scholarly journals The TFF Peptides xP1 and xP4 Appear in Distinctive Forms in the Xenopus laevis Gastric Mucosa: Indications for Different Protective Functions

2019 ◽  
Vol 20 (23) ◽  
pp. 6052 ◽  
Author(s):  
Stürmer ◽  
Reising ◽  
Hoffmann
Keyword(s):  
Gastric Mucosa ◽  
Xenopus Laevis ◽  
Molecular Mass ◽  
Molecular Forms ◽  
Binding Studies ◽  
Mucus Barrier ◽  
A Minor ◽  
Tff Peptides ◽  
Mass Form

The gastric secretory trefoil factor family (TFF) peptides xP1 and xP4 are the Xenopus laevis orthologs of mammalian TFF1 and TFF2, respectively. The aim of this study was to analyze the molecular forms of xP1 and xP4 in the X. laevis gastric mucosa by FPLC. xP1 mainly occurred in a monomeric low-molecular-mass form and only a minor subset is associated with the mucus fraction. The occurrence of monomeric xP1 is unexpected because of its odd number of cysteine residues. Probably a conserved acidic residue flanking Cys55 allows monomeric secretion. Furthermore, Cys55 is probably post-translationally modified. For the first time, we hypothesize that the free thiol of monomeric xP1-and probably also its mammalian ortholog TFF1-could have a protective scavenger function, e.g., for reactive oxygen/nitrogen species. In contrast, xP4 mainly occurs in a high-molecular-mass form and is non-covalently bound to a mucin similarly as TFF2. In vitro binding studies with radioactively labeled porcine TFF2 even showed binding to X. laevis gastric mucin. Thus, xP4 is expected to bind as a lectin to an evolutionary conserved sugar epitope of the X. laevis ortholog of mucin MUC6 creating a tight mucus barrier. Taken together, xP1 and xP4 appear to have different gastric protective functions.

scholarly journals Different Forms of TFF2, A Lectin of the Human Gastric Mucus Barrier: In Vitro Binding Studies

2019 ◽  
Vol 20 (23) ◽  
pp. 5871 ◽  
Author(s):  
Franziska Heuer ◽  
René Stürmer ◽  
Jörn Heuer ◽  
Thomas Kalinski ◽  
Antje Lemke ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Gastric Mucus ◽  
Binding Studies ◽  
Binding Characteristics ◽  
Duodenal Glands ◽  
Vitro Binding ◽  
Trefoil Factor Family ◽  
Mucus Barrier ◽  
Mass Form

Trefoil factor family 2 (TFF2) and the mucin MUC6 are co-secreted from human gastric and duodenal glands. TFF2 binds MUC6 as a lectin and is a constituent of the gastric mucus. Herein, we investigated human gastric extracts by FPLC and identified mainly high- but also low-molecular-mass forms of TFF2. From the high-molecular-mass forms, TFF2 can be completely released by boiling in SDS or by harsh denaturing extraction. The low-molecular-mass form representing monomeric TFF2 can be washed out in part from gastric mucosa specimens with buffer. Overlay assays with radioactively labeled TFF2 revealed binding to the mucin MUC6 and not MUC5AC. This binding is modulated by Ca2+ and can be blocked by the lectin GSA-II and the monoclonal antibody HIK1083. TFF2 binding was also inhibited by Me-β-Gal, but not the α anomer. Thus, both the α1,4GlcNAc as well as the juxtaperipheral β-galactoside residues of the characteristic GlcNAcα1→4Galβ1→R moiety of human MUC6 are essential for TFF2 binding. Furthermore, there are major differences in the TFF2 binding characteristics when human is compared with the porcine system. Taken together, TFF2 appears to fulfill an important role in stabilizing the inner insoluble gastric mucus barrier layer, particularly by its binding to the mucin MUC6.

scholarly journals Trefoil Factor Family (TFF) Modules Are Characteristic Constituents of Separate Mucin Complexes in the Xenopus laevis Integumentary Mucus: In Vitro Binding Studies with FIM-A.1

2020 ◽  
Vol 21 (7) ◽  
pp. 2400 ◽  
Author(s):  
René Stürmer ◽  
Jana Reising ◽  
Werner Hoffmann
Keyword(s):  
Molecular Mass ◽  
Complex I ◽  
Size Exclusion ◽  
Trefoil Factor ◽  
Binding Studies ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
Exclusion Chromatography ◽  
Trefoil Factor Family

The skin of the frog Xenopus laeevis is protected from microbial infections by a mucus barrier that contains frog integumentary mucins (FIM)-A.1, FIM-B.1, and FIM-C.1. These gel-forming mucins are synthesized in mucous glands consisting of ordinary mucous cells and one or more cone cells at the gland base. FIM-A.1 and FIM-C.1 are unique because their cysteine-rich domains belong to the trefoil factor family (TFF). Furthermore, FIM-A.1 is unusually short (about 400 amino acid residues). In contrast, FIM-B.1 contains cysteine-rich von Willebrand D (vWD) domains. Here, we separate skin extracts by the use of size exclusion chromatography and analyze the distribution of FIM-A.1 and FIM-C.1. Two mucin complexes were detected, i.e., a high-molecular-mass Complex I, which contains FIM-C.1 and little FIM-A.1, whereas Complex II is of lower molecular mass and contains the bulk of FIM-A.1. We purified FIM-A.1 by a combination of size-exclusion chromatography (SEC) and anion-exchange chromatography and performed first in vitro binding studies with radioactively labeled FIM-A.1. Binding of 125I-labeled FIM-A.1 to the high-molecular-mass Complex I was observed. We hypothesize that the presence of FIM-A.1 in Complex I is likely due to lectin interactions, e.g., with FIM-C.1, creating a complex mucus network.

scholarly journals Evidence that there are two copies of subunit c″ in V0 complexes in the vacuolar H+-ATPase

2002 ◽  
Vol 366 (3) ◽  
pp. 911-919 ◽  
Author(s):  
Lucien C.D. GIBSON ◽  
Graham CADWALLADER ◽  
Malcolm E. FINBOW
Keyword(s):  
Copy Number ◽  
Transmembrane Domain ◽  
Free Form ◽  
Binding Studies ◽  
Subunit C ◽  
Dimeric Form ◽  
A Minor ◽  
Mutant Forms ◽  
Vacuolar Membranes

The proton-translocating core of eukaryotic vacuolar H+-ATPase (V-ATPase), V0 consists of a hexameric arrangement of transmembrane α-helices formed from the related polypeptides, subunit c and subunit c′′. The former is comprised of four transmembrane α-helices, whilst the latter has an extra transmembrane domain at its N-terminus. In addition, the fungal form of V0 contains a minor subunit c-related polypeptide, subunit c′. All three are required for activity of the proton pump in Saccharomyces cerevisiae. We have introduced cysteine residues in the N-terminal extension of subunit c′′ in a cysteine-free form. All mutant forms are active in the V-ATPase from S. cerevisiae. Oxidation of vacuolar membranes containing the cysteine-replaced forms gave a cross-linked product of 42000Da. Analysis of this species showed it to be a dimeric form of subunit c′′, and further studies confirmed there are two copies of subunit c′′ in the V-ATPases in which it is present. Co-expression of double cysteine-replaced forms of both subunit c and c′′ gave rise to only homotypic cross-linked forms. Also, subunit c oligomeric complexes are present in vacuolar membranes in the absence of subunit c′′, consistent with previous observations showing hexameric arrangements of subunit c in gap-junction-like membranes. In vitro studies showed subunit c′′ can bind to subunit c and itself. The extent of binding can be increased by removal of the N-terminal domain of subunit c′′. This domain may therefore function to limit the copy number of subunit c′′ in V0. A deletion study shows that the domain is essential for the activity of subunit c′′. The results can be combined into a model of V0 which contains two subunit c′′ protomers with the extra transmembrane domain located toward the central pore. Thus the predicted stoichiometry of V0 in which subunit c′′ is present is subunit c3:subunit c′1 :subunit c′2. On the basis of the mutational and binding studies, it seems likely that two copies of subunit c′′ are next to each other.

scholarly journals The Tumor Suppressor TFF1 Occurs in Different Forms and Interacts with Multiple Partners in the Human Gastric Mucus Barrier: Indications for Diverse Protective Functions

2020 ◽  
Vol 21 (7) ◽  
pp. 2508 ◽  
Author(s):  
Jörn Heuer ◽  
Franziska Heuer ◽  
René Stürmer ◽  
Sönke Harder ◽  
Hartmut Schlüter ◽  
...  
Keyword(s):  
Gastric Mucosa ◽  
Tumor Suppressor ◽  
Disulfide Bonds ◽  
Thiol Group ◽  
Molecular Forms ◽  
Human Gastric Mucosa ◽  
Monomeric Form ◽  
Gastric Mucus ◽  
Multiple Partners

TFF1 is a protective peptide of the Trefoil Factor Family (TFF), which is co-secreted with the mucin MUC5AC, gastrokine 2 (GKN2), and IgG Fc binding protein (FCGBP) from gastric surface mucous cells. Tff1-deficient mice obligatorily develop antropyloric adenoma and about 30% progress to carcinomas, indicating that Tff1 is a tumor suppressor. As a hallmark, TFF1 contains seven cysteine residues with three disulfide bonds stabilizing the conserved TFF domain. Here, we systematically investigated the molecular forms of TFF1 in the human gastric mucosa. TFF1 mainly occurs in an unusual monomeric form, but also as a homodimer. Furthermore, minor amounts of TFF1 form heterodimers with GKN2, FCGBP, and an unknown partner protein, respectively. TFF1 also binds to the mucin MUC6 in vitro, as shown by overlay assays with synthetic 125I-labeled TFF1 homodimer. The dominant presence of a monomeric form with a free thiol group at Cys-58 is in agreement with previous studies in Xenopus laevis and mouse. Cys-58 is likely highly reactive due to flanking acid residues (PPEEEC58EF) and might act as a scavenger for extracellular reactive oxygen/nitrogen species protecting the gastric mucosa from damage by oxidative stress, e.g., H2O2 generated by dual oxidase (DUOX).

scholarly journals ADP-ribosylation in isolated nuclei of Physarum polycephalum

1988 ◽  
Vol 253 (3) ◽  
pp. 859-867 ◽  
Author(s):  
G Golderer ◽  
R Schneider ◽  
B Auer ◽  
P Loidl ◽  
P Gröbner
Keyword(s):  
Cell Cycle ◽  
Molecular Mass ◽  
Physarum Polycephalum ◽  
High Mobility ◽  
Nuclear Proteins ◽  
Isolated Nuclei ◽  
Adp Ribosylation ◽  
Adp Ribosyltransferase ◽  
Mass Form

ADP-ribosylation of histones and non-histone nuclear proteins was studied in isolated nuclei during the naturally synchronous cell cycle of Physarum polycephalum. Aside from ADP-ribosyltransferase (ADPRT) itself, histones and high mobility group-like proteins are the main acceptors for ADP-ribose. The majority of these ADP-ribose residues is NH2OH-labile. ADP-ribosylation of the nuclear proteins is periodic during the cell cycle with maximum incorporation in early to mid G2-phase. In activity gels two enzyme forms with Mr of 115,000 and 75,000 can be identified. Both enzyme forms are present at a constant ratio of 3:1 during the cell cycle. The higher molecular mass form cannot be converted in vitro to the low molecular mass form, excluding an artificial degradation during isolation of nuclei. The ADPRT forms were purified and separated by h.p.l.c. The low molecular mass form is inhibited by different ADPRT inhibitors to a stronger extent and is the main acceptor for auto-ADP-ribosylation. The high molecular mass form is only moderately auto-ADP-ribosylated.

The Identification and Significance of a Thr→Pro Polymorphism in Kringle IV Type 8 of Apolipoprotein(a)

1997 ◽  
Vol 77 (05) ◽  
pp. 0949-0954 ◽  
Author(s):  
J Prins ◽  
F R Lues ◽  
Y Y van der Hoek ◽  
J J.P Kastelein ◽  
B N Bouma ◽  
...  
Keyword(s):  
Case Control Study ◽  
Amino Acid Position ◽  
Case Control ◽  
Binding Studies ◽  
Binding Characteristics ◽  
Apolipoprotein A ◽  
Significant Independent Risk Factor ◽  
And Gender ◽  
Control Study

SummaryElevated plasma levels of lipoprotein(a) [Lp(a)] represent a significant independent risk factor for the development of atherosclerosis. Interindividual levels of apo(a) vary over 1000-fold and are mainly due to inheritance that is linked to the locus of the apolipoprotein(a) [apo(a)] gene. The apo(a) gene encodes multiple repeats of a sequence exhibiting up to 85% DNA sequence homology with plasminogen kringle IV (K.IV), a lysine binding domain. In our search for sequence polymorphisms in the K.IV coding domain, we identified a polymorphism predicting a Thr→Pro substitution located at amino acid position 12 of kringle IV type 8 of apo(a). The functional and clinical significance of this polymorphism was analysed in a case-control study and by comparing the in vitro lysine binding characteristics of the two Lp(a) subtypes.The case-control study (involving 153 subjects having symptomatic atherosclerosis and 153 age and gender matched normolipidemic controls) revealed an overall allele frequency for the Thr12-→Pro substitution in kringle IV type 8 of 14% and a negative association between presence of the Pro12-subtype and symptomatic atherosclerosis (p <0.03). The in vitro lysine binding studies, using Lp(a) isolated from subjects homozygous for either Thr12 or Pro12 in K.IV type 8, revealed comparable lysine-Sepharose binding fractions for the two subtypes. The binding affinity (Kd) for immobilised plasmin degraded des- AA-fibrin (DesafibTM-X) was also comparable for the two subtypes, however a decreased maximal attainable binding (Bmax) for immobilised desafibTM-X was observed for the Pro12-subtype Lp(a).

scholarly journals In-Vitro Evaluation of the Antioxidant and Anti-Inflammatory Activity of Volatile Compounds and Minerals in Five Different Onion Varieties

Separations ◽  
2021 ◽  
Vol 8 (5) ◽  
pp. 57
Author(s):  
Rokayya Sami ◽  
Abeer Elhakem ◽  
Mona Alharbi ◽  
Manal Almatrafi ◽  
Nada Benajiba ◽  
...  
Keyword(s):  
Volatile Compounds ◽  
Dimethyl Sulfide ◽  
Antioxidant Activities ◽  
Dry Weight ◽  
No Production ◽  
Oxidative Stresses ◽  
Anti Inflammatory ◽  
Red Variety ◽  
A Minor

Onions contain high antioxidants compounds that fight inflammation against many diseases. The purpose was to investigate some selected bioactive activities of onion varieties (Yellow, Red, Green, Leek, and Baby). Antioxidant assays and anti-inflammatory activities such as NO production with the addition of some bioactive components were determined and analyzed by using a spectrophotometer. Gas chromatography and mass spectrometry (GC–MS) was used for the volatile compounds, while an Atomic absorption spectrometer was used for mineral determinations. Red variety achieved the highest antioxidant activities. The total flavonoids were between (12.56 and 353.53 mg Quercetin/gin dry weight) (dw) and the total phenol was (8.75–25.73 mg/g dw). Leek, Yellow and Green extracts achieved highly anti-inflammatory values (3.71–4.01 μg/mL) followed by Red and Baby extracts, respectively. The highest contents of sodium, potassium, zinc, and calcium were established for Red onions. Furfuraldehyde, 5-Methyl-2-furfuraldehyde, 2-Methyl-2-pentenal, and 1-Propanethiol were the most predominant, followed by a minor abundance of the other compounds such as Dimethyl sulfide, Methyl allyl disulfide, Methyl-trans-propenyl-disulfide, and Methyl propyl disulfide. The results recommend that these varieties could act as sources of essential antioxidants and anti-inflammatories to decrease inflammation and oxidative stresses, especially red onions that recorded high activities.

Structural and Genetic Aspects of Proline-rich Proteins

1987 ◽  
Vol 66 (2) ◽  
pp. 457-461 ◽  
Author(s):  
A. Bennick
Keyword(s):  
General Structure ◽  
Single Gene ◽  
Conclusive Evidence ◽  
In Vitro Translation ◽  
Binding Studies ◽  
Nmr Studies ◽  
Post Translational Modifications ◽  
Single Precursor ◽  
Made In

Considerable advances have been made in the genetics of salivary proline-rich proteins (PRP). The genes for acidic, basic, and glycosylated PRP have been cloned. They code for precursor proteins that all have an acidic N-terminal followed by proline-rich repeat sequences. Structural studies on secreted proteins have demonstrated that not only acidic but also some basic PRPs have this general structure. It is possible that mRNA for different PRP may have originated from a single gene by differential mRNA splicing, but post-translational cleavages of the primary translation product apparently also occur. In vitro translation of salivary gland mRNA results in a single precursor protein for acidic PRP. Such in vitro translated protein can be cleaved by salivary kallikrein, giving rise to two commonly secreted acidic PRPs, and kallikrein or kallikrein-like enzymes may be responsible for other post-translational cleavages of PRPs. Acidic as well as some basic PRPs are phosphorylated. A protein kinase has been demonstrated in salivary glands which phosphorylates the PRPs and other secreted salivary proteins in a cAMP and Ca2+-calmodulinindependent manner. Knowledge of the conformation of PRPs is limited. There is no conclusive evidence of polyproline-like structure in the proline-rich part of PRPs. Ca2+ binding studies on acidic PRPs indicate that there is interaction between the Ca2+ binding N-terminal end and the proline-rich C-terminal part. This interaction is relieved by modification of arginine side-chains. 1H, 32P, and 43Ca NMR studies have further elucidated the conformation of acidic PRPs in solution. Present evidence shows that salivary PRPs constitute a unique superfamily of proteins which pose a number of interesting questions concerning gene structure, pre- and post-translational modifications, and protein conformation.

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