Strain-specific diversity of mucus-binding proteins in the adhesion and aggregation properties of Lactobacillus reuteri

Mucus-binding proteins (MUBs) have been revealed as one of the effector molecules involved in mechanisms of the adherence of lactobacilli to the host; mub, or mub-like, genes are found in all of the six genomes of Lactobacillus reuteri that are available. We recently reported the crystal structure of a Mub repeat from L. reuteri ATCC 53608 (also designated strain 1063), revealing an unexpected recognition of immunoglobulins. In the current study, we explored the diversity of the ATCC 53608 mub gene, and MUB expression levels in a large collection of L. reuteri strains isolated from a range of vertebrate hosts. This analysis revealed that the MUB was only detectable on the cell surface of two highly related isolates when using antibodies that were raised against the protein. There was considerable variation in quantitative mucus adhesion in vitro among L. reuteri strains, and mucus binding showed excellent correlation with the presence of cell-surface ATCC 53608 MUB. ATCC 53608 MUB presence was further highly associated with the autoaggregation of L. reuteri strains in washed cell suspensions, suggesting a novel role of this surface protein in cell aggregation. We also characterized MUB expression in representative L. reuteri strains. This analysis revealed that one derivative of strain 1063 was a spontaneous mutant that expressed a C-terminally truncated version of MUB. This frameshift mutation was caused by the insertion of a duplicated 13 nt sequence at position 4867 nt in the mub gene, producing a truncated MUB also lacking the C-terminal LPxTG region, and thus unable to anchor to the cell wall. This mutant, designated 1063N (mub-4867i), displayed low mucus-binding and aggregation capacities, further providing evidence for the contribution of cell-wall-anchored MUB to such phenotypes. In conclusion, this study provided novel information on the functional attributes of MUB in L. reuteri, and further demonstrated that MUB and MUB-like proteins, although present in many L. reuteri isolates, show a high genetic heterogeneity among strains.

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Involvement of LFA-1 in lymphoma invasion and metastasis demonstrated with LFA-1-deficient mutants.

The Journal of Cell Biology ◽

10.1083/jcb.108.5.1979 ◽

1989 ◽

Vol 108 (5) ◽

pp. 1979-1985 ◽

Cited By ~ 74

Author(s):

F F Roossien ◽

D de Rijk ◽

A Bikker ◽

E Roos

Keyword(s):

Cell Surface ◽

Surface Protein ◽

Metastatic Potential ◽

Surface Expression ◽

Cell Surface Expression ◽

Lymphocyte Function ◽

Lymphoma Cells ◽

Mutant Cells

Lymphocyte function-associated antigen-1 (LFA-1) is a leukocyte and lymphoma cell surface protein that promotes intercellular adhesion. We have previously shown that the invasion of hepatocyte cultures by lymphoma cells is inhibited by anti-LFA-1 antibodies (Roos, E., and F. F. Roossien. 1987. J. Cell Biol. 105:553-559). In addition, we now report that LFA-1 is also involved in invasion of lymphoma cells into fibroblast monolayers. To investigate the role of LFA-1 in metastasis of these lymphoma cells, we have generated mutants that are deficient in LFA-1 cell surface expression because of impaired synthesis of either the alpha or beta subunit precursor of LFA-1. We identified at least three distinct mutant clones. The invasive potential of the mutant cells in vitro, in both hepatocyte and fibroblast cultures, was considerably lower than that of parental cells. The metastatic potential of the mutants was much reduced, indicating that LFA-1 expression is required for efficient metastasis formation by certain lymphoma cells.

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A high-molecular-mass cell-surface protein from Lactobacillus reuteri 1063 adheres to mucus components The GenBank accession number for the sequence reported in this paper is AF120104.

Microbiology ◽

10.1099/00221287-148-2-433 ◽

2002 ◽

Vol 148 (2) ◽

pp. 433-442 ◽

Cited By ~ 228

Author(s):

Stefan Roos ◽

Hans Jonsson

Keyword(s):

Cell Surface ◽

Lactobacillus Reuteri ◽

Surface Protein ◽

Surface Proteins ◽

Cell Surface Protein ◽

Intestinal Mucus ◽

A Cell ◽

Molecular Masses ◽

Ph Range

A gene from Lactobacillus reuteri 1063 encoding a cell-surface protein, designated Mub, that adheres to mucus components in vitro has been cloned and sequenced. The deduced amino acid sequence of Mub (358 kDa) shows the presence of 14 approximately 200 aa repeats and features typical for other cell-surface proteins of Gram-positive bacteria. Fusion proteins consisting of different repeats of Mub and the maltose-binding protein (MBP) were produced. These proteins adhered to pig mucus components, with molecular masses ranging from <0·1 to >2 MDa, to pig gastric mucin and to hen intestinal mucus. The binding of Mub to mucus components occurred in the pH range 3–7·4, with maximum binding at pH 4–5 and could be partly inhibited by the glycoprotein fetuin. Affinity-purified antibodies against recombinant Mub were used in immunofluorescence microscopy to demonstrate the presence of Mub on the cell surface of strain 1063. By using the antibodies in a Western blot analysis, Mub could also be detected in the growth medium. The results implicate Mub as a cell-surface protein that is involved in Lactobacillus interactions with mucin and in colonization of the digestive tract.

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Role of Lactobacillus reuteri cell and mucus-binding protein A (CmbA) in adhesion to intestinal epithelial cells and mucus in vitro

Microbiology ◽

10.1099/mic.0.073551-0 ◽

2014 ◽

Vol 160 (4) ◽

pp. 671-681 ◽

Cited By ~ 41

Author(s):

Hanne Jensen ◽

Stefan Roos ◽

Hans Jonsson ◽

Ida Rud ◽

Stine Grimmer ◽

...

Keyword(s):

Gastrointestinal Tract ◽

Epithelial Cells ◽

Lactobacillus Reuteri ◽

Intestinal Epithelial Cells ◽

Protein A ◽

Surface Protein ◽

Null Mutation ◽

Intestinal Epithelial

Lactobacillus reuteri, a symbiotic inhabitant of the gastrointestinal tract in humans and animals, is marketed as a probiotic. The ability to adhere to intestinal epithelial cells and mucus is an interesting property with regard to probiotic features such as colonization of the gastrointestinal tract and interaction with the host. Here, we present a study performed to elucidate the role of sortase (SrtA), four putative sortase-dependent proteins (SDPs), and one C-terminal membrane-anchored cell surface protein of Lactobacillus reuteri ATCC PTA 6475 in adhesion to Caco-2 cells and mucus in vitro. This included mutagenesis of the genes encoding these proteins and complementation of mutants. A null mutation in hmpref0536_10255 encoding srtA resulted in significantly reduced adhesion to Caco-2 cells and mucus, indicating involvement of SDPs in adhesion. Evaluation of the bacterial adhesion revealed that of the five putative surface protein mutants tested, only a null mutation in the hmpref0536_10633 gene, encoding a putative SDP with an LPxTG motif, resulted in a significant loss of adhesion to both Caco-2 cells and mucus. Complementation with the functional gene on a plasmid restored adhesion to Caco-2 cells. However, complete restoration of adhesion to mucus was not achieved. Overexpression of hmpref0536_10633 in strain ATCC PTA 6475 resulted in an increased adhesion to Caco-2 cells and mucus compared with the WT strain. We conclude from these results that, among the putative surface proteins tested, the protein encoded by hmpref0536_10633 plays a critical role in binding of Lactobacillus reuteri ATCC PTA 6475 to Caco-2 cells and mucus. Based on this, we propose that this LPxTG motif containing protein should be referred to as cell and mucus binding protein A (CmbA).

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CD133 Promotes Adhesion to the Ovarian Cancer Metastatic Niche

Cancer Growth and Metastasis ◽

10.1177/1179064418767882 ◽

2018 ◽

Vol 11 ◽

pp. 117906441876788 ◽

Cited By ~ 16

Author(s):

Lynn Roy ◽

Alexander Bobbs ◽

Rachel Sattler ◽

Jeffrey L Kurkewich ◽

Paige B Dausinas ◽

...

Keyword(s):

Ovarian Cancer ◽

Cancer Metastasis ◽

Ex Vivo ◽

Surface Protein ◽

Ovarian Cancer Cells ◽

Metastatic Niche ◽

Attractive Therapeutic Target ◽

Peritoneal Mesothelium

Cancer stem cells (CSCs) are an attractive therapeutic target due to their predicted role in both metastasis and chemoresistance. One of the most commonly agreed on markers for ovarian CSCs is the cell surface protein CD133. CD133+ ovarian CSCs have increased tumorigenicity, resistance to chemotherapy, and increased metastasis. Therefore, we were interested in defining how CD133 is regulated and whether it has a role in tumor metastasis. Previously we found that overexpression of the transcription factor, ARID3B, increased the expression of PROM1 (CD133 gene) in ovarian cancer cells in vitro and in xenograft tumors. We report that ARID3B directly regulates PROM1 expression. Importantly, in a xenograft mouse model of ovarian cancer, knockdown of PROM1 in cells expressing exogenous ARID3B resulted in increased survival time compared with cells expressing ARID3B and a control short hairpin RNA. This indicated that ARID3B regulation of PROM1 is critical for tumor growth. Moreover, we hypothesized that CD133 may affect metastatic spread. Given that the peritoneal mesothelium is a major site of ovarian cancer metastasis, we explored the role of PROM1 in mesothelial attachment. PROM1 expression increased adhesion to mesothelium in vitro and ex vivo. Collectively, our work demonstrates that ARID3B regulates PROM1 adhesion to the ovarian cancer metastatic niche.

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Interaction between Poly(ADP-ribose) and NuMA Contributes to Mitotic Spindle Pole Assembly

Molecular Biology of the Cell ◽

10.1091/mbc.e09-06-0477 ◽

2009 ◽

Vol 20 (21) ◽

pp. 4575-4585 ◽

Cited By ~ 58

Author(s):

Paul Chang ◽

Margaret Coughlin ◽

Timothy J. Mitchison

Keyword(s):

Binding Proteins ◽

Magnetic Beads ◽

Spindle Pole ◽

Covalent Modification ◽

Mitotic Spindles ◽

Spindle Poles ◽

Tankyrase 1 ◽

Mitotic Spindle Pole

Poly(ADP-ribose) (pADPr), made by PARP-5a/tankyrase-1, localizes to the poles of mitotic spindles and is required for bipolar spindle assembly, but its molecular function in the spindle is poorly understood. To investigate this, we localized pADPr at spindle poles by immuno-EM. We then developed a concentrated mitotic lysate system from HeLa cells to probe spindle pole assembly in vitro. Microtubule asters assembled in response to centrosomes and Ran-GTP in this system. Magnetic beads coated with pADPr, extended from PARP-5a, also triggered aster assembly, suggesting a functional role of the pADPr in spindle pole assembly. We found that PARP-5a is much more active in mitosis than interphase. We used mitotic PARP-5a, self-modified with pADPr chains, to capture mitosis-specific pADPr-binding proteins. Candidate binding proteins included the spindle pole protein NuMA previously shown to bind to PARP-5a directly. The rod domain of NuMA, expressed in bacteria, bound directly to pADPr. We propose that pADPr provides a dynamic cross-linking function at spindle poles by extending from covalent modification sites on PARP-5a and NuMA and binding noncovalently to NuMA and that this function helps promote assembly of exactly two poles.

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Neural induction and the structure of the target cell surface

Development ◽

10.1242/dev.70.1.171 ◽

1982 ◽

Vol 70 (1) ◽

pp. 171-187

Author(s):

A. M. Duprat ◽

L. Gualandris ◽

P. Rouge

Keyword(s):

Cell Surface ◽

Structural Integrity ◽

Neural Induction ◽

Active Role ◽

Target Cells ◽

Similar Treatment ◽

Structural Modifications ◽

Pleurodeles Waltl

Lectins (SBA and PSA) were used to provoke crowding and structural modifications of the presumptive ectoderm cell surface in order to investigate the role of the membrane organization of the competent target cells in neural induction. Are specific characteristics of the cell surface essential for this phenomenon to occur? From amphibian gastrulae, it is possible to obtain neural induction in vitro by association of presumptive ectoderm (target cells) with chordamesoderm (inductor tissue): 4 h of contact is sufficient in Pleurodeles waltl for transmission of the inductive signal. Very quickly, the treatment of the normal ectoderm by lectins (SBA-FITC or PSA-FITC) provoked surface modifications. Lectin-treatment (50 µg ml1−, 30 min) of presumptive ectoderm did not result in any neural induction. Lectin-treatment (50 µg ml1−, 30 min) of presumptive ectoderm previous to its association with the natural inductor for 4 h, disturbed the phenomenon: no induction. Similar treatment followed by association with the inductor for 24 h: induction. Treatment of SBA or PSA with their respective hapten inhibitors prior to addition to ectodermal cells completely blocked the suppressive effects on induction. The structural integrity of the membrane of competent target cells is necessary for neural induction to occur. The cell membrane could thus play, directly or indirectly, an active role in the specificity of this process

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Role of kallikrein-kinin system in pathogenesis of bacterial cell wall-induced inflammation

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1991.260.2.g213 ◽

1991 ◽

Vol 260 (2) ◽

pp. G213-G219 ◽

Cited By ~ 4

Author(s):

R. A. DeLa Cadena ◽

K. J. Laskin ◽

R. A. Pixley ◽

R. B. Sartor ◽

J. H. Schwab ◽

...

Keyword(s):

Cell Wall ◽

Acute Phase ◽

Bacterial Cell ◽

Chronic Phase ◽

Bacterial Cell Wall ◽

Kinin System ◽

Kallikrein Kinin System ◽

Bacterial Products

The plasma kallikrein-kinin system is activated in Gram-negative sepsis and typhoid fever, two diseases in which bacterial products have been shown to initiate inflammation. Because a single intraperitoneal injection of bacterial cell wall peptidoglycan-polysaccharide polymers from group A steptococci (PG-APS) into a Lewis rat produces a syndrome of relapsing polyarthritis and anemia, we investigated changes in the role of the kallikrein-kinin system in this model of inflammation. Coagulation studies after injection of PG-APS revealed an immediate and persistent decrease in prekallikrein levels. High-molecular-weight kininogen levels decreased significantly during the acute phase and correlated with the severity of arthritis. Factor XI levels were decreased only during the acute phase. Antithrombin III levels remained unchanged, indicating that neither decreased hepatic synthesis nor disseminated intravascular coagulation caused the decreased plasma contact factors. Plasma T-kininogen (an acute phase protein) was significantly elevated during the chronic phase. PG-APS failed to activate the contact system in vitro. Thus the kallikrein-kinin system plays an important role in this experimental model of inflammation, suggesting that activation of this system may play a role in the pathogenesis of inflammatory bowel disease and rheumatoid arthritis in which bacterial products might be etiologically important.

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MiR-26a-5p inhibits GSK3β expression and promotes cardiac hypertrophy in vitro

PeerJ ◽

10.7717/peerj.10371 ◽

2020 ◽

Vol 8 ◽

pp. e10371

Author(s):

Liqun Tang ◽

Jianhong Xie ◽

Xiaoqin Yu ◽

Yangyang Zheng

Keyword(s):

Cell Surface ◽

Cardiac Hypertrophy ◽

Surface Area ◽

Myocardial Cell ◽

Immunofluorescence Staining ◽

Cell Surface Area ◽

Direct Target

Background The role of miR-26a-5p expression in cardiac hypertrophy remains unclear. Herein, the effect of miR-26a-5p on cardiac hypertrophy was investigated using phenylephrine (PE)-induced cardiac hypertrophy in vitro and in a rat model of hypertension-induced hypertrophy in vivo. Methods The PE-induced cardiac hypertrophy models in vitro and vivo were established. To investigate the effect of miR-26a-5p activation on autophagy, the protein expression of autophagosome marker (LC3) and p62 was detected by western blot analysis. To explore the effect of miR-26a-5p activation on cardiac hypertrophy, the relative mRNA expression of cardiac hypertrophy related mark GSK3β was detected by qRT-PCR in vitro and vivo. In addition, immunofluorescence staining was used to detect cardiac hypertrophy related mark α-actinin. The cell surface area was measured by immunofluorescence staining. The direct target relationship between miR-26a-5p and GSK3β was confirmed by dual luciferase report. Results MiR-26a-5p was highly expressed in PE-induced cardiac hypertrophy. MiR-26a-5p promoted LC3II and decreased p62 expression in PE-induced cardiac hypertrophy in the presence or absence of lysosomal inhibitor. Furthermore, miR-26a-5p significantly inhibited GSK3β expression in vitro and in vivo. Dual luciferase report results confirmed that miR-26a-5p could directly target GSK3β. GSK3β overexpression significantly reversed the expression of cardiac hypertrophy-related markers including ANP, ACTA1 and MYH7. Immunofluorescence staining results demonstrated that miR-26a-5p promoted cardiac hypertrophy related protein α-actinin expression, and increased cell surface area in vitro and in vivo. Conclusion Our study revealed that miR-26a-5p promotes myocardial cell autophagy activation and cardiac hypertrophy by regulating GSK3β, which needs further research.

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Lectin Staining of Peritoneal Mesothelial Cells in Vitro

Peritoneal Dialysis International ◽

10.1177/089686089101100403 ◽

1991 ◽

Vol 11 (4) ◽

pp. 307-316 ◽

Cited By ~ 8

Author(s):

J. Thomas Hjelle ◽

Barbara T. Golinska ◽

Diane C. Waters ◽

Kevin R. Steidley ◽

Marcia A. Miller ◽

...

Keyword(s):

Tissue Culture ◽

Cell Surface ◽

Lectin Binding ◽

Mesothelial Cells ◽

Peritoneal Mesothelial Cells ◽

Lectin Staining ◽

Intracellular Vesicles ◽

Intracellular Fluorescence

A survey of lectin-binding specificities present on rodent and human mesothelial cells propagated and maintained in tissue culture was made using fluorescein isothiocynate conjugated (FITC) lectins. Rodent and human cells exhibited cell-associated fluorescence following exposure to the FITC-Iectins from C. ensiformis, T. vulgaris, A. hypogaea, E. cristagalli and B. simplicifolia, but not with lectins from G. max and D. biflorus. Rodent cells were also positive for FITC-M. pomifera lectin binding. Human, but not rodent, cells were positive for FITC T. purpureas lectin binding. Exposure of rabbit mesothelial cells in vitro to FITC-Iectins that bound to the cell surface resulted in the appearance of discrete loci of putatively intracellular fluorescence. Exposure of cells to ferritin-Iabelled T. vulgaris lectin at 37°C for as little as 7.5 minutes resulted in the appearance of ferritin-size particles in intracellular vesicles. These results demonstrate 1. the presence of lectinbinding sites in and on peritoneal mesothelial cells from rodents and humans and 2. a possible role of such sites in mediating the entry of lectin-Iike endogenous molecules into the vacuolar apparatus of these cells.

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Compartmentalization of protein traffic in insulin-sensitive cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1996.271.1.e1 ◽

1996 ◽

Vol 271 (1) ◽

pp. E1-E14 ◽

Cited By ~ 16

Author(s):

K. V. Kandror ◽

P. F. Pilch

Keyword(s):

Cell Surface ◽

Physiological Role ◽

Fat Cells ◽

Protein Traffic ◽

Glut 1 ◽

Glut 4 ◽

Factor Ii ◽

Nutritional Needs

Insulin-sensitive cells, adipocytes and myocytes, translocate a number of intracellular proteins to the cell surface in response to insulin. Among these proteins are glucose transporters 1 and 4 (GLUT-1 and GLUT-4, respectively), receptors for insulin-like growth factor II (IGF-II)/mannose 6-phosphate (Man-6-P) and transferrin, the aminopeptidase gp 160, caveolin, and a few others. In the case of insulin-activated glucose transport, this translocation has been proven to be the major, if not the only regulatory mechanism of this process. It seems likely that the cell surface recruitment of the IGF-II/Man-6-P and transferrin receptors also serves the nutritional needs of cells, whereas the physiological role of the aminopeptidase gp160 remains uncertain. Analysis of the compartmentalization and trafficking pathways of translocatable proteins in fat cells identified more than one population of recycling vesicles, although all have identical sedimentation coefficients and buoyant densities in vitro. GLUT-4-containing vesicles include essentially all the intracellular GLUT-4, gp160, and the acutely recycling populations of receptors for IGF-II/Man-6-P and transferrin. Besides these proteins, which can be considered as vesicle “cargo”, GLUT-4-containing vesicles have other components, like secretory carrier-associated membrane proteins (SCAMP), Rab(s), and vesicle-associated membrane protein (VAMP)/cellubrevin, which are ubiquitous to secretory vesicles and granules from different tissues. GLUT-1 and caveolin are excluded from GLUT-4-containing vesicles and form different vesicular populations of unknown polypeptide composition. In skeletal muscle, two independent populations of GLUT-4-containing vesicles are found, insulin sensitive and exercise sensitive, which explains the additive effect of insulin and exercise on glucose uptake. Both vesicular populations are similar to each other and to analogous vesicles in fat cells.

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