- Email: [email protected]
Lipoprotein lipase enhanced binding of human triglyceride-rich lipoproteins to heparan sulphate: Modulation by apolipoproteins E, C and apolipoprotein glycosylation
$30
Oral session abstracts / A therosclerosis 115 (Suppl.) (1995) $3-$42 019 Proteoglycans
O19 PROTEOGLYCANS 109 Withdrawn
111 LIPOPROTEIN LIPASE ENHANCED BINDING OF HUMAN TRIGLYCERIDE-R1CH L1POPROTEINS TO HEPARAN SULPHATE: MODULATION BY APOLIPOPROTEINS E, C AND APOLIPOPROTE1N GLYCOSYLATION H.J.J. van Barlingen, H. de Jong, D.W. Erkelens, T.W.A. de Bruin Department of Internal Medicine, University Hospital, Utrecht University. The Netherlands Catabolism of triglyceride-rich lipoproteins /TRLP) requires interaction with heparan sulfate proteoglycans (HSPG) on the vessel wall, complexed with lipnprotein lipase (LPL), or in the liver. This study investigated the effect of enriching human TRLP with apolipoprotein lapol E and apoCs and the effect of deglycosylation of apolipoproteins on the binding of heparan sulphate (HS). TRLPs, were isolated by ultracentrifugation and incubated with purified apoE, apoCs, neuraminidase, or O-glycosidase. Modified and control TRLP were coated on microtiter plates. The ability to bind biotin-conjugated heparan sulphate (b-HS), in the presence or absence of heat inactivated LPL was measured. ApoE enhanced the binding of b-HS to TRLP. both in the presence and absence of LPL. The increased binding of b-HS to TRLP elicited by apoE or LPL involved different binding interactions that were complementary. ApoC decreased the binding of b-HS to TRLP only in the precence of LPL. Enzymatic treatmelu of TRLP resulted in increased b-HS binding. Our results provide evidence that the docking of TRLP to LPL. anchored by HSPG to the vascular wall. is regulated by the apoC content of the TRLP Lipoprotein binding to HSPG in the space of Disse, which is necessary for the binding and degradation of TRLP remnants, is dependent on apoE and requires apoC depletion.
110
112
CHLORATE-INDUCED UNDERSULFATION OF PROTEOHEPARAN SULFATE STIMULATES BASIC FIBROBLAST GROWTH FACTOR EXPRESSION AND PROTEIN SYNTHESIS BUT SUPPRESSES DIVISION OF CORONARY SMOOTH MUSCLE CELLS Ch. Schriever, A. Schmidt, G. Breithardt, E. Buddecke Institute of Arteriosclerosis Research, Division of Molecular Cardiology, University of Miinster, Germany
GROWTH STATUS-DEPENDENT CHANGES OF bFGF COMPARTMENTALIZATION AND HEPARAN SULFATE STRUCTURE IN ARTERIAL SMOOTH MUSCLE CELLS A. Schmidt, A. Skaletz-Rorowski, G. Breithardt, E. Buddecke Institute for Arteriosclerosis Research, Division of Molecular Cardiology, University of MOnster, Germany
Proteoheparan sulfate (HSPG) is known to form a trimolecular complex with basic fibroblast growth factor (bFGF) and the FGF receptor (FGF-RI) that supports mitogenesis of several cell lines. Cell mutants deficient in HSPG synthesis do not respond to bFGF but are reconstituted by low doses of heparin. Sodium chlorate is known to block sulfate transfer to growing glycosaminoglycan chains and leads to the formation of an undersulfated HSPG, that reduces bFGF triggered signal transduction for cell division. We have adressed the question whether chlorate-induced suppression of HSPG sulfation influences the bFGF expression and compartmentalisation and found significant alterations of cell growth and endogenous bFGF production When sodium chlorate (lOmM) was added to cultured bovine coronary smooth muscle ceils (cSMC) we observed a decrease by 66% in cell number after 96h compared to a control and a corresponding decrease in DNA synthesis as judged from the [~H]thymidine incorporation. Furthermore we observed a twofold increase of intracellular and pericellular bFGF expression and protein synthesis under chlorate. Chlorate reduced the [35S] incorporation into total cell-associated proteoglyeans by 90% but affected the sulfate incorporation of chondmitin/dermatan containing proteoglycans and heparan containing pmteoglycans to a different extent. These results would suggest that undersulfation of HSPG modulates bFGFmediated transcellular signaling pathways which leads to an increase of bFGF- and protein-synthesis but not to subsequent cell division The results further indicate that bFGF is produced in the early Gl-phase because bFGF increases correspondingly with the protein content of cSMC although the DNA-synthesis is not elevated.
Cultured bovine arterial smooth muscle cells express 6-7 ng basic fibroblast growth factor (bFGF)/mg cell protein and distribute it to several compartments. About 80% of total bFGF remain intracellulady, 15% are present in cell associated (pericenular) form and 5% can be recovered from the subcellular matrix. No bFGF can be detected in the culture medium. All bFGF fractions have full biological activity. They are quantified by a highly specific immunoassay system and identified after SDS polyacrylamide electrophoresis as a 18 kD double band by immunoblotting. During exponential growth the intracellular concentration of bFGF decreases from about 130 pg to 20-40 pg/lOs cells. Simultaneously the pericellular bFGF increases to 60-70% of total bFGF, but declines continuously with increasing cell density whereas the intracellular bFGF reincreases under these conditions, The pericellular bFGF is known to form complexes with (membrane integrated) proteoheparan sulfate which undergoes structural changes during transition from subconfluent to confluent growth status. After metabolic labelling of the cells with [~S]sulfate and [3H]glucosamine the 3~S/3H ratio of heparan sulfate oligosaccharides increases from 1.58 during proliferation to 2.47 in growthinhibited cells. The results indicate that the bFGF-induced proliferation of arterial smooth muscle cells depends on the pericellular localization of bFGF and on a specific molecular organization of the cell surface heparan sulfate. Depending on its specific structural characteristics heparan sulfate may promote or inhibit bFGF receptor binding and signal transduction.
Oral session abstracts / A therosclerosis 115 (Suppl.) (1995) $3-$42 019 Proteoglycans
O19 PROTEOGLYCANS 109 Withdrawn
111 LIPOPROTEIN LIPASE ENHANCED BINDING OF HUMAN TRIGLYCERIDE-R1CH L1POPROTEINS TO HEPARAN SULPHATE: MODULATION BY APOLIPOPROTEINS E, C AND APOLIPOPROTE1N GLYCOSYLATION H.J.J. van Barlingen, H. de Jong, D.W. Erkelens, T.W.A. de Bruin Department of Internal Medicine, University Hospital, Utrecht University. The Netherlands Catabolism of triglyceride-rich lipoproteins /TRLP) requires interaction with heparan sulfate proteoglycans (HSPG) on the vessel wall, complexed with lipnprotein lipase (LPL), or in the liver. This study investigated the effect of enriching human TRLP with apolipoprotein lapol E and apoCs and the effect of deglycosylation of apolipoproteins on the binding of heparan sulphate (HS). TRLPs, were isolated by ultracentrifugation and incubated with purified apoE, apoCs, neuraminidase, or O-glycosidase. Modified and control TRLP were coated on microtiter plates. The ability to bind biotin-conjugated heparan sulphate (b-HS), in the presence or absence of heat inactivated LPL was measured. ApoE enhanced the binding of b-HS to TRLP. both in the presence and absence of LPL. The increased binding of b-HS to TRLP elicited by apoE or LPL involved different binding interactions that were complementary. ApoC decreased the binding of b-HS to TRLP only in the precence of LPL. Enzymatic treatmelu of TRLP resulted in increased b-HS binding. Our results provide evidence that the docking of TRLP to LPL. anchored by HSPG to the vascular wall. is regulated by the apoC content of the TRLP Lipoprotein binding to HSPG in the space of Disse, which is necessary for the binding and degradation of TRLP remnants, is dependent on apoE and requires apoC depletion.
110
112
CHLORATE-INDUCED UNDERSULFATION OF PROTEOHEPARAN SULFATE STIMULATES BASIC FIBROBLAST GROWTH FACTOR EXPRESSION AND PROTEIN SYNTHESIS BUT SUPPRESSES DIVISION OF CORONARY SMOOTH MUSCLE CELLS Ch. Schriever, A. Schmidt, G. Breithardt, E. Buddecke Institute of Arteriosclerosis Research, Division of Molecular Cardiology, University of Miinster, Germany
GROWTH STATUS-DEPENDENT CHANGES OF bFGF COMPARTMENTALIZATION AND HEPARAN SULFATE STRUCTURE IN ARTERIAL SMOOTH MUSCLE CELLS A. Schmidt, A. Skaletz-Rorowski, G. Breithardt, E. Buddecke Institute for Arteriosclerosis Research, Division of Molecular Cardiology, University of MOnster, Germany
Proteoheparan sulfate (HSPG) is known to form a trimolecular complex with basic fibroblast growth factor (bFGF) and the FGF receptor (FGF-RI) that supports mitogenesis of several cell lines. Cell mutants deficient in HSPG synthesis do not respond to bFGF but are reconstituted by low doses of heparin. Sodium chlorate is known to block sulfate transfer to growing glycosaminoglycan chains and leads to the formation of an undersulfated HSPG, that reduces bFGF triggered signal transduction for cell division. We have adressed the question whether chlorate-induced suppression of HSPG sulfation influences the bFGF expression and compartmentalisation and found significant alterations of cell growth and endogenous bFGF production When sodium chlorate (lOmM) was added to cultured bovine coronary smooth muscle ceils (cSMC) we observed a decrease by 66% in cell number after 96h compared to a control and a corresponding decrease in DNA synthesis as judged from the [~H]thymidine incorporation. Furthermore we observed a twofold increase of intracellular and pericellular bFGF expression and protein synthesis under chlorate. Chlorate reduced the [35S] incorporation into total cell-associated proteoglyeans by 90% but affected the sulfate incorporation of chondmitin/dermatan containing proteoglycans and heparan containing pmteoglycans to a different extent. These results would suggest that undersulfation of HSPG modulates bFGFmediated transcellular signaling pathways which leads to an increase of bFGF- and protein-synthesis but not to subsequent cell division The results further indicate that bFGF is produced in the early Gl-phase because bFGF increases correspondingly with the protein content of cSMC although the DNA-synthesis is not elevated.
Cultured bovine arterial smooth muscle cells express 6-7 ng basic fibroblast growth factor (bFGF)/mg cell protein and distribute it to several compartments. About 80% of total bFGF remain intracellulady, 15% are present in cell associated (pericenular) form and 5% can be recovered from the subcellular matrix. No bFGF can be detected in the culture medium. All bFGF fractions have full biological activity. They are quantified by a highly specific immunoassay system and identified after SDS polyacrylamide electrophoresis as a 18 kD double band by immunoblotting. During exponential growth the intracellular concentration of bFGF decreases from about 130 pg to 20-40 pg/lOs cells. Simultaneously the pericellular bFGF increases to 60-70% of total bFGF, but declines continuously with increasing cell density whereas the intracellular bFGF reincreases under these conditions, The pericellular bFGF is known to form complexes with (membrane integrated) proteoheparan sulfate which undergoes structural changes during transition from subconfluent to confluent growth status. After metabolic labelling of the cells with [~S]sulfate and [3H]glucosamine the 3~S/3H ratio of heparan sulfate oligosaccharides increases from 1.58 during proliferation to 2.47 in growthinhibited cells. The results indicate that the bFGF-induced proliferation of arterial smooth muscle cells depends on the pericellular localization of bFGF and on a specific molecular organization of the cell surface heparan sulfate. Depending on its specific structural characteristics heparan sulfate may promote or inhibit bFGF receptor binding and signal transduction.