Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The kinetics of attachment and ingestion of Chlamydia trachomatis serotype L1 by monolayers of McCoy cells were studied by using a method that discriminated between attachment and uptake. When about 1% of the McCoy cells was infected, the proteinase K-resistant chlamydial fraction, regarded as ingested chlamydiae, reached a constant value after about 3 h of incubation at 37 degrees C. Uptake of chlamydiae at 4 degrees C could not be demonstrated. The attached and ingested chlamydial fractions were constant over an eightfold increase in chlamydial inoculum. Chitobiose and chitotriose, the di- and trisaccharides of N-acetyl-D-glucosamine, reduced the association of C. trachomatis serotype L1 with McCoy cells. Higher concentrations of chitobiose also selectively inhibited ingestion of chlamydiae. A corresponding effect of chitobiose was also observed on the number of chlamydial inclusions. Wheat germ agglutinin, specific for N-acetyl-D-glucosamine residues, reduced the association of chlamydiae when incubated at 4 degrees C, but not at 37 degrees C. A small inhibiting effect of concanavalin A on association of chlamydiae, but no effect of the corresponding carbohydrates, indicates a nonspecific effect on chlamydial attachment of this lectin. These results suggest that beta 1 leads to 4-linked oligomers of N-acetyl-D-glucosamine are important in the specificity of attachment of C. trachomatis to McCoy cells.
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PMID:Attachment and internalization of a Chlamydia trachomatis lymphogranuloma venereum strain by McCoy cells: kinetics of infectivity and effect of lectins and carbohydrates. 664 70

A 15,000-dalton protein was purified from HeLa cells infected with adenovirus type 2. Proteins solubilized from a membrane fraction of lytically infected cells was used as the starting material for purification. Subsequent purification steps involved lentil-lectin, phosphocellulose, hydroxyapatite, DEAE-cellulose, and aminohexyl-Sepharose chromatographies. A monospecific antiserum, raised against the purified protein, immunoprecipitated a 15,000-dalton protein encoded in early-region E1B (E1B/15K protein) of the adenovirus type 2 DNA. Tryptic finger print analysis revealed that the purified protein was identical to the E1B/15K protein encoded in the transforming part of the viral genome. The antiserum immunoprecipitated the E1B/15K protein from a variety of viral transformed cell lines isolated from humans, rats, or hamsters. The E1B/15K protein was associated with the membrane fraction of both lytically and virus-transformed cell lines and could only be released by detergent treatment. Furthermore, a 11,000- to 12,000-dalton protein that could be precipitated with the anti-E1B/15K serum was recovered from membranes treated with trypsin or proteinase K, suggesting that a major part of the E1B/15K protein is protected in membrane vesicles. Translation of early viral mRNA in a cell-free system, supplemented with rough microsomes, showed that this protein was associated with the membrane fraction also in vitro.
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PMID:Purification of a native membrane-associated adenovirus tumor antigen. 709 63

All methods described in the literature that allow quantitative measurements of protein expression at the cell surface are applicable to subsets of surface-exposed proteins only. We developed a new method, involving 3,3'-diaminobenzidine (DAB) cytochemistry, which allowed determination of cell-surface expression of all plasma membrane proteins measured, in at least three different cell lines. Adherent cells were first brought into suspension by proteinase K and EDTA treatment at 0 degrees C removing many, but not all, surface-exposed proteins. Subsequently, horseradish peroxidase (HRP) was linked by means of its glycosyl residues to specific cell-surface-exposed sugar moieties using the multivalent lectin concanavalin A (ConA). The suspended cells were encapsulated by polymerized DAB, a process that was catalysed by plasma membrane-bound HRP. After cell lysis, and removal of nuclei and most of the DAB polymer by centrifugation, proteins were analysed by SDS-PAGE. Surface proteins encapsulated by non-pelleted DAB polymer were retained on top of the stacking gel. After 125I-labelling the cell surface, protease-resistant 125I-labelled proteins could be quantitatively coupled to DAB polymer. This process was completely dependent on the presence of ConA, HRP, DAB and H2O2. Surface 125I-labelled beta-Na+,K(+)-ATPase was resistant to proteinase K but could be completely removed using DAB cytochemistry. Intracellular ConA binding proteins were not affected. Other intracellular proteins, including endosomal asialoglycoprotein receptor and cation-independent mannose 6-phosphate/insulin-like growth factor II receptor were also not affected.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A novel method for measuring protein expression at the cell surface. 812 1

Helicobacter pylori is a microaerophilic bacterium found in the stomach of asymptomatic humans as well as patients with acid peptic disease and gastric adenocarcinoma. We have developed an in situ adherence assay to examine the cell lineage-specific nature of binding of this organism and to characterize the nature of cell surface receptors that recognize its adhesin. Fluorescein isothiocyanate-labeled H. pylori strains were bound to surface mucous cells present in the pit region of human and rat gastric units but not to mucous neck, parietal, or chief cell lineages present in the glandular domains of these units. Binding was abolished by proteinase K treatment of tissue sections and by pretreatment of the bacteria with bovine submaxillary gland mucin, a rich source of fucosylated and sialylated carbohydrates. Several lines of evidence suggest that binding to surface mucous cells is not dependent upon terminal nonsubstituted alpha 2,3- and alpha 2,6-linked sialic acids in the adhesin receptor: (i) binding was not inhibited by incubating H. pylori strains with sialylated glycoconjugates such as fetuin and free sialyllactose; (ii) immunohistochemical stainings using the sialic acid-specific Sambucus nigra and Maackia amurensis lectins and the cholera toxin B subunit did not detect any sialylated glycoconjugates in these epithelial cells; and (iii) binding was not sensitive to metaperiodate under conditions that selectively cleaved carbons 8 and 9 of terminal nonmodified sialic acids. A role for fucosylated epitopes in the glycoprotein(s) that mediate binding of H. pylori to surface mucous cells was suggested by the facts that this lineage coexpresses the adhesin receptor and major fucosylated histo-blood group antigens, that monoclonal antibodies specific for histo-blood group antigens H, B, and Leb block binding, and that the lectin Ulex europaeus type 1 agglutinin, which is specific for alpha-L-fucose, also bound to the same cells that bound the bacteria. Furthermore, human colostrum secretory IgA inhibited adhesion in a metaperiodate- and alpha-L-fucosidase-sensitive but neuraminidase-independent fashion. The in situ adherence assay should be useful in further characterizing the H. pylori adhesin and its receptor and for identifying therapeutically useful compounds that inhibit strain-specific and cell lineage-specific binding of this human pathogen.
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PMID:An in vitro adherence assay reveals that Helicobacter pylori exhibits cell lineage-specific tropism in the human gastric epithelium. 838 33

A macromolecule with a molecular weight of 90-100 kDa was purified from normal human pregnancy urine. The molecule was proved to be the Tamm-Horsfall glycoprotein (THG) by Western blot analysis. The macromolecule contains carbohydrate as detected by an enzyme immunoassay. Functionally, the glycoprotein can adhere to and stimulate the thymidine incorporation of human mononuclear cells (MNC) in modest degree via its membranotropic property. In addition to MNC, the protein can also bind to the surface of human polymorphonuclear neutrophils (PMN), red blood cells (RBC) and rat glomerular mesangial cells (RMC). Western blot analysis of various cell lysates with/without proteinase K pretreatment before cell lysis revealed that a 60 kDa and a molecule larger that 94 kDa on the surface of PMN, a 60 kDa protein on MNC and a 32 kDa protein on RBC are the binding molecules for THG. In contrast, many proteins on the surface of RMC could be bound by THG. Immunoprecipitation of membranous iodinated MNC lysates also confirmed that the 60 kDa molecule on MNC is the binding protein for THG. A number of monosaccharide including N-acetylneuraminic acid, N-acetyl-galactosamine, N-acetyl-glucosamine and alpha-methyl-D-mannoside could not inhibit the mitogenic effect of THG on human mononuclear cells. These results suggest that THG is capable of reacting with surface membrane proteins on different cells, but not through the specific carbohydrate-containing lectin-like receptors on the cell surface.
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PMID:Tamm-Horsfall glycoprotein (THG) is a binder for surface membrane proteins on blood cells and glomerular mesangial cells. 904 37

Previous studies on murine T cell IgD-R have shown that these receptors recognize N-glycans of murine IgD, and not of other Ig isotypes. We have now studied the specificity of IgD-R on human T cells. Human IgD digested with proteinase K to fragments of < 5 kDa inhibit the ability of T cells to form rosettes with IgD-coated ox erythrocytes. The same amount of digested IgG does not. We tested all the human Ig isotypes: IgG1, -2, -3, -4, IgA2, IgE and IgM fail to inhibit significantly at 20 microg/assay. However, IgA1 is as effective as IgD itself, showing approximately 60 % and 80 % inhibition at 5 microg and 10 microg/assay. Human IgA1 and IgD both contain Gal-1 --> 3-GalNac-rich O-linked glycans, and on this basis are both bound to ricin and jacalin. The O-linked glycans may therefore also represent the common moiety binding to IgD-R. Disaccharides Gal-1 --> 3-GalNac, and Gal-1 --> 4-Glc at 10 microg/assay blocked IgD rosetting while Gal-1 --> 6-Glc did not. We conclude that the human IgD-R is a lectin, differing from the murine IgD-R in that it has both IgA1 and IgD as ligands.
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PMID:Human T cell IgD receptors react with O-glycans on both human IgD and IgA1. 971 Feb 14

Loxosceles spp. (brown spider) envenomation has been reported to provoke dermonecrosis and haemorrhage at the bite site (a hallmark of accidents) and, to a lesser extent, thrombocytopenia, hemolysis and disseminated intravascular coagulation in some cases. Using lectin-immunolabeling, lectin-affinity chromatography, glycosidase and proteinase K treatments we were able to identify several venom N-glycosylated proteins with high-mannose oligosaccharide structures, complex-type glycoconjugates such as fucosylated glycans, but no galactose or sialic acid residues as complex sugars or glycosaminoglycan residues. Working with enzymatically or chemically deglycosylated venom we found that platelet aggregation (thrombocytopenic activity) as well as the fibronectinolytic and fibrinogenolytic (haemorrhagic) effects of the venom were sugar-independent when compared to glycosylated venom. Nevertheless, zymograph analysis in co-polymerized gelatin gels showed that enzymatic N-deglycosylation of loxolysin-B, a high-mannose 32-35 kDa glycoprotein of the venom with gelatinolytic metalloproteinase activity, caused a reduction of approximately 2 kDa in its molecular weight and a reduction of the gelatinolytic effect to a residual activity of 28% when compared to the glycosylated molecule, indicating a post-translational glycosylation-dependent gelatinolytic effect. Analysis of the dermonecrotic effect of the chemically or enzymatically N-deglycosylated venom detected only residual activity when compared with the glycosylated control. Thus, the present report suggests that oligosaccharide moieties play a role in the destructive effects of brown spider venom and opens the possibility for a carbohydrate-based therapy.
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PMID:Oligosaccharide residues of Loxosceles intermedia (brown spider) venom proteins: dependence on glycosylation for dermonecrotic activity. 1008 60

Gene A40R from vaccinia virus (VV) strain Western Reserve has been characterized. The open reading frame (ORF) was predicted to encode a 159 amino acid, 18152 Da protein with amino acid similarity to C-type animal lectins and to the VV A34R protein, a component of extracellular enveloped virus (EEV). Northern blotting and S1 nuclease mapping showed that gene A40R is transcribed early during infection from a position 12 nucleotides upstream of the ORF, producing a transcript of approximately 600 nucleotides. Rabbit anti-sera were raised against bacterial fusion proteins containing parts of the A40R protein. These were used to identify an 18 kDa primary translation product and N- and O-glycosylated forms of 28, 35 and 38 kDa. The A40R proteins were detected early during infection, formed higher molecular mass complexes under non-reducing conditions and were present on the cell surface but absent from virions. The proteins partitioned with integral membrane proteins in Triton X-114. Canine pancreatic microsomal membranes protected in vitro-translated A40R from proteinase K digestion, suggesting the A40R protein has type II membrane topology. A mutant virus with the A40R gene disrupted after amino acid 50, so as to remove the entire lectin-like domain, and a revertant virus were constructed. Disruption of the A40R gene did not affect virus plaque size, in vitro growth rate and titre, EEV formation, or virus virulence in a murine intranasal model.
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PMID:The vaccinia virus A4OR gene product is a nonstructural, type II membrane glycoprotein that is expressed at the cell surface. 1046 13

Human plasma PAF-AH (platelet-activating factor-acetylhydrolase) is a Ca(2)+-independent phospholipase A2 of hematopoietic origin associated with LDL and HDL; it degrades PAF and oxidizes phospholipids. We show that human macrophages synthesize PAF-AH as a premedial Golgi precursor containing high mannose N-linked glycans. Secreted PAF-AH possesses a molecular mass of approximately 55 kDa and contains mature N-linked glycans. Secreted PAF-AH activity (90 +/- 4% of the total) bound to a wheat germ lectin column and could be eluted with N-acetylglucosamine, whereas digestion with N-acetylneuraminidase II completely abolished enzyme absorption. Tunicamycin significantly reduced cell-associated PAF-AH activity and inhibited enzyme secretion; but it did not alter the ratio of secreted to cell-associated enzyme (1.8 at 6 h and 3.1 at 24 h), suggesting that glycosylation is not essential for PAF-AH secretion. Digestion of cell-associated PAF-AH or secreted PAF-AH with peptide N-glycosidase F affected neither catalytic activity nor its resistance to proteolysis with trypsin or proteinase K; in addition, it did not affect PAF-AH association with LDL, but significantly increased its association with HDL. We suggest that macrophage-derived PAF-AH contains heterogeneous asparagine-conjugated sugar chain(s) involving sialic acid, which hinders its association with HDL but does not influence the secretion, catalytic activity, or resistance of PAF-AH to proteases.
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PMID:N-linked glycosylation of macrophage-derived PAF-AH is a major determinant of enzyme association with plasma HDL. 1159 Feb 21

Lectin activity, agglutinating sheep erythrocytes, was associated with parasporal inclusion proteins from a Lepidoptera-specific isolate of Bacillus thuringiensis serovar galleriae (H5ab). The activity was generated when parasporal inclusions were solubilized in an alkaline condition. Proteolytic processing was not required for generation of the lectin activity; the activity level was not affected by the presence/absence of the three proteases (trypsin, chymotrypsin, and proteinase K). SDS-PAGE analysis revealed that (1) alkali-solubilized parasporal inclusion proteins consisted of two major components of 130 kDa and 65 kDa, and (2) proteinase K treatment of alkali-solubilized proteins yielded a single major protein of 60 kDa. Lectin activity of our isolate was strongly inhibited by preincubation with D-mannose, but not with the six other monosaccharides: D-galactose, D-glucose, L-fucose, N-acetyl- D-glucosamine, N-acetyl- D-galactosamine, and N-acetylneuraminic acid. In contrast, D-mannose did not inhibit the in vivo larvicidal activity of the proteins against the silkworm, Bombyx mori.
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PMID:Mannose-specific lectin activity of parasporal proteins from a lepidoptera-specific Bacillus thuringiensis strain. 1243 63


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