Two alkaline motifs in the Lactobacillus salivarius Lv72 OppA surface are important to its adhesin function

2019 ◽  
Vol 10 (1) ◽  
pp. 101-109 ◽  
Author(s):  
C. Martín ◽  
S. Escobedo ◽  
G. Pérez-Martínez ◽  
J.M. Coll-Marqués ◽  
R. Martín ◽  
...  
Keyword(s):  
Cell Surface ◽  
Hela Cell ◽  
Cell Cultures ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Eukaryotic Cell ◽  
Cloning And Expression ◽  
Lactobacillus Salivarius ◽  
Normal Microbiota

Glycosaminoglycans are involved in the attachment of Lactobacillus salivarius Lv72, a strain of vaginal origin, to HeLa cell cultures, indicating that they play a fundamental role in the attachment of mutualistic bacteria to the epithelium lining cavities where the normal microbiota thrives. The bacterial OppA protein has been proposed as an adhesin involved in this adherence since, once purified, it significantly interferes with attachment of the lactobacilli to HeLa cell cultures. In this article, the role of OppA is confirmed through the determination of its location at the cell surface and its ability to promote Lactobacillus casei and Lactococcus lactis adherence to eukaryotic cell cultures upon cloning and expression of oppA in these bacteria. The OppA sequence showed five potential domains for glycosaminoglycan-binding, and structural modelling of the protein showed that two of them were located in the vicinity of an OppA superficial groove whose width approached the diameter of the helical form of heparin in solution. Their involvement in the binding was demonstrated through substitution of critical basic amino acids by acidic ones, which resulted in loss of affinity for heparan sulphate and chondroitin sulphate depending on the domain mutated, suggesting that there might be a certain degree of specialisation. In addition, circular dichroism analysis showed that the spectrum changes induced by OppA-heparan sulphate binding were attenuated by the variant proteins, indicating that these motifs are the OppA recognition domains for the eukaryotic cell surface.

Widespread use of Lactobacillus OppA, a surface located protein, as an adhesin that recognises epithelial cell surface glycosaminoglycans

2019 ◽  
Vol 10 (4) ◽  
pp. 463-472 ◽  
Author(s):  
C. Martín ◽  
S. Escobedo ◽  
J.E. Suárez ◽  
L.M. Quirós
Keyword(s):  
Cell Surface ◽  
Hela Cell ◽  
Cell Cultures ◽  
Lactobacillus Reuteri ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Surface Protein ◽  
Epithelial Cell Surface ◽  
Surface Proteome ◽  
Reduced Adherence

Specific adherence is the first requisite that a microorganism has to fulfil to become established onto a mucosal surface. It was previously shown that the OppA surface protein of Lactobacillus salivarius Lv72 bound HeLa cell cultures through interaction with glycosaminoglycans (GAGs). To determine whether this is a peculiarity of that strain or whether it can be extended to other lactobacilli, 12 strains, belonging to six species, were confronted with HeLa-cell cultures in the presence of soluble GAGs. Interference was observed to six of them, heparan sulphate and chondroitin sulphate C being more interfering than chondroitin sulphate A or chondroitin sulphate B. Furthermore, inhibition of the biosynthesis of GAGs or their elimination from the cell surface with specific enzymes also resulted in reduced adherence. Analysis of the surface proteome of Lactobacillus crispatus Lv25 and of Lactobacillus reuteri RC14 revealed single proteins that immunoreacted with antibodies raised against OppA, the main adhesin of L. salivarius Lv72. Upon MALDI-TOF-TOF analysis, they were identified as OppA-like proteins, thus indicating that these proteins participate as adhesins in attachment of diverse lactobacilli to the surface of human epithelial cells.

Glycosaminoglycan expression in intestinal epithelial skin-fibroblastic cell cocultures. Fibroblastic cell-mediated effects of glucocorticoids

1989 ◽  
Vol 92 (4) ◽  
pp. 679-685
Author(s):  
F. Bouziges ◽  
P. Simon-Assmann ◽  
C. Leberquier ◽  
K. Haffen ◽  
M. Kedinger
Keyword(s):  
Molecular Weight ◽  
Cell Surface ◽  
Cell Cultures ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Cell Types ◽  
Molecular Forms ◽  
Fibroblastic Cell ◽  
Endodermal Cells ◽  
Molecular Weight Form

The nature and distribution of newly synthesized glycosaminoglycans (GAGs) were studied in foetal rat skin fibroblasts, in rat intestinal endodermal cells and in cocultures of both cell types. The data show that fibroblasts synthesize and secrete hyaluronic acid (HA), heparan sulphate (HS) and chondroitin sulphate molecules (CS). Our data focus on HA, which is found as two different molecular forms, the smallest hydrodynamic-sized species being mostly recovered within the cell or associated with the cell surface, and the largest one secreted into the medium, whatever the cell type. Endodermal cells synthesize only two types of GAGs: the low molecular weight form of HA and HS. Cocultures of rat intestinal endodermal and skin fibroblastic cells in the presence of dexamethasone (Dx), allow optimal epithelial cytodifferentiation (Kedinger et al. 1987a). The main changes in the GAGs synthesized under these conditions as compared to skin fibroblastic cell cultures concern: (1) the enhancement of the lowest molecular weight form of HA to the detriment of the highest form in the cellular, pericellular and extracellular compartments; (2) the increase in the proportion of HS molecules associated with the cell surface. Interestingly, similar modifications are obtained by addition of Dx to the skin fibroblastic cell cultures. The data are discussed with reference to the constitution of a basement membrane at the epithelial-fibroblast interface in the cocultures, to the fibroblastic-dependent induction of epithelial differentiation and to the glucocorticoid response.

scholarly journals Glycosaminoglycans on fibroblasts accelerate thrombin inhibition by protease nexin-1

1987 ◽  
Vol 245 (2) ◽  
pp. 543-550 ◽  
Author(s):  
D H Farrell ◽  
D D Cunningham
Keyword(s):  
Complex Formation ◽  
Cell Surface ◽  
Plasma Membranes ◽  
Chondroitin Sulphate ◽  
Human Fibroblasts ◽  
Heparan Sulphate ◽  
Order Rate Constant ◽  
Subsequent Treatment ◽  
Protease Nexin ◽  
Inhibition Of Thrombin

Protease nexin-1 (PN-1) is a proteinase inhibitor that is secreted by human fibroblasts in culture. PN-1 inhibits certain regulatory serine proteinases by forming a covalent complex with the catalytic-site serine residue; the complex then binds to the cell surface and is internalized and degraded. The fibroblast surface was recently shown to accelerate the rate of complex-formation between PN-1 and thrombin. The present paper demonstrates that the accelerative activity is primarily due to cell-surface heparan sulphate, with a much smaller contribution from chondroitin sulphate. This conclusion is supported by the effects of purified glycosaminoglycans on the second-order rate constant for the inhibition of thrombin by PN-1. Also, treatment of 35SO4(2-)-labelled cells with heparitin sulphate lyase or chondroitin sulphate ABC lyase demonstrated two discrete pools of 35S-labelled glycosaminoglycans; subsequent treatment of plasma membranes with these glycosidases showed that heparitin sulphate lyase treatment abolished about 80% of the accelerative activity and chondroitin sulphate ABC lyase removed the remaining 20%. These results show that two components are responsible for the acceleration of PN-1-thrombin complex-formation by human fibroblasts. Although dermatan sulphate is also present on fibroblasts, it did not accelerate the inhibition of thrombin by PN-1.

scholarly journals Rapid determination of general cell status, cell viability, and optimal harvest time in eukaryotic cell cultures by impedance flow cytometry

2019 ◽  
Vol 103 (20) ◽  
pp. 8619-8629 ◽  
Author(s):  
Christian Opitz ◽  
Grit Schade ◽  
Silvan Kaufmann ◽  
Marco Di Berardino ◽  
Marcel Ottiger ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Viability ◽  
Cell Cultures ◽  
Rapid Determination ◽  
Eukaryotic Cell ◽  
Harvest Time ◽  
Optimal Harvest

scholarly journals Cell surface hydrophobicity, adherence to HeLa cell cultures and haemagglutination pattern of pyelonephritogenicEscherichia colistrains

1990 ◽  
Vol 105 (2) ◽  
pp. 255-263 ◽  
Author(s):  
A. Brauner ◽  
M. Katouli ◽  
K. Tullus ◽  
S. H. Jacobson
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Hela Cell ◽  
Cell Cultures ◽  
Hela Cells ◽  
Acute Pyelonephritis ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
Hydrophobic Properties ◽  
E Coli

SUMMARYCell surface hydrophobicity, haemagglutination pattern and adherence to HeLa cells were examined in 230 strains ofEscherichia colicollected from women (n= 61 strains) and children (n= 65 strains) with non-obstructive acute pyelonephritis and in 104 faecal control strains ofE. colifrom healthy adults (n= 71 strains) and children (n= 33 strains). PyelonephritogenicE. colistrains showed a significantly increased incidence of hydrophobic properties (90%) and mannose resistant haemagglutination (MRHA) of human erythrocytes (83%) than faecal control strains (64 and 23% respectively,P< 0·001 in both cases). Mannose sensitive haemagglutination (MSHA) was observed in 48% of the pyelonephritogenicE. colistrains and in 50% of the faecal control strains (NS). The incidence of adherence to HeLa cells was low both in pyelonephritogenic and faecal control strains, 6 and 7% respectively (NS). The bacterial phenotypes MRHA + MSHA + and MRHA + MSHA− appeared significantly more often in pyelonephritogenicE. colistrains (35 and 48% respectively) than in faecal control strains (5 and 17% respectively,P< 0·001 in both cases). The phenotype MRHA − MSHA + occurred significantly more often in control strains (45%) than in pyelonephritogenic strains (13%,P< 0·001). Eighty-three per cent of the pyelonephritogenicE. colistrains expressing hydrophobic properties showed MRHA and 50% of the hydrophobic strains showed MSHA. There were no significant correlations between cell surface hydrophobic properties and haemagglutination pattern or adherence to HeLa cells in pyelonephritogenicE. colistrains nor in faecal control strains.

scholarly journals Comparative study on glycosaminoglycans synthesized in peripheral and peritoneal polymorphonuclear leucocytes from guinea pigs

1984 ◽  
Vol 217 (1) ◽  
pp. 199-207 ◽  
Author(s):  
Y Ohhashi ◽  
F Hasumi ◽  
Y Mori
Keyword(s):  
Cell Surface ◽  
Guinea Pigs ◽  
Lysosomal Enzymes ◽  
Culture Medium ◽  
Chondroitin Sulphate ◽  
Alkali Treatment ◽  
Heparan Sulphate ◽  
Polymorphonuclear Leucocytes ◽  
Dermatan Sulphate ◽  
Cell Cell

Glycosaminoglycans synthesized in polymorphonuclear (PMN) leucocytes isolated from blood (peripheral PMN leucocytes) and in those induced intraperitoneally by the injection of caseinate (peritoneal PMN leucocytes) were compared. Both peripheral and peritoneal PMN leucocytes were incubated in medium containing [35S]sulphate and [3H]glucosamine. Each sample obtained after incubation was separated into cell, cell-surface and medium fractions by trypsin digestion and centrifugation. The glycosaminoglycans secreted from peripheral and peritoneal PMN leucocytes were decreased in size by alkali treatment, indicating that they existed in the form of proteoglycans. Descending paper chromatography of the unsaturated disaccharides obtained by the digestion of glycosaminoglycans with chondroitinase AC and chondroitinase ABC identified the labelled glycosaminoglycans of both the cell and the medium fractions in peripheral PMN leucocytes as 55-58% chondroitin 4-sulphate, 16-19% chondroitin 6-sulphate, 16-19% dermatan sulphate and 6-8% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found only in the medium fraction. In peritoneal PMN leucocytes there is a difference in the composition of glycosaminoglycans between the cell and the medium fractions; the cell fraction was composed of 60% chondroitin 4-sulphate, 5.5% chondroitin 6-sulphate, 16.8% dermatan sulphate and 13.9% heparan sulphate, whereas the medium fraction consisted of 24.5% chondroitin 4-sulphate, 28.2% chondroitin 6-sulphate, 33.7% dermatan sulphate and 10% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found in the cell, cell-surface and medium fractions. On the basis of enzymic assays with chondro-4-sulphatase and chondro-6-sulphatase, the positions of sulphation in the disulphated disaccharides were identified as 4- and 6-positions of N-acetylgalactosamine. Most of the 35S-labelled glycosaminoglycans synthesized in peripheral PMN leucocytes were retained within cells, whereas those in peritoneal PMN leucocytes were secreted into the culture medium. Moreover, the amount of glycosaminoglycans in peritoneal PMN leucocytes was significantly less than that in peripheral PMN leucocytes. Assay of lysosomal enzymes showed that these activities in peritoneal PMN leucocytes were 2-fold higher than those in peripheral PMN leucocytes.

scholarly journals Evidence for the stimulation of dolichol and mannolipid synthesis by glucocorticoids in HeLa cells

1981 ◽  
Vol 198 (1) ◽  
pp. 23-28 ◽  
Author(s):  
C K Ramachandran ◽  
C E Hignite ◽  
S L Gray ◽  
G Melnykovych
Keyword(s):  
Cell Surface ◽  
Hela Cell ◽  
Cell Cultures ◽  
Hela Cells ◽  
Surface Glycoprotein ◽  
Dexamethasone Treatment ◽  
Cell Surface Glycoprotein ◽  
Dolichyl Phosphate ◽  
Stimulation Of

The effects of addition of 1 microM-dexamethasone on the rate of transfer of mannose from GDP-mannose into mannolipid have been studied in HeLa cell cultures. Concurrent with an increase in incorporation of mannose into glycoproteins, the incorporation of mannose from GDP-mannose in vitro into mannolipid and dolichol-linked oligosaccharides was increased after dexamethasone treatment. Stimulation of mannolipid synthesis showed a correlation with the 11 beta, 17 alpha, 21-trihydroxy structure of C21 steroids. Dexamethasone treatment also resulted in an increased incorporation of acetate into dolichol and dolichyl phosphate. The results suggest that the effect of dexamethasone on the cell-surface glycoprotein accumulation is related to increased sugar-linked dolichol synthesis.

scholarly journals Proteoglycan forms of the lymphocyte homing receptor CD44 are alternatively spliced variants containing the v3 exon.

1995 ◽  
Vol 128 (4) ◽  
pp. 673-685 ◽  
Author(s):  
D G Jackson ◽  
J I Bell ◽  
R Dickinson ◽  
J Timans ◽  
J Shields ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Cell Surface ◽  
Inflammatory Responses ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Surface Glycoprotein ◽  
Alternative Exon ◽  
Cell Surface Glycoprotein ◽  
Cd44 Isoforms ◽  
Alternatively Spliced

The CD44 cell surface glycoprotein is expressed on a broad range of different tissues as multiple isoforms containing from one to ten alternatively spliced exons v1-v10 inserted within the extracellular domain. Differential glycosylation generates still further variability, yielding both N- and O-glycan-modified forms of CD44 in addition to proteoglycan-like variants containing chondroitin sulphate and heparan sulphate. These high molecular mass proteoglycan-like variants, previously identified in lymphocytes, melanomas, and keratinocytes have been implicated in cell-matrix adhesion, cell motility, and invasiveness. More recently, monocyte CD44 molecules presumed to carry glycosaminoglycan chains were shown to bind the chemokine MIP-1 beta (Tanaka, Y.,D. H. Adams, S. Hubscher, H. Hirano, U. Siebenlist, and S. Shaw. 1993. Nature (Lond). 361:79-82.) raising the intriguing possibility that proteoglycan-like CD44 variants might play a role in regulating inflammatory responses. Here we have investigated the molecular identity of these proteoglycan-like CD44 variants by generating a panel of recombinant CD44 isoforms using a novel cassette cloning strategy. We show that both chondroitin and heparan sulphate modifications are associated specifically with isoforms (CD44v3-10 and CD44v3,8-10) containing the v3 alternative exon which encodes a consensus motif SGXG for GAG addition. Other isoforms (CD44v10, CD44v8-10, CD44v7-10, and CD44v6-10) are shown to lack these GAG chains but to carry extensive O-glycan modifications, most likely within the mucin-like alternative exon inserts. We also demonstrate that the majority of endogenous GAG-modified CD44 isoforms present in epithelial cells constitute v3 isoforms thus establishing that in these cells the majority of proteoglycan-like CD44 variants are generated by alternative splicing. Finally we present evidence using transfected B lymphoma cells that the GAG-modified CD44 isoforms CD44v3-10 and CD44v3,8-10, unlike CD44H, bind only weakly to hyaluronan. Together with the demonstration in the accompanying paper (Bennett, K., D. G. Jackson, J.C. Simon, E. Tanczos, R. Peach, B. Modrell, I. Stamenkovic, G. Plowman, and A. Aruffo. 1995. J. Cell Biol. 128:687-698.), that CD44 molecules containing the v3 exon bind growth factors, these results highlight a new and potentially important role for CD44 alternative splicing in the control of cell-surface proteoglycan expression.

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