scholarly journals Degradation of heparin proteoglycan in cultured mouse mastocytoma cells

1987 ◽  
Vol 246 (2) ◽  
pp. 409-415 ◽  
Author(s):  
K G Jacobsson ◽  
U Lindahl
Keyword(s):  
Mast Cells ◽  
Substrate Specificity ◽  
Cell Cultures ◽  
Alkali Treatment ◽  
Degradation Pathway ◽  
Gel Chromatography ◽  
Pulse Labelling ◽  
Intracellular Degradation ◽  
Mastocytoma Cells ◽  
Intracellular Polysaccharide

Pulse-labelling of mouse mastocytoma cell cultures, established from ascites fluid, with inorganic [35S]sulphate for 1 h yielded labelled heparin proteoglycan containing polysaccharide chains of Mr 60,000-100,000. After chase incubation for 24 h most of the 35S appeared in intracellular polysaccharide fragments similar in size to commercially available heparin, Mr 5000-25,000, as indicated by gel chromatography. Products isolated from cultures after 6 h of chase incubation consisted of partially degraded free polysaccharide chains and, in addition, residual proteoglycans that were of smaller size than the proteoglycans initially pulse-labelled. The polysaccharide chains released by alkali treatment from the residual chase-incubated proteoglycans were of the same size as the chains derived from proteoglycans after 1 h of pulse labelling. These results suggest that the intracellular degradation of heparin proteoglycan to polysaccharide fragments is initiated by release of intact polysaccharide chains, probably by action of a peptidase, and is pursued through cleavage of these chains by an endoglycosidase. An endoglucuronidase with stringent substrate specificity [Thunberg, Bäckström, Wasteson, Ogren & Lindahl (1982) J. Biol. Chem. 257, 10278-10282] has previously been implicated in the latter step. Cultures of more purified mastocytoma cells (essentially devoid of macrophages) did not metabolize [35S]heparin proteoglycan to polysaccharide fragments, but instead accumulated free intact polysaccharide chains, i.e. the postulated intermediate of the complete degradation pathway. When such purified cells were co-cultured with adherent mouse peritoneal cells, presumably macrophages, formation of polysaccharide fragments was observed. It is tentatively proposed that the expression of endoglucuronidase activity by the mast cells depends on collaboration between these cells and macrophages.

scholarly journals Comparative biosynthesis, covalent post-translational modifications and efficiency of prosegment cleavage of the prohormone convertases PC1 and PC2: glycosylation, sulphation and identification of the intracellular site of prosegment cleavage of PC1 and PC2

1993 ◽  
Vol 294 (3) ◽  
pp. 735-743 ◽  
Author(s):  
S Benjannet ◽  
N Rondeau ◽  
L Paquet ◽  
A Boudreault ◽  
C Lazure ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Brefeldin A ◽  
Pulse Labelling ◽  
Prohormone Convertases ◽  
Post Translational Modifications ◽  
Intracellular Degradation ◽  
Golgi Compartment ◽  
Carbonyl Cyanide ◽  
Insulinoma Cells

We present herein the pulse-chase analysis of the biosynthesis of the prohormone convertases PC1 and PC2 in the endocrine GH4C1 cells infected with vaccinia virus recombinants expressing these convertases. Characterization of the pulse-labelled enzymes demonstrated that pro-PC1 (88 kDa) is cleaved into PC1 (83 kDa) and pro-PC2 (75 kDa) into PC2 (68 kDa). Secretion of glycosylated and sulphated PC1 (84 kDa) occurs about 30 min after the onset of biosynthesis, whereas glycosylated and sulphated PC2 (68 kDa) is detected in the medium after between 1 and 2 h. Furthermore, in the case of pro-PC2 only, we observed that a fraction of this precursor escapes glycosylation. A small proportion (about 5%) of the intracellular glycosylated pro-PC2 (75 kDa) is sulphated, and it is this glycosylated and sulphated precursor that is cleaved into the secretable 68 kDa form of PC2. Major differences in the carbohydrate structures of PC1 and PC2 are demonstrated by the resistance of the secreted PC1 to endoglycosidase H digestion and sensitivity of the secreted PC2 to this enzyme. Inhibition of N-glycosylation with tunicamycin caused a dramatic intracellular degradation of these convertases within the endoplasmic reticulum, with the net effect of a reduction in the available activity of PC1 and PC2. These results emphasize the importance of N-glycosylation in the folding and stability of PC1 and PC2. Pulse-labelling experiments in uninfected mouse beta TC3 and rat Rin m5F insulinoma cells, which endogenously synthesize PC2, showed that, as in infected GH4C1 cells, pro-PC2 predominates intracellularly. In order to define the site of prosegment cleavage, pulse-chase analysis was performed at low temperature (15 degrees C) or after treatment of GH4C1 cells with either brefeldin A or carbonyl cyanide m-chlorophenylhydrazone. These results demonstrated that the onset of the conversions of pro-PC1 into PC1 and non-glycosylated pro-PC2 into PC2 (65 kDa) occur in a pre-Golgi compartment, presumably within the endoplasmic reticulum. In contrast, pulse labelling in the presence of Na(2)35SO4 demonstrated that the processing of glycosylated and sulphated pro-PC2 occurs within the Golgi apparatus. In order to test the possibility that zymogen processing is performed by furin, we co-expressed this convertase with either pro-PC1 or pro-PC2. The data demonstrated the inability of furin to cleave either proenzyme.

scholarly journals Identification of glyA (Encoding Serine Hydroxymethyltransferase) and Its Use Together with the Exporter ThrE To Increase l-Threonine Accumulation by Corynebacterium glutamicum

2002 ◽  
Vol 68 (7) ◽  
pp. 3321-3327 ◽  
Author(s):  
Petra Simic ◽  
Juliane Willuhn ◽  
Hermann Sahm ◽  
Lothar Eggeling
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Corynebacterium Glutamicum ◽  
Serine Hydroxymethyltransferase ◽  
Acid Formation ◽  
Initial Activity ◽  
Intracellular Degradation ◽  
Cleavage Activity ◽  
Substrate Reduction

ABSTRACT l-Threonine can be made by the amino acid-producing bacterium Corynebacterium glutamicum. However, in the course of this process, some of the l-threonine is degraded to glycine. We detected an aldole cleavage activity of l-threonine in crude extracts with an activity of 2.2 nmol min−1 (mg of protein)−1. In order to discover the molecular reason for this activity, we cloned glyA, encoding serine hydroxymethyltransferase (SHMT). By using affinity-tagged glyA, SHMT was isolated and its substrate specificity was determined. The aldole cleavage activity of purified SHMT with l-threonine as the substrate was 1.3 μmol min−1 (mg of protein)−1, which was 4% of that with l-serine as substrate. Reduction of SHMT activity in vivo was obtained by placing the essential glyA gene in the chromosome under the control of P tac , making glyA expression isopropylthiogalactopyranoside dependent. In this way, the SHMT activity in an l-threonine producer was reduced to 8% of the initial activity, which led to a 41% reduction in glycine, while l-threonine was simultaneously increased by 49%. The intracellular availability of l-threonine to aldole cleavage was also reduced by overexpressing the l-threonine exporter thrE. In C. glutamicum DR-17, which overexpresses thrE, accumulation of 67 mM instead of 49 mM l-threonine was obtained. This shows that the potential for amino acid formation can be considerably improved by reducing its intracellular degradation and increasing its export.

scholarly journals Biosynthesis of heparin. Relationship between the polymerization and sulphation processes

1989 ◽  
Vol 261 (3) ◽  
pp. 999-1007 ◽  
Author(s):  
K Lidholt ◽  
L Kjellén ◽  
U Lindahl
Keyword(s):  
Microsomal Fraction ◽  
Alkali Treatment ◽  
Deae Cellulose ◽  
Molecular Size ◽  
Chain Elongation ◽  
Gel Chromatography ◽  
Sulphated Polysaccharide ◽  
Transient Interactions ◽  
Sulphated Proteoglycan

Incubation of a mouse mastocytoma microsomal fraction with UDP-[3H]GlcA and UDP-GlcNAc yielded proteoglycans containing non-sulphated polysaccharide chains. Similar incubations performed in the presence of sulphate donor 3′-phosphoadenosine 5′-phosphosulphate (PAPS) produced both sulphated and non-sulphated proteoglycans, which were separated by chromatography on DEAE-cellulose Analysis by gel chromatography of single polysaccharide chains, released from the proteoglycans by alkali treatment, showed that the non-sulphated chains produced during incubation for 5 min or 25 min, either in the absence or in the presence of PAPS, were of fairly small molecular size, with an average peak Mr of approx. 10 x 10(3)-15 x 10(3). In contrast, the sulphated chains exceeded Mr 100 x 10(3) Pulse-chase experiments suggested that sulphated chains were capable of further elongation. These results indicate that sulphation promotes, by so far unknown mechanisms, further chain elongation. Sulphated proteoglycan (retarded on DEAE-cellulose chromatography) isolated after similar incubation of the microsomal fraction for 1 min only was found to contain a mixture of sulphated and virtually non-sulphated polysaccharide chains. However, when [35S]PAPS was included in the incubations, some 35S was found to be associated, essentially as N-sulphate groups, also with the latter type of chains, preferentially the high-Mr fraction. These results are interpreted in terms of a biosynthetic model by which the heparin proteoglycan is generated through transient interactions of macromolecular intermediates with distinctly separate complexes of membranebound enzymes.

scholarly journals Constitutive activation of c-kit in FMA3 murine mastocytoma cells caused by deletion of seven amino acids at the juxtamembrane domain

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 273-283 ◽  
Author(s):  
T Tsujimura ◽  
M Morimoto ◽  
K Hashimoto ◽  
Y Moriyama ◽  
H Kitayama ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mast Cells ◽  
Cell Line ◽  
Point Mutation ◽  
Coding Region ◽  
Juxtamembrane Domain ◽  
Base Pairs ◽  
Constitutive Activation ◽  
Mastocytoma Cells ◽  
Type C

Abstract A peculiar point mutation results in constitutive activation of c-kit receptor tyrosine kinase (KIT) in three different tumor mast cell lines; ie, the HMC-1, P-815, and RBL-2H3. Because constitutive activation of KIT was also observed in the FMA3 mouse mastocytoma cell line, we investigated the molecular mechanism. Sequencing of the whole coding region of the c-kit showed that the point mutation found in HMC- 1, P-815, and RBL-2H3 cells was absent in FMA3 cells and that the c-kit cDNA of FMA3 cells carried an in-frame deletion of 21 base pairs (bp) encoding Thr-Gln-Leu-Pro-Tyr-Asp-His at codons 573 to 579 at the juxtamembrane domain. The FMA3-type c-kit cDNA with 21 bp deletion was introduced into the IC-2 cell line, which was derived from murine cultured mast cells. IC-2 cells were dependent on interleukin (IL)-3 and did not express KIT on the surface. In IC-2 cells introduced with the FMA3-type c-kit cDNA, KIT was constitutively phosphorylated on tyrosines and activated. Moreover, the FMA3-type KIT was dimerized without the stimulation by stem cell factor (SCF), a ligand for KIT. The spontaneously dimerized FMA3-type KIT without SCF binding was not internalized even after the activation. IC-2 cells expressing the FMA3- type KIT grew in suspension culture without IL-3 and SCF and became leukemic in nude athymic mice. The deletion of seven amino acids at the juxtamembrane domain appeared to be a new activating mutation of KIT that might be involved in neoplastic growth of mast cells.

Spectroscopic studies of rat mast cells, mouse mastocytoma cells, and compound 48/80—I

1981 ◽  
Vol 30 (4) ◽  
pp. 277-282 ◽  
Author(s):  
Mary J. Ortner ◽  
Norbert Turek ◽  
Colin F. Chignell
Keyword(s):  
Mast Cells ◽  
Spectroscopic Studies ◽  
Mastocytoma Cells ◽  
Rat Mast Cells

scholarly journals Differentiation in Murine Mastocytoma Induced by Macrophage Gangliosides

2000 ◽  
Vol 55 (11-12) ◽  
pp. 1004-1010 ◽  
Author(s):  
Lars Schaade ◽  
Reiner Thomssen ◽  
Klaus Ritter
Keyword(s):  
Cell Division ◽  
Mast Cells ◽  
Tumor Cells ◽  
Morphological Changes ◽  
Complete Inhibition ◽  
Mouse Macrophages ◽  
Mastocytoma Cells

In the membrane of mouse macrophages two gangliosides were detected which inhibit the division of murine mastocytoma P815 tumor cells. The two gangliosides were incorporated into the cytoplasmatic membrane of mastocytoma cells. The concentration necessary to achieve a complete inhibition of P815 tum or cell division is about 1 [_im for both effective gangliosides. Macrophage ganglioside-induced inhibition of cell division is accompanied by morphological changes of the mastocytoma cells. While the cells are rounding, their diameter increases and serotonin and granules appear in the cytoplasm of the enlarged cells. Our findings suggest that macrophage gangliosides may differentiate mastocytoma cells into mast cells.

Regulation of autophagy by mTOR-dependent and mTOR-independent pathways: autophagy dysfunction in neurodegenerative diseases and therapeutic application of autophagy enhancers

2013 ◽  
Vol 41 (5) ◽  
pp. 1103-1130 ◽  
Author(s):  
Sovan Sarkar
Keyword(s):  
Neurodegenerative Diseases ◽  
Small Molecules ◽  
Mammalian Target Of Rapamycin ◽  
Degradation Pathway ◽  
Therapeutic Application ◽  
Mutant Proteins ◽  
Chemical Inducers ◽  
Intracellular Degradation ◽  
Wide Range ◽  
Potential Therapeutic Application

Autophagy is an intracellular degradation pathway essential for cellular and energy homoeostasis. It functions in the clearance of misfolded proteins and damaged organelles, as well as recycling of cytosolic components during starvation to compensate for nutrient deprivation. This process is regulated by mTOR (mammalian target of rapamycin)-dependent and mTOR-independent pathways that are amenable to chemical perturbations. Several small molecules modulating autophagy have been identified that have potential therapeutic application in diverse human diseases, including neurodegeneration. Neurodegeneration-associated aggregation-prone proteins are predominantly degraded by autophagy and therefore stimulating this process with chemical inducers is beneficial in a wide range of transgenic disease models. Emerging evidence indicates that compromised autophagy contributes to the aetiology of various neurodegenerative diseases related to protein conformational disorders by causing the accumulation of mutant proteins and cellular toxicity. Combining the knowledge of autophagy dysfunction and the mechanism of drug action may thus be rational for designing targeted therapy. The present review describes the cellular signalling pathways regulating mammalian autophagy and highlights the potential therapeutic application of autophagy inducers in neurodegenerative disorders.

scholarly journals Chymase and tryptase in dog mastocytoma cells: asynchronous expression as revealed by enzyme cytochemical staining.

1988 ◽  
Vol 36 (8) ◽  
pp. 1053-1060 ◽  
Author(s):  
G H Caughey ◽  
N F Viro ◽  
L D Calonico ◽  
D M McDonald ◽  
S C Lazarus ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Staining Technique ◽  
Staining Intensity ◽  
Cell Development ◽  
Cytochemical Staining ◽  
Mast Cell Tumors ◽  
General Utility ◽  
Developmental Relationship ◽  
Mastocytoma Cells

Mast cell populations can be distinguished by differences in the content and substrate specificity of their two major cytoplasmic granule proteases, the chymases and the tryptases. To explore the origins of differences in the types of proteases present in mast cells, we used a double cytochemical staining technique to reveal both chymase and tryptase in cells from four lines of dog mast cell tumors containing both enzymes. We expected that if chymase and tryptase were expressed together during cell development the relative staining intensity of chymase compared to tryptase would be constant among different cells of each tumor. Instead, we found substantial variation in the relative intensity of chymase and tryptase staining among cells of a given mastocytoma line, each of which contained cells presumed to be monoclonal in origin but heterogeneous with respect to cell development. The overall staining intensity for chymase or tryptase correlated with the amount of protease activity in extracts of tumor homogenates. Staining specificity was established by use of selective inhibitors and competitive substrates and was tested on various types of dog cells obtained by bronchoalveolar lavage. The results suggest that active chymase and tryptase may be expressed differently during mast cell differentiation and support the possibility of a close developmental relationship between mast cells differing in protease phenotype. Moreover, the success of the staining procedures applied to mastocytoma cells suggests that they may be of general utility in phenotyping of mast cells according to the protease activities present in their granules.

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