Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The adherence of Treponema denticola GM-1, TD-4, and MS25 to human gingival fibroblasts (HGFs) was studied to serve as an introduction to investigations into the interactions of these oral bacteria with human host cells. Under both aerobic (5% CO2) and anaerobic (85% N2 plus 10% H2 plus 5% CO2) environments, the interactions with the HGFs were such that strains GM-1 and MS25 were consistently more adherent than strain TD-4. Polyclonal antibodies to GM-1 inhibited GM-1 adherence by 70%, while MS25 and TD-4 showed differing degrees of cross-reactive inhibition, indicative of common but not identical epitopes on the surface of the three T. denticola strains. Pretreatment of the three strains with trypsin did not inhibit adherence; proteinase K did, however, inhibit this interaction by 80%. Trypsin pretreatment of the HGFs resulted in increases in adherence of 50 and 86% for GM-1 and MS25, respectively, while a decrease of 41% was noted for TD-4. Exposure of the T. denticola strains to sugars and lectin pretreatment of the HGFs implicated adherence mediation by mannose and galactose residues on the HGF surface. Periodate treatment of HGFs resulted in a 50% drop in adherence for GM-1 and MS25, but did not decrease that of TD-4. Addition of fetal bovine serum inhibited adherence of the three strains to differing degrees, with TD-4 being the most susceptible. Addition of purified fibronectin (100 micrograms/ml) resulted in greater than 50% inhibition in GM-1 and MS25 adherence, while a 25% increase occurred with TD-4. While strain differences were noted in some of the parameters studied, the results indicate two possibilities for T. denticola-HGF adherence: a lectinlike adhesin(s) on the T. denticola surface with affinity for galactose and mannose on the HGF surface, and a serum host factor(s) bridging T. denticola and HGFs.
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PMID:Interaction of Treponema denticola TD-4, GM-1, and MS25 with human gingival fibroblasts. 216 Apr 30

Giardia lamblia, a protozoan parasite that causes widespread diarrheal disease, expresses a surface membrane associated lectin, taglin, which is specifically activated by limited proteolysis with trypsin, a protease that is present in abundance at the site of infection. When activated, taglin agglutinates enterocytes which are the cells to which the parasite adheres in vivo, and in addition, binds to isolated brush border membranes of these cells. These findings suggest that this lectin may be involved in the host-parasite interaction. Taglin is most specific for terminal phosphomannosyl residues and its binding to red cells is mediated by cell surface phosphate residues. Hemagglutinating activity induced by taglin is most active at pH 6.5 and is dependent on divalent cations. A monoclonal antibody to taglin reacts with the surface membrane of live trophozoites and recognizes a protein of 28/30 kDa in lysates of Giardia trophozoites, by immunoblotting. This finding is confirmed by direct demonstration of lectin activity by erythrocyte binding to proteins electroblotted to nitrocellulose, which revealed specific red cell binding to giardial protein bands in the same molecular weight range as those recognized by the monoclonal antibody.
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PMID:Induction of a phosphomannosyl binding lectin activity in Giardia. 219 51

Rat and human colonic mucin glycoproteins bind to the Gal/GalNAc adherence lectin on the surface of Entamoeba histolytica in vitro, thus inhibiting the organism from adhering to and lysing the target cells. Human colonic mucin glycoproteins were isolated by Sepharose 4B gel filtration chromatography, they were proteolytically degraded with trypsin, pronase, and papain, and the glycoprotein fractions were reisolated by Sephacryl S-200 gel filtration chromatography. Binding of the mucin glycoprotein fractions to amoebae was quantitated by the inhibition of adherence of Chinese hamster ovary cells to the surface of the amoebae. Trypsin and papain digests caused 40 and 20% reductions, respectively, in the excluded fractions (void volume) that contained all the carbohydrates; pronase digests resulted in extensive degradation of the mucin glycoprotein with the carbohydrate fractions eluting over 40% of the gel bed volume. 3H-labelled mucin glycoprotein and sodium dodecylsulfate-polyacrylamide gel electrophoresis confirmed the presence of the high molecular weight carbohydrate-rich glycoproteins with no subunits in the excluded fractions and the absence of sugars in the included peptides. Only the high molecular weight carbohydrate-containing fractions bind amoebae and inhibit amoebic adherence to Chinese hamster ovary cells. The trypsin digested mucins in the excluded volume were more efficient than the native undigested mucins in binding amoebae. The carbohydrate-containing fractions of the pronase digests were the least effective in binding amoebae and inhibiting adherence of Chinese hamster ovary cells. This suggests that proteolytically-degraded colonic mucins that are glycosylated, as well as the undegraded native mucin glycoproteins of the gut, may play a protective role in binding to amoebae, thus preventing contact of amoebae with mucosal epithelial cells and potential invasion.
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PMID:Binding of proteolytically-degraded human colonic mucin glycoproteins to the Gal/GalNAc adherence lectin of Entamoeba histolytica. 220 83

A major problem encountered when isolating human microvascular endothelium is the presence of contaminating cells such as fibroblasts that rapidly over-grow the endothelial cells. We describe here a simple, rapid technique for purifying endothelial cells derived from the microvasculature of neonatal foreskin and osteoarthritic and rheumatoid arthritic synovium. This technique is based on the selective binding of the lectin Ulex europaeus I (UEA I) to the endothelial cell surface via fucose residues. Initially UEA I was covalently bound to tosyl-activated super-paramagnetic polystyrene beads (Dynabeads) by incubation for 24 h at room temperature. Cells were isolated by extracting microvascular segments from enzyme-treated (trypsin and Pronase) cubes of tissue. The mixed population of cells obtained were purified by incubating them at 4 degrees C for 10 min with the UEA I-coated Dynabeads. Endothelium bound to the beads whilst contaminating cells were removed by five washes with HBSS using a magnetic particle concentrator. The endothelial cells thus obtained grew to confluence as a cobblestone-like monolayer and expressed von Willebrand factor antigen. The cells were released from the Dynabeads by the competitive binding of fucose (10 min at 4 degrees C). This new method is simple and reproducible and allows pure human microvascular endothelial cells to be cultured within 2 h of obtaining a specimen.
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PMID:Binding of human endothelium to Ulex europaeus I-coated Dynabeads: application to the isolation of microvascular endothelium. 221 66

This study examined the effect of immunoglobulin A (IgA) and the IgA-binding lectin jacalin on the phagocytosis of type II group B streptococci (GBS). Strains possessing the trypsin-sensitive and trypsin-resistant components of the c protein (II/c) and type II GBS lacking the c protein (II) were examined by radiolabeled bacterial uptake, bactericidal assays, and electron microscopy. Type II/c GBS resisted phagocytosis by monocytes (4.9% +/- 0.8% uptake, mean +/- SE, n = 25) compared with type II GBS (8.5% +/- 1.4% uptake, n = 14, P = 0.03). Phagocytic killing by polymorphonuclear leukocytes was also less for the type II/c strain 78-471 than for the type II strain 79-176 (68% +/- 5% versus 86% +/- 4% reduction in CFU at 45 min, P = 0.03). IgA binding did not explain the resistance of type II/c GBS to phagocytosis. The uptake of type II/c GBS was not significantly different after opsonization in cord sera lacking endogenous IgA (5.93% +/- 1.4%) than in the same cord sera after addition of exogenous IgA (5.48% +/- 1.4%, P = 0.69, n = 9). Attempts to remove serum IgA with the IgA-binding lectin jacalin resulted in the binding of IgA-jacalin complexes to II/c GBS. This combination of nonspecific IgA and jacalin increased uptake of II/c GBS from 4.9% +/- 0.8% to 11.8% +/- 1.9% (P = 0.002). Jacalin also combined with specific, immune, monoclonal IgA bound to the surface of Haemophilus influenzae and promoted the uptake of these bacteria. Jacalin and IgA mediated phagocytosis of II/c GBS via receptors that were not dependent on divalent cations and that were not modulated by plating monocytes on antigen-antibody complexes.
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PMID:Opsonic effect of jacalin and human immunoglobulin A on type II group B streptococci. 222 38

Oral isolates of Streptococcus milleri were examined for their ability to coaggregate with actinomyces. Of the 68 S. milleri strains tested, including 3 reference strains, 40 strains coaggregated with Actinomyces naeslundii WVU45 (actinomyces coaggregation group B) and 36 strains coaggregated with Actinomyces viscosus T14V (actinomyces coaggregation group A). All S. milleri strains of serotypes b (4 strains), e (2 strains), and f (24 strains) coaggregated with both of the actinomyces. The coaggregation reactions between the S. milleri cells and A. naeslundii WVU45 cells were optimal at about pH 7.0 and were Ca2+ or Mg2+ dependent, but they were not inhibited by the presence of simple sugars or amino sugars, including lactose (up to 0.5 M). Treatment of the S. milleri cells with heat (100 degrees C, 3 min) or proteases (trypsin, 1.0 mg/ml; pronase, 0.25 mg/ml; 37 degrees C; 3 h) and of the actinomyces cells with periodate (0.01 M, 4 degrees C, 16 h) destroyed their coaggregating abilities. The coaggregations between cells of the S. milleri strains, we well as cells of the Streptococcus sanguis H1 (reference strain for streptococcus coaggregation group 2) and the actinomyces strains (WVU45 and T14V), were inhibited by AFH1 (a carbohydrate receptor on T14V cells for a lectin on H1 cells). These interactions were also inhibited by anti-AFH1 immunoglobulin G (IgG) and by anti-b, anti-e, and anti-f S. milleri IgG or anti-f IgG Fab fragments. These results suggest that S. milleri, at least strains of serotypes b, e, and f, belongs to streptococcus coaggregation group 2.
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PMID:Cellular coaggregation of oral Streptococcus milleri with actinomyces. 229 47

A lectin was isolated from the homogenate of the tunicate Polyandrocarpa misakiensis by heat treatment, ammonium sulfate fractionation, gel filtration, and high-performance ion-exchange chromatography. Analytical gel filtration on Superose 12 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the lectin is a monomeric protein with a molecular mass of approximately 15 kDa. The lectin bound to an immobilized D-galactose column in the presence of calcium ion with a threshold of 500 microM and eluted completely with 5 mM EDTA. It did not bind to an immobilized D-mannose or N-acetyl-D-galactosamine column. Thus, Polyandrocarpa lectin was found to be a calcium-dependent galactose-binding lectin. The complete amino acid sequence of Polyandrocarpa lectin was determined by automated or manual Edman sequencing of the peptides derived by digestion with trypsin, endoproteinase Asp-N, Staphylococcus aureus V8 protease, and pepsin. It is composed of 125 residues, contains no carbohydrate group, and has a calculated molecular mass of 14,034 Da. The lectin contains four half-cystines, and Cys-21 and Cys-119 and also Cys-96 and Cys-111 form intrachain disulfide bridges, respectively. The amino acid sequence of Polyandrocarpa lectin shows about 20-30% homology with those of fly, barnacle, sea urchin, and several vertebrate lectins that belong to C-type lectin (Drickamer, K. (1988) J. Biol. Chem. 263, 9557-9560). Although the physiological role of Polyandrocarpa lectin is not clear, preliminary experiments suggest that the lectin may be related to defense mechanisms because it has a strong antibacterial activity.
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PMID:A calcium-dependent galactose-binding lectin from the tunicate Polyandrocarpa misakiensis. Isolation, characterization, and amino acid sequence. 229 29

Echinonectin (EN) is a 230-kDa extracellular matrix glycoprotein found in the hyaline layer of sea urchin embryos. Dissociated embryonic cells attached strongly to EN-coated microtiter wells in a centrifugal-based in vitro adhesion assay, suggesting that EN is one of the hyaline layer proteins to which cells adhere in vivo (Alliegro et al., 1988). The present study examines the molecular properties of that adhesion using monoclonal antibodies as probes to block cell attachment, and also demonstrates that EN possesses lectin activity. EN binds tenaciously to agarose-based chromatography resins, such as Sepharose. The sugar-binding activity is associated with the polypeptide component of EN, and not with the carbohydrate moiety. Binding is inhibited with galactose and fucoidan, but not with glucose or locust bean gum. Although functional sites both for polysaccharide binding and for cell attachment are present on each subunit of the EN molecule, the sites appear to be functionally distinct because galactose and fucoidan are completely without effect on cell attachment in vitro. Proteolytic digestion of EN yields a highly limited set of immunoreactive peptides. Digestion with trypsin yields a 20-kDa fragment, chymotrypsin, a doublet at 20 kDa, and 20- and 23-kDa fragments with thermolysin. McAb's directed against these peptides block cell adhesion in vitro, suggesting that they possess the cell attachment domain of EN. This is supported by the observations that trypsin-digested EN is an effective substrate in adhesion assays and that adhesion to the tryptic fragments is also blocked by McAb's to the 20-kDa domain.
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PMID:In vitro biological activities of echinonectin. 232 45

1. An anti-N lectin was extracted from Vicia unijuga leaves with phosphate-buffered saline (PBS). Purification of the lectin was achieved, after pretreatment of the PBS extract by ammonium sulfate fractionation and absorption with human M erythrocytes, by using a combination of conventional chromatographic techniques with asialoglycophorin AN-Sepharose CL-4B affinity chromatography. Purification steps were followed by increase of specific activity. 2. Homogeneity of the purified lectin was demonstrated by HPLC and SDS-PAGE. The purified lectin was a glycoprotein with 11.4% carbohydrate and relatively high percentages of serine, threonine and aspartic acid residues and had a Mw of 120,000 Da. 3. This lectin agglutinated human N and MN erythrocytes, but did not agglutinate M erythrocytes. Hemagglutination of the lectin was inhibited by glycophorin AN and N-active sialoglycopeptide released from human N erythrocytes by treatment with Pronase or trypsin. However, it was not inhibited by any of mono- and di-saccharides, ABH-active glycoproteins, glycophorin AM and M-active sialoglycopeptide liberated from human M erythrocytes by treatment with Pronase or trypsin.
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PMID:Purification and characterization of anti-N lectin from Vicia unijuga leaves. 232 20

In this report, we use 8 different mouse monoclonal antibodies to define 3 immunodominant determinants of the human leukocyte common (LC) antigen, and by defining the relative location and the stability to proteolytic enzymes of these determinants we make some structural predictions about the LC family of molecules. One lineage-restricted determinant is expressed predominantly on B lymphocytes and is located on the two higher (210 and 195) kDa bands of LC, with possibly some expression on the 180-kDa band. The two other determinants, one of which is a complex of 3 partially overlapping sites, are expressed on all leukocytes and found on all four (210, 195, 180, 160 kDa) LC bands. Pronase and trypsin treatment of peripheral blood lymphocytes and of Daudi cells gave complete cleavage of the lineage-restricted determinant from the cells, but the two common determinants were, surprisingly, left intact on the cell surface. By contrast, pronase and trypsin treatment of pure, detergent-solubilized LC resulted in rapid degradation of the common determinants, while the restricted determinant was sensitive to pronase but not to trypsin. Purification of the LC molecule from pronase-treated Daudi cells yielded a 130-kDa lentil lectin-binding protein, while undegraded LC from Daudi cells has a molecular mass of 210 kDa. Our results demonstrate that both common and restricted determinants are at least partly protein in nature and that the restricted determinant is external to and therefore on the presumed amino terminal side of the common determinants. Moreover, because LC has been reported to have a large (approximately 100 kDa) intracellular component, our data suggest the presence of a relatively small (approximately 25 kDa) membrane proximal domain containing both of the common LC determinants and at least one N-linked oligosaccharide chain. This domain is potentially susceptible to proteolytic cleavage, but it is interesting that on the intact cell it is both protected from proteolytic degradation and unable to be cleaved from the cell surface.
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PMID:Structural implications of the location and stability to proteolytic enzymes of immunodominant determinants of the human leukocyte common molecule. 242 43


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