Polysaccharides of Crithidia fasciculata: Identification and partial characterization of a cell surface constituent

A carbohydrate-containing fraction was extracted from the trypanosomatid Crithidia fasciculata by a phenol-water procedure. Ion-exchange chromatography separated this fraction into three components: a polysaccharide which was not retained on the column; RNA which eluted upon addition of salt; and, another polysaccharide which eluted upon addition of detergent. The unretained fraction was shown to be composed solely of d-mannose. The mannan, which was heterodisperse on Sephadex G-100, had an average molecular weight of approx. 14 000 as based on analysis of reducing groups. The detergent-eluted material yielded arabinose and galactose upon acid hydrolysis. The arabinogalactan was excluded from Sephadex G-100 and Sephacryl S-200 molecular sieve columns, suggesting a molecular weight ≥ 200 000. Cell fractionation studies showed the bulk of extractable polysaccharide was associated with a particulate fraction. Further determination of the cellular localization of the polysaccharide was accomplished by employing a specific antiserum prepared from rabbits immunized with the polysaccharide extract. The cell surface localization of the arabinogalactan was demonstrated by cell agglutination studies as well as immunocytochemical techniques using fluorescein and ferritin conjugated antibodies.     

Identification and partial characterization of a pheromone-induced adhesive surface antigen of Streptococcus faecalis

A surface protein antigen that is produced only during the induction of aggregation of Streptococcus faecalis was shown to contribute to and perhaps be primarily responsible for aggregation. The antigen is an immunodominant surface component of induced cells. F(ab) fragments of immunoglobulins specific for this antigen prevented aggregation, providing direct evidence that the antigen is an adhesin. Consistent with this proposed association was the coincident timing of appearance of the antigen and the timing of aggregation after induction.     

Detection and partial characterization of lymphoid cell surface proteases

Cultures of viable thymocytes and lymph node cells (LNC) were found to exhibit neutral protease activity toward radiolabeled protein substrates. Proteases were not actively secreted in serum-free culture. Thymocyte surface proteases were not affected by incubation of the cells in 1 mM ethylenediaminetetraacetic acid (EDTA) or 1 mM ethylene glycol bis(aminoethyl ether) N, N'-tetraacetic acid (EGTA); however, approximately 25% of lymph node cell surface protease activity was released from the cells by EDTA. It was concluded that the majority of protease activity displayed by both cell types was tightly associated with the cell surface. The inhibitor sensitivity of the cell surface proteases detected on hamster thymocytes and LNC and rat thymocytes was very similar. Cell surface protease activity was inhibited (85%) by the serine protease inhibitors diisopropylfluorophosphate (DFP) and phenylmethylsulfonylfluoride (PMSF) and was partially inhibited by l-1-tosylamide-2-phenylethylchloromethyl ketone(TPCK) and soybean trypsin inhibitor (SBTI), but not by N-α-p-tosyl-l-lysine-chloromethyl ketone (TLCK) or ?-aminocaproic acid (EACA). The bacterial protease inhibitor antipain was strongly inhibitory whereas leupeptin was less effective and elastinal did not inhibit cell surface protease activity. Thymocyte surface proteases were also inhibited (65%) by ZnCl₂, but not be several other divalent cations. In LNC, both ZnCl₂ and NiCl₂ were inhibitory to a lesser extent (32% inhibition). At least one surface protease in both thymocytes and LNC could function as a plasminogen activator.     

Identification and characterization of the endothelial cell surface lipoprotein lipase receptor.

The hydrolysis of triglycerides in plasma lipoproteins is mediated by lipoprotein lipase (LPL) that is bound to vascular endothelial cells. The specific endothelial cell surface protein(s) with which LPL associates has not been characterized. To identify this LPL binding protein(s), radioiodinated cell surface proteins from cultured bovine aortic endothelial cells were chromatographed using bovine LPL-Sepharose. A single radioiodinated protein of apparent molecular mass 220 kDa was specifically retained by the gel and eluted with 0.4 M NaCl. A LPL-binding protein of similar size was obtained after metabolic labeling of the cellular proteoglycans with 35SO4, indicating that the 220-kDa protein is a proteoglycan. After heparitinase or nitrous acid treatments the molecular mass of the LPL-binding protein decreased to approximately 50 kDa, suggesting that it contains heparin sulfate chains. A 220-kDa protein from the basal cell surface was also identified using LPL-Sepharose chromatography. 125I-LPL was cross-linked to the endothelial cell surface using ethylene glycobis (succinimidylsuccinate). A single ligand-receptor complex, approximately 350 kDa, was obtained. Heparin and unlabeled LPL decreased the cross-linking of radioiodinated LPL to the cell surface receptor. To examine whether the receptor mediates the internalization of cross-linked 125I-LPL, cells containing 125I-LPL complexed to the surface were incubated at either 37 or at 4 degrees C. The amount of 125I-LPL internalized by the cells was 74% greater at 37 degrees C than at 4 degrees C. This suggested that LPL cross-linked to the receptor was internalized in a temperature-dependent manner. Thus, a 220-kDa heparan sulfate proteoglycan functions as an endothelial cell surface receptor for LPL.     

Assay and partial characterization of the solubilized cell surface receptor for immunoglobulin E.

The cell surface component (receptor) which specifically binds immunoglobulin E (IgE) presumably forms an integral part of the functional chain involved in the antigen-induced IgE-mediated degranulation of histamine-containing mast cells and basophils. This paper describes a simple (NH4)2SO4 predipitation assay with which the interaction of IgE with detergent-solubilized receptors can be reproducibly quantitated. Receptor saturation was demonstrated and a linear response to receptor concentration over at least a 30-fold rang obtained. By means of the assay it was shown that (a) all assayable receptors of rat basophil leukemia cells are cell surface expressed; (b) receptor specificity remains intact during solubilization; (c) the binding constants of the solubilized IgE receptors are similar to those determined on intact cells. Utilizing agarose gel filtration, preliminary estimates of the molecular weight of the active free solubilized receptor and of its complex with IgE suggest that the receptor is univalent.     

O2''-Methylinosine, a constituent of the ribosomal RNA of Crithidia fasciculata.

A novel nucleoside, O2'-methylinosine (Im), has been identified as a constituent of the ribosomal RNA of Crithidia fasciculata, a hemoflaggelate protozoan. The nucleoside is released as part of an alkali-stable dinucleotide, Im-Up, by alkaline hydrolysis of Crithidia rRNA, and as a 5'-nucleotide, pIm, by snake venom hydrolysis of the same RNA. The Im-containing derivatives isolated from Crithidia rRNA were characterized by comparison with marker compounds prepared by chemical deamination of the corresponding adenosine analogues. O2'-Methylinosine prepared from either natural Im-Up or natural pIm had the same ultraviolet absorption spectra and chromatographic properties as marker Im. Characterization of the base and sugar components of Im as hypoxanthine and 2-O-methylribose, respectively, provided final confimration of structure. Control experiments have eliminated the possibility that Im arises from O2'-methyladenosine (Am), a known constituent of ribosomal RNA, by chemical or enzymatic deamination during hydrolysis of Crithidia rRNA.     

Identification of a macrophage-specific cell surface antigen.

A mouse-specific macrophage antigen (MSMA) was identified in NP-40 extracts of 125I-radiolabeled mouse preitoneal macrophages by using a rabbit anti-mouse macrophage serum (AMS) and SDS-polyacrylamide gel electrophoresis. The antigen was shown to have a m.w. of 83,000 daltons and was present on both normal and "activated" peritoneal macrophages. MSMA was also present on syngeneic adherent spleen cells, allogeneic peritoneal macrophages, a mouse macrophage cell line (P388D1), and exhibited some cross-reactivity with peritoneal macrophages from closely related species (rats and hamsters). MSMA was not present on nonadherent peritoneal exudate cells, spleen cells, erythrocytes, thymocytes, or bone marrow cells. Extensive absorptions of AMS with thymocytes and erytrocytes from mice were necessary to remove other antibodies that reacted with other mouse membrane antigens. An antiserum directed against a specific membrane antigen has great potential in elucidating structure-function relationships with regard to a number of macrophage activities.     

Identification and characterization of the murine cell surface receptor for the urokinase-type plasminogen activator.

Cell-binding experiments have indicated that murine cells on their surface have specific binding sites for mouse urokinase-type plasminogen activator (u-PA). In contrast to the human system, chemical cross-linking studies with an iodinated ligand did not yield any covalent adducts in the murine system, but in ligand-blotting analysis, two mouse u-PA-binding proteins could be visualized. To confirm that these proteins are the murine counterpart of the human u-PA receptor (u-PAR), a peptide was derived from the murine cDNA clone assigned to represent the murine u-PAR due to cross-hybridization and pronounced sequence similarity with human u-PAR cDNA [Kristensen, P., Eriksen, J., Blasi, F. & Dan?, K. (1991) J. Cell Biol. 115, 1763-1771]. A rabbit antiserum raised against this peptide specifically recognized two polypeptide bands with electrophoretic mobilities identical to those identified by ligand-blotting analysis. Binding of mouse u-PA to its receptor showed species specificity in ligand-blotting analysis, since mouse u-PA did not bind to human u-PAR and human u-PA did not bind to mouse u-PAR. The apparent M(r) of mouse u-PAR varied between different mouse cell lines and ranged over M(r) 45,000-60,000. In four of the cell lines, mouse u-PA bound to two mouse u-PAR variant proteins, whereas in the other two cell lines studied, there was only one mouse u-PA-binding protein. In the monocyte macrophage cell line P388D.1, trypsin-treatment of intact cells could remove only the large mouse u-PAR variant (M(r) 60,000) indicating that only this type was a cell-surface-exposed molecule. The smaller mouse u-PAR variant (M(r) 45,000), was deglycosylated by the enzyme endo-beta-N-acetylglucosaminidase H and is probably an intracellular precursor form carrying only high-mannose carbohydrate. Deglycosylation of this variant yielded a polypeptide with an apparent M(r) of about 30,000, which corresponds to the Mr calculated from the cDNA derived protein sequence of mouse u-PAR. Receptor-bound mouse u-PA could be released by phosphatidylinositol-specific phospholipase C treatment, indicating that mouse u-PAR is attached to the cell surface by glycosylphosphatidylinositol. Purification of the two mouse u-PAR variant proteins by diisopropylfluorophosphate-inactivated mouse u-PA-Sepharose affinity chromatography yielded two silver-stained bands when analysed by SDS/PAGE, corresponding in electrophoretic mobility to those seen by ligand-blotting analysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Detection and partial characterization of a human mast cell carboxypeptidase

Using a high performance liquid chromatography assay that detects the cleavage of the C-terminal leucine from angiotensin I, we have identified a carboxypeptidase activity in mast cells from human lung and in dispersed mast cell preparations from human skin. The enzyme activity was detected in a preparation of dispersed human mast cells from lung of greater than 99% purity and was released with histamine after stimulation with goat anti-human IgE. In nine preparations of dispersed human mast cells from lung of 10 to 99% purity, net percentage of release of carboxypeptidase correlated with the release of histamine, localizing carboxypeptidase to mast cell secretory granules. The enzyme activity was also detected in preparations of dispersed human mast cells from skin and in extracts of whole skin. The inhibitor profile and m.w. of carboxypeptidase activity from preparations of dispersed mast cells from skin was similar to that from dispersed mast cells from lung. Mast cell carboxypeptidase had a m.w. on gel filtration of 30,000 to 35,000. The enzyme in crude lysates of dispersed mast cell preparations had optimal activity between pH 8.5 and 9.5 and was inhibited by potato inhibitor, which distinguished it from carboxypeptidase in cultured human foreskin keratinocytes and adult fibroblasts, and from other proteolytic mast cell enzymes. The enzyme activity was also inhibited by EDTA, o-phenanthroline, and, to a small extent, by 8-OH quinoline, but not by Captopril, soybean trypsin inhibitor, or pepstatin. These findings demonstrate that human mast cell secretory granules contain carboxypeptidase in addition to tryptase and chymase. It appears that mast cells from skin may have a higher content of carboxypeptidase than do mast cells from lung.     

Identification, immunoaffinity purification and partial characterization of a human decidua-associated protein

A crude extract of pooled early-pregnancy decidual tissue was enriched for soluble decidual proteins by exhaustive affinity absorption with antibodies to human serum proteins immobilized on Eupergit C. The partly purified extract was used to prepare monoclonal antibodies. A monoclonal antibody was obtained recognizing an antigen present in extract of decidual tissue and not in extract of proliferative endometrium. The monoclonal antibody was used for immunoaffinity purification of the decidua-associated protein. By SDS-PAGE analysis, under reducing conditions it yielded 2 bands at apparent molecular weights of 55,000 and 25,000. Under non-reducing conditions a single protein band at apparent molecular weight of 200,000 was observed. The Mr 200,000 protein was named hDP200 and the Mr 55,000 protein was named hDP55. It is suggested that hDP55 is a subunit of the hDP200. The hDP200 did not react with polyclonal antibodies specific for PP12 and PP14. PP14 has been shown to be immunologically indistinguishable from PEP and alpha 2-PEG. Our data therefore suggest that hDP200 is a novel human decidua-associated protein.     

Identification of a cell surface glycoprotein involved in cell aggregation in D. discoideum.

Analysis of the composition of cell surface-associated glycoproteins of D. discoideum by lactoper-oxidase-catalyzed radioiodination, followed by isolation by Con A-Sepharose chromatography, revealed that the developmentally regulated cell surface expression of a certain glycoprotein (gp150) parallels the onset of mutual cellular cohesiveness (Geltosky, Siu and Lerner, 1976). We have purified gp150 and raised specific antibodies to it. Through utilization of the specific antibody and a fluorescence-activated cell sorter, the expression of gp150 on the cell surface has been studied. Starting from a low level in noncohesive (vegetative) cells, there is a rapid accumulation of gp150 on the surfaces of aggregating cells. A peak level of expression is achieved by 10 hr and maintained at least until the steps of terminal differentiation. Most significantly, monovalent Fa'b derived from anti-gp150, when added to aggregation-competent cells, blocks the cells' ability to reaggregate. Fab's derived from antisera with different specificities were ineffective inhibitors of cell aggregation. These results suggest that gp150 serves an intimate role in cell adhesion.     

Identification and partial characterization of two eukaryotic UDP-galactopyranose mutases

Galactofuranose metabolism is valued as an important target for the development of new antituberculosis drugs. UDP-galactopyranose mutase, a central enzyme in galactofuranose biosynthesis, is essential for the growth and viability of mycobacteria. This enzyme catalyzes the conversion of UDP-galactopyranose into UDP-galactofuranose, the donor used by various galacto-furanosyltransferases. While D-galactofuranose residues are often found in important surface glycoconjugates of pathogenic bacteria, fungi and protozoan parasites, they are absent in the mammalian host, and thus their biosynthesis is an attractive target for the development of novel therapeutic strategies. In contrast to mycobacteria, the importance of galactofuranose for eukaryotic pathogens has not been ascertained because the enzymes involved in galactofuranose metabolism are unknown. Here, we report the identification and characterization of the first eukaryotic UDP-galactopyranose mutases. The genes encoding the enzymes were cloned from two different human pathogens: the parasite Leishmania major and the opportunistic fungus Aspergillus fumigatus. The newly identified eukaryotic enzymes exhibit 51% sequence identity, but are less than 20% identical to the prokaryotic counterparts. The sequence identity between pro- and eukaryotic enzymes is concentrated at amino acid residues that are involved in substrate and cofactor binding. Therefore, an inhibitor of UDP-galactopyranose mutase might be effective against a wide range of pathogenic organisms.     

Identification and characterization of a leptin-responsive neuroblastoma cell line

The adipocyte-derived hormone leptin plays a critical role in a variety of physiological and pathological actions. As such the determination of leptin signal transduction pathways are important both for understanding the molecular mechanisms of leptin action and for identifying sites for possible therapeutic intervention. Since the hypothalamus is the primary site of leptin action, we sought to identify a neuronal-derived human cell line containing the long form of the leptin receptor (OBRb). To this end, we screened several neuroblastoma cell lines and isolated a sub-line of SH-SY5Y cells, which we designated as SH-OBRb, for further studies. We characterized the transduction pathways induced by leptin in SH-OBRb cells and demonstrated that OBRb mediates tyrosine phosphorylation of STAT3, phosphorylation of ERK1/2, but not SAPK/JNK and p38 MAPK, in a dose and time dependent fashion. In addition, Akt appears to be phosphorylated in the basal state and to be insensitive to further activation by leptin. In summary, we have isolated a unique cell line that can be utilized as a model for use in the study of leptin action and molecular mechanisms.     

Identification and partial characterization of proteins and proteoglycans encrusting the secondary cell walls of flax fibres

 Four proteins were isolated from depectinised elementary fibres of flax (Linum usitatissimum L.), using either alkali or cellulase digestion treatments. All the four proteins were characterized by a deficiency or low contents of hydroxyproline and by high levels of glutamic acid/glutamine and/or aspartic acid/asparagine. The two proteoglycans solubilized with cellulase strongly reacted with β-glucosyl Yariv reagent but not with α-glucosyl Yariv reagent and contained appreciable amounts of alanine, glycine, serine and threonine, suggesting a relationship with cell wall hydroxyproline-deficient arabinogalactan-proteins. The two alkali-extracted proteins did not show any reaction with β-glucosyl Yariv dye. Due to the harsh treatment, they might only partially represent the original proteins. Due to its high level of glycine (41%), one of these proteins might be classified as a glycine-rich protein. The latter polypeptide, of low molecular molar mass, contained 14.6% leucine and might consist of a domain related to leucine-rich proteins. The data show that these proteins and arabinogalactan-protein-like proteoglycans were strongly associated with the secondary walls of flax fibres. Their presence in small amounts (0.1–0.4%), raises the problem of their putative structural role. Received: 22 October 1999 / Accepted: 17 January 2000     

Caenorhabditis briggsae and C. elegans: partial characterization of cuticle surface carbohydrates.

The presence of galactose, glucose, mannose, and N-acetylglucosamine on the exposed surface of the nematodes Caenorhabditis briggsae and C. elegans was indicated by specific binding of three iodinated plant lectins. Proteolysis experiments suggested the absence of digestible glycoproteins on the exposed surfaces of the two nematode species. High resolution micrographs of cuticle surface preparations labeled with cationized ferritin indicated that the negative charge-bearing molecules are more densely packed on the nematode surface than on animal plasma membranes.     

Identification and partial characterization of a retinal pigment epithelial membrane receptor for plasma retinol-binding protein.

We have developed a membrane binding assay by which we have been able to characterize the interaction between 125I-labeled retinol-binding protein and its receptor in microsome fractions derived from retinal pigment epithelial cells. The binding of retinol-binding protein to the membranes was fast, with a dissociation constant in the range of 31-72 nM, and maximum binding occurred at neutral pH. Receptor binding sites were also found in microsome fractions of liver and kidney, whereas lung and muscle contained few, if any. Chemical cross-linking of retinol-binding protein to the microsomal membranes yielded a major molecular complex of Mr 86,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein responsible for binding of retinol-binding protein was identified as a Mr 63,000 protein using a label transfer cross-linking technique. Further characterization demonstrated that the receptor for retinol-binding protein is a terminally glycosylated membrane protein noncovalently associated with a high molecular weight complex.     

Leishmania donovani: purification and partial characterization of a glycophosphosphingolipid antigen expressed on promastigote surface.

A ceramide-anchored glycophosphosphingolipid antigen was isolated from the lipid extract of Leishmania donovani promastigotes. The affinity-purified glycolipid antigen contained galactose, mannose, myo-inositol, phosphate, ceramide, and hexosamine but no sialic acid. The phosphate group was present internally at the core of the structure: inositol (1-O)-phosphorylceramide. The phosphate group became susceptible to alkaline phosphatase only after alkali-catalyzed hydrolysis of the glycolipid.     

Identification of a cell surface receptor common to germ and somatic cells.

The plasma membrane forms of guanylyl cyclase constitute a diverse family of cell surface receptors. An mRNA for the enzyme/receptor was first cloned from sea urchin testis after cross-linking studies suggested that guanylyl cyclase was a sperm receptor for egg peptides. The enzyme/receptor was shown to contain a single putative transmembrane domain, a large extracellular region that presumably binds peptide ligands, and an intracellular region that contains a protein kinase-like and a cyclase catalytic domain. The sea urchin cDNA was then used to isolate positive-hybridizing clones from mammalian tissues. At least two forms recognize natriuretic peptides and one form recognizes the heat-stable enterotoxins. In the case of the enterotoxin receptor, it remains to be shown whether or not an endogenous ligand exists that regulates enzyme activity. The discovery of this cell surface receptor family presents a new paradigm for second messenger signalling in that a low-molecular weight second messenger (cyclic GMP) is produced by the same protein that binds the extracellular ligand.