| 1. |
- Andersson, Emma, et al.
(författare)
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Antimicrobial activities of heparin-binding peptides.
- 2004
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956. ; 271:6, s. 1219-1226
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Tidskriftsartikel (refereegranskat)abstract
- Antimicrobial peptides are effector molecules of the innate immune system. We recently showed that the human antimicrobial peptides alpha-defensin and LL-37 bind to glycosaminoglycans (heparin and dermatan sulphate). Here we demonstrate the obverse, i.e. structural motifs associated with heparin affinity (cationicity, amphipaticity, and consensus regions) may confer antimicrobial properties to a given peptide. Thus, heparin-binding peptides derived from laminin isoforms, von Willebrand factor, vitronectin, protein C inhibitor, and fibronectin, exerted antimicrobial activities against Gram-positive and Gram-negative bacteria. Similar results were obtained using heparin-binding peptides derived from complement factor C3 as well as consensus sequences for heparin-binding (Cardin and Weintraub motifs). These sequence motifs, and additional peptides, also killed the fungus Candida albicans. These data will have implications for the search for novel antimicrobial peptides and utilization of heparin-protein interactions should be helpful in the identification and purification of novel antimicrobial peptides from complex biological mixtures. Finally, consensus regions may serve as templates for de novo synthesis of novel antimicrobial molecules.
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| 2. |
- Frick, Inga-Maria, et al.
(författare)
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Interactions between M proteins of Streptococcus pyogenes and glycosaminoglycans promote bacterial adhesion to host cells.
- 2003
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956. ; 270:10, s. 2303-2311
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Tidskriftsartikel (refereegranskat)abstract
- Several microbial pathogens have been reported to interact with glycosaminoglycans (GAGs) on cell surfaces and in the extracellular matrix. Here we demonstrate that M protein, a major surface-expressed virulence factor of the human bacterial pathogen, Streptococcus pyogenes, mediates binding to various forms of GAGs. Hence, S. pyogenes strains expressing a large number of different types of M proteins bound to dermatan sulfate (DS), highly sulfated fractions of heparan sulfate (HS) and heparin, whereas strains deficient in M protein surface expression failed to interact with these GAGs. Soluble M protein bound DS directly and could also inhibit the interaction between DS and S. pyogenes. Experiments with M protein fragments and with streptococci expressing deletion constructs of M protein, showed that determinants located in the NH2-terminal part as well as in the C-repeat region of the streptococcal proteins are required for full binding to GAGs. Treatment with ABC-chondroitinase and HS lyase that specifically remove DS and HS chains from cell surfaces, resulted in significantly reduced adhesion of S. pyogenes bacteria to human epithelial cells and skin fibroblasts. Together with the finding that exogenous DS and HS could inhibit streptococcal adhesion, these data suggest that GAGs function as receptors in M protein-mediated adhesion of S. pyogenes.
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| 3. |
- Schmidtchen, Artur, et al.
(författare)
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Analysis of glycosaminoglycan chains from different proteoglycan populations in human embryonic skin fibroblasts
- 1992
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 208:2, s. 537-546
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Tidskriftsartikel (refereegranskat)abstract
- 1. The structure of chondroitin/dermatan and heparan-sulphate chains from various proteoglycan populations derived from cultured human skin fibroblasts have been examined. Confluent cell cultures were biosynthetically labelled with [3H]-glucosamine and 35SO4(2-), and proteoglycans were purified according to buoyant density, size and charge density [Schmidtchen, A., Carlstedt, I., Malmstrom, A. & Fransson, L.-A. (1990) Biochem. J. 265, 289-300]. Some proteoglycan fractions were further fractionated according to hydrophobicity on octyl-Sepharose in Triton X-100 gradients. The glycosaminoglycan chains, intact or degraded by chemical or enzymic methods were then analysed by gel chromatography on Sepharose CL-6B, Bio-Gel P-6, ion exchange HPLC and gel electrophoresis. 2. Three types of dermatan-sulphate chains were identified on the basis of disaccharide composition and chain length. They were derived from the large proteoglycan, two small proteoglycans and a cell-associated proteoglycan with core proteins of 90 kDa and 45 kDa. Intracellular, free dermatan-sulphate chains were very similar to those of the small proteoglycans. 3. Heparan-sulphate chains from different proteoglycans had, in spite of small but distinct differences in size, strikingly similar compositional features. They contained similar amounts of D-glucuronate, L-iduronate (with or without sulphate) and N-sulphate groups. They all displayed heparin-lyase-resistant domains with average molecular mass of 10-15 kDa. The heparan-sulphate chains from proteoglycans with 250-kDa and 350-kDa cores were the largest greater than 50 kDa), containing an average of four or five domains, in contrast to heparan-sulphate chains from the small heparan-sulphate proteoglycans which had average molecular mass of 45 kDa and consisted of three or four such domains. Free, cell-associated heparan-sulphate chains were heterogeneous in size (5-45 kDa). 4. These results suggest that the core protein may have important regulatory functions with regard to dermatan-sulphate synthesis. On the other hand, synthesis of heparan sulphate may be largely controlled by the cell that expresses a particular proteoglycan core protein.
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| 4. |
- Schmidtchen, Artur, et al.
(författare)
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Analysis of heparan-sulphate chains and oligosaccharides from proliferating and quiescent fibroblasts. A proposed model for endoheparanase activity
- 1994
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 223:1, s. 211-221
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Tidskriftsartikel (refereegranskat)abstract
- Human skin fibroblasts in different growth states were incubated with [3H]glucosamine and/or Na(2)35SO4 and extracted with Triton X-100 for various periods of time. Free heparan-sulphate oligosaccharides and protein-bound heparan-sulphate chains were separated by chromatography on octyl-Sepharose and analyzed. A pool of endogenously produced oligosaccharides, present in the cultured cells and isolated after brief extraction, contained fragments of uniform size (approximately 7-10 kDa corresponding to approximately 14-20 disaccharides). Analysis by heparinase I and heparinase III degradations followed by electrophoretic separation (oligosaccharide mapping) showed that the oligosaccharides were rich in glucuronic acid but had a few sulphated iduronic acid residues at the periphery of each molecule. These results indicated that endoheparanase cleavage points were located close to linkages between N-sulphated glucosamine and sulphated iduronic acid, generating fragments that comprise a major portion of the unmodified segments and a minor portion of the highly modified segments. Prolonged extraction (24-48 h) of cells with Triton X-100 at 4 degrees C in the presence of proteinase inhibitors resulted in further degradation. There was an increase in the amount of heparan-sulphate oligosaccharides and a concomitant decrease in the amount of protein-bound heparan-sulphate chains present in the same extract. The heparan-sulphate oligosaccharides obtained after prolonged extraction were more heterogeneous in size comprising, in addition to the major species of approximately 7-10 kDa, intermediate and larger fragments of approximately 17 kDa and 30-40 kDa. This observation suggests that endoheparanase acted at periodically appearing, specific regions in the intact heparan-sulphate chain. Furthermore, the enzyme and substrate should remain closely associated during cold Triton X-100 extraction. To determine if the endogenously produced heparan-sulphate oligosaccharides were derived from a particular heparan-sulphate species degraded during the growth phase, proteoglycan-derived heparan-sulphate chains obtained from proliferating or quiescent fibroblasts were also examined. These chains showed similar oligosaccharide maps, except for a small increase in the amount of glucuronic acid as cell growth was arrested. Hence, an endoheparanase with restricted specificity may generate slightly different oligosaccharides in the various growth states.
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| 5. |
- Westergren-Thorsson, Gunilla, et al.
(författare)
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Transforming growth factor-beta induces selective increase of proteoglycan production and changes in the copolymeric structure of dermatan sulphate in human skin fibroblasts
- 1992
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 205:1, s. 277-286
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Tidskriftsartikel (refereegranskat)abstract
- Human embryonic skin fibroblasts were pretreated with transforming growth factor-beta (TGF-beta) for 6 h and then labeled with [35S]sulphate and [3H]leucine for 24 h. Radiolabeled proteoglycans from the culture medium and the cell layer were isolated and separated by isopycnic density-gradient centrifugation, followed by gel, ion-exchange and hydrophobic-interaction chromatography. The major proteoglycan species were examined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate before and after enzymatic degradation of the polysaccharide chains. The results showed that TGF-beta increased the production of several different 35S-labelled proteoglycans. A large chondroitin/dermatan sulphate proteoglycan (with core proteins of approximately 400-500 kDa) increased 5-7-fold and a small dermatan sulphate proteoglycan (PG-S1, also termed biglycan, with a core protein of 43 kDa) increased 3-4-fold both in the medium and in the cell layer. Only a small effect was observed on another dermatan sulphate proteoglycan, PG-S2 (also named decorin). These observations are generally in agreement with results of other studies using similar cell types. In addition, we have found that the major heparan sulphate proteoglycan of the cell layer (protein core approximately 350 kDa) was increased by TGF-beta treatment, whereas all the other smaller heparan sulphate proteoglycans with protein cores from 250 kDa to 30 kDa appeared unaffected. To investigate whether TGF-beta also influences the glycosaminoglycan (GAG) chain-synthesizing machinery, we also characterized GAGs derived from proteoglycans synthesized by TGF-beta-treated cells. There was generally no increase in the size of the GAG chains. However, the dermatan sulphate chains on biglycan and decorin from TGF-beta treated cultures contained a larger proportion of D-glucuronosyl residues than those derived from untreated cultures. No effect was noted on the 4- and 6-sulphation of the GAG chains. By the use of p-nitrophenyl beta-D-xyloside (an initiator of GAG synthesis) it could be demonstrated that chain synthesis was also enhanced in TGF-beta-treated cells (approximately twofold). Furthermore, the dermatan sulphate chains synthesized on the xyloside in TGF-beta-treated fibroblasts contained a larger proportion of D-glucuronosyl residues than those of the control. These novel findings indicate that TGF-beta affects proteoglycan synthesis both quantitatively and qualitatively and that it can also change the copolymeric structure of the GAG by affecting the GAG-synthesizing machinery. Altered proteoglycan structure and production may have profound effects on the properties of extracellular matrices, which can affect cell growth and migration as well as organisation of matrix fibres.
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