Biomedical subjects

U Rauch

Publications and source records attributed to U Rauch.

54 records · Page 3Linked to original sources

PubMed1997

Adenine nucleotide translocator in dilated cardiomyopathy: pathophysiological alterations in expression and function.

Several findings pointed to an insufficient energy supply in heart muscle tissue of patients suffering from dilated cardiomyopathy (DCM). We found a lowered ANT transport capacity of the adenine nucleotide translocator (ANT), the only transport system for ATP and ADP in eucaryotic cells, in explanted hearts of DCM patients. The reduced ANT transport rate was accompanied by a marked elevation in total ANT protein caused by an increase in ANT 1 isoform protein. Simultaneously, a reduction in ANT 2 transcripts and an unchanged ANT 3 expression was observed. In contrast, patients with ischemic or valvular heart disease showed no alteration in ANT function or expression, which indicates the disease-specificity of these findings. With regard to autoimmunological and viral processes, which are thought to play an important role in the pathogenesis of DCM, we could show that the ANT function is reduced in the hearts of A.SW/Sn-J mice infected with the enterovirus Cox-sackie B3, and in those of guinea pigs immunized with purified myocardial ANT. Both treatments led to autoimmunological reactions against the ANT protein, that reduce the myocardial ANT transport capacity, thus disturbing energy metabolism and consequently depressing heart function. In contrast to these animal models, no restriction in ANT capacity was observed in hypoxic hearts of guinea pigs, which corresponds to the findings of unaffected ANT function in ischemic human hearts.

Animals↗

PubMed1996

Analysis of neurocan structures interacting with the neural cell adhesion molecule N-CAM.

Neurocan is a brain-specific chondroitin sulfate proteoglycan, which has been shown to bind to the neural cell adhesion molecule N-CAM and to inhibit its homophilic interaction. To study in more detail the structures of neurocan responsible for this interaction, various recombinant neurocan fragments were generated. The ability of these fragments to interact with N-CAM was investigated in several different in vitro assay systems, enzyme-linked immunosorbent assay-type binding assays, Covasphere-aggregation assays, and assays based on an optical biosensor (BIAcoreTM) system. The analysis of the homophilic N-CAM interaction in the BIAcore system revealed a KD of 64 nM. This homophilic interaction could be reduced by preincubation of soluble N-CAM with neurocan. Direct binding of N-CAM to immobilized neurocan core protein and recombinant neurocan fragments could also be demonstrated, and KD values between 25 and 100 nM were obtained. In addition, direct binding of N-CAM to chondroitin sulfate could be demonstrated. Binding of N-CAM to the immobilized neurocan core protein could be inhibited with all recombinant fragments containing chondroitin sulfate or major parts of the mucin-like central region of neurocan. For the inhibition of homophilic N-CAM interactions, however, a combination of globular and extended structures was required.

Animals↗

PubMed1996

Structural and electron microscopic analysis of neurocan and recombinant neurocan fragments.

Neurocan, a nervous tissue-specific chondroitin sulfate proteoglycan of the aggrecan family which has been shown to interact with neural cell adhesion molecules and tenascin, could be visualized by rotary shadowing electron microscopy as two globular domains interconnected by an extended flexible filament of 60-90 nm. Several recombinant neurocan fragments generated in the human embryonic kidney cell line 293 represent as observed by electron microscopy the expected parts of this structure, which indicates a correct folding of these molecules. Biological activity of the recombinant N-terminal globular domain could be demonstrated by its coelution with hyaluronan in gel permeation chromatography. In addition, the modification of the recombinant fragments with certain carbohydrate structures was analyzed. High mannose oligosaccharides could be mapped to the N-terminal globular domain of the brain-derived molecule. Only recombinant fragments containing parts of the central region of the molecule were modified with chondroitin sulfate chains and with the HNK-1 epitope, and could be considerably altered in their migratory behavior on SDS-polyacrylamide gel electrophoresis by neuraminidase treatment. These findings and the electron microscopic shape indicate a mucin-like character for the central neurocan region.

Amino Acid Sequence↗

PubMed1996

Neurocan and phosphacan: two major nervous tissue-specific chondroitin sulfate proteoglycans.

Neurocan is a multidomain hyaluronan-binding chondroitin sulfate proteoglycan that is synthesized by neurons, whereas the astroglial proteoglycan phosphacan is an mRNA splice variant representing the entire extracellular portion of a receptor-type protein tyrosine phosphatase. A glycoform of phosphocan (phosphocan-KS) that contains both chondroitin sulfate and keratan sulfate is present in the postnatal rat central nervous system (CNS). The concentration of neurocan in brain increases during late embryonic development but then declines steeply during the early postnatal period together with hyaluronan, and neurocan also undergoes extensive proteolytic processing during the course of brain development. In contrast, the concentrations of both phosphocan and phosphocan-KS rise steadily after embryonic day 20 to reach a plateau at about 2 weeks postnatally. In the embryonic CNS the distribution of neurocan mRNA is more widespread than that of phosphocan, which is primarily present in regions of active cell proliferation. Neurocan mRNA is also present in areas where the proteoglycan is not expressed, and there is evidence that the short open reading frame in its 5'-leader may function as a cis-acting regulatory signal for the modulation of neurocan expression in the developing CNS. Neurocan and phosphocan bind saturably, reversibly, and with high affinity to neural cell adhesion molecules (Ng-CAM/L1, NCAM, TAG-1/axonin-1) and to tenascin-C. The proteoglycans and their ligands have overlapping localizations in the CNS, and binding of phosphocan to Ng-CAM/L1, NCAM, and tenascin-C is mediated by complex-type N-linked oligosaccharides on the proteoglycan. Neurocan and phosphocan also bind to neurons and are potent inhibitors of neuronal and glial adhesion and neurite outgrowth. Through their interactions with neural cell adhesion and extracellular matrix molecules, these proteoglycans may play a major role in modulating cell adhesion, neurite growth, and signal transduction across the plasma membrane during the development of the CNS.

Aging↗

PubMed1995

Brevican, a chondroitin sulfate proteoglycan of rat brain, occurs as secreted and cell surface glycosylphosphatidylinositol-anchored isoforms.

cDNA clones encoding proteins related to the aggrecan/versican family of proteoglycan core proteins have been isolated with antisera against rat brain synaptic junctions. Two sets of overlapping cDNAs have been characterized that differ in their 3'-terminal regions. Northern analyses with probes derived from unique regions of each set were found to hybridize with two brain-specific transcripts of 3.3 and 3.6 kilobases (kb). The 3.6-kb transcript encodes a polypeptide that exhibits 82% sequence identity with bovine brevican and is thought to be the rat ortholog of brevican. Interestingly, the polypeptide deduced from the open reading frame of the 3.3-kb transcript is truncated just carboxyl-terminal of the central domain of brevican and instead contains a putative glypiation signal. Antibodies raised against a bacterially expressed glutathione S-transferase-brevican fusion protein have been used to show that both soluble and membrane-bound brevican isoforms exist. Treatment of the crude membrane fraction and purified synaptic plasma membranes with phosphatidylinositol-specific phospholipase C revealed that isoforms of brevican are indeed glycosylphosphatidylinositol-anchored to the plasma membrane. Moreover, digestions with chondroitinase ABC have indicated that rat brevican, like its bovine ortholog, is a conditional chondroitin sulfate proteoglycan. Immunohistochemical studies have shown that brevican is widely distributed in the brain and is localized extracellularly. During postnatal development, amounts of both soluble and phosphatidylinositol-specific phospholipase C-sensitive isoforms increase, suggesting a role for brevican in the terminally differentiating and the adult nervous system.

Amino Acid Sequence↗

PubMed1995

Structure and chromosomal localization of the mouse neurocan gene.

Cosmid clones containing the mouse neurocan gene were isolated from a genomic library using rat neurocan cDNA fragments as probe. The murine gene has a size of approximately 25 kb and contains the coding sequence for the mRNA on 15 exons. The exon-intron structure reflected the structural organization of neurocan, which is a multidomain protein belonging to the aggrecan/versican proteoglycan family. All introns between conserved modular protein domains are phase I introns. Primer extension experiments indicate a transcriptional start point 28 bases downstream of a consensus TATA sequence. Further analysis of 1 kb of 5' flanking sequence revealed in addition to AP1, AP2, and SP1 consensus binding sites multiple E-box elements and a glucocorticoid responsive element. Single-strand conformation polymorphism was used to map neurocan to chromosome 8 between the microsatellite markers D8Mit29 and D8Mit78. Among mouse mutants that have been mapped around this region are the three allelic neurological diseases tottering, leaner, and rolling. The multidomain structure and the preferential expression of neurocan in the brain suggest a potential involvement in these diseases.

Animals↗

PubMed1995

[Arachnoid cyst as the differential diagnosis of ischemic optic neuropathy in type-II diabetes].

HISTORY AND CLINICAL FINDINGS: A 71-year-old woman, a diabetic (type IIb) for 27 years, developed bilateral hemianopsia over a period of about 2 years. A few weeks before hospital admission the defect in her visual fields increased more rapidly and double vision occurred intermittently. The hemianopsia was demonstrated by finger perimetry. There was no evidence of heart failure or peripheral vascular disease. Muscle reflexes were normal, but there was a decrease in vibratory sensation in both feet. The cause of the visual disturbance was at first thought to be an ischaemic optic neuropathy. INVESTIGATIONS: Biochemical tests showed an HbA1 of 12.8%, blood sugar levels were between 230 and 359 mg/dl, and there was increased intravascular platelet activation. Ophthalmological examination confirmed bitemporal hemianopsia and early retinopathy. Magnetic resonance imaging of the skull revealed an intra- and suprasellar cystic space-occupying lesion extending to the right optic chiasma. These findings, taken together, indicated an arachnoidal cyst. TREATMENT AND COURSE: After the diabetic metabolic state had been normalized with insulin treatment (average of 30 IU of an intermediary insulin) and dietary measures, the cyst was evacuated stereotactically. The hemianopsia quickly improved markedly and the patient was discharged 4 days after the operation with her vision nearly fully restored.

Aged↗

PubMed1994

Phosphacan, a chondroitin sulfate proteoglycan of brain that interacts with neurons and neural cell-adhesion molecules, is an extracellular variant of a receptor-type protein tyrosine phosphatase.

We have identified cDNA clones encoding a chondroitin sulfate proteoglycan of rat brain (previously designated 3F8 and now named phosphacan) that binds to neurons and neural cell-adhesion molecules. A sequence of 1616 amino acids deduced from a 4.8-kb open reading frame contains the N-terminal amino acid sequence of the 3F8 core glycoprotein as well as four internal CNBr, tryptic, and endoproteinase Lys-C peptide sequences from the proteoglycan. The deduced amino acid sequence, beginning with a 24-amino acid signal peptide, reveals an N-terminal domain of 255 amino acids homologous to carbonic anhydrases. The entire amino acid sequence deduced from our cDNA clones corresponds to the extracellular portion of a human receptor-type protein tyrosine phosphatase (RPTP zeta/beta) with which it has 76% identity, and the proteoglycan may represent an mRNA splicing variant of the larger transmembrane protein. RNA analysis demonstrated that a probe to the N-terminal carbonic anhydrase domain of the proteoglycan hybridizes with rat brain mRNA of 9.5, 8.4, and 6.4 kb, whereas probes to the phosphatase domains hybridize with only the 9.5-kb message and with the 6.4-kb message (which corresponds to a previously identified variant of the transmembrane protein in which half of the extracellular domain is deleted). The 30 N-terminal amino acids of the 3H1 chondroitin/keratan sulfate proteoglycan of brain are identical to those of the 3F8 proteoglycan, and six internal tryptic peptide sequences also matched those found in sequenced peptides of the 3F8 proteoglycan and/or amino acid sequences deduced from the cDNA clones. We therefore conclude that the 3H1 chondroitin/keratan sulfate proteoglycan and the 3F8 chondroitin sulfate proteoglycan represent glycosylation and possible extracellular splicing variants of a receptor-type protein tyrosine phosphatase. These proteoglycans may modulate cell interactions and other developmental processes in nervous tissue through heterophilic binding to cell-surface and extracellular matrix molecules, and by competition with ligands of the transmembrane phosphatase.

Alternative Splicing↗

PubMed1994

Alteration of the cytosolic-mitochondrial distribution of high-energy phosphates during global myocardial ischemia may contribute to early contractile failure.

Depending on its duration, temporary myocardial ischemia leads to a disturbance of myocardial function before irreversible cellular necrosis is developed. Mechanical, electrical, and metabolic disturbances were suggested to be possible mechanisms accounting for the altered mechanical performance in ischemic hearts. To further investigate the alteration of myocardial energy metabolism on the subcellular level, we determined, by means of nonaqueous fractionation, the cytosolic-mitochondrial distribution of high-energy phosphates and other metabolites (ATP, ADP, phosphocreatine, creatine, and inorganic phosphate) in ischemic (zero-flow) guinea pig hearts after isolated perfused working heart preparation. Additional experiments using 31P nuclear magnetic resonance spectroscopy were performed to determine pHi and [Mg2+]i changes during global ischemia. The total ATP content of myocardial tissue dropped only slowly to 76% of control ATP at 10 minutes and to 51% at 30 minutes and reached almost zero at 60 minutes of ischemia. However, striking differences were observed on the subcellular level: While cytosolic phosphocreatine was almost completely consumed after 3 minutes of ischemia (from 19.1 +/- 1.6 to 3.3 +/- 0.5 mmol/L), ATP concentration in the cytosol decreased within 30 minutes from 8.4 +/- 0.6 to only 5.4 +/- 0.9 mmol/L. Mitochondrial ATP was rapidly and linearly reduced to 60% after 5 minutes of ischemia and was nearly unmeasurable after a further 20 minutes. Thus, in contrast to the breakdown of phosphocreatine in cytosol, the only slight alteration of cytosolic ATP reveals a reduction in cytosolic ATP utilization. Moreover, the unaffected cytosolic-mitochondrial difference in the phosphorylation potential of ATP demonstrates the intact function of the ADP/ATP carrier during early ischemia. These results might indicate a disturbance of the functional coupling between carrier and phosphocreatine kinase (phosphocreatine shuttle), which could be of importance for the early contractile failure in myocardial ischemia.

Adenosine Diphosphate↗

PubMed1993

Continuous hormone replacement therapy with estradiol valerate and chlormadinone acetate in adjustable dosages. A preliminary study.

A group of 62 peri- and postmenopausal women suffering from vasomotor disturbances and a variety of other symptoms were treated with estradiol valerate and chlormadinone acetate continuously using an adjustable dosage regimen. They obtained complete relief from vasomotor symptoms. The continuation rate was 81% after 1 year. In 18 patients the dose had to be adjusted because of breakthrough bleeding (n = 12), mastodynia (n = 3), and for the prevention of bone loss. In 11/12 patients breakthrough bleeding could be stopped by adjusting the dosage. This regimen seems to offer a more flexible approach to hormone replacement therapy (HRT) in the postmenopause than presently available combined preparations for continuous use.

Adult↗

PubMed1992

Cloning of a major heparan sulfate proteoglycan from brain and identification as the rat form of glypican.

We have obtained the complete coding sequence of a highly conserved heparan sulfate proteoglycan which we previously characterized biochemically after isolation from rat brain. An open reading frame of 558 amino acids encodes a protein with a molecular mass of 62 kDa containing three peptide sequences present in the isolated proteoglycan. The total sequence obtained is 3.5 kb long, including 1.6 kb of 3'-untranslated sequence and 0.2 kb of 5'-untranslated sequence. The deduced amino acid sequence and the 3'- and 5'-untranslated sequences have 89% and 66-80% identity, respectively, with those of a phosphatidylinositol-anchored human lung fibroblast heparan sulfate proteoglycan (glypican) for which mRNA is detectable in a large number of human cell lines. Our data therefore demonstrate that this major heparan sulfate proteoglycan of brain is the rat form of glypican.

Amino Acid Sequence↗

PubMed1992

Cloning and primary structure of neurocan, a developmentally regulated, aggregating chondroitin sulfate proteoglycan of brain.

We have obtained the complete coding sequence of neurocan, a chondroitin sulfate proteoglycan of rat brain which is developmentally regulated with respect to its molecular size, concentration, carbohydrate composition, sulfation, and immunocytochemical localization. Two degenerate oligonucleotides, based on amino acid sequence data from the proteoglycan isolated from adult brain by immunoaffinity chromatography with the 1D1 monoclonal antibody, were used as sense and antisense primers in the polymerase chain reaction with a brain cDNA library as template to generate an unambiguous cDNA probe. A second probe for the N-terminal portion of the early postnatal form of the proteoglycan was obtained by reverse transcription/polymerase chain reaction. The composite sequence of overlapping cDNA clones is 5.2-kilobases (kb) long, including 1.3 kb of 3'-untranslated sequence and 76 base pairs of 5'-untranslated sequence. An open reading frame of 1257 amino acids encodes a protein with a molecular mass of 136 kDa containing 10 peptide sequences present in the adult and/or early postnatal brain proteoglycans. The deduced amino acid sequence revealed a 22-amino acid signal peptide followed by an immunoglobulin domain, tandem repeats characteristic of the hyaluronic acid-binding region of aggregating proteoglycans, and an RGDS sequence. The C-terminal portion (amino acids 951-1215) has approximately 60% identity to regions in the C termini of the fibroblast and cartilage proteoglycans, versican and aggrecan, including two epidermal growth factor-like domains, a lectin-like domain, and a complement regulatory protein-like sequence. The central 595-amino acid portion of neurocan has no homology with other reported protein sequences. The proteoglycan contains six potential N-glycosylation sites and 25 potential threonine O-glycosylation sites. In the adult form of the proteoglycan (which represents the C-terminal half of neurocan) a single 32-kDa chondroitin 4-sulfate chain is linked at serin-944, whereas three additional potential chondroitin sulfate attachment sites (only two of which are utilized) are present in the larger proteoglycan species. A probe corresponding to a region of neurocan having no homology with versican or aggrecan hybridized with a single band at approximately 7.5 kb on Northern blots of mRNA from both 4-day and adult rat brain (but not with muscle, kidney, liver, or lung mRNA), indicating that the 1D1 proteoglycan of adult brain, containing a 68-kDa core protein, is generated by a developmentally regulated in vivo proteolytic processing of the 136-kDa species which is predominant in early postnatal brain.(ABSTRACT TRUNCATED AT 400 WORDS)

Aggrecans↗

PubMed1991

Isolation and characterization of developmentally regulated chondroitin sulfate and chondroitin/keratan sulfate proteoglycans of brain identified with monoclonal antibodies.

A panel of monoclonal antibodies prepared to the chondroitin sulfate proteoglycans of rat brain was used for their immunocytochemical localization and isolation of individual proteoglycan species by immunoaffinity chromatography. One of these proteoglycans (designated 1D1) consists of a major component with an average molecular size of 300 kDa in 7-day brain, containing a 245-kDa core glycoprotein and an average of three 22-kDa chondroitin sulfate chains. A 1D1 proteoglycan of approximately 180 kDa with a 150-kDa core glycoprotein is also present at 7 days, and by 2-3 weeks postnatal this becomes the major species, containing a single 32-kDa chondroitin 4-sulfate chain. The concentration of 1D1 decreases during development, from 20% of the total chondroitin sulfate proteoglycan protein (0.1 mg/g brain) at 7 days postnatal to 6% in adult brain. A 45-kDa protein which is recognized by the 8A4 monoclonal antibody to rat chondrosarcoma link protein copurifies with the 1D1 proteoglycan, which aggregates to a significant extent with hyaluronic acid. A chondroitin/keratan sulfate proteoglycan (designated 3H1) with a size of approximately 500 kDa was isolated from rat brain using monoclonal antibodies to the keratan sulfate chains. The core glycoprotein obtained after treatment of the 3H1 proteoglycan with chondroitinase ABC and endo-beta-galactosidase decreases in size from approximately 360 kDa at 7 days to approximately 280 kDa in adult brain. In 7-day brain, the proteoglycan contains three to five 25-kDa chondroitin 4-sulfate chains and three to six 8.4-kDa keratan sulfate chains, whereas the adult brain proteoglycan contains two to four chondroitin 4-sulfate chains and eight to nine keratan sulfate chains, with an average size of 10 kDa. The concentration of 3H1 increases during development from 3% of the total soluble proteoglycan protein at 7 days to 11% in adult brain, and there is a developmental decrease in the branching and/or sulfation of the keratan sulfate chains. A third monoclonal antibody (3F8) was used to isolate a approximately 500-kDa chondroitin sulfate proteoglycan comprising a 400-kDa core glycoprotein and an average of four 28-kDa chondroitin sulfate chains. In the 1D1 and 3F8 proteoglycans of 7-day brain, 20 and 33%, respectively, of the chondroitin sulfate is 6-sulfated, whereas chondroitin 4-sulfate accounts for greater than 96% of the glycosaminoglycan chains in the adult brain proteoglycans.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1989

Endocytosis of a small dermatan sulphate proteoglycan. Identification of binding proteins.

Endosomal preparations from human osteosarcoma cells and from fibroblasts contain 51,000- and 26,000-Mr proteins which bind a small dermatan sulphate proteoglycan after SDS/polyacrylamide-gel electrophoresis and Western blotting. Binding can be inhibited by unlabelled proteoglycan core protein. The proteins co-precipitate with a proteoglycan core protein-antibody complex. Scatchard analysis of immobilized endosomal proteins yielded a KD of about 37 nM for the proteoglycan. In intact cells proteins of the same size can be found. They are sensitive to trypsinization. A 51,000-Mr protein is the predominant membrane protein with strong binding to immobilized dermatan sulphate proteoglycan. There are additional proteoglycan-binding proteins with Mr values of around 30,000 and 14,000 which are insensitive to trypsin treatment. In contrast with the 51,000- and 26,000-Mr proteins, they resist deoxycholate/Triton X-100 extraction several days after subcultivation.

Aggrecans↗

PubMed1988

Degradation of endocytosed dermatan sulfate proteoglycan in human fibroblasts.

Endocytosis and subsequent degradation of iduronic acid-rich small dermatan sulfate proteoglycan from fibroblast secretions were studied in human fibroblasts. Upon endocytosis of [3H]leucine- and [35S]sulfate-labeled proteoglycan release of free leucine was 10 to 15 times more rapid than that of inorganic sulfate. Within approximately 3 h a steady state was approached between transport of proteoglycan to the compartment of core protein degradation and release of free leucine. No such steady state could be found with respect to the dermatan sulfate chains. In the presence of benzyloxycarbonyl-Phe-Ala-diazomethylketone or of other SH-protease inhibitors the degradation of the protein moiety of endocytosed proteoglycan was much less inhibited than the degradation of the polysaccharide chain. Benzyloxycarbonyl-Phe-Ala-diazomethylketone did not affect the degradation of dermatan sulfate chains taken up by fluid phase endocytosis and the activities of all known dermatan sulfate-degrading enzymes. Percoll gradient centrifugation indicated that also in the presence of the protease inhibitor the partially degraded proteoglycan accumulated in dense lysosomes. The isolation of intracellular dermatan sulfate peptides and molecular size determinations of endocytosed dermatan sulfate proteoglycan supported the conclusion that a critical proteolytic step is required before the dermatan sulfate chain becomes accessible to hydrolytic enzymes.

Ammonium Chloride↗

PubMed1988

Tyrosine O-sulfate ester in proteoglycans.

Tyrosine O-sulfate residues were detected in the protein core of sulfated proteoglycans. When cultured skin fibroblasts and arterial smooth muscle cells were incubated in the presence of [35S]sulfate, dermatan sulfate proteoglycan and chondroitin sulfate proteoglycan isolated from the culture medium contained tyrosine [35S]sulfate ester which accounted for 0.03%-0.82% of total 35S radioactivity incorporated into the sulfated proteoglycans. This corresponds to a tyrosine sulfation of every second (fibroblasts) and every 10th (smooth muscle cells) dermatan sulfate proteoglycan molecule. [3H]Tyrosine labeling of fibroblast dermatan sulfate proteoglycan gave a similar stoichiometry. However, the relative proportion of tyrosine [35S]sulfate in proteoglycans from arterial tissue was about 10 times higher than in that from cultured arterial cells. Pulse chase experiments with [35S]sulfate revealed that tyrosine sulfation is a late event in the biosynthesis of dermatan sulfate proteoglycan from fibroblasts and occurs immediately prior to secretion. Cultured skin fibroblasts from a patient with a progeroid variant (Kresse et al. 1987, Am. J. Hum. Gen. 41, 436-453) which exhibit a partial deficiency to synthesize dermatan sulfate proteoglycan were shown to form and to secrete a tyrosine-sulfated but glycosaminoglycan-free protein core, thus confirming a selective and independent [35S]sulfate labeling of the protein core.

Animals↗

PubMed1986

Comparison of small proteoglycans from skin fibroblasts and vascular smooth-muscle cells.

Physicochemical and chemical properties of small proteoglycans containing galactosaminoglycan chains from cultured human skin fibroblasts and human smooth-muscle cells were compared to determine the extent of structural similarity. The proteoglycan secreted by smooth-muscle cells was of larger molecular size and of higher buoyant density, due to longer glycosaminoglycan chains, than the secretion product of skin fibroblasts. Additionally, both proteoglycans differed in the ratio of iduronic acid and glucuronic acid residues. On the other hand, degradation of secreted [3H]leucine-labelled proteoglycans with chondroitin ABC lyase followed by SDS/polyacrylamide-gel electrophoresis resulted in the appearance of core protein bands of identical size (Mr 48,000 and 45,000, depending on the number of asparagine-bound oligosaccharides). An Mr value of 40,000 was determined for the core protein of cells pretreated with tunicamycin. An antibody against the core protein from fibroblast secretions was cross-reactive with the core protein from smooth-muscle cells. Core protein accumulating intracellularly after treatment with carbonyl cyanide m-chlorophenylhydrazone exhibited, on reduction and alkylation, an isoelectric point of 7.8 in both cell types. Limited proteolysis by staphylococcal V8 serine proteinase or endoproteinase Lys-C led in both instances to the formation of peptides of identical size. Peptides bearing asparagine-bound oligosaccharides were free of glycosaminoglycan chains. Similar peptide patterns were obtained when 125I-labelled core proteins were digested with either trypsin or chymotrypsin. Thus small proteoglycans from fibroblasts and smooth-muscle cells can be differentiated by their glycosaminoglycan moieties but not by the nature of their core proteins.

Cells, Cultured↗

PubMed1986

Biosynthesis and processing of proteodermatan sulphate.

The biosynthesis and processing of the small iduronic acid-rich proteodermatan sulphate (PDS) was studied in cultured human skin fibroblasts and arterial smooth muscle cells (SMC) with the aid of core-directed antibodies and various inhibitors of protein synthesis, intracellular transport, and glycosylation. Components of the linkage region became attached to the core protein most likely in a pre-Golgi compartment. Phosphorylation of PDS precursors also occurred in the endoplasmic reticulum with a minor contribution by the Golgi complex. Serine residues and the linkage region were identified as phosphorylated species in secreted PDS. Blockade of transport by monensin did not affect 6-sulphation but affected uronic acid epimerization and 4-sulphation. On relief from the monensin block, additional sulphation along the glycosaminoglycan chain was possible, whereas chain elongation was as in the continuous presence of the drug. Asparagine-bound oligosaccharides or glycosaminoglycan chains were not required for secretion of PDS or core protein. PDS from fibroblast and SMC secretions differed markedly in the composition of the glycosaminoglycan chains. No significant difference, however, was found on isoelectric focusing of core protein and after limited proteolysis of chondroitin ABC lyase-treated core protein. Tryptic and chymotryptic peptide maps of iodinated core proteins were similar.

Arteries↗