scholarly journals Isolation and characterization of the integral glycosaminoglycan constituents of human amyloid A and monoclonal light-chain amyloid fibrils

1991 ◽  
Vol 275 (1) ◽  
pp. 67-73 ◽  
Author(s):  
S R Nelson ◽  
M Lyon ◽  
J T Gallagher ◽  
E A Johnson ◽  
M B Pepys
Keyword(s):  
Light Chain ◽  
Amyloid Fibrils ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Heparan Sulphate ◽  
Cellulose Acetate Electrophoresis ◽  
Amyloid A ◽  
Isolation And Characterization ◽  
Cscl Density Gradient ◽  
A Minor

Amyloid fibrils were isolated by extraction in water from the livers and spleens of four patients who had died of monoclonal, light-chain (AL)-type, systemic amyloidosis and one with reactive systemic, amyloid A protein (AA)-type amyloidosis. Each fibril preparation contained 1-2% by weight of glycosaminoglycan (GAG) which was tightly associated with the fibrils and not just co-isolated from the tissues with them. After exhaustive digestion of the fibrils with papain and Pronase, the GAGs were specifically precipitated with cetylpyridinium chloride and were identified by cellulose acetate electrophoresis and selective susceptibility to specific glycosidases. All the preparations contained approximately equal amounts of heparan sulphate and dermatan sulphate. There was no evidence for the presence of chondroitin sulphate or other GAGs. Fine structural analysis by oligosaccharide mapping in gradient polyacrylamide gels, following partial digestion with specific glycosidases, showed very similar structures among the heparan sulphates and the dermatan sulphates, respectively. GAGs were also extracted by solubilizing amyloid fibrils in 4 M-guanidinium chloride followed by CsCl density-gradient ultracentrifugation. Although a minor proportion of the GAG material obtained in this way was apparently in the form of proteoglycan molecules, most of it was free GAG chains. The presence in amyloid fibrils of different types, in different organs and from different patients of particular GAG classes with similar structures supports the view that these molecules may be of pathogenic significance.

Localisation and characterization of acid mucopolysaccharides in the early chick blastoderm

Development ◽  
1980 ◽  
Vol 56 (1) ◽  
pp. 169-178
Author(s):  
Ch. Vanroelen ◽  
L. Vakaet ◽  
L. Andries
Keyword(s):  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Middle Layer ◽  
Ventral Side ◽  
Chick Blastoderm ◽  
Cellulose Acetate Electrophoresis ◽  
Acid Mucopolysaccharides ◽  
Extracellular Compartment ◽  
Early Specialization ◽  
Testicular Hyaluronidase

Acid mucopolysaccharides in the extracellular compartment of early chick blastoderms (16 h of incubation) were labelled with tritiated glucosamine and/or ]35S]sulphate. The incorporation pattern was studied autoradiographically. Treatment with testicular hyaluronidase revealed a testicular hyaluronidase-sensitive fraction, mainly at the periphery of Middle Layer and Deep Layer cells, and a testicular hyaluronidase-resistant fraction, mainly at the ventral side of the Upper Layer. A biochemical analysis, utilizing chondroitinase ABC and nitrous acid, followed by cellulose acetate electrophoresis, demonstrated the synthesis of a non-sulphated fraction, i.e. hyaluronic acid and/or chondroitin, and a sulphated fraction, comprising two undersulphated components, i.e. chondroitin sulphate, and heparan sulphate or heparin. The appearance of different AMPS in specific areas of the early chick blastoderm is regarded as an early specialization of the extracellular compartment.

scholarly journals Properties of heparan sulphate and chondroitin sulphate from young and old human aortae

1969 ◽  
Vol 114 (1) ◽  
pp. 89-96 ◽  
Author(s):  
G. Manley ◽  
R. N. Mullinger ◽  
P. H. Lloyd
Keyword(s):  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Heparan Sulphate ◽  
Total Amino Acid ◽  
Cross Linking ◽  
Molecular Weights ◽  
Consistent Increase ◽  
Salt Gradient ◽  
Alkali Hydrolysis ◽  
Dowex 1

1. Glycosaminoglycans were liberated from old and young human ascending aortae by digestion with papain. Heparan sulphate and chondroitin sulphate were separated by the different solubilities of their complexes with cetylpyridinium chloride in solutions of sodium chloride. Final fractionation was achieved by salt-gradient column chromatography on Dowex 1 (Cl−form). 2. Heparan sulphate from old aortae showed a slight, but consistent, increase in sulphation compared with heparan sulphate from young aortae. 3. The major amino acids associated with aortic heparan sulphate and chondroitin sulphate were serine, glycine, glutamic acid and aspartic acid. Heparan sulphate and chondroitin sulphate from old aortae contained about twice as much total amino acid as heparan sulphate and chondroitin sulphate from young aortae. Alkali hydrolysis resulted in the destruction of more serine in chondroitin sulphate from old, compared with young, aortae. 4. Molecular weights of glycosaminoglycans from old and young aortae were found to be similar, and in the region of 35000. 5. It is suggested that there is an increased degree of protein–glycosaminoglycan cross-linking in old aortae.

scholarly journals Human Serum and Urine Glycosaminoglycans in Health and in Patients with Chronic Renal Failure

1992 ◽  
Vol 29 (2) ◽  
pp. 190-195 ◽  
Author(s):  
L Bower ◽  
C Warren ◽  
G Manley
Keyword(s):  
Renal Failure ◽  
Chronic Renal Failure ◽  
Uronic Acid ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Heparan Sulphate ◽  
Normal Urine ◽  
Glycosaminoglycan Metabolism ◽  
Normal Controls

Quantitation of uronic acid precipitable by cetylpyridinium chloride (CPC) and electrophoretic separation of glycosaminoglycans were performed on sera from patients with chronic renal failure and compared to normal controls. Serum CPC-precipitable uronic acid (CpUA) levels in patients with renal failure were significantly higher (mean 13·7 mg/L, range 7·1–23·6 mg/L) than normal controls (mean 9·6 mg/L, range 5·1–13·9 mg/L) due to increased concentrations of low sulphated chondroitin sulphate. A positive correlation between serum CpUA and creatinine was found in renal failure patients. Urine CpUA excretion was raised in renal failure patients compared to normal controls with an increased excretion of chondroitin sulphate (Ch-S) of reduced electrophoretic mobility. Heparan sulphate (HS), a major glycosaminoglycan in normal urine, was absent from the urine of these patients. The possible origin of urine glycosaminoglycans and the role of the kidney in glycosaminoglycan metabolism are discussed.

scholarly journals Colocalization of Apolipoprotein AI in Various Kinds of Systemic Amyloidosis

2005 ◽  
Vol 53 (2) ◽  
pp. 237-242 ◽  
Author(s):  
Naohiro Sakata ◽  
Yoshinobu Hoshii ◽  
Tomomi Nakamura ◽  
Makiko Kiyama ◽  
Hirofumi Arai ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Amyloid Fibrils ◽  
Senile Plaques ◽  
Systemic Amyloidosis ◽  
High Density Lipoproteins ◽  
Apolipoprotein Ai ◽  
Aa Amyloidosis ◽  
Amyloid A ◽  
Amyloid Deposits ◽  
A Minor

Apolipoprotein AI (apoAI), a major component of high-density lipoproteins, is one of the major amyloid fibril proteins and a minor constituent of the senile plaques observed in Alzheimer's disease. We examined colocalization of apoAI in various kinds of systemic amyloidosis in this study. Forty-three of 48 formalin-fixed paraffin-embedded heart specimens with various forms of systemic amyloidosis reacted immunohistochemically with anti-human apoAI antibody. ApoAI was also detected in water-extracted amyloid material by immunoblotting. In addition, we observed colocalization of apoAI and murine amyloid A (AA) amyloidosis in human apoAI transgenic mice. This is the first report of colocalization of apoAI with amyloid deposits in various forms of human systemic amyloidosis and murine AA amyloidosis in human apoAI transgenic mice. ApoAI may not always be a major component of amyloid fibrils, even when it is present in systemic amyloid deposits.

Separate Effects of Urinary Chondroitin Sulphate and Heparan Sulphate on the Crystallization of Urinary Calcium Oxalate: Differences between Stone Formers and Normal Control Subjects

1993 ◽  
Vol 85 (1) ◽  
pp. 33-39 ◽  
Author(s):  
D. K. Y. Shum ◽  
M. D. I. Gohel
Keyword(s):  
Calcium Oxalate ◽  
Normal Control ◽  
Uronic Acid ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Heparan Sulphate ◽  
Urinary Calcium ◽  
Crystal Nucleation ◽  
Control Subjects ◽  
Stone Formers

1. Urinary glycosaminoglycans were recovered from the papain digest of polyanions precipitated sequentially by cetylpyridinium chloride and sodium acetate-saturated ethanol. Those from the early morning urine of 48 stone formers and 43 normal control subjects measured 11 and 16 μg of uronic acid/ml of urine, respectively. 2. Preparative agarose gel electrophoresis of the recovered glycosaminoglycans in barium acetate buffer (pH 5.8) yielded fractions containing purely chondroitin sulphate, co-polymeric chondroitin/dermatan sulphates and heparan sulphate. Identification was based on the susceptibility of the fractions to chondroitinase or nitrous acid treatment. Similar compositions of glycosaminoglycan classes were observed in samples from stone formers and normal control subjects. 3. The fractionated glycosaminoglycans were dissolved in urine ultrafiltrate to assay for nucleation-promoting and growth-inhibiting activities towards crystallization of urinary calcium oxalate. When compared at the same uronic acid concentration, both the urinary chondroitin sulphate isomers and heparan sulphates of stone formers demonstrated the capacity to enhance crystal nucleation from calcium oxalate endogenous in urine ultrafiltrates, whereas only urinary heparan sulphates of normal control subjects demonstrated this capacity. 4. Tissue-derived reference chondroitin sulphate, dermatan sulphate and heparin, when similarly tested, showed negligible crystal nucleation-promoting activity. The tissue-derived heparan sulphate was similar to the urinary heparan sulphates in showing marked crystal nucleation-promoting activity. 5. Crystal-growth inhibitory activity was evident in all urinary glycosaminoglycan fractions studied. In particular, urinary heparan sulphate of normal control subjects showed higher activity than that of stone formers or the chondroitin sulphate isomers of both stone formers and normal control subjects (P <0.005).

scholarly journals Characterization of proteoglycans associated with mouse splenic AA amyloidosis

1994 ◽  
Vol 303 (2) ◽  
pp. 663-670 ◽  
Author(s):  
T Stenstad ◽  
J H Magnus ◽  
G Husby
Keyword(s):  
Amyloid Fibrils ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Pulse Labelling ◽  
Sulphate Proteoglycan ◽  
Amyloid Deposits ◽  
Amyloid Fibril Formation

We here report for the first time on the chemical characteristics of proteoglycans associated with mouse splenic reactive AA amyloid. Amyloid was induced in CBA/J mice by two different procedures; conventional casein treatment and by employing Freund's complete adjuvant, accelerated by Trypan Blue. Pulse-labelling was employed at distinct stages during amyloid development, followed by [35S]proteoglycan characterization of organ extracts. Repetitive 35S injections were also administered during the phase where amyloid deposition occurred most rapidly. Proteoglycans were extracted with guanidine in the presence of protease inhibitors and purified. The results showed that the production of proteoglycans is dramatically enhanced during amyloidogenesis, the glycosaminoglycan and proteoglycan accumulation being not only dependent on alterations in proteoglycan catabolism, but rather on increased synthesis. The increment could be demonstrated even at the stage before microscopic detection of amyloid deposits, clearly suggesting that the upregulation of proteoglycan expression precedes amyloid fibril formation. Two major proteoglycans were found to accumulate in advanced splenic amyloid; one a heparan sulphate proteoglycan of approx. 200 kDa with a core protein of 70 kDa, the other a chondroitin sulphate proteoglycan of smaller size. Moreover, free dermatan sulphate chains seemed to specifically accumulate in the organs during amyloid fibrillogenesis. We suggest that free glycosaminoglycans may be a specific feature of amyloidosis and that different proteoglycans and glycosaminoglycans play a role in formation and stabilization of amyloid fibrils in vivo.

scholarly journals Isolation and characterization of proteoglycans synthesized by mouse osteoblastic cells in culture during the mineralization process

1990 ◽  
Vol 266 (1) ◽  
pp. 15-24 ◽  
Author(s):  
Y Takeuchi ◽  
T Matsumoto ◽  
E Ogata ◽  
Y Shishiba
Keyword(s):  
Core Protein ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Osteoblastic Cell ◽  
Guanidinium Chloride ◽  
Mineralization Process ◽  
Osteoblastic Cell Line ◽  
Isolation And Characterization

Proteoglycans in mineralized (0.5 M-EDTA/4 M-guanidinium chloride-extractable) and non-mineralized (4 M-guanidinium chloride-extractable) matrices synthesized by a mouse osteoblastic-cell line MC3T3-E1 were characterized at different phases of mineralization in vitro. Cell cultures were labelled with [35S]sulphate and either [3H]glucosamine or 3H-labelled amino acids. At the mineralization phase a large majority of proteoglycans were extracted with 4 M-guanidinium chloride (G extract), and at least five species of labelled proteoglycans were identified; dermatan sulphate proteoglycans (DSPG), apparent Mr approx. 120,000 and 70,000), heparan sulphate proteoglycans (HSPG, apparent Mr approx. 200,000 and 120,000) and DS chains with very little core protein. DSPGs weakly bound to an octyl-Sepharose CL-4B column and HSPGs bound more tightly, whereas DS chains did not bind to the column. Amounts of labelled proteoglycans extracted with 0.5 M-EDTA/4 M-guanidinium chloride (EDTA extract) were much less than those in G extract. Although the predominant species in the EDTA extract were comparable with the DS or DSPGs in the G extract, none of them bound to octyl-Sepharose CL-4B, indicating their lack of hydrophobicity. At the nonmineralizing phase a large chondroitin sulphate proteoglycan (Mr greater than 600,000) was found in the matrix in addition to the five proteoglycan species similar to those at the mineralization phase. Although DS chains at the early phase were similar in size to those at the mineralization phase, the ratio of 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulpho-D-galactose to 2-acetamido-2-deoxy-3-O-(beta-D-gluculo-4-enepyranosyluronic acid)-6-O-sulpho-D-galactose was less than that at the mineralization phase. These results agree with those of previous studies performed in vivo and suggest that alteration in the synthesis of proteoglycans is involved in the mineralization process. They also suggest that at the osteoblastic mineralization front proteoglycans undergo partial degradation and lose their hydrophobicity.

scholarly journals Evidence for the existence of a multichain proteoglycan of heparan sulphate

1970 ◽  
Vol 117 (4) ◽  
pp. 699-702 ◽  
Author(s):  
L. Jansson ◽  
U. Lindahl
Keyword(s):  
Potassium Chloride ◽  
Aortic Wall ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Heparan Sulphate ◽  
Gel Chromatography ◽  
Dermatan Sulphate ◽  
Fraction I ◽  
Two Peaks ◽  
Testicular Hyaluronidase

1. Glycosaminoglycans were extracted with 2m-potassium chloride from bovine aorta and purified by precipitation with cetylpyridinium chloride from 0.5m-potassium chloride. The yield amounted to 24% of the total glycosaminoglycan content of the tissue. 2. After removal of chondroitin sulphate by digestion with testicular hyaluronidase, the residual glycosaminoglycan material (11% of the extracted polysaccharide) was fractionated by gel chromatography on Sephadex G-200. Two peaks (I and II) were obtained, the more retarded of which (II) corresponded to single polysaccharide chains. 3. The macromolecular properties of fraction I were investigated by repeated gel chromatography, after treatment of the fraction with alkali or digestion with papain. In both cases the elution position of fraction I was shifted towards that of the single polysaccharide chains. 4. Analysis of fraction I showed approximately equal amounts of heparan sulphate and dermatan sulphate. It is concluded that these glycosaminoglycans both occur in the aortic wall as multichain proteoglycans.

scholarly journals Phosphorylation of a membrane-intercalated proteoglycan, syndecan-2, expressed in a stroma-inducing clone from a mouse Lewis lung carcinoma

1996 ◽  
Vol 315 (3) ◽  
pp. 925-930 ◽  
Author(s):  
Naoki ITANO ◽  
Kayoko OGURI ◽  
Yuko NAGAYASU ◽  
Yuri KUSANO ◽  
Hayao NAKANISHI ◽  
...  
Keyword(s):  
Lung Carcinoma ◽  
Lewis Lung Carcinoma ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Specific Binding ◽  
Heparan Sulphate ◽  
Major Constituent ◽  
Lewis Lung ◽  
The Core ◽  
Cscl Density Gradient

We previously reported that a mouse Lewis lung carcinoma-derived stroma-inducing clone, P29, highly expresses a syndecan-like proteoglycan exhibiting specific binding to fibronectin, a major constituent of the interstitial matrix formed by the induced stromal cells, via its heparan sulphate chains [Itano, Oguri, Nakanishi and Okayama (1993) J. Biochem. (Tokyo) 114, 862–873]. On metabolic labelling of the proteoglycan with [32P]Pi, followed by identification of the radiolabelled material using glycanases, almost all the isotope was found to have been incorporated into a core portion of molecular mass 48 kDa, which was generated by digestion with heparan sulphate lyase I plus chondroitin ABC lyase. Immunoblotting of the core protein with a monoclonal antibody, F58-6G12, demonstrated that the proteoglycan was mouse syndecan-2. CsCl-density-gradient centrifugation after mild treatment of liposome-intercalated 32P-labelled syndecan-2 with trypsin resulted in clear separation of the radioactivity into a bottom fraction containing all the glycosaminoglycans (accounting for 40% of the total radioactivity) and a top fraction containing liposome-associated peptides (60%). The former isotope was shown to be linked covalently to both heparan sulphate and chondroitin sulphate chains, probably at their bridge regions. The latter was mostly attributed to phosphoserine, the one and only phosphorylated amino acid released on acid hydrolysis of this proteoglycan, strongly suggesting that the phosphorylation occurs at a specific serine residue(s) in the cytoplasmic domain of the core protein.

scholarly journals Circular-dichroism studies on two murine serum amyloid A proteins

1988 ◽  
Vol 256 (3) ◽  
pp. 775-783 ◽  
Author(s):  
W D McCubbin ◽  
C M Kay ◽  
S Narindrasorasak ◽  
R Kisilevsky
Keyword(s):  
Secondary Structure ◽  
Structure Prediction ◽  
Amyloid Fibrils ◽  
Secondary Structure Prediction ◽  
Sequence Data ◽  
Heparan Sulphate ◽  
Alpha Helix ◽  
Serum Amyloid ◽  
Amyloid A ◽  
Helical Content

C.d. studies have shown that mouse SAA2 (serum amyloid A2) protein has about one-half of the alpha-helix content of the SAA1 (serum amyloid A1) analogue (15 as against 32%), although secondary-structure prediction analyses based on sequence data do not suggest such a large difference between the forms. The decreased helical content may be a reflection or indication of a stronger propensity to aggregation of the SAA2 form compared with SAA1. The main elements of secondary structure in both proteins are beta-sheets/turns. Interactions with Ca2+ are accompanied by small losses in alpha-helix content, whereas binding to chondroitin-6-sulphate in the presence of millimolar Ca2+ also decreases the amount of secondary structure. However, SAA2 binding to heparan sulphate increases its beta-sheet structure, whereas with SAA1 secondary structure is not apparently altered by its interaction with heparan sulphate. Computer-generated surface profiles show slight differences in accessibility, hydrophilicity and flexibility between the proteins. Understanding these differences may help to explain why SAA2 is found in amyloid fibrils whereas SAA1 is not. In particular, a stronger tendency to aggregation might be the reason why SAA2 is deposited exclusively in these fibrils.

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