In this study, by the generation of a specific monoclonal antibody, we identified p75/AIRM1 (for adhesion inhibitory receptor molecule 1), a novel inhibitory receptor that is mostly confined to human natural killer cells. p75/AIRM1 is a 75-kD glycoprotein that, upon sodium pervanadate treatment, becomes tyrosine phosphorylated and associates to src homology 2 domain-bearing protein tyrosine phosphatase (SHP)-1. The p75/AIRM1 gene is located on human chromosome 19 and encodes a novel member of the sialoadhesin family characterized by three immunoglobulin-like extracellular domains (one NH(2)-terminal V-type and two C2-type) and a classical immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic portion. The highest amino acid sequence similarity has been found with the myeloid-specific CD33 molecule and the placental CD33L1 protein. Similar to other sialoadhesin molecules, p75/AIRM1 appears to mediate sialic acid-dependent ligand recognition.

Tumor necrosis can be induced in transplanted mouse methylcholanthrene-induced sarcoma by a tumor necrosis factor in the serum of mice infected with bacillus Calmette-Guérin and given bacterial endotoxin. Sera from normal mice, endotoxin-treated mice, and mice infected with bacillus Calmette-Guérin do not contain this factor. A 20- to 30-fold purification of the serum factor has been achieved by (NH4)2SO4 fractionation, Sephadex G-100 and G-200 gel filtration, and preparative polyacrylamide electrophoresis. Tumor necrosis factor is not bacterial endotoxin. It migrates with alpha-globulins, is made up of at least four subunits, and has a molecular weight of about 150,000. The active factor is a glycoprotein that contains sialic acid and galactosamine.

CD33 is a myeloid specific member of the sialic acid-binding receptor family and is expressed highly on myeloid progenitor cells but at much lower levels in differentiated cells. Human CD33 has two tyrosine residues in its cytoplasmic domain (Y340 and Y358). When phosphorylated, these tyrosines could function as docking sites for the phosphatases, SHP-1 and/or SHP-2, enabling CD33 to function as an inhibitory receptor. Here we demonstrate that CD33 is tyrosine phosphorylated in the presence of the phosphatase inhibitor, pervanadate, and recruits SHP-1 and SHP-2. Co-expression studies suggest that the Src-family kinase Lck is effective at phosphorylating Y340, but not Y358, suggesting that these residues may function in the selective recruitment of adapter molecules and have distinct functions. Further support for overlapping, but nonredundant, roles for Y340 and Y358 comes from peptide-binding studies that revealed the recruitment of both SHP-1 and SHP-2 to Y340 but only SHP-2 to Y358. Analysis using mutants of SHP-1 demonstrated that binding Y340 of CD33 was primarily to the amino Src homology-2 domain of SHP-1. The potential of CD33 to function as an inhibitory receptor was demonstrated by its ability to down-regulate CD64-induced calcium mobilization in U937. The dependence of this inhibition on SHP-1 was demonstrated by blocking CD33-mediated effects with dominant negative SHP-1. This result implies that CD33 is an inhibitory receptor and also that SHP-1 phosphatase has a significant role in mediating CD33 function. Further studies are essential to identify the receptor(s) that CD33 inhibits in vivo and its function in myeloid lineage development. (Blood. 2000;96:483-490)

Influenza virus is a global health concern due to its unpredictable pandemic potential. This potential threat was realized in 2009 when an H1N1 virus emerged that resembled the 1918 virus in antigenicity but fortunately was not nearly as deadly. 5J8 is a human antibody that potently neutralizes a broad spectrum of H1N1 viruses, including the 1918 and 2009 pandemic viruses. Here, we present the crystal structure of 5J8 Fab in complex with a bacterially expressed and refolded globular head domain from the hemagglutinin (HA) of the A/California/07/2009 (H1N1) pandemic virus. 5J8 recognizes a conserved epitope in and around the receptor binding site (RBS), and its HCDR3 closely mimics interactions of the sialic acid receptor. Electron microscopy (EM) reconstructions of 5J8 Fab in complex with an HA trimer from a 1986 H1 strain and with an engineered stabilized HA trimer from the 2009 H1 pandemic virus showed a similar mode of binding. As for other characterized RBS-targeted antibodies, 5J8 uses avidity to extend its breadth and affinity against divergent H1 strains. 5J8 selectively interacts with HA insertion residue 133a, which is conserved in pandemic H1 strains and has precluded binding of other RBS-targeted antibodies. Thus, the RBS of divergent HAs is targeted by 5J8 and adds to the growing arsenal of common recognition motifs for design of therapeutics and vaccines. Moreover, consistent with previous studies, the bacterially expressed H1 HA properly refolds, retaining its antigenic structure, and presents a low-cost and rapid alternative for engineering and manufacturing candidate flu vaccines.

Lysosomal membrane proteins act at several crucial steps of the lysosome life cycle, including lumen acidification, metabolite export, molecular motor recruitment and fusion with other organelles. This review summarizes the molecular mechanisms of lysosomal storage diseases caused by defective transport of small molecules or ions across the lysosomal membrane, as well as Danon disease. In cystinosis and free sialic acid storage diseases, transporters for cystine and acidic monosaccharides, respectively, are blocked or retarded. A putative cobalamin transporter and a hybrid transporter/transferase of acetyl groups are defective in cobalamin F type disease and mucopolysaccharidosis type IIIC, respectively. In neurodegenerative forms of osteopetrosis, mutations of a proton/chloride exchanger impair the charge balance required for sustained proton pumping by the V-type ATPase, thus resulting in bone-resorption lacuna neutralization. However, the mechanism leading to lysosomal storage and neurodegeneration remains unclear. Mucolipidosis type IV is caused by mutations of a lysosomal cation channel named TRPML1; its gating properties are still poorly understood and the ion species linking this channel to lipid storage and membrane traffic defects is debated. Finally, the autophagy defect of Danon disease apparently arises from a role of LAMP2 in lysosome/autophagosome fusion, possibly secondary to a role in dynein-based centripetal motility.

We investigated the interactions of rotaviruses with glycoproteins and cells that support rotaviral replication. We found that a wide range of naturally occurring glycoproteins, including ovalbumins and ovomucoids from chicken and turkey eggs, and mucin derived from bovine submaxillary glands, inhibit the replication of rotaviruses in MA-104 cells. Our studies further indicated that the glycoproteins bind directly to rotaviruses and that virus-glycoprotein binding is dependent largely upon interactions with sialic acid oligosaccharides. We found that accessible sialic acid oligosaccharides are required for efficient rotavirus infection of MA-104 cells, thus demonstrating that sialic acid oligosaccharides play an important role in the interactions of rotaviruses with both glycoproteins and cells that support rotaviral replication. Bovine submaxillary mucin and chicken ovoinhibitor can also prevent the shedding of rotavirus antigen and the development of rotavirus gastroenteritis in a mouse model of rotavirus infection. Our findings document that a range of glycoproteins inhibit the in vivo and in vitro replication of rotaviruses and suggest that the alteration in the quantity or chemical composition of intestinal glycoproteins is a potential means for the modulation of enteric infections.

The distal portion of rotavirus (RV) VP4 spike protein (VP8*) is implicated in binding to cellular receptors, thereby facilitating viral attachment and entry. While VP8* of some animal RVs engage sialic acid, human RVs often attach to and enter cells in a sialic acid-independent manner. A recent study demonstrated that the major human RVs (P[4], P[6], and P[8]) recognize human histo-blood group antigens (HBGAs). In this study, we performed a phylogenetic analysis of RVs and showed further variations of RV interaction with HBGAs. On the basis of the VP8* sequences, RVs are grouped into five P genogroups (P[I] to P[V]), of which P[I], P[IV], and P[V] mainly infect animals, P[II] infects humans, and P[III] infects both animals and humans. The sialic acid-dependent RVs (P[1], P[2], P[3], and P[7]) form a subcluster within P[I], while all three major P genotypes of human RVs (P[4], P[6], and P[8]) are clustered in P[II]. We then characterized three human RVs (P[9], P[14], and P[25]) in P[III] and observed a new pattern of binding to the type A antigen which is distinct from that of the P[II] RVs. The binding was demonstrated by hemagglutination and saliva binding assay using recombinant VP8* and native RVs. Homology modeling and mutagenesis study showed that the locations of the carbohydrate binding interfaces are shared with the sialic acid-dependent RVs, although different amino acids are involved. The P[III] VP8* proteins also bind the A antigens of the porcine and bovine mucins, suggesting the A antigen as a possible factor for cross-species transmission of RVs. Our study suggests that HBGAs play an important role in RV infection and evolution.

When cultured in broth to the transmissive phase, Legionella pneumophila infects macrophages by inhibiting phagosome maturation, whereas replicative-phase cells are transported to the lysosomes. Here we report that the ability of L. pneumophila to inhibit phagosome-lysosome fusion correlated with developmentally regulated modifications of the pathogen's surface, as judged by its lipopolysaccharide profile and by its binding to a sialic acid-specific lectin and to the hydrocarbon hexadecane. Likewise, the composition of membrane vesicles shed by L. pneumophila was developmentally regulated, based on binding to the lectin and to the lipopolysaccharide-specific monoclonal antibody 3/1. Membrane vesicles were sufficient to inhibit phagosome-lysosome fusion by a mechanism independent of type IV secretion, since only approximately 25% of beads suspended with or coated by vesicles from transmissive phase wild type or dotA secretion mutants colocalized with lysosomal probes, whereas approximately 75% of beads were lysosomal when untreated or presented with vesicles from the L. pneumophila letA regulatory mutant or E. coli. As observed previously for L. pneumophila infection of mouse macrophages, vesicles inhibited phagosome-lysosome fusion only temporarily; by 10 h after treatment with vesicles, macrophages delivered approximately 72% of ingested beads to lysosomes. Accordingly, in the context of the epidemiology of the pneumonia Legionnaires' disease and virulence mechanisms of Leishmania and Mycobacteria, we discuss a model here in which L. pneumophila developmentally regulates its surface composition and releases vesicles into phagosomes that inhibit their fusion with lysosomes.

BACKGROUND

Sargramostim, granulocyte macrophage colony-stimulating factor (GM-CSF), a hematopoietic growth factor, stimulates cells of the intestinal innate immune system. Clinical trials show that sargramostim induces clinical response and remission in patients with active Crohn's disease. To study the mechanism, we examined the effects of GM-CSF in the dextran sulfate sodium (DSS)-induced acute colitis model. We hypothesized that GM-CSF may work through effects on dendritic cells (DCs).

METHODS

Acute colitis was induced in Balb/c mice by administration of DSS in drinking water. Mice were treated with daily GM-CSF or phosphate-buffered saline (PBS). To probe the role of plasmacytoid DCs (pDCs) in the response to GM-CSF, we further examined the effects of monoclonal antibody 440c, which is specific for a sialic acid-binding immunoglobulin (Ig)-like lectin expressed on pDCs.

RESULTS

GM-CSF ameliorates acute DSS-induced colitis, resulting in significantly improved clinical parameters and histology. Microarray analysis showed reduced expression of proinflammatory genes including TNF-alpha and IL1-beta; the results were further confirmed by real-time reverse-transcriptase polymerase chain reaction and serum Bio-plex analysis. GM-CSF treatment significantly expands pDCs and type 1 IFN production. Administration of mAb 440c completely blocked the therapeutic effect of GM-CSF. GM-CSF is also effective in RAG1(-/-) mice, demonstrating activity-independent effects on T and B cells. IFN-beta administration mimics the therapeutic effect of GM-CSF in DSS-treated mice. GM-CSF increases systemic and mucosal type 1 IFN expression and exhibits synergy with pDC activators, such as microbial cytosine-phosphate-guanosine (CpG) DNA.

CONCLUSIONS

GM-CSF is effective in the treatment of DSS colitis in a mechanism involving the 440c(+) pDC population.

BACKGROUND

Pigs are considered susceptible to influenza A virus infections from different host origins because earlier studies have shown that they have receptors for both avian (sialic acid-alpha-2,3-terminal saccharides (SA-alpha-2,3)) and swine/human (SA-alpha-2,6) influenza viruses in the upper respiratory tract. Furthermore, experimental and natural infections in pigs have been reported with influenza A virus from avian and human sources.

METHODS

This study investigated the receptor distribution in the entire respiratory tract of pigs using specific lectins Maackia Amurensis (MAA) I, and II, and Sambucus Nigra (SNA). Furthermore, the predilection sites of swine influenza virus (SIV) subtypes H1N1 and H1N2 as well as avian influenza virus (AIV) subtype H4N6 were investigated in the respiratory tract of experimentally infected pigs using immunohistochemical methods.

RESULTS

SIV antigen was widely distributed in bronchi, but was also present in epithelial cells of the nose, trachea, bronchioles, and alveolar type I and II epithelial cells in severely affected animals. AIV was found in the lower respiratory tract, especially in alveolar type II epithelial cells and occasionally in bronchiolar epithelial cells. SA-alpha-2,6 was the predominant receptor in all areas of the respiratory tract with an average of 80-100% lining at the epithelial cells. On the contrary, the SA-alpha-2,3 was not present (0%) at epithelial cells of nose, trachea, and most bronchi, but was found in small amounts in bronchioles, and in alveoli reaching an average of 20-40% at the epithelial cells. Interestingly, the receptor expression of both SA-alpha-2,3 and 2,6 was markedly diminished in influenza infected areas compared to non-infected areas.

CONCLUSIONS

A difference in predilection sites between SIV and AIV virus was found, and this difference was in accordance with the distribution of the SA-alpha-2,6 and SA-alpha-2,3 receptor, respectively. The results indicated that the distribution of influenza A virus receptors in pigs are similar to that of humans and therefore challenge the theory that the pig acts as a mixing vessel between human and avian influenza viruses. Furthermore, it was shown that AIV prefers to infect alveolar type II epithelial cells in pigs. This corresponds with findings in humans emphasising the resemblance between the two species.

GM2 and GD2 gangliosides are sialic acid-containing glycosphingolipids expressed in some normal tissues such as brain and in various tumors such as neuroblastomas, astrocytomas, and malignant melanomas. We used a eukaryotic cell transient expression system to isolate cDNA clones that determine GM2 expression. We developed a new cell line from murine melanoma line B16 by transfecting with the polyoma T antigen gene that was suitable for this purpose. Two cDNA clones, both of which have a continuous open reading frame of 1683 base pairs, were isolated. Although the cloned cDNAs had no primary sequence similarity to reported glycosyltransferases, the deduced amino acid sequence predicted a type II transmembrane protein with an overall structure similar to other glycosyltransferases. The cDNA clones, when stably transfected, determined the expression of GM2 in B16 cells and GM2 and GD2 in the human melanoma line MeWo. Northern blot analysis revealed two transcripts in all cells that expressed either GM2 or GD2 or both. These findings indicate that the cDNAs catalyze the transfer of GalNAc onto GM3 and GD3 by a beta 1,4 linkage, resulting in the synthesis of GM2 and GD2, respectively. Namely they suggest that these cDNAs derive from the UDP-GalNAc: GM3/GD3 beta 1,4 N-acetylgalactosaminyltransferase (EC 2.4.1.92) gene.

Two terminal glycosyltransferases, a sialyltransferase and the blood group A alpha 1,3 N-acetylgalactosaminyltransferase, were found to exhibit differential subcompartmentation in the Golgi apparatus of intestinal goblet and absorptive cells. As expected from their role in terminal glycosylation, the two glycosyltransferases and their products, sialic acid residues and blood group A substance, were localized in the trans cisternae of the Golgi apparatus of goblet cells. In contrast, however, they were found throughout the Golgi apparatus stack of adjacent absorptive cells, with the exception of the fenestrated first cis cisterna. The results are in contrast to the general view that enzymes in the glycosylation pathway are arranged in a cis to trans gradient across the Golgi apparatus and that such polarized distributions may instead be cell type-specific.

An insertional mutation in hsa, the gene encoding the sialic acid-binding adhesin of Streptococcus gordonii DL1, resulted in a significant reduction of the infection rate of the organism and an inflammatory reaction in the rat aortic valve with experimental endocarditis, suggesting that the adhesin contributes to the infectivity of the organism for heart valves.

Roles for UDP-GlcNAc 2-epimerase/ManNAc 6-kinase (GNE) beyond controlling flux into the sialic acid biosynthetic pathway by converting UDP-GlcNAc to N-acetylmannosamine are described in this report. Overexpression of recombinant GNE in human embryonic kidney (HEK AD293) cells led to an increase in mRNA levels for ST3Gal5 (GM3 synthase) and ST8Sia1 (GD3 synthase) as well as the biosynthetic products of these sialyltransferases, the GM3 and GD3 gangliosides. Conversely, down-regulation of GNE by RNA interference methods had the opposite, but consistent, effect of lowering ST3Gal5 and ST8Sia1 mRNAs and reducing GM3 and GD3 levels. Control experiments ensured that GNE-mediated changes in sialyltransferase expression and ganglioside biosynthesis were not the result of altered flux through the sialic acid pathway. Interestingly, exogenous GM3 and GD3 also changed the expression of GNE and led to reduced ST3Gal5 and ST8Sia1 mRNA levels, demonstrating a reciprocating feedback mechanism where gangliosides regulate upstream biosynthetic enzymes. Cellular responses to the GNE-mediated changes in ST3Gal5 and ST8Sia1 expression and GM3 and GD3 levels were investigated next. Conditions that led to reduced ganglioside production (e.g. short hairpin RNA exposure) stimulated proliferation, whereas conditions that resulted in increased ganglioside levels (e.g. recombinant GNE and exogenous gangliosides) led to reduced proliferation with a concomitant increase in apoptosis. Finally, changes to BiP expression and ERK1/2 phosphorylation consistent with apoptosis and proliferation, respectively, were observed. These results provide examples of specific biochemical pathways, other than sialic acid metabolism, that are influenced by GNE.

On the basis of the conservation of neuraminidase (N) active-site residues in influenza virus N and paramyxovirus hemagglutinin-neuraminidase (HN), it has been suggested that the three-dimensional (3D) structures of the globular heads of the two proteins are broadly similar. In this study, details of this structural similarity are worked out. Detailed multiple sequence alignment of paramyxovirus HN proteins and influenza virus N proteins was based on the schematic representation of the previously proposed structural similarity. This multiple sequence alignment of paramyxovirus HN proteins was used as an intermediate to align the morbillivirus hemagglutinin (H) proteins with neuraminidase. Hypothetical 3D structures were built for paramyxovirus HN and morbillivirus H, based on homology modelling. The locations of insertions and deletions, glycosylation sites, active-site residues, and disulfide bridges agree with the proposed 3D structure of HN and H of the Paramyxoviridae. Moreover, details of the modelled H protein predict previously undescribed enzymatic activity. This prediction was confirmed for rinderpest virus and peste des petits ruminants virus. The enzymatic activity was highly substrate specific, because sialic acid was released only from crude mucins isolated from bovine submaxillary glands. The enzymatic activity may indicate a general infection mechanism for respiratory viruses, and the active site may prove to be a new target for antiviral compounds.

The infectivity of trachoma-inclusion conjunctivitis (TRIC) organisms (TW-5) was enhanced by pretreatment of HeLa cell monolayers before inoculation with diethylaminoethyl (DEAE)-dextran (30 mug/ml) and poly-l-lysine (10 mug/ml) and inhibited by dextran sulphate (250 mug/ml), fetuin (4%), ovomucoid (5%), N-acetyl neuraminic acid (0.5%), and Cholera vibrio neuraminidase (100 U/ml). The infectivity of lymphogranuloma venereum organisms (434) was not affected by DEAE-dextran, fetuin, and neuraminidase, was slightly inhibited by poly-l-lysine, and was inhibited by dextran-sulphate, ovomucoid, and N-acetyl neuraminic acid. The study suggested that sialic acid residues on the cell surface may be specific receptors for TRIC organisms. The receptors for TRIC organisms (TW-5 and TW-3) could be specifically blocked with inactivated (56 C for 30 min) TRIC organisms at the ratio of one live to 100 inactivated TRIC organisms, but not by inactivated lymphogranuloma venereum (434) or influenza virus (A(2)/Jap 305).

Population studies have established that one of the common isoforms of apolipoprotein E, the apoE4, is associated with higher incidence and earlier age of onset of late onset familial Alzheimer's disease (AD), whereas apoE2 may have the opposite effect. The apoE3 and apoE4 isoforms were shown to display different binding reactivities with amyloid beta peptide (Abeta) and tau protein in vitro. On the basis of these findings, it has been proposed that the apoE isoforms may modulate positively or negatively the formation of either the neurofibrillary tangles or the amyloid deposits in the brain of patients with AD. To study the interaction of Abeta with nascent apoE isoforms we have expressed their cDNAs in baby hamster kidney (BHK-21) cells using the Semliki Forest Virus expression system. Analysis of the secreted apoE by one- and two-dimensional gel electrophoresis and immunoblotting showed that the nascent apoE is heavily modified with carbohydrate chains containing sialic acid. A dimeric form of apoE is formed with apoE2 and apoE3 but not with apoE4 isoforms. Analysis of the interaction of nascent apoE2, apoE3, and apoE4 produced by BHK-21 cells with Abeta (1-40) under physiological conditions (pH 7.4, 37 degrees C) showed that the efficiency of the apoE monomer-Abeta complex formation follows the order apoE2>> apoE3>>) apoE4. In addition, the apoE2 dimer formed a complex with Abeta more efficiently than the apoE3 dimer. The isoform-specific differences in binding were temperature-dependent and are attenuated upon decrease of the temperature. The binding behavior of the monomeric apoE is different from that reported for plasma apoE3 and apoE4 or commercially available apoE3 and apoE4 preparations and similar to that described for apoE3 and apoE4 produced by human embryonic kidney (HEK-293) cells. It appears that the efficiency of binding between each of three main apoE isoforms and Abeta correlates inversely with the risk of developing late-onset familial AD and may indicate possible involvement of apoE in the binding and clearance of Abeta in vivo.

The x-ray structure of a complex of sialic acid (Neu5Ac) with neuraminidase N9 subtype from A/tern/Australia/G70C/75 influenza virus at 4 degrees C has revealed the location of a second Neu5Ac binding site on the surface of the enzyme. At 18 degrees C, only the enzyme active site contains bound Neu5Ac. Neu5Ac binds in the second site in the chair conformation in a similar way to which it binds to hemagglutinin. The residues that interact with Neu5Ac at this second site are mostly conserved in avian strains, but not in human and swine strains, indicating that it has some as-yet-unknown biological function in birds.

In the alternative pathway of complement (APC) factor H is the primary control factor involved in discrimination between potential pathogens. The APC deposits C3b on possible Ags, and the interaction with factor H determines whether the initial C3b activates the APC. Factor H is composed of a linear array of 20 homologous short consensus repeats (SCR) domains with many functional sites. Three of these sites are involved in binding C3b and regulating complement activation; others bind to sialic acid and/or heparin and are responsible for host recognition. Using site-directed mutations we have examined the contributions of each of these sites to target discrimination and to functional activities of factor H. Decay acceleration by SCR1-4 of C3/C5 convertases bound to nonactivators was strongly dependent on SCR domains 11-15 and 16-20. Loss of these regions caused a 97% loss of activity, with SCR16-20 being the most critical (>90% loss). On APC activators the pattern of site usage was different and unique on each. On yeast, deletion of the 10 C-terminal domains (SCR11-20) had no effect on specific activity. On rabbit erythrocytes, this deletion caused loss of 75% of the specific activity. An examination of binding affinity to C3b on the four cell types demonstrated that factor H exhibits a unique pattern of SCR involvement on each cell. The results reveal a complex molecular mechanism of discrimination between microbes and host in this ancient innate defense system and help explain the different rates and intensities of APC activation on different biological particles.

The highly glycosylated domains of rat small intestinal mucins were isolated after reduction and trypsin digestion and separated into two populations (A and B) by gel chromatography. The molecular mass values were 650 and 335 kDa, respectively, and the relative yields suggest that the two glycopeptides occur in equimolar proportions. Electron microscopy revealed linear structures with weight average lengths of 230 nm (A) and 110 nm (B) corresponding to a mass/unit length of about 3 kDa/nm. The protein cores (17-19%) contain large amounts of threonine (over 40%), serine (17-24%), and proline (18-19%). Carbohydrate and sulfate account for approximately 80 and 0.5%, respectively, and gas chromatography-mass spectrometry showed that the patterns of neutral and sialic acid-containing glycans are very similar in the two glycopeptides. Both contain a significant amount (7-10 mol %) of single GalNAc residues, the average oligosaccharide is about 4 sugar residues long, and the largest species observed are heptasaccharides. The major neutral and sialic acid-containing oligosaccharides are Fuc1-2Gal1-3GalNAcol and GlcNAc1-6(NeuGc2-Gal1-3)GalNAcol, respectively. Sialic acid is present as both N-acetyl- and N-glycoloyl-neuraminic acid. Repeated extractions of the tissue with guanidinium chloride left approximately 80% of the mucus glycoproteins as an insoluble glycoprotein complex whereas exposure to dithiothreitol or high speed homogenization accomplished complete solubilization. The "subunits" obtained after reduction with dithiothreitol are larger than glycopeptides A and B, and fragments corresponding in size to the latter are obtained after cleavage with trypsin. Most of the mucins from rat small intestine thus occurs as an insoluble glycoprotein complex composed of subunits joined with disulfide bonds. The subunits contain two highly glycosylated regions with different lengths substituted with very similar oligosaccharides.

E- and P-selectin are inducible cell adhesion molecules on endothelial cells, which function as Ca(2+)-dependent lectins and mediate the binding of neutrophils and monocytes. We have recently identified a 150-kD glycoprotein ligand for E-selectin on mouse myeloid cells, using a recombinant antibody-like form of mouse E-selectin. Here, we report that this ligand does not bind to an analogous P-selectin fusion protein. Instead, the chimeric P-selectin-IgG protein recognizes a 160-kD glycoprotein on the mouse neutrophil progenitor 32D cl 3, on mature mouse neutrophils and on human HL60 cells. The binding is Ca(2+)-dependent and requires the presence of sialic acid on the ligand. This P-selectin-ligand is not recognized by E-selectin. Removal of N-linked carbohydrate side chains from the 150-kD and the 160-kD monospecific selectin ligands abolishes the binding of both ligands to the respective selectin. Treatment of HL60 cells with Peptide: N-glycosidase F inhibited cell binding to P- and E-selectin. In addition, glycoproteins of 230 and 130 kD were found on mature mouse neutrophils, which bound both to E- and P-selectin in a Ca(2+)-dependent fashion. The signals detected for these ligands were 15-20-fold weaker than those for the monospecific ligands. Both proteins were heavily sialylated and selectin-binding was blocked by removal of sialic acid, but not by removal of N-linked carbohydrates. Our data reveal that E- and P-selectin recognize two categories of glycoprotein ligands: one type requires N-linked carbohydrates for binding and is monospecific for each of the two selectins and the other type binds independent of N-linked carbohydrates and is common for both endothelial selectins.

A monoclonal antibody (mAb 10A8), derived from mice immunized with fractions of the Golgi apparatus from rat brain neurons, was exploited to isolate and partially characterize a novel glycoprotein of 160 kDa apparent molecular mass which was localized by immunoelectron microscopy in medial cisternae of the Golgi apparatus of neurons, glia, pituitary cells, and rat pheochromocytoma (PC 12). The yield of immunoaffinity purified protein was 0.9 microgram/g of rat brain and represented 3% of the Golgi protein; the protein contained asparagine-linked carbohydrates and sialic acid and N-acetylglucosamine residues; unreduced protein had a greater electrophoretic mobility (130 kDa) consistent with the presence of intrachain disulfide bonds. The bulk of the glycoprotein resided within the membrane and/or luminal face of the Golgi cisternae. After extraction with Triton X-114, the glycoprotein was found in both aqueous and detergent phases. The monoclonal antibody did not inhibit the activities of Golgi enzymes or the uptake of nucleotide sugars by intact Golgi vesicles. The findings indicate that the 160-kDa glycoprotein is a specific constituent of medial Golgi cisternae. The results of this study lend support to the hypothesis that the distributions of glycosyltransferases in the Golgi apparatus are cell specific, since in neurons this sialic acid containing glycoprotein is found in medial rather than in trans and/or in the trans Golgi reticulum cisternae, where sialyltransferases have been localized in other cells. Alternatively, resident neuronal Golgi sialoglycoproteins may acquire sialic acid in trans elements of the apparatus and then shuttle back in medial cisternae.