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Involvement of Heparan Sulphate Biosynthesis and Turnover in Cell Proliferation. A Novel Role for Nitric Oxide in Recycling of Heparan Sulphate Proteoglycans.

Mani, Katrin LU (2001)
Abstract (Swedish)
Popular Abstract in Swedish

Proteoglykaner (PG), framför allt sådana som bär glykosaminoglykan-sidokedjor av heparansulfat (HS) typ, utgör multifunktionella receptorer på cellytan och i pericellulär matrix hos alla adherenta celler i högre organismer. HS-proteoglykaner är inblandade i många olika cellprocesser, bl.a. koagulation, celltillväxt, och aktivering av enzymer, antienzymer samt bindning till olika matrixproteiner. HS påverkar och reglerar cell tillväxt genom att binda olika tillväxtfaktorer (Bernfield et al., 1999). Olika HS varianter har beroende på sin struktur hämmande eller stimulerande effekt. Speciell intressant är att FGF binder till vissa sekvenser i HS-kedjorna, vilket stimulerar FGF att aktivera den... (More)
Popular Abstract in Swedish

Proteoglykaner (PG), framför allt sådana som bär glykosaminoglykan-sidokedjor av heparansulfat (HS) typ, utgör multifunktionella receptorer på cellytan och i pericellulär matrix hos alla adherenta celler i högre organismer. HS-proteoglykaner är inblandade i många olika cellprocesser, bl.a. koagulation, celltillväxt, och aktivering av enzymer, antienzymer samt bindning till olika matrixproteiner. HS påverkar och reglerar cell tillväxt genom att binda olika tillväxtfaktorer (Bernfield et al., 1999). Olika HS varianter har beroende på sin struktur hämmande eller stimulerande effekt. Speciell intressant är att FGF binder till vissa sekvenser i HS-kedjorna, vilket stimulerar FGF att aktivera den signalförmedlande receptorn (Guimond et al., 1993). Det har visats att vissa former av HS kan utöva en direkt antiproliferativ effekt (Westergren-Thorsson et al., 1991; 1993).



Tumörceller bär också på sin yta proteoglykaner av HS-typ. En familj som kallas glypikan uttrycks i ökad mängd vid malign transformation (Bernfield et al., 1999). En annan karakteristisk tumörcellsprodukt är enzymet endoheparanas (HS-as) som partiellt fragmenterar HS. Detta underlättar nedbrytningen av basalmembran, som innehåller en annan HSPG, perlekan, samt frisättningen av FGF från sin vävnadsreservoir. Denna faktor verkar både chemotaktiskt och tillväxtstimulerande på vaskulära endotelceller.



Proteoglykaner kan vara integrerade i plasmamembranen via penetrerande hydrofoba peptidsegment (t.ex. syndekaner och betaglykan) eller kovalent bundna till plasmamembranen via ett glykosylfosfatidylinositol-ankare, kallat GPI (en sådan PG familj är glypikanerna med 6 isoformer till dags dato). De flesta cellyte-PG är substituerade med HS men även chondroitinsulfat (CS) förekommer. Glykanerna är kovalent kopplade till proteinet via en gemensam GlcA-Gal-Gal-Xyl sekvens där Xyl är bundet till OH-gruppen på serin. HS-kedjan byggs på som en polymer av (-4GlcAb1-4GlcNAca1-)n där n varierar från ca 50 till kanske 200. Genom olika modifikationer, såsom utbyte av NAc mot NSO3, epimerisering av GlcA till IdoA och substitutioner med OSO3 på C3/C6 i GlcN och C2/C3 i GlcA/IdoA, vilket sker i varierande grad i olika sektioner av polymeren, skapas ett stort antal sackaridsekvenser (Lindahl et al., 1998). HS degraderas av speciella endoheparanaser lokaliserade till endosomer och dessa enzymer är speciellt aktiva i tumörceller (Nakajima et al., 1988). HS i extrakt av humana umbilikalvenendotelceller (HUVEC) degraderas under betingelser som pekar på en icke-enzymatisk degradation (Lindblom et al., 1989; Lindblom, 1991). Endotelceller producerar kväveoxid (NO) från arginin med hjälp av NO-syntas (eNOS). NO, som är en välkänd signalssubstans har kort livslängd eftersom den oxideras vid kontakt med syre, varvid bl.a. nitrit (NO2-) bildas (Wink et al., 1996). NO2- i sin tur degraderar HS genom deaminativ klyvning. Vilar et al. (1997) fann att HUVEC genererar tillräckliga mängder NO2- för att kunna degradera exogent tillfört HS. Man har också påvisat förhöjd NO-produktion i tumörer (Doi et al., 1996; Thomsen and Miles, 1998).



Glypikan-1 (HSPG) kan internaliseras och recirkulera tillbaka till cellytan. Under passagen genom cellen (troligen via endosomer och Golgi) sker en degradering av HS-kedjorna följt av en återuppbyggnad av nya HS-kedjor (Fransson et al., 1995; Edgren et al., 1997). Vi har funnit att transformerade vaskulära endotelceller (ECV) uttrycker glypikan-1. Den förekommer huvudsakligen som protein med korta HS-stumpar, samtidigt som en betydande pool av HS-oligosackarider finns i cellerna (delarbete II). Vid blockering av recirkulationen med (a) brefeldin A, (b) heparanas-hämmaren suramin eller (c) NOS-hämmare och sulfamat (nitritförstörare) ansamlas (a) intakt glypikan-PG respektive (b och c) partiellt HS-degraderade former. Den inledande klyvningen synes ske intill GlcN med fri aminogrupp. Vi har också funnit att recirkulation av glypikan, efter endogen degradation av HS-delen, uteblir om ECV behandlas med NOS-hämmare eller sulfamat. Efter tillförsel av NO återbildas glypikan-PG från de degraderade formerna (delarbete II). Fortsatta studier har visat att de fria NH2-grupperna är samlade till en region nära proteinändan (delarbete III).



Vi har även funnit att en viss naphtol-innehållande xylosid kan selektivt hämma celldelningen i tumörceller och transformerade celler (delarbete I). Varken motsvarande L-xylosid, som inte initierar HS-syntes, eller xylosider som enbart initierar CS-syntes, har någon effekt på celldelningen inom motsvarande koncentrationsintervall. I ett samarbete med avd. för org. kemi, LTH har vi testat en rad syntetiska xylosider där aglykonens struktur varieras på systematisk sätt. En förutsättning för tillväxthämmande effekten synes vara att xylosiden bär hydroxylgrupper i aglykondelen och att den leder till syntes av HS som senare degraderas av nitrit (delarbete IV). (Less)
Abstract
The present investigation focuses on the role of HS metabolism in cell proliferation. The effect of the HS priming ß-D-xyloside, 2-(6-hydroxynaphthyl)-O-ß-D-xylopyranoside (Xyl-2-Nap-6-OH) on the proliferation of normal and transformed cells was studied. Xyl-2-Nap-6-OH inhibited growth of transformed cells to a significantly greater extent than normal cells. The growth inhibition exerted by the xyloside was believed to be due to its ability to prime GAG synthesis since the non-priming L-isomer did not possess any antiproliferative activity. The molecular structure of the aglycone and its ability to prime HS were other features assumed to be of importance for its antiproliferative activity.



Recycling of HSPG may be a... (More)
The present investigation focuses on the role of HS metabolism in cell proliferation. The effect of the HS priming ß-D-xyloside, 2-(6-hydroxynaphthyl)-O-ß-D-xylopyranoside (Xyl-2-Nap-6-OH) on the proliferation of normal and transformed cells was studied. Xyl-2-Nap-6-OH inhibited growth of transformed cells to a significantly greater extent than normal cells. The growth inhibition exerted by the xyloside was believed to be due to its ability to prime GAG synthesis since the non-priming L-isomer did not possess any antiproliferative activity. The molecular structure of the aglycone and its ability to prime HS were other features assumed to be of importance for its antiproliferative activity.



Recycling of HSPG may be a vehicle for endo/exocytosis of HS-binding growth factors and polyamines and thereby regulating cell proliferation. HSPG metabolism/recycling was therefore extensively studied in transformed endothelial cells. The major focus was on the role of NO-derived nitrite in the turnover of HSPG. As N-unsubstituted GlcN in HS chains are potential sites for NO-generated cleavage, the content and location of such residues was investigated. Transformed endothelial cells expressed a HSPG with a core protein of 60-70 kDa, which was recognised by a polyclonal antiserum raised against recombinant glypican-1 protein. In unperturbed cells, most of the radiolabelled glypican-1 carried truncated HS chains accompanied by HS oligosaccharides. Treatment with brefeldin A, which inhibits transport from the ER to the Golgi and also exit from the TGN and/or endocytosis in polarised cells, resulted in accumulation of glypican PG with full-size side chains while oligosaccharides disappeared. Treatment with suramin, an inhibitor of endoheparanase, led to partial inhibition of degradation of HS. Glypican-1 glycoforms in brefeldin A-treated cells contained long HS chains with GlcNH2 residues in multiple places, whereas unperturbed cells, suramin-treated cells, and nitrite-deprived cells contained short HS chains with only a few GlcNH2 residues. The number of GlcNH2 residues increased by combined suramin treatment and nitrite deprivation. This suggested that GlcNH2 residues and endoheparanase cleavage sites were closely located in HS chains. Pulse-chase studies clearly indicated that suramin arrested chains were the precursor of large-size PGs in the absence of de novo glypican-1 core protein synthesis. Recycling of suramin-arrested chains back to brefeldin A-arrested large PGs was precluded by nitrite deprivation. Formation of brefeldin A-arrested large glypican PGs was restored when NO-donor was supplied to nitrite-deprived cells. Taken together our data suggest a recycling of glypican-1 in transformed endothelial cells. During recycling, there is endoglycosidic cleavage of HS at or near GlcNH2 residues, and removal of these short nonreducing terminal GlcNH2 containing saccharides by NO derived nitrite would provide fresh acceptor-sites for HS chain extension.



In order to assess the relationship between HS priming and antiproliferative activity of naphthol-containing ß-D-xylosides, cell proliferation assays and GAG priming studies were performed in the presence of nitrite depriving drugs. Different xylosides were synthesised and tested for growth inhibition and priming of HS synthesis. The selective growth-inhibitory effect of Xyl-2-Nap-6-OH appeared to be quite specific for this compound. Xylosides without the 6-hydroxyl, with an O-methylated 6-hydroxyl, with a free hydroxyl in a different position, or with non-fused aromatic rings were not antiproliferative. All of the tested naphthol-based xylosides were capable of priming HS synthesis. Inhibition of degradation by suramin or nitrite-deprivation resulted in increased intracellular accumulation of HS chains. Interestingly, nitrite-deprivation abrogated the growth inhibitory effect of Xyl-2-Nap-6-OH. We propose that Xyl-2-Nap-6-OH initiates synthesis of HS chains with occasional N-unsubstituted GlcN residues and that these chains can be taken up by the cells and degraded to bioactive compounds in the presence of nitrite. (Less)
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author
opponent
  • Prof. Kjellén, Lena
organization
publishing date
type
Thesis
publication status
published
subject
keywords
nitrite., nitric oxide, growth-inhibition, xyloside, glypican, heparan sulphate, Proteoglycan, glycosaminoglycan, Clinical biology, Klinisk biologi
pages
105 pages
publisher
BMC Biomedical Centre, Lund University
defense location
Rune Grubb salen, BMC
defense date
2001-04-24 10:15
language
English
LU publication?
yes
id
3cde4df0-7367-4fb1-9075-ca357c345b7f (old id 41457)
date added to LUP
2007-07-31 10:36:48
date last changed
2016-09-19 08:45:15
@phdthesis{3cde4df0-7367-4fb1-9075-ca357c345b7f,
  abstract     = {The present investigation focuses on the role of HS metabolism in cell proliferation. The effect of the HS priming ß-D-xyloside, 2-(6-hydroxynaphthyl)-O-ß-D-xylopyranoside (Xyl-2-Nap-6-OH) on the proliferation of normal and transformed cells was studied. Xyl-2-Nap-6-OH inhibited growth of transformed cells to a significantly greater extent than normal cells. The growth inhibition exerted by the xyloside was believed to be due to its ability to prime GAG synthesis since the non-priming L-isomer did not possess any antiproliferative activity. The molecular structure of the aglycone and its ability to prime HS were other features assumed to be of importance for its antiproliferative activity.<br/><br>
<br/><br>
Recycling of HSPG may be a vehicle for endo/exocytosis of HS-binding growth factors and polyamines and thereby regulating cell proliferation. HSPG metabolism/recycling was therefore extensively studied in transformed endothelial cells. The major focus was on the role of NO-derived nitrite in the turnover of HSPG. As N-unsubstituted GlcN in HS chains are potential sites for NO-generated cleavage, the content and location of such residues was investigated. Transformed endothelial cells expressed a HSPG with a core protein of 60-70 kDa, which was recognised by a polyclonal antiserum raised against recombinant glypican-1 protein. In unperturbed cells, most of the radiolabelled glypican-1 carried truncated HS chains accompanied by HS oligosaccharides. Treatment with brefeldin A, which inhibits transport from the ER to the Golgi and also exit from the TGN and/or endocytosis in polarised cells, resulted in accumulation of glypican PG with full-size side chains while oligosaccharides disappeared. Treatment with suramin, an inhibitor of endoheparanase, led to partial inhibition of degradation of HS. Glypican-1 glycoforms in brefeldin A-treated cells contained long HS chains with GlcNH2 residues in multiple places, whereas unperturbed cells, suramin-treated cells, and nitrite-deprived cells contained short HS chains with only a few GlcNH2 residues. The number of GlcNH2 residues increased by combined suramin treatment and nitrite deprivation. This suggested that GlcNH2 residues and endoheparanase cleavage sites were closely located in HS chains. Pulse-chase studies clearly indicated that suramin arrested chains were the precursor of large-size PGs in the absence of de novo glypican-1 core protein synthesis. Recycling of suramin-arrested chains back to brefeldin A-arrested large PGs was precluded by nitrite deprivation. Formation of brefeldin A-arrested large glypican PGs was restored when NO-donor was supplied to nitrite-deprived cells. Taken together our data suggest a recycling of glypican-1 in transformed endothelial cells. During recycling, there is endoglycosidic cleavage of HS at or near GlcNH2 residues, and removal of these short nonreducing terminal GlcNH2 containing saccharides by NO derived nitrite would provide fresh acceptor-sites for HS chain extension.<br/><br>
<br/><br>
In order to assess the relationship between HS priming and antiproliferative activity of naphthol-containing ß-D-xylosides, cell proliferation assays and GAG priming studies were performed in the presence of nitrite depriving drugs. Different xylosides were synthesised and tested for growth inhibition and priming of HS synthesis. The selective growth-inhibitory effect of Xyl-2-Nap-6-OH appeared to be quite specific for this compound. Xylosides without the 6-hydroxyl, with an O-methylated 6-hydroxyl, with a free hydroxyl in a different position, or with non-fused aromatic rings were not antiproliferative. All of the tested naphthol-based xylosides were capable of priming HS synthesis. Inhibition of degradation by suramin or nitrite-deprivation resulted in increased intracellular accumulation of HS chains. Interestingly, nitrite-deprivation abrogated the growth inhibitory effect of Xyl-2-Nap-6-OH. We propose that Xyl-2-Nap-6-OH initiates synthesis of HS chains with occasional N-unsubstituted GlcN residues and that these chains can be taken up by the cells and degraded to bioactive compounds in the presence of nitrite.},
  author       = {Mani, Katrin},
  keyword      = {nitrite.,nitric oxide,growth-inhibition,xyloside,glypican,heparan sulphate,Proteoglycan,glycosaminoglycan,Clinical biology,Klinisk biologi},
  language     = {eng},
  pages        = {105},
  publisher    = {BMC Biomedical Centre, Lund University},
  school       = {Lund University},
  title        = {Involvement of Heparan Sulphate Biosynthesis and Turnover in Cell Proliferation. A Novel Role for Nitric Oxide in Recycling of Heparan Sulphate Proteoglycans.},
  year         = {2001},
}