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J T Gallagher

Publications and source records attributed to J T Gallagher.

At least 73 records · Page 4Linked to original sources
PubMed1992

Heparan sulphate in the binding and activation of basic fibroblast growth factor.

Heparan sulphate proteoglycans (HSPGs) are widely distributed in animal tissues, but their most prominent locations are cell surface membranes and basement membranes. Their influence on various fundamental aspects of cell behaviour (e.g. cell adhesion, growth and morphogenesis) are dependent on the specific binding properties of the heparan sulphate (HS) chains. These polysaccharides are complex structures in which N-sulphated glucosamine and ester sulphate groups tend to be clustered in discrete regions of the chain separated by sequences enriched in N-acetylglucosamine residues, but with a low sulphate concentration. The sulphated domains contain the sugar residue sequences for interaction with specific proteins essential for HS function. In this review, we describe the plasma membrane HSPGs and their role in regulating the activity of basic fibroblast growth factor (bFGF).

Animals↗
PubMed1991

Sequence analysis of heparan sulphate indicates defined location of N-sulphated glucosamine and iduronate 2-sulphate residues proximal to the protein-linkage region.

A strategy that we originally used to identify an N-acetylated domain adjacent to the protein-linkage sequence of heparan sulphate proteoglycan (HSPG) [Lyon, Steward, Hampson & Gallagher (1987) Biochem. J. 242, 493-498] has been adapted for analysis of the location of GlcNSO3-HexA and GlcNSO3(+/- 6S)-IdoA(2S) units most proximal to the core protein. [3H]Glucosamine-labelled HSPG from human skin fibroblasts was depolymerized by using HNO2 or heparinase under conditions that allowed cleavage of all susceptible linkages. The degraded PG was coupled to Sepharose beads through the protein component, enabling specific recovery of protein-linked resistant oligosaccharides. These were released by treatment with alkaline borohydride and analysed by gel filtration and gradient PAGE. This strategy allowed investigation of the sequence of sugar residues along the chain relative to a common reference point (i.e. the reducing end of the chain). HNO2 scission confirmed the presence of a well-defined N-acetylated sequence predominantly 9-12 disaccharide units in length proximal to the core protein. Heparinase scission produced two classes of oligosaccharides (Mr approx. 7000 and 15,000) with the general formula: IdoA(2S)-GlcNSO3-[HexA-GlcNR]n-HexA-GlcNSO3-[Hex A-GlcNAc]9 12-GlcA-Gal-Gal-Xyl in which the average value for n is 1-2 for the 7000-Mr species and approx. 22 for the 15,000-Mr species. The latter oligosaccharides extend to about one-third of the total length of the HS chains (Mr approx. 45,000). HNO2 scission of these oligosaccharides enabled hypothetical models for their sequence to be proposed. The general arrangement of N-sulphated and N-acetylated disaccharides between the proximal GlcNSO3 and terminal IdoA(2S) residues of the 15,000-Mr fragment was similar to that in the original polysaccharide, suggesting the possibility of a tandemly repeating pattern in the sequence of HS.

Carbohydrate Sequence↗
PubMed1991

Mucins in cat airway secretions.

Mucous secretions were obtained from cat tracheas that had received [3H]glucose and [35S]sulphate to radiolabel mucus glycoproteins biosynthetically. Samples were collected under resting ('basal') conditions as well as after pilocarpine stimulation and were separated into gel and sol phases by centrifugation. Macromolecules were partially purified by using gel chromatography on Sepharose CL-4B, and the species that were eluted with the void volume were then separated into two major populations with isopycnic density-gradient centrifugation in CsCl. The major component from the gel phase of pilocarpine-induced secretions had a buoyant density typical of mucins and was observed as linear and apparently flexible chains by electron microscopy. Reduction of disulphide bonds gave subunits that could be further cleaved by trypsin digestion into components of approximately the same size as the high-Mr glycopeptides obtained from other mucins after this treatment. In contrast, the dominant species in the gel phase of the 'basal' secretion had a significantly higher buoyant density than expected for mucins and was largely unaffected by reduction, as studied by gel chromatography. The macromolecules were fragmented by trypsin, suggesting that they contain a polypeptide backbone. This more dense component also predominated in the sol phase both from the 'basal' secretions and from the pilocarpine-released secretions. Digestion with DNAase, chondroitin ABC lyase or heparan sulphate lyase had no effect, which shows that this component is not DNA, a dermatan sulphate/chondroitin sulphate or a heparan sulphate proteoglycan. In contrast, endo-beta-galactosidase and keratanase caused some fragmentation, suggesting that the molecules contain some linkages of the poly-(N-acetyl-lactosamine) type, although the degradation was not as extensive as expected for keratan sulphate. Treatment with alkaline borohydride resulted in extensive fragmentation of the high-Mr glycopeptides from both components, indicating that the glycans were oligosaccharides that were probably O-linked. The monosaccharide compositions of both components were consistent with that expected for mucins. The data are in keeping with the major component from the pilocarpine-stimulated gel secretions being a mucus glycoprotein and the more dense component being a mucin-like molecule, possibly related to the keratanase-sensitive material isolated from canine trachea by Varsano, Basbaum, Forsberg, Borson, Caughey & Nadel [(1987) Exp. Lung Res. 13, 157-184].

Animals↗
PubMed1991

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

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.

Amyloidosis↗
PubMed1991

Distribution of iduronate 2-sulphate residues in heparan sulphate. Evidence for an ordered polymeric structure.

The structure of human skin fibroblast heparan sulphate has been examined by depolymerization with heparinase, which specifically cleaves highly sulphated disaccharides of structure GlcNSO3 (+/-6S)-alpha 1,4IdoA(2S) [N-sulphated glucosamine (6-sulphate)-alpha 1,4-iduronic acid 2-sulphate]. Heparan sulphate contained only a small proportion (approximately 10%) of linkages susceptible to this enzyme. The major products of depolymerization with heparinase were large oligosaccharides with an average molecular mass of 10 kDa (dp approximately 40, where dp is degree of polymerization; for disaccharides, dp = 2 etc.) as assessed by gel filtration on Sepharose CL-6B, compared with a molecular mass of 45 kDa (dp approximately 200) for the intact chains. The large heparinase-resistant oligosaccharides were highly susceptible to depolymerization with the enzyme heparitinase, which cleaves heparan sulphate in areas of low sulphation, where N-acetylated disaccharides [GlcNAc-alpha 1,4GlcA (N-acetylglucosaminyl-alpha 1,4-glucuronic acid)] are the predominant structural unit. Further analysis of the location of the heparinase cleavage sites indicated that they were predominantly found in a central position in GlcNSO3-alpha 1,4IdoA repeat sequences of average length four to seven disaccharides (dp 8-14). These results indicate that heparinase cleaves heparan sulphate in approximately four or five N-sulphated domains, each domain containing a cluster of two or three susceptible disaccharides; the domains are separated by long N-acetyl-rich sequences that are markedly deficient in sulphate groups. On the basis of these findings a model is proposed which depicts heparan sulphate as an ordered polymeric structure composed of an alternate arrangement of sulphate-rich and sulphate-poor regions. The sulphate-rich regions are likely to be flexible areas of the chain because of their high content of the conformationally versatile IdoA and IdoA(2S) residues. The model has important implications for the biosynthesis and functions of heparan sulphate.

Adult↗
PubMed1991

Purification and partial characterization of the major cell-associated heparan sulphate proteoglycan of rat liver.

Heparan sulphate proteoglycans were solubilized from whole rat livers by homogenization and dissociative extraction with 4 M-guanidinium chloride containing Triton X-100 and proteinase inhibitors. The extract was subjected to trichloroacetic acid precipitation and the proteoglycan remained soluble. This was then purified to apparent homogeneity by a combination of (a) DEAE-Sephacel chromatography, (b) digestion with chondroitinase ABC followed by f.p.l.c. Mono Q ion-exchange chromatography, and (c) density-gradient centrifugation in CsCl and 4 M-guanidinium chloride. Approx. 1.5 mg of proteoglycan was obtained from 30 livers with an estimated recovery of 25%. The purified proteoglycan was eluted from Sepharose CL6B as an apparently single polydisperse population with a Kav. of 0.19 and displayed a molecular mass of greater than or equal to 200 kDa (relative to protein standards) by SDS/PAGE. Its heparan sulphate chains were eluted with a Kav. of 0.44 and have an estimated molecular mass of 25 kDa. Digestion of the proteoglycan with a combination of heparinases yielded core proteins of 77, 49 and 44 kDa. Deglycosylation using trifluoromethanesulphonic acid, though slightly decreasing the sizes, gave an identical pattern of core proteins. Electrophoretic detergent blotting demonstrated that all of the core proteins were hydrophobic and are probably integral plasma membrane molecules. The peptide maps generated by V8 proteinase digestion of the two major core proteins (77 and 49 kDa) were very similar, suggesting that these two core proteins are structurally related.

Animals↗
PubMed1991

Production of heparan sulphate proteoglycans by human bone marrow stromal cells.

Haemopoietic progenitors from human bone marrow bind strongly to human marrow stromal cell cultures but the interaction only occurs if the stromal cells are maintained in methyl prednisolone. Heparan sulphate has been implicated in this interaction and in the binding of haemopoietic cell growth factors. In the present study we have compared the molecular structures of the heparan sulphate proteoglycans, metabolically labelled with [35S]sulphate, produced by methyl prednisolone-treated and untreated human marrow stromal cells in vitro. [35S]proteoglycans were examined in the cell layers (extracted with 1% (v/v) Triton X-100 in 6 M urea) and in the culture medium. Fractionation of proteoglycans by ion-exchange chromatography indicated that the heparan sulphate produced by the treated cultures eluted at a higher NaCl concentration than the counterpart from untreated cells. The heparan sulphate appeared to be mainly expressed on the cell surface, since it was efficiently extracted by treatment with dilute trypsin (50 micrograms ml-1 for 10 min). All cultures contained two heparan sulphate proteoglycan species, the major component eluted from a Sepharose CL-4B column with a median Kav of 0.33 and apparently contained an average of only one heparan sulphate chain. Small quantities of a larger proteoglycan, which was eluted in the void volume from the CL-4B column, was also detected, mainly in the cell layer extracts. The molecular structure of the heparan sulphate chains was analysed by oligosaccharide mapping, following specific enzymic depolymerisation, and separation of breakdown products by gradient PAGE. The maps revealed significant differences in overall enzyme susceptibilities and sulphation patterns of polysaccharides produced by methyl prednisolone-treated and untreated cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone Marrow↗
PubMed1990

Proteoglycans in haemopoietic cells.

Proteoglycans are produced by all types of haemopoietic cells including mature cells and the undifferentiated stem cells. The proteinase-resistant secretory granule proteoglycan (serglycin; Ref. 14), is the most prevalent and best characterised of these proteoglycans. Although its complete pattern of distribution in the haemopoietic system is unknown, serglycin has been identified in the mast cells, basophils and NK cells, in which secretion is regulated, and in HL-60 cells and a monocytoid cell line (Kolset, S.O., unpublished data) in which secretion is constitutive. Proteinase-resistant proteoglycans have been detected in human T-lymphocytes and murine stem cells (FDCP-mix) and the core proteins may be closely related to serglycin. A variety of glycosaminoglycan chains are assembled on the serglycin protein and it is likely that this class of proteoglycan can carry out a wide variety of functions in haemopoietic cells including the regulation of immune responses, inflammatory reactions and blood coagulation. There is strong evidence that in mast cells, NK cells and platelets, the proteoglycans are complexed to basic proteins (including enzymes and cytolytic agents) and amines in secretory granules and such complexes may dissociate following secretion from the cell. The stability of the complexes may be regulated by the ambient pH which may be acidic in the granules and neutral or above in the external medium. However, proteinase-proteoglycan complexes in mast cell granules seem to remain stable after secretion and it has been proposed that the proteoglycan regulates activity of proteinases released into the pericellular domain. The functions of proteoglycans which are constitutively secreted from cells are less clear. If cells have no requirement for storage of basic proteins why do they utilise the same design of proteoglycan as cells which accumulate secretory material prior to regulated release? We should stress that the so-called constitutive secretory pathway has been identified in haemopoietic cells in culture, which are usually maintained and grown in the presence of mitogenic factors (e.g., IL-2, IL-3). the cells are therefore activated and it has not been established that continuous proteoglycan secretion occurs in quiescent cells circulating in the peripheral blood. It is possible that lymphocytes, monocytes and macrophages, in which the constitutive secretion pathway operates in vitro, may store proteoglycan in vivo unless stimulated by mitogens or other activating agents.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

The synthesis of proteoglycans by human T lymphocytes.

We have examined the proteoglycans produced by highly-purified cultures of human T-lymphocytes. The proteoglycans were metabolically labelled with [35S]sulphate and analysed in cellular and medium fractions using DEAE-cellulose chromatography, gel filtration and specific enzymatic and chemical degradations. The results showed that the T cells synthesized a relatively homogeneous, proteinase-resistant chondroitin 4-sulphate proteoglycan that accumulated in the culture medium during a 48 h incubation period. The cellular fraction contained a significant amount of free chondroitin sulphate chains that were not secreted into the medium. These polysaccharides were formed by intracellular degradation of proteoglycan in a chloroquine-sensitive process, indicating a requirement for an acidic environment. In contrast to chondroitin sulphate derived from proteoglycan, chondroitin sulphates synthesized on the exogenous primer, beta-D-xyloside, were mainly secreted by the cells. beta-D-Xylosides caused an 8-fold stimulation in the synthesis of chondroitin sulphate, but decreased the synthesis of proteoglycan by about 50%. These proteoglycans contained shorter chondroitin sulphate chains than their normal counterparts. The results indicate that although proteoglycans are mainly secretory components in human T-cell cultures, a specific metabolic step leads to the intracellular accumulation of free glycosaminoglycans. Separate functions are likely to be associated with the intracellular and secretory pools of chondroitin sulphate.

Cells, Cultured↗
PubMed1990

Examination of the substrate specificity of heparin and heparan sulfate lyases.

We have examined the activities of different preparations of heparin and heparan sulfate lyases from Flavobacterium heparinum. The enzymes were incubated with oligosaccharides of known size and sequence and with complex polysaccharide substrates, and the resulting degradation products were analyzed by strong-anion-exchange high-performance liquid chromatography and by oligosaccharide mapping using gradient polyacrylamide gel electrophoresis. Heparinase (EC 4.2.2.7) purified in our laboratory and a so-called Heparinase I (Hep I) from a commercial source yielded similar oligosaccharide maps with heparin substrates and displayed specificity for di- or trisulfated disaccharides of the structure----4)-alpha-D-GlcNp2S(6R)(1----4)-alpha-L-IdoAp2S( 1----(where R = O-sulfo or OH). Oligosaccharide mapping with two different commercial preparations of heparan sulfate lyase [heparitinase (EC 4.2.2.8)] indicated close similarities in their depolymerization of heparan sulfate. Furthermore, these enzymes only degraded defined oligosaccharides at hexosaminidic linkages with glucuronic acid:----4)-alpha-D-GlcNpR(1----4)-beta-D-GlcAp(1----(where R = N-acetamido or N-sulfo). The enzymes showed activity against solitary glucuronate-containing disaccharides in otherwise highly sulfated domains including the saccharide sequence that contains the antithrombin binding region in heparin. A different commercial enzyme, Heparinase II (Hep II), displayed a broad spectrum of activity against polysaccharide and oligosaccharide substrates, but mapping data indicated that it was a separate enzyme rather than a mixture of heparinase and heparitinase/Hep III. When used in conjunction with the described separation procedures, these enzymes are powerful reagents for the structural/sequence analysis of heparin and heparan sulfate.

Carbohydrate Conformation↗
PubMed1990

A general method for the detection and mapping of submicrogram quantities of glycosaminoglycan oligosaccharides on polyacrylamide gels by sequential staining with azure A and ammoniacal silver.

A sensitive method has been developed for the visualization of nonradiolabeled glycosaminoglycan oligosaccharides resolved by polyacrylamide gel electrophoresis using fixation with azure A followed by staining with ammoniacal silver. This method, which can detect as little as 1-2 ng of a single oligosaccharide species, can be used to stain a few micrograms of a complex oligosaccharide mixture. The combination of gradient polyacrylamide gel electrophoresis and sequential azure A/silver staining can be applied to the analysis of all the complex glycosaminoglycans (i.e., heparin, heparan sulfate, chondroitin/dermatan sulfate, keratan sulfate) and hyaluronate, as well as to comparisons of specificities of the glycosaminoglycan-degrading enzymes. This procedure may be particularly valuable in situations where the availability of glycosaminoglycan is very limited and/or where radiolabeling is impractical or undesirable.

Azure Stains↗
PubMed1990

Molecular organization of heparan sulphate from human skin fibroblasts.

The molecular structure of human skin fibroblast heparan sulphate was examined by specific chemical or enzymic depolymerization and high-resolution separation of the resulting oligosaccharides and disaccharides. Important features of the molecular organization, disaccharide composition and O-sulphate disposition of this heparan sulphate were identified. Analysis of the products of HNO2 hydrolysis revealed a polymer in which 53% of disaccharide units were N-acetylated and 47% N-sulphated, with an N-/O-sulphate ratio of 1.8:1. These two types of disaccharide unit were mainly located in separate domains. Heparitinase and heparinase scission indicated that the iduronate residues (37% of total hexuronate) were largely present in contiguous disaccharide sequences of variable size that also contained the majority of the N-sulphate groups. Most of the iduronate residues (approx. 70%) were non-sulphated. About 8-10% of disaccharide units were cleaved by heparinase, but only a minority of these originated from contiguous sequences in the intact polymer. Trisulphated disaccharide units [alpha-N-sulpho-6-sulphoglucosaminyl-(1----4)-iduronate 2-sulphate], which are the major structural units in heparin, made up only 3% of the disaccharide units in heparan sulphate. O-Sulphate groups (approx. 26 per 100 disaccharide units) were distributed almost evenly among C-6 of N-acetylglucosamine, C-2 of iduronate and C-6 of N-sulphated glucosamine residues. The results indicate that the sulphated regions of heparan sulphate have distinctive and potentially variable structural characteristics. The high content of non-sulphated iduronate in this heparan sulphate species suggests a conformational versatility that could have important implications for the biological properties of the polymer.

Carbohydrate Sequence↗
PubMed1989

Metabolic properties of a homogeneous proteoglycan of a haemopoietic stem cell line, FDCP-mix.

A biochemical analysis has been carried out of metabolically labelled proteoglycans and glycosaminoglycans synthesized by a haemopoietic multipotential stem cell line, FDCP-mix. The only proteoglycan identified in these multipotential cells was a homogeneous component that contained chondroitin 4-sulphate chains (Mr approximately 10,000) arranged in close proximity in a proteinase-resistant domain of the protein core. Small quantities of free chondroitin 4-sulphate were also detected. Following a 48 h incubation with Na2 35SO4 the majority of the 35S-radiolabelled proteoglycans (approximately 80%) were associated with the cells, mainly in an intracellular compartment, and the remaining 20% were in the culture medium. Pulse-chase studies demonstrated two turnover pathways for the newly synthesized cellular proteoglycans. In the minor pathway, the proteoglycans were secreted rapidly into the medium without any discernable structural modification. In the major pathway the proteoglycans seemed to be transferred into a storage compartment from which the intact macromolecules were not secreted. Eventually, these proteoglycans were degraded to yield free polysaccharide chains and these chains were then released into the medium, but only at a relatively slow rate. There was very little intracellular degradation of chondroitin sulphate chains. The pathway to polysaccharide secretion was a slow stepwise process with a time-lag of about 5 h between proteoglycan synthesis and the appearance of free chondroitin sulphate and a second time-lag, also of about 5 h, before these chains began to be secreted. The existence of separate secretory pathways for proteoglycans and chondroitin sulphate chains is an interesting characteristic that seems to distinguish proteoglycan metabolism in primitive multipotent stem cells from related metabolic processes in mature haemopoietic cells.

Animals↗
PubMed1988

Oligosaccharide mapping of heparan sulphate by polyacrylamide-gradient-gel electrophoresis and electrotransfer to nylon membrane.

A new method that we have called 'oligosaccharide mapping' is described for the analysis of radiolabelled heparan sulphate and other glycosaminoglycans. The method involves specific enzymic or chemical scission of polysaccharide chains followed by high-resolution separation of the degradation products by polyacrylamide-gradient-gel electrophoresis. The separated oligosaccharides are immobilized on charged nylon membranes by electrotransfer and detected by fluorography. A complex pattern of discrete bands is observed covering an oligosaccharide size range from degree of polymerization (d.p.) 2 (disaccharide) to approximately d.p. 40. Separation is due principally to differences in Mr, though the method also seems to detect variations in conformation of oligosaccharide isomers. Resolution of oligosaccharides is superior to that obtained with isocratic polyacrylamide-gel-electrophoresis systems or gel chromatography, and reveals structural details that are not accessible by other methods. For example, in this paper we demonstrate a distinctive repeating doublet pattern of iduronate-rich oligosaccharides in heparitinase digests of mouse fibroblast heparan sulphate. This pattern may be a general feature of mammalian heparan sulphates. Oligosaccharide mapping should be a valuable method for the analysis of fine structure and sequence of heparan sulphate and other complex polysaccharides, and for making rapid assessments of the molecular distinctions between heparan sulphates from different sources.

Chromatography, Gel↗
PubMed1988

Heparan sulphate-degrading endoglycosidase in liver plasma membranes.

An endoglycosidase is described in isolated liver plasma membranes that brings about a rapid and selective degradation of membrane-associated heparan sulphate, pre-labelled biosynthetically with Na2(35)SO4. The enzyme attacked mainly the polysaccharide chains of a hydrophobic membrane proteoglycan and it had little effect on a proteoglycan that could be displaced from the membranes with 1.0 M-NaCl. The highest activity was measured in the pH range 7.5-8.0, and the enzyme was almost completely inhibited below pH 5.5. Breakdown of susceptible polysaccharide chains was fast, being complete in 20-30 min. The major oligosaccharide fraction (Mr approx. 6000) produced by the enzyme was considerably smaller than the intact heparan sulphate chains. Enzyme activity was retained in membranes solubilized in 1% (v/v) Triton X-100. The high pH optimum and plasma-membrane association distinguish this enzyme from other heparan sulphate-degrading endoglycosidases that have acid pH optima and may be of lysosomal origin. A plasma-membrane endoglycosidase could modulate cellular interactions mediated by heparan sulphate, and/or release biologically active fragments of the polysaccharide from the cell periphery.

Animals↗