibrinogen-like recognition domain that bind to pathogen- and apoptotic cell-associated molecular patterns. To investigate their carbohydrate binding specificities, serum-derived L-ficolin and recombinant H- and M-ficolins were fluorescently labeled, and their carbohydrate binding ability was analyzed by glycan array screening. L-ficolin preferentially recognized disulfated N-acetyllactosamine and tri- and tetrasaccharides containing terminal galactose or N-acetylglucosamine. Binding was sensitive to the position and orientation of the bond between N-acetyllactosamine and the adjacent carbohydrate. No significant binding of H-ficolin to any of the 377 glycans probed could be detected, providing further evidence for its poor lectin activity. M-ficolin bound preferentially to 9-O-acetylated 2-6-linked sialic acid derivatives and to various glycans containing sialic acid engaged in a 2-3 linkage. To further investigate the structural basis of sialic acid recognition by M-ficolin, point mutants were produced in which three residues of the fibrinogen domain were replaced by their counterparts in L-ficolin. Mutations G221F and A256V inhibited binding to the 9-O-acetylated sialic acid derivatives, whereas Y271F abolished interaction with all sialic acid-containing glycans. The crystal structure of the Y271F mutant fibrinogen domain was solved, showing that the mutation does not alter the structure of the ligand binding pocket. These analyses reveal novel ficolin ligands such as sulfated N-acetyllactosamine (L-ficolin) and gangliosides (M-ficolin) and provide precise insights into the sialic acid binding specificity of M-ficolin, emphasizing the essential role of Tyr²⁷¹ in this respect.     

The cell surface protein MUL_3720 confers binding of the skin pathogen Mycobacterium ulcerans to sulfated glycans and keratin

Mycobacterium ulcerans is the causative agent of the chronic, necrotizing skin disease Buruli ulcer. Modes of transmission and molecular mechanisms involved in the establishment of M. ulcerans infections are poorly understood. Interactions with host glycans are often crucial in bacterial pathogenesis and the 22 kDa M. ulcerans protein MUL_3720 has a putative role in host cell attachment. It has a predicted N-terminal lectin domain and a C-terminal peptidoglycan-binding domain and is highly expressed on the surface of the bacilli. Here we report the glycan-binding repertoire of whole, fixed M. ulcerans bacteria and of purified, recombinant MUL_3720. On an array comprising 368 diverse biologically relevant glycan structures, M. ulcerans cells showed binding to 64 glycan structures, representing several distinct classes of glycans, including sulfated structures. MUL_3720 bound only to glycans containing sulfated galactose and GalNAc, such as glycans known to be associated with keratins isolated from human skin. Surface plasmon resonance studies demonstrated that both whole, fixed M. ulcerans cells and MUL_3720 show high affinity interactions with both glycans and human skin keratin extracts. This MUL_3720-mediated interaction with glycans associated with human skin keratin may contribute to the pathobiology of Buruli ulcer.     

Quantitative determination of sulfated glycopeptide by two-dimensional electrophoresis on cellulose acetate membrane

Quantitative determination of the sulfated glycoproteins present in tissue and secretion fluid was performed. After digestion of the specimen with pronase in order to convert glycoproteins to glycopeptides, the sulfated glycopeptides were separated from a mixture of acidic glycans (glycosaminoglycans, sialoglycopeptides and sulfated glycopeptides) by two-dimensional electrophoresis on cellulose acetate membrane [(1986) J. Biochem. Biophys. Methods 12, 239-246]. After staining with alcian blue, the spot of sulfated glycopeptide on the cellulose acetate membrane was cut out, and then only the dye bound to the sulfated glycopeptide was extracted with a 5% cetylpyridinium chloride solution at 100 degrees C for 15 min. The extract was then measured by absorbance at 615 nm using an authentic sulfated glycopeptide as a standard. This method facilitated the determination of sulfated glycopeptides, which were separated from other acidic glycans, within the range 0-25 micrograms.     

NMR structural biology of sulfated glycans

Sulfated fucans, sulfated galactans, and glycosaminoglycans are extensively studied worldwide in terms of both structure and biomedical functions. Liquid-state nuclear magnetic resonance (NMR) spectroscopy is the most employed analytical technique in structural analysis of these sulfated glycans. This is due to the fact that NMR-based analyses enable a series of achievements such as (i) accurate structure characterization/determination; (ii) measurements of parameters regarding molecular motion (dynamics); (iii) assessment of the 3D structures (usually assisted by computational techniques of Molecular Modeling and/or Molecular Dynamics) of the composing monosaccharides (ring conformers) and the overall conformational states of the glycan chains either free in solution or bound to proteins; and (iv) analysis of the resultant intermolecular complexes with functional proteins through either the protein or the carbohydrate perspective. In this review, after a general introduction about the principal NMR parameters utilized for achieving this set of structural information, discussion is given on NMR-based studies of some representative sulfated fucans, sulfated galactans, and glycosaminoglycans. Due to the growing number of studies concerning both structure and function of sulfated glycans and the widely use of NMR spectroscopy in such studies, a review paper discussing (i) the most experiments employed for analysis, (ii) procedures used in data interpretation, and (iii) the general aspects of the sulfated glycans, is timely in the literature.     

Immunohistochemical and immunoelectron microscopic analyses of alpha-amylase isozymes in human intrahepatic biliary epithelium and hepatocytes.

The expression and localization of the pancreatic and salivary isozymes of alpha-amylase in the intrahepatic biliary epithelium and hepatocytes were examined by the immunohistochemical method with polyclonal and monoclonal antibodies in 45 normal autopsied human livers. Immunoelectron microscopic studies with the protein A-gold method were performed with the monoclonal antibodies (MAb) on seven of the livers. The intrahepatic biliary system was divided into large ducts, septal ducts, interlobular ducts, bile ductules, and peribiliary glands. Immunohistochemically, pancreatic isozyme was observed in the supranuclear cytoplasm of the epithelium of large ducts, septal ducts, and peribiliary glands in almost all livers. Interlobular ducts expressed pancreatic isozyme in only four (9%) livers. Bile ductules and hepatocytes were negative for pancreatic isozyme in all cases. Expression of salivary isozyme was observed in the supranuclear cytoplasm of the epithelium of large ducts, septal ducts, interlobular ducts, bile ductules, and peribiliary glands in almost all livers, although the expression in interlobular ducts and bile ductules was weak. Hepatocytes were weakly positive for salivary isozyme. Immunoelectron microscopy revealed that both pancreatic and salivary isozymes were located in the supranuclear cytoplasm of the epithelium of large ducts, septal ducts, and peribiliary glands, and that hepatocytes had no pancreatic isozyme but contained salivary isozyme. These data suggest that pancreatic and salivary isozymes of alpha-amylase are produced by the intrahepatic biliary epithelium and secreted into intrahepatic biliary lumens, and that they may play an important role in the physiology of the intrahepatic biliary tree and hepatic bile. It is also suggested that hepatocytes produce a small amount of salivary alpha-amylase that may be secreted into the biliary tree.     

Immunoexpression of intermediate filaments and morphological changes in the liver and bile duct of rats infected with Fasciola hepatica

We investigated the immunoexpression of the intermediate filament proteins, cytokeratin and desmin, and the morphological changes in the liver of rats during experimental fasciolosis at 4, 7 and 10 weeks post-infection. Rats were infected with 30 Fasciola hepatica metacercariae. Paraffin sections of the liver were stained using H & E, PAS and azan stains. Immunohistochemical reactions were performed using antibodies against cytokeratin and desmin. The experimental F. hepatica infection led to fibrosis and cirrhosis of the liver, and to inflammation of the common bile ducts. The expression of cytokeratin was increased in the epithelial cells of both the liver bile ductules at 4, 7 and 10 weeks post-infection and in the common bile ducts at 7 and 10 weeks post-infection compared to uninfected rats; expression in the common bile ducts was more intense. The myofibroblasts of the liver and smooth myocytes of the interlobular bile ducts and common bile ducts, showed a slight increase in desmin expression compared to the uninfected rats. The increased expression of cytokeratins in the hyperplastic rat common bile duct epithelium during the biliary phase of fasciolosis at 7 and 10 weeks post-infection may be explained by mechanical irritation by the parasite and an inflammatory reaction in the bile duct epithelium and in periductal fibrous tissue.     

Hemocytes and Plasma of the Eastern Oyster (Crassostrea virginica) Display a Diverse Repertoire of Sulfated and Blood Group A-modified N-Glycans

The eastern oyster (Crassostrea virginica) has become a useful model system for glycan-dependent host-parasite interactions due to the hijacking of the oyster galectin CvGal1 for host entry by the protozoan parasite Perkinsus marinus, the causative agent of Dermo disease. In this study, we examined the N-glycans of both the hemocytes, which via CvGal1 are the target of the parasite, and the plasma of the oyster. In combination with HPLC fractionation, exoglycosidase digestion, and fragmentation of the glycans, mass spectrometry revealed that the major N-glycans of plasma are simple hybrid structures, sometimes methylated and core α1,6-fucosylated, with terminal β1,3-linked galactose; a remarkable high degree of sulfation of such glycans was observed. Hemocytes express a larger range of glycans, including core-difucosylated paucimannosidic forms, whereas bi- and triantennary glycans were found in both sources, including structures carrying sulfated and methylated variants of the histo-blood group A epitope. The primary features of the oyster whole hemocyte N-glycome were also found in dominin, the major plasma glycoprotein, which had also been identified as a CvGal1 glycoprotein ligand associated with hemocytes. The occurrence of terminal blood group moieties on oyster dominin and on hemocyte surfaces can account in part for their affinity for the endogenous CvGal1.     

In vivo and in vitro tyrosine sulfation of a membrane glycoprotein

A431 cells incorporate 35SO4 into a protein of Mr 61,000 (P61). We examined sulfation of P61 by cells (in vivo) and by a cell-free system (in vitro) which requires only addition of A431 cell membranes and a 3'-phosphoadenosine 5'-phospho[35S]sulfate-generating system prepared from Krebs ascites cells. Sulfate is found exclusively in the form of tyrosine SO4 by two-dimensional high voltage electrophoresis following Pronase digestion. Endoglycosidase F digestion reduces the Mr by 2,000 but does not release the sulfate, indicating that P61 is a glycoprotein but that sulfate is not incorporated into the carbohydrate. Sulfated P61 is not found in the medium from cultured cells and remains associated with the membrane fraction following cell lysis. Treatment of membranes with 0.4 M NaCl, 0.3 M KCl, 15 mM EDTA, or pH 11.0 does not release sulfated P61. P61 is solubilized by Triton X-114 treatment of membranes and partitions into the detergent phase upon warming. Based on these characteristics, we conclude that P61 is an integral membrane protein. Trypsin digestion experiments with intact cells suggest that sulfated P61 is predominantly located in the plasma membrane. This is the first example of an integral membrane protein which is sulfated on tyrosine. The properties of the sulfation reaction are distinct from those reported for secreted proteins and are consistent with the possibility that this modification occurs at the plasma membrane rather than in the Golgi.     

The apical plasma membrane of chitin‐synthesizing epithelia

Abstract Chitin is the second most abundant polysaccharide on earth. It is produced at the apical side of epidermal, tracheal, fore‐, and hindgut epithelial cells in insects as a central component of the protective and supporting extracellular cuticle. Chitin is also an important constituent of the midgut peritrophic matrix that encases the food supporting its digestion and protects the epithelium against invasion by possibly ingested pathogens. The enzyme producing chitin is a glycosyltransferase that resides in the apical plasma membrane forming a pore to extrude the chains of chitin into the extracellular space. The apical plasma membrane is not only a platform for chitin synthases but, probably through its shape and equipment with distinct factors, also plays an important role in orienting and organizing chitin fibers. Here, I review findings on the cellular and molecular constitution of the apical plasma membrane of chitin‐producing epithelia mainly focusing on work done in the fruit fly Drosophila melanogaster.     

Horizontal Gene Transfer Contributed to the Evolution of Extracellular Surface Structures: The Freshwater Polyp Hydra Is Covered by a Complex Fibrous Cuticle Containing Glycosaminoglycans and Proteins of the PPOD and SWT (Sweet Tooth) Families

The single-cell layered ectoderm of the fresh water polyp Hydra fulfills the function of an epidermis by protecting the animals from the surrounding medium. Its outer surface is covered by a fibrous structure termed the cuticle layer, with similarity to the extracellular surface coats of mammalian epithelia. In this paper we have identified molecular components of the cuticle. We show that its outermost layer contains glycoproteins and glycosaminoglycans and we have identified chondroitin and chondroitin-6-sulfate chains. In a search for proteins that could be involved in organising this structure we found PPOD proteins and several members of a protein family containing only SWT (sweet tooth) domains. Structural analyses indicate that PPODs consist of two tandem β-trefoil domains with similarity to carbohydrate-binding sites found in lectins. Experimental evidence confirmed that PPODs can bind sulfated glycans and are secreted into the cuticle layer from granules localized under the apical surface of the ectodermal epithelial cells. PPODs are taxon-specific proteins which appear to have entered the Hydra genome by horizontal gene transfer from bacteria. Their acquisition at the time Hydra evolved from a marine ancestor may have been critical for the transition to the freshwater environment.     

Characterization of N-glycans from mouse brain neural cell adhesion molecule.

The N-glycosylation pattern of the neural cell adhesion molecule (NCAM), isolated from brains of newborn mice, has been analyzed. Following digestion with trypsin, generated glycopeptides were fractionated by serial immunoaffinity chromatography using immobilized monoclonal antibodies specifically recognizing polysialic acid (PSA) units or the HNK1-carbohydrate epitope. Subsequent analyses of the resulting (glyco)peptides by Edman degradation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) revealed polysialylated glycans to be exclusively linked to glycosylation sites 5 (Asn(431)) and 6 (Asn(460)), whereas glycans carrying the HNK1-epitope could be assigned to sites 2 (Asn(297)), 5, 6, and, to a lesser extent, site 3 (Asn(329)). PSA-, HNK1-, and non-PSA/HNK1-glycan fractions were characterized by carbohydrate constituent and methylation analyses as well as MALDI-TOF-MS in conjunction with chromatographic fractionation techniques. The results revealed that the core structures of PSA-glycans represented predominantly fucosylated, partially sulfated 2,6-branched isomers of triantennary as well as tetraantennary complex-type glycans, whereas carbohydrate chains bearing the HNK1-epitope were dominated by diantennary species carrying in part bisecting GlcNAc residues. Non-PSA/HNK1-glycans exhibited a highly heterogeneous pattern of partially truncated, mostly diantennary structures being characterized by the presence of additional fucose, bisecting GlcNAc and/or sulfate residues. In conclusion, our results revealed that the glycosylation pattern of murine NCAM displays high structural and regional selectivity, which might play an important role in controlling the biological activities of this molecule.     

Apical membrane rupture and backward bile flooding in acetaminophen-induced hepatocyte necrosis

Morphological changes of hepatocyte death have so far only been described on cells in culture or in tissue sections. Using a high-resolution and high-magnification multiphoton microscopic system, we recorded in living mice serial changes of acetaminophen (APAP)-induced hepatocyte necrosis in relevance to metabolism of a fluorogenic bile solute. Initial changes of hepatocyte injury included basal membrane disruption and loss of mitochondrial membrane potential. An overwhelming event of rupture at adjacent apical membrane resulting in flooding of bile into these hepatocytes might ensue. Belbs formed on basal membrane and then dislodged into the sinusoid circulation. Transmission electron microscopy disclosed a necrotic hepatocyte depicting well the changes after apical membrane rupture and bile flooding. Administration of the antidote N-acetylcysteine dramatically reduced the occurrence of apical membrane rupture. The present results demonstrated a hidden but critical step of apical membrane rupture leading to irreversible APAP-induced hepatocyte injury.     

Altered alkaline phosphatase activity in obese Zucker rats liver respect to lean Zucker and Wistar rats discussed in terms of all putative roles ascribed to the enzyme

Biliary complications often lead to acute and chronic liver injury after orthotopic liver transplantation (OLT). Bile composition and secretion depend on the integrated action of all the components of the biliary tree, starting from hepatocytes. Fatty livers are often discarded as grafts for OLT, since they are extremely vulnerable to conventional cold storage (CS). However, the insufficiency of donors has stimulated research to improve the usage of such marginal organs as well as grafts. Our group has recently developed a machine perfusion system at subnormothermic temperature (20°C; MP20) that allows a marked improvement in preservation of fatty and even of normal rat livers as compared with CS. We sought to evaluate the response of the biliary tree of fatty liver to MP20, and a suitable marker was essential to this purpose. Alkaline phosphatase (AlkP, EC 3.1.3.1), frequently used as marker of membrane transport in hepatocytes and bile ducts, was our first choice. Since no histochemical data were available on AlkP distribution and activity in fatty liver, we have first settled to investigate AlkP activity in the steatotic liver of fatty Zucker rats (fa/fa), using as controls lean Zucker (fa/+) and normal Wistar rats. The AlkP reaction in Wistar rats was in accordance with the existing data and, in particular, was present in bile canaliculi of hepatocytes in the periportal region and midzone, in the canals of Hering and in small bile ducts but not in large bile ducts. In lean ZR liver the AlkP reaction in Hering canals and small bile ducts was similar to Wistar rat liver but hepatocytes had lower canalicular activity and besides presented moderate basolateral reaction. The difference between lean Zucker and Wistar rats, both phenotypically normal animals, could be related to the fact that lean Zucker rats are genotypically heterozygous for a recessive mutated allele. In fatty liver, the activity in ductules and small bile ducts was unchanged, but most hepatocytes were devoid of AlkP activity with the exception of clusters of macrosteatotic hepatocytes in the mid-zone, where the reaction was intense in basolateral domains and in distorted canaliculi, a typical pattern of cholestasis. The interpretation of these data was hindered by the fact that the physiological role of AlkP is still under debate. In the present study, the various functions proposed for the role of the enzyme in bile canaliculi and in cholangiocytes are reviewed. Independently of the AlkP role, our data suggest that AlkP does not seem to be a reliable marker to study the initial step of bile production during OLT of fatty livers, but may still be used to investigate the behaviour of bile ductules and small bile ducts.