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Hypothesis of Mechanistically Related Nitric Oxide Involvement of Heparan

Sulphate in Degenerative Diseases & Animal Evolution

Chronic stress may alter RNA and DNA via an ascorbate modulated heparan sulphate/nitric oxide

dependent mechanism.

David Grant*

To bottom *Address for correspondence Ashbank, 2a High Street, New Deer, Turriff, AB53 6SX,United Kingdom,

This literature-based research (part funded by the Carnegie Foundation for the Universities of

Scotland) was conducted after tenure of a Research Fellowship with Dr FB Williamson and Dr WF

Long at the University of Aberdeen Department of Molecular and Cell Biology. Prof. KEL McColl

(Univeristy of Glasgow, Department of Medicine & Therapeutics) is thanked for discussions.

Draft by DG 7/02, 8/02

For Medical Hypothesis

Post 30/7/02 modifications

1. Summary

A necessarily tentative hypothesis is presented in which the already large range of of controls known to

be exerted by the system of sulphated polysaccharides heparin/heparan sulphateproteoglycans(HSPGs) is extrapolated to include animal evolution and degenerative diseases,

including cancer. These are suggested to be similarly promoted by stress events which stimulate a

nitric oxide (NO)-dependent, postsynthetic non-enzymic processing of heparan sulphate (HS) allowing

this, together with interactive effects generated with a parallel system dependent on enzymic

postsynthetic HS processing, to enable cross reaction between the two HS processsing systems to

generate amplification of long-term feedback loops. This enables altered Golgi HS biosynthesis for

alteration genetic control element modulation (e.g. by HS fragmentbinding to clusters of basic amino

acids in nucleic acid control elements).

It is suggested that the existence of this dual control mechanism of HS processing, while permitting

directed animal evolution, also permits, by disfunction of this system, the occurrence of various

degenerative diseases. Directed alteration for stress minimization may be especially directed at in HS

processing, sulphatase enzymes implicated in tissue morphogenesis.

Additional discussion of HS-related controls includes a role for ascorbate in tissue protection both in

by its effect modulating NO activity and by directly influencing heparin/HS biosynthsis and interaction

of HS systems with cysteine and tyrosine based signalling systems.

2.Introduction. HS, biosynthesised exclusively as heparan sulphate proteoglycans (HSPGs) seems to

have evolved coincidentally with the initital appearance of multicellular animals. This hint that the

evolution of multicellular animals could somehow be dependent on HSPG biochemistry can now been

seen to be consistent with the emerging knowledge of wide-ranging HSPG functional roles in cell-to-cell interactions in developmnetal biology.

Proposals for debate include the notion that HSPGs contibute to the response of genes to stress.

An initial result of evolutionary pressures by NO and its metabolites is suggested to be an alteration in

HS signalling producing a prompted targeted modulation of HS processing enzymes (which are known

control the formation of animal development HS morphogens) via feedback loops linking genes with

their eventual products via HS oligosaccharides which are formed both by the non-enzymic but highly

stress-dependent NO processing and by parallel enzymic processing.

Additional possible key mechanisms relevant to transmission of oxidative stress to genes, however, may

include those proposed to be tranmitted by switching systems containing tyrosine and cysteine which

are capable of interacting both with HS (including fragment) and with NO (and its metabolite)

facilitating environmental and genetic interfacing. Cysteine and tyrosine (CY) groupings e.g. in

SH2 protein domains are implicated in intracellular signalling. C..Y systems also occur in RNA

processing enzymes including RNAase.

Such (CY) systems are proposed to be subject to HS as well as a NO (and metabolites) dependence

on (oxidative stress related) including trace redox metal catalysis of the formation of tyrosine nitrate.


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3. General Background3-1 Importance of HS in Multicellular Animal Biochemistry.

HSPG isinvolved in multicelluar animal development, haematopoiesis and for mRNA control.

The HS system of sulphated polysaccharides (cf Dietrich et al, 1983) occurring at all adherent animalcell surfaces as HSPGs (which an emerging consensus of recent work suggests to be precise-sugar-

sequence-dependent information carriersspecifying each of cell type by unique HS patterns) has beenso highly conserved throughout animal evolution, that this suggests that there mustexist some major

currently unknown function for HS, in addition to the growing number of known activities. It is now

proposed, that this is the faculty of directly influencing animal evolution by the provision of feedback

loops involving an influence of HSPG and its fragments upon selective alteration in gene activity.

Such an HS-driven model of animal evolution is consistent with the HSPG modulation of kinase

activities in growth factors and their receptors, involved in the processing of extracellular signalling,

that are now known to pattern fields of cells in the developmental biology of animals (Perrimon &

Bernfield 2000), including a dependence of embryogenesis on extracellular HSPG sulphatase activity

(Dhoot et al 2001).

(A corollary to this idea is that degenerative diseases including cancer are aberrant consequences of a

similar process. Uncontrolled tumour growth may be caused by an alteration in HS gene controlfunction (cf Dundas et al 2000) which is potentially subject to influence by the effects of oxidant/nitrant

stress).

3-2 Effects of Reactive Oxygen Species

Multicellular animal evolution seems to have been stimulated by the emergence of photosynthetic

organisms capable of producing sufficient amounts of atmospheric oxygen and the possible formation

of nitric oxides to promote the necessary stress factor-related catalysis of the generation of reactive

oxygen and nitrogen intermediates capable of prompting environmental-directed alteration in RNA for

eventual reverse transcription into genetic DNA.

3-3 Effects of Nitric Oxide & its Metabolites

Oxidant stress has long been a central theme in medicine and biology, this having likely effects on

animal lifespan and the occurrence of degenerative diseases such as cancer. In recent years, however,additional related effects of nitric oxide and its metabolites have been discovered which seem to

uniquely involve HSPG biochemistry and which are also implicated in anti-pathogen, anti-crystal, and

antioxidant defenses which all additionally potentially involve the effects of induced immunologically

produced NO biochemical activity. A NO-dependent HSPG scission reaction is specifically directed

towards HS chains in GAG mixtures. This reaction was employed in laboratory protocols for

evaluating HS by oligosaccharide formation in GAG mixtures long before the discovery that such

reactions had any physiological relevance.

3-4 Ascorbate in Control of HS and NO

Critical control of reactive oxygen and reactive nitrogen oxide chemical species is clearly necessary

for the maintenance of cellular integrity, evidently accomplished in animals by a number of

complimentary systems which include important activities of superoxide dismutases (extracellularly

bound to HSPG), as well as the major antioxidant ascorbate, which both demonstrates majorinfluences on HSPG biochemistry, directly via enhancing the rate of biosynthesis and degree of

sulphation, as well, indirectly, by controlling the rate of postsythetic deaminative cleavage by nitric

oxide and its metabolites. NO itself is also a powerful antioxidant (e.g. Kanner et al 1991), it

preventing the oxidative degradation of cell surface lipids more effectively than does alpha tocopherol

(Rubbo et al 2000). Ascorbate efficiently reduces nitrite back to NO.

3.4.1 Effects of Lipids

Lipids also signal for altered HSPG biosynthesis evidently by affecting Golgi protein transport.

Deleterious effects of e.g. saturated lipids may be at least partly accounted for, it is suggested, by such

effects, which may also explain beneficial effects of e.g. omega three lipids on alteration of HSPG in an

anti-cancer role may partly be due to such HSPG alteration. In a related scenario oxidised lipids may

signal for a deleterious pro-alteraiosclerosis alteration in HS biosynthesis at blood vessel walls.

Excess serum glucose may similarly produce deleterious HS-dependent lipid procssing via biosynthetic

modulatoin by excess glucose for reduction of sulphation of HS.


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3-5 Effects of Polyamines.

Another function of NO which together with ascorbate is apparently to boost Golgi-controlled HS

biosynthesis and increase sulphation, which further contibute to a complex HS- antioxidant-related

biofeedback mechanisms, apparently includes the scission of heparan sulphate at specific sites by NO

metabolites specified by polyamines (Ding et al 2001)).

3-6 Effects of Cysteine-Tyrosine Coupling.A potential mode of coupling between oxidantstress signals, HSPG and genes is via (CY) signalling

systems (cf Nakashima, 1996). Such coupled (CY) redox metal systems for the facilitation ofsignalling likely even preceded genetic use of DNA since ribonucleotide reductase which requires for

its biosynthesis the pre-existence of this type of redox-based activity. RNAases also contain conserved

(CY) groupings.

The (CY) systems are also chemically related to the industrial antioxidant phenolic and bivalent

sulphur systems, which can interact synergistically, and also to alpha tocopherol, glutathione and, it is

believed, various clusters of cysteine and tyrosine (CY) in proteins including growth factors

(discussed by Grant et al (1989)). An important antioxidant function proposed for(CY) containing

apoliprotein a which apparently is uniquely elevated in animals such as man which lack the ability to

synthesise ascorbic acid (Rath & Pauling 1990) but which also suggests that ascorbate-like HSPG

control feedback signalling may also be an inherent property of such (C..Y) systems.HSPG/NO-ascorbate/(CY)- based systems are therefore suggested to be capable ot inducing initial

alterations in Golgi apparatus biosynthesis of those HSPG sequences which are potentially designated,

following fragmentation to competetivly bind to basic amino acid clusters in targeted RNA and DNA

control elements and especially specifying those proteins which are most affected by the applied stress.

Those genes specifiying e.g. heparanse but perhaps more especially sulfatase enzymes involved directly

in organism development may be sensitive to such directed rearrangements.

3-7 HSPG Genetic Transfection?

Genetic transfection processes may also potentially involve related HSPG activities since cell surface

HSPG-gene polycation complexes facilitate the entry of genetic material into distant target cells

(Mislick et al 1996; Mounkes et al 1998) provide a possible route for evolutionary directed change

e.g. following apoptosis, to germ cells. Related effects of pathogens include HSPG binding bacterial

(e.g. Helicobacter pylori) and HSPG binding viruses, which are further potential contributors totranfer of genetic material via HSPG dependent pathways including reverse transcriptase activities of

retroviruses.

3.7.1

Prion biochemistry seems to have a fundemental HSPG linked perspective, the details of which are yet,

however, incompletely understod, but may be relevant to this discussion. The use by host organisms of

viral reverse transcriptase activities also seems a suitable potential mechanism of gene modulation.

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Such newly proposed heparin/HSPG functions, as well as the long-established roles in haemostasis,lipid processing, immune responsesandangiogenesiswill potentially be dependenton signalling by

specific heparin/HS microstructures,especially sulphationpatterns, which are apparently cell type

specific but are subject to an evidently finely contolled alterationduring biosynthesis beingapparently potentially affected by lactate/pyruvate ratio, and the extracellular levels of glucose

glucosamine, ascorbate, toxins, trace metal ions, types of lipids, lipid peroxidation products and

perhaps oncogenic products (since transformed cells exhibt major alteration in HSPGs). Post-syntheticHSPGprocessing is also known to occur directly by action of reactive oxygen species as well

as by the specific enzymic actions of heparanases and sulfatases. This is in addition to the well known,

highly heparin/HSPG-specific non-enymic processing facilitated by NO (and itsmetabolite especially

nitrous acid) affordedGlcNH2 deaminative cleavage reaction and which recent work has indicated,

may be programmed via polyamine signalling (Ding et al 2001) but isprobably hypersensitive to a

directcatalytic influence by stress factors (especially by trace metals) and augmented by additional NOformation induced by immune responses. Acidosis, a form of stress is known to promote the

deaminative cleavage of HS as is the (additional) presence of unprotected trace amounts of redox

metal ions (such as those of iron and copper). These situations directly increases unsulphonated

glucosamine moieties in HSPG primed for deaminative cleavage. These situations also likely increase


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the incidence of tyrosine nitration (which although a hallmark of degenerative processes does not form

by reaction of tyrosine with peroxynitrite at pH 7.4 in vitro in absence of catalysis).

The individually specificfied cellular dependence of arranged anionic charge arrays present in HS

polysaccharide chains seems to provide a system of extracellular communication allowing an unique

specification of individual cell typing including patterning for organ and organism speciation.

Rather than DNA biochemistry being the sole determinant of the aetilogy of degenerative diseases suchas cancer and in determining how directed evolution might be possible, HSPG biochemistry is now

suggested to participate in stress-directed controlled intracelluar nucleic acid rearrangements via the

effect of NO modulated HSPG-dependent morphogenesis processes which are capable by amplified

feedback signalling via alteration in the Golgi biosynthesis of HS to eventually influencing genetic

controls.

Since postsynthetic alteration of HSPG seems most higly perturable and an almost haphazard process,

such rections are suggested be a useful focus of future research directed at more fully understanding

the aetiology of degenerative diseases suvh as cancer.

3-9 Osmotic Stress & Hofmeister Effects.

In addition to oxidant stress, osmotic stress may be countered by HSPG-related effects.

There are hints (cf Graham, 1994) of an interrelationship between HSPG biochemistry and that of heatshock proteins (HSP) which share with HS the common property of having been conserved over long

evolutionary times but have uncertain, but surely major, biological functions. Acute heat shock intiates

the phenomenon of stress prompted glycosylation of proteins (cf Jethmalean et al 1994). Similar

considerations perhaps also apply to the glucose regulation of gene expression.

Modulation of aqueous solutions activities is relevant to their possible chaperone duties of prevention

protein denaturation. Hofmeister ranked salts for such abilities. The aggregate structure of aqueoussolutions follows the Hofmeister series (Luck 1965) of relevance to the nature of hydrated heparin

HSPG/polysaccharides/counterion /water aggregate associations which may form with osmotic-related

stress factors.

Part 2

More Specific Discussion of Research Reports

An example of possible feedback opportunity between HSPG and gene structure is apparent in the

extracellular sulfatase HSPG processing genes which are involved in postsynthetic HSPG processing

and therefore openly exposed to stress factor effects. Extracellular processing of HSPG is known

(Dhoot et al (2001) to occur by desulphation of GlcNAc,C6(-O-SO3-) for specifying Wnt signalling in

avian embryo patterning. Qsulf sulphatase which contains a conserved C-Y motif at the active site

cysteine (which when post-translationally modified to N-formylglycine, hydrolyses HSPG GlNAc, C6-

O-SO3-groups). C-Y motifs could also further permit modulation of Qsulf activity by ligand binding

e.g. by heparin and related molecules which are known to bind to the C-Y motifs in epidermal growth

factor (EGF) and related molecules (vide infra).

Abnormal heparanase activity (experimentally induced) completely prevented CNS and mesodermal

tissue development in Xenopus embryos (Furuya et al 1995). It is apparent that less severe effects of

modulated heparanse activities could induce non-fatal perhaps beneficial alterations in tissue of

relevance to evolutionary situations.

An example of stress factor cellular activity subject to postsynthetic alteration in HSPG is provided by

the cellular adhesion of alveolar cells which is dependent on cultured cell density, implicating

interactions between HSPG GlcNSO3- groupings (Lin et al, 2000) which are subject to extracellular

modification e.g. by NO or nitrous acid deaminative cleavage and/or desulphonation by acidosis.

Both processes are also believed (Grant et al, unpublished) to be catalysed by those trace redox ions

elevated during stress or intoxicantion which are suspected to promote cancer by induced alteration in

DNA.


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Regulatory oligosaccharides for angiogenic signalling evidently derive from severe post synthetic

degradation of endothelial HSPG. These include unsulphated glucuronic acid rich fragments

resembling a putative ancestral primitive bacterial capsular polysaccharide persisting in mature

HSPGs. Hyaluronic acid with isomeric beta 1,3 and beta 1,4 linkages was inactive (Hahnenberger et

al 1993).

Metal Ion HSPG Interactions.

Divalent Cation Modulation of HSPG ActivityFibroblast growth factors (FGFs), which have accorded a particular focus of intensive research activity,

are known to employ heparan sulphates in complex control mechanisms, including the action of the

heparin-like segments for a putative divalent cation dependent control of autophosphorylation of

tyrosine (Kan et al 1996, 1999) in receptors. Such divalent cation dependence suggests that possible

stress via pathological alteration in the Ca/Mg ratio or Mn intoxication may influence such growth

factor activity and be relevant to considerations of mechanisms of transmission of environmental stress

signals.

The adhesion of cells likely also includes HSPG, Ca2+-dependent coupling between receptors in

different cells (cf Richard 1995) as well as HSPG-Ca2+

-HSPGeffects

(cf transfection studies of

syndecan-1 by Stanley et al 1995), a Ca2+

dependent interacton of L-selectin with heparin-like ligands(Norgard-Sumnicht et al 1993) and a Ca2+ dependent binding to immunoblogin superfamily

platelet/endothelial cell adhesion molecule (PECAM) to HSPGs (and chondroitin sulphate) (DeLisser

et al 1993) - Extracellular Ca2+ directs HSPG biosynthesis, at least for parathyroid cells (cf Takeuchi et

al 1992). Such involvement of Ca2+ in HSPG biochemistry is consistent with the prediction (Long &

Williamson 1979) of a focus for HSPG biochemistry involving iduronate conformation dependent

bivalent cation signalling consistent with the original nmr results (Boyd et al 1979) which have beengenerally confirmed by later research. Anion effects include possible relevant effects of F- intoxication

(reported by Susheela & Jha (1981) which were reported to increase the overall degree of sulphation

but diminished the ratio of gluosamine/galactosamine incorporation into the glycosaminoglycans of

bones). Chlorate is used in laboratory techniques to biosynthetically reduce HS sulphation . Selenate

also alters HS biosynthesis (Dietrich et al DATA.) However, attempts to change the sulphation of

BHK HS by alteration of cell culture medium sulphate concentration failed to show an effect (Grant et

al unpublished).

A mechanism whereby conformational effects of Na+/Ca

2+metal ion interaction at endothelial wall

heparan sulphates may be used by the organism for flow sensing has been described (Siegel et al 1998);

fluid shear forces (as applicable to blood flow) have also been found to alter heparan sulphate

proteoglycan biosynthsis (Elhadj et al 2002) and nitric oxide production by bovine endothelial cells

(Korenaga et al 1994)

Such a mechanism may permit stress factors causeing abnormal flow patterns to alter HSPG

biosythesis.

HSPG in Cell Nuclei

The occurrence of glycosaminoglycans at the cell nuclei (e.g. Bhavanandan & Davidson, 1975), the

alteration of nucleosome conformation and activity by heparin (Brotherton et al 1989) and the bindingof nucleosomes at the cell surface byHSPGs (Watson et al 1999) provide evidence for a likely

involvement of HSPGs in gene processing consistent with the known involvement of heparin/HSPG in

fertilisation (including a chromatin decondensation effect of heparin) and embryology (Perrimon &Bernfield 2000) which may implicate conseved cysteines Zn fingers (Zn binding and transport might

be facilitated by heparin like fragments since heparin has been known for some time to preferentially

bind Zn (Parrish & Fair,1981)) (Zn also facilitates heparin kininogen binding (Renne/ et al, 2001).

Reduced ability to control nuclear transcription activators (e.g. AP-1. SP-2, ETS-1 and nuclear factor

kappa B) by defective HS in tumour cells may contibute to their uncontrolled proliferation (Dundas et

al 2000).

Interactions between HSPG and RNA involving destabilisation of the latter have been reported to

modulate cytokine expression in primate bone marrow stromal fibroblasts (Yang et al 1998).

Hence an involvement in multifunctional roles utilising a repertoire of uniquely flexible potentialresponses capable of generating biofeedback signals suggests that for the heparin/HSPG system a


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functional capability exists similar to an assumed required complex fuzzy logic processor capable of

organism stress minimisationover long-term coupled interactions between the environment,HSPG and

DNA

Such a smart sulphated polysaccharide physiochemical property is proposed to allow responsive

gene restructuring directed by the unique HSPGs systems which contribute to long range

communication and generate effective logical response to stress throughout the whole organism oreven amongst groups of organisms (e.g. via the effect of genetic alteration produced by host

organisms).

Addendum.

Heparin binds with moderate strength under physiological saline conditions to the C-Y kringle domain

in urokinase (Stephen et al 1992) suggesting that a related C-Y, but moderate strength binding to

HS/HSPG may facilitate the storage and release of FGF from reservoirs in the extracellular matrix

(Bashkin et al 1989) and in other growth factors related to epidermal growth factor (EGF which

demonstrate well defined C-Y motifs (Grant et al in 1989) and demonstrate GAG enhancement of its

angiogenic activity (Sato et al 1991) as well as heparin-dependent human cancinoma cell growth

inhibition (Halpern & Carter 1989). Such C-Y structures are also present in the immunoglobin

superfamily and may participate in relevant HSPG interactions.

Heparin binding also occurs with the heparin binding epidermal like growth factor (HB-EGF) of

macrophage origin, a smooth muscle cell mitogen, the transcription of which is markedly increased by

TNFalpha and IL1, perhaps directly promoting atherosclerosis (as suggested by Yoshizumi et al 1992),

however, since the expression of inducible NO synthase is also promoted by TNFalpha and IL-1 (as

well as by IFN gamma, and IL-2 but suppressed by TGFbeta, IL4, IL8, IL10 and IL13) which mightsuggest that NO and its metabolites are also involved in the aetiology of this disease since NO is

implicated in the promotion of related diseases (Stichentoch & Frolich 1998), the aetiology of which

may include an inappropriate degradation of HSPG by excessive NO production. NO is established to

be widely employed in various immune and signalling responses (which include inputs from

heparin/HS biochemistry and gene activation. A characteristic marker of such NO activity in

rheumatic diseases is the nitration of tyrosine (e.g. altering CY) dependent switching.

Perhaps the least evolved effects ofstress on HSPG is via NO and its metabolites and other oxidative

stresses which are also generated by chronic inflammatory situations. The latter are associated with

acidic pH conditions which cause inappropriate degradation of heparin and HS. Deaminative cleavage

under acid conditions often associated with inflammation is primed by a removal of N-sulphonate

groups, which normally stabilise HSPG against the occurrence of this process.

Deleterious effects of excessive nitric oxide metabolite activity might be countered by inhibiting

acidosis, or by glucosamine or glucosamine sulphate dietary supplementation, which tend to increase

the rate of HSPG biosynthesis. (Reported benefit of dietary glucosamine supplementation in

osteoarthritis patients therefore may arise by HSPG replacement therapy as discussed by McCarty

1995, 1997).

Serum ferritin (related to iron overload) has been strongly correlated (Salonen et al 1990) with human

mortality attributable to cardiovascular disfunction but this iron storage protein also (Jarrett et al 1989)increases with age in the absence of disease but its presence is associated with an increased risk of pro-

oxidant damage. The deaminative cleavage of HSPG, is thought to be subject to catalysis by trace

redox metals such as iron (Grant et al unpublished) made available under excess ferritin conditions, orby copper ions (Sorenson, 1984) augmented under stress response conditions (Grant 1998). This is

relevant to HSPG-linked endothelial cell disfunction under iron overload conditions and may also

account for the reported, unexpected and unexplained, in vitro deaminative cleavage observed (Vilar et

al 1997) by direct reaction of NO with heparin (usually the formation of nitrous acid is required) at pH

7.4 (usually acidic pH is required) in phosphate but not in imidazole buffer). Later work by Fransson

and coworkers has confirmed the endogenous utilisation of nitric oxide for deaminative cleavage ofHSPG within endothelial cells, this process being putatively linked to polyamine signalling which

alters HSPG biosynthesis by inserting unsulphonated glucosamine residues, pre-primed for rapid

deaminative cleavage; these process are subject to control in laboratory studies via an alteration of the

availabity of copper ions (which release nitric oxide bound at thiol storgage sites) via provision ofexogenous copper chelating ligands (Mani et al 2000).


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The induction of mouth cancer by betel nuts (Trivedy et al 1997) has been attributed to specific effects

of copper. This may perhaps arise, it is now suggested, promoted via catalysis of the deaminativecleavage of HSPG.

In the presence of EDTA or ascorbate copper and iron ions may stimulate redox cycling generating

reactive nitrogen and oxygen species capable of degrading HSPG and DNA. A degradative acitivity ofascorbate on GAGs e.g. in vitreous humour was soon attributed to reactive oxygen species generated

by the presence of trace copper ions (Robertson et al 1941).ESR detection of the extracellular release of ascorbyl radicals is a very reliable marker of the severity

of myocardial ischemia and reperfusion injury (Pietri et al 1990) in which reactive oxygen free radicals

are implicated.

Ascorbate efficiently reduces nitrous acid back to NO. This may be an important biological function of

ascorbate. The NO metabolite nitrous acid is a likely candidate as the major reactive agent for the

deaminative cleavage of HS and hence ascorbate may spare HSPG from inappropriate degradation by

this mechanism.

The use of ascorbate dietary supplmentation for the inhibition of viral infections and combatting

degenerative diseases such as cancer (Pauling discussed by Grant in 2000) may, be especially

dependent on a specific signalling by ascorbate for a direct modulation of HSPG biosynthesis. Anincrease in sulphation of HS is produced under the influence of ascorbate for human fibroblasts

(Edward & Oliver 1983, confirmed by Kao et al, 1990). (This is likely related to a wider effect of

redox factors on HSPG biosynthsis as chlorate produces decreased sulphation). More highly sulphated

heparin like fragments of HS demonstrate more potent antiviral (e.g. De Clercq E (1993), Choay et al

1993) as well as antiprion (Diringer 1985, Perez et al 1998) and anti-cancer activities (Engelberg in

2000). At least two members of the EXT family of tumour suppressors were found to encode D-glucosyl and N-acetylglucuronyl transferases required for the chain elongation of HS (Lin & Lindahl,

1998). HSPGs and their fragments are known to participate in antioxidant defence and defective HSPG

antioxidative protection may therefore potentially promote tumorigenesis if depleted of this activity.

Graham (1994) discussed a recently evolved heat shock protein (HSP) with putative heparanase

activity. The puzzling detection of a 23 amino acid residue terminal fragment from a mammalian

endo-heparanse recently incorporated into an anti-tumour heat shock protein (which only affectedmouse but not analogous bacterial or yeast HSPs) and the putative role of heparin/HS in HSP70 (Furcht

& McCarthy 1995) however concurs with the notion of a focus in animal evolution of mutually

interactive HS and HSPG systems involved in gene alteration. HSPs and HSPGs, both strongly

conserved throughout animal evolution and are implicated in the aetiology of degenerative diseases,

which implies that coupled HSP HSPG effects, as well as post synthetic processing of GAGs and

proteins, may be linked to stress response effects on genes induced by pathogenic microorganisms.

This is consistent with known pro-cancer perturbation of heparan sulphate dependent growth factor

activity generated as a consequence of binding to HSPG by Helicobacter pylori (associated with

stomach cancer in a heparan sulphate binding-virulence correlated manner (Ascencio et al 1995,

Chmiela et al 1995, Berq et al 1998).

Alteration in heparan sulphate microstructure via the effects of (saturated/polyunsaturated) lipids isapparent from reported effects of lipids on heparan sulphate biosynthesis (e.g. Paka et al 1999).

(Oxidised lipids evidently signal for altered heparan sulphate production this being a possible major

mechanism of cardiovascular damage (cf Paka et al 1999) perhaps partly due to the effects of suchproducts (OBrien et al 1988) on the perturbation of heparin-like oligosaccharide modulation (Herbet &

Maffrand 1989) of protein kinase C activity.

Heparan sulphates including fragments evidently function in vivo for the inhibition of pathological

crystallization (preventing calcification of blood and urinary systems, normally supersaturated with

respect to Ca2+

salt precipitation) (cf Yamaguchi et al 1993, Grant et al 1992). Pathological plaqueformation may include a related functionality failure of defective heparan sulphate proteoglycan

systems (e.g. at artertial walls and in neurogenerative diseases and amyloidosis) perhaps due to

aberrant self assembly of shed heparan sulphate proteoglycan fragments and their altered interaction

with collagen, fibronectin etc under pathologcial conditons (including in cell culture models of cancer).


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{FootnotesThe original biochemical interest in heparin/ heparan sulphate biochemisty stemmed from the medical

use of heparin as a blood anticoagulant. Haemostasis activity typically resides in proteolytic

modulation via a highly sulphated heparin microstructure containing Glc C3,6(OSO3-)2NSO3

-moieties

which alter the conformation and activity of key proteins, especially antithrombin(III). Numerousadditional activities of heparin/heparan sulphate have ultimately been discovered involving heparan

sulphate proteoglycan dependent systems including an involvement in lipid metabolism and growthfactor control. The latter was originally discovered by during research into angiogenesis by Folkman

and coworkers for which a bivalent cation (e.g. Cu2+) dependence was also indicated. A wide variety

of growth factors and cell types are now known to employ heparan sulphate-dependent control

mechanisms (cf Bernfield et al 1999)}.

A general statement of the mode of action of heparan sulphates in growth factor control is for

recruitment of the growth factors and the assembly of cell surface receptors which includes the

modulation of autophosphorylation kinase activity by the promotion growth factor receptor interaction.

Such HSPG dependent systems are now known to critically influence haematopoeiesis (Gupta et al

1998, Drzeniek et al 1999) embryo development (Perrimon & Bernfield 1990) and wound healing.

Environmentally-dependent effects transmitted by reactive oxygen and reactive nitrogen species duringintracellular and extracellular stress coupling to C-Y signalling, enhanced by helper heparan sulphate

proteoglycan (HSPGs) activities, agrees with the long-held notion that extracellular polysaccharides are

involved in long range intracellular communication. This is in addition to their involvement in

haemostasis, fibrinolysis, capillary permeability, matrix assembly, potentiation of mitotic, chemotactic,

neurotrophic and angiogenic activities, cellular recognition, immunological signalling, adhesion,,

differentation, proliferation, wound healing, apoptosis, gene expression, capacitation, morphogenesis,embryogenesis, nerve and brain development and function (including neurite outgrowth, synaptic

function and myelination) phosphtidyl inositide anchoring and cytoskeletal function, lipid processing,

glomerular filtration and antioxidant protection involving both the intact polymers and in some

instances, fragments. Thus, although the biochemical details are far from clear, involvement in such

wide-ranging processes could allow arrayed events affecting whole organisms to influence intracellular

activity. Such processes could also allow complex extracellular communication events to occur over

long time periods to stimulate altered gene structuring in a manner not producing lethal consequences.Signalling (by fragments of or by some other method dependent on cell type specific charge density

array signalling) by heparin/heparan sulphate might fulfil this function which may involve nulcear

elements such as nucleosomes bound to heparan sulphate proteoglycans at the cell surface and/or

additional helper function of symbiotic organisms to facilitate non-lethal introduction of genetic

material into host immunological cells communicating to stem cells. These types of cells involve

HSPG directed recruitment and activation.

Sensitivity of HS systems to various extracellular situations also includes direct effects of redox metal

ions in the depolymerisation of heparin by complexed ferrous ions (Lahiri et al 1992) and induced bio-

feedback including a direct influence on heparan sulphate proteoglycan biosynthesis (both in causing

altered biosynthesis producing altered microstructure and degree of glycosylation) as well as affectingpost synthetic cleavage) such as the direct perturbation by hyperbaric oxygen of heparan sulphate

biosynthsis (Karlinsky 1992). There is a possible heparan sulphate proteoglycan /iron biochemistry

analogous to calcium biochemistry, including a helper function for tranferrin receptors, provision of analternative ligand for iron entry into the cell as well as for modulation of morpholgical control of iron-

rich particles. Redox activity of Cu ions involved in immunological signalling (cf Sorenson 1984) may

include a function of heparin/heparan processing including catalysis of nitric oxide reactions withheparin/HS.

Nitric oxide and its metabolites, including tyrosine nitrate from peroxynitrite, activities are dependent

on redox status (e.g. via ascorbate and thiol activities) and further subject to modulation by metal ion

catalysis and osmotic stress. HS biosynthesis can be directly influenced by feedback from fragmented

HS, redox status, metal ions and osmotic effects and additional polyamine signalling directs

unsubstituted glucosamine positional biosynthesis (pre-primed for deaminative cleavage to specificoligosaccharides) allowing a fine-tuning of such HS structural modifications. Highly evolved

heparanases and some heat shock proteins which seem likely also to generate HS oligosaccharides may

provide a higher order of controlled HS oligosaccharide production and signalling relevant to gene

modification


9/30

Nitric oxide is normally stored intracellularly at porphyrins and cysteine thiol groups where additional

coordination to metal ions may occur. Release from thiol groups is subject to copper ion (Cu+ being

more effective than Cu2+

Singh et al (1992)) catalysis which can be diminished by the use of copperchelators in the laboratory to modulate in vivo intracellular HSPG processing by active nitric oxide

metabolites (Mani et al 2000)) A further relevant related aspect of such nitric oxide dependent HSPG

biochemistry is an important interlinking with polyamine signalling, recently established by Ding et al

(2001).

Clustering of acetyl choline receptors and related effects by heparin/ heparan sulphate proteoglycan(Baker et al 1992) suggests a related fundamental involvement of such biochemistry in neurological

activity.

The existence of biofeedback processes are the suggested from

1) the effect of exogenous heparin and nitric oxide in stimulation of the biosynthesis of heparin likeHSPG by endothelial cells (Pinal et al 1994);

2) from the effects of extracellular metal ions on heparan sulphate biosynthesis (e.g. Ca2+ (forparathyroid cell heparan sulphate metabolic processing (Takeuchi et al 1990, 1992), the effects of

toxic metal ions on kidney heparan sulphate proteoglycan biosynthesis, e.g. Pb (Fujiwara et al et

al (1999) which diminishes HSPG sulphation much more than of other GAGs; cf also Kaji et al

1991), Cd (Cardenas et al 1992 ) Cu and Ni (Turnbull 1992);Different divalent metal ions are potentially required for two NacetylGlcN transferases TII and TII

which initiate of polymerisation of HS. TI uses both Ca2+

and Mn2+ but TII uses only Mn

2+which

effect may then influence HSPG structure.

3) Augmented extracellular glucose was found to markedly decrease heparan sulphate biosynthesisin glomerular epithelial cells (Morano et al 1999).

4) Ascorbate, on the other hand, markedly stimulated the biosynthesis of highly sulphated heparansulphates (particularly those remaining at the cell surface following trypsinisation) in human

embryonic epithelial cells (Edward & Oliver 1984) and in fibroblasts (Kao et al, 1990).

There is also believed to be involvement in cytoskeletal biochemistry and HSPG has been found at the

mitotic spindle. (DATA)

References

Abate C Patel L Rauscher FJ III Curran T (1990)

Redox regulation of Fos and Jun DNA-binding activity in vitro Science

1157 DATA

[The proto-oncogenes c-fos and c-jun function cooperatively in signal transduction via a heterdimericcomplex of their protein products Fos and Jun which interact with the DNA regulators activator

protein-1 (AP-1) leucine zipper domains direct positioning of clustered basic amino acid domains that

bind to the DNA to which binding is modulated by redox alteration of a single conserved cysteineresidue]

cf Imai et al (1993) loc cit Heparin inhibits c-fos expression and endothelium 1 (ET-1) production in

bovine endothelial cells

Ascencio F Hansson HA Larm O Wadstroem T (1995)

Helicobacter pylori interacts with heparin and heparin-depedent growth factors

FEMS Immunol Med Microbiol 12 (3-4) 265-272 CA 124 109845f

Cf Chmiela M et al (1995) and Berq DJ et al (1998)

Baker LP Chen Q Peng HB (1992)

Induction of acetylcholine receptor clustering by native polystyrene beads. Implication of an

endogenous muscle-derived signalling system J Cell Sci 102 543-555

[A role of FGF2 -HSPG was implicated in the clustering of acetyl choline receptor clusters]cf Bullock S Rose SPR (1992)


10/30

Glycoproteins modulate changes in synaptic connectivity in memory function

Biochem Soc Trans 20 412-414]

Bashkin P Doctrow S Klagsburn M Svahn CM Folkman J Vlodavsky I (1989)

Basic fibroblast growth factor binds to subendothelial extracellular matrix and is released by

heparitinase and heparin-like molecules

Biochemistry 28 1737-1743

Beckman JS Beckman TW Chen J Marshall PA Freeman BA (1990)Apparent hydroxyl radical production by peroxynitritie: implication for endothelial injury from nitric

oxide and superoxide

Proc Natl Acad Sci USA 87 1620-1624

[Peroxynitrite is a major reactive nitrogen reagent producing effects previously ascribed to hydroxyl

radicals] cf however Pfeiffer S Mayer B (1998)

Lack of tyrosine nitration by peroxynitrite generated at physiological pH

J Biol Chem 273 (42) 27280-27285 high level of nitration of tyrosine a hallmark several disease

degenerative diseases had been proudced by an additional factor ro by a different reagent (perhaps

enhanced by CO2 {also redox metals such as trace active iron or copper ions as with the anomalous

deaminative cleavage of HSPG by NO at pH 7.4}]

Belting M Petersson S (CHECK) Fransson L-A (1999)

Proteoglycan involvement in polyamine uptake

Biochem j 338 317-323

[A specific role including sequence specific binding of HSPGs for the uptake of spermine by

fibroblasts]

Berq DJ et al (1998)

Rapid development of severe hyperplastic gastritis with gastric epitheilial dedifferentiation in

Helicobacter felis-infected IL-10(-/-) mice

Am J Pathol 152 (5) 1377 CA 129 107408q

Bernfield M Gotte M Park PW Reizes O Marilyn L Fitzgerald JL Zako M (1999)

Functions of cell surface heparan sulfate proteoglycansAnnu Rev Biochem 68 729-777

[Conserved cysteines in (GPI-anchored) glypicans and tyrosine(s) in (transmembrane cytoplasmic

linked) syndecans may interact in growth factor regulation, cellular adhesion, activation, differentiation

and motility in development, wound healing and immunolgical signalling in feedback loops involving

kinase activities linked to post synthetic processing of HSPGs before and after shedding and re-uptake

of HSPG units by cells. These events are known to be affected by mechanical, osmotic and redox

stress.]

Bhavanandan VP Davidson EA (1975)

Mucopolysaccharides associated with nuclei of cultured mammalian cellsProc Natl Acad Sci USA 72 (6) 2032-2036

Boyd Long Williamson Getting DATA (1979)J Mol Biol DATA

Bralet J Schreiber L Bouvier CEffect of acidosis and anoxia on iron delocalization from brain homogenates

Brotherton TW Jagannadham MV Ginder GD (1989)

Heparin binds to intact mononuleosomes and induces a novel unfolded structure

Biochemistry 238 3518-3525

Brown GC (2000)Nitric oxide as a competetive inhibitor of oxygen consumption in the mitochoindrial respiratory chain

Acta Physiol Scand 168 (4) 667-674 CA 133 149755y

{NO diminsihes NF kappa beta which with the protein complex with MCP1 is believed to play an

important role in atherosclerosis cf Takeshita A & Eto K CA 133 56741z}


11/30

Brown DR Qin K Herms JW Madlung A Manson J Strome R Fraser PE Kruck T von Boheln A

Schulz-Schaeffer W Giese A Westaway D Kretzschmar H (1997)

The cellular prion protein binds copper in vivoNature 390 684-687

Cardenas A Bernard A Lauwerys R (1992)

Incorporation of [35]sulfate into glomerular membranes of rats chronically exposed to cadmium and itsrelation with urinary glycosaminoglycans and proteinuria

Toxicology 76 219-231

CechowskaPasko M Palka J (2000)

Age-dependent changes in glycosaminoglycan content in the skin of fasted rats. A possible mechanism

Exp Toxicol Pathol 52 (2) 127-131 CA 133 264382k

[NAD/NADH and lactate/pyruvate ratios, changed during ageing and fasting and lactate inhibits at

least some GAG biosynthesis; the lactate content of blood and skin and lactate/pyruvate ratio was

correlated with a 40% reduction in GAGs in the skin of old rats,and a redction in young but not old rats

occurred with fasting. Lactate content was doubled in the skin of young fasted rats. An increase in the

skin lactate/pyruvate and a decrease in this ratio in the blood was accompanied by a decrease in the

skin GAG content suggesting that the effects were due to regulation of glucose (and glucosamine?

CHE CK) for GAG biosynthesis (cori cycle? CHECK)

Challis BC Edwards A Hunma RR Kyrtopoulos SA Outram JR (1978)

IARC Sci Publ 19 (Environ. Aspects N-Nitroso Compd.) 127-142 CA 89 196583x

[Fe and Cu etc catlayse nitrosamine formation]

Chan RW (1992)Transparent ferromagnetic nonostructures

Nature 359 591

[Fe2+

held at colloidal organic sulphonate(SO3-)n groups are hydrolysed and oxisised to give

gamma Fe2O3 (cf Ziolo et a (1992)l Science 257 217-223)

suggesting that Fe2+ bound by sulphated polysaccharides may behave similarly]

Chmiela M Paziak-Domanska B Rudnicka W Wadstroem T (1995)The role of heparan sulphate-binding acitivity of Helicobacter pyroli in their adhesion to murine

macrophages

APMIS 103 (6) 469-474 CA 124 53455n

[Pathologenic activity increases with strength of binding]

Colburn P Dietrich CP Buonassisi T CHECK (1996)

Arch Biochim Biophys 325 (1) 129 DATA CA 124 79179a

[Endotoxin altered HSPG structure in endothelial cells by loss in a region rich in sulphate located at the

non-reducing end of the GAG chain]

Colin S Jeanny J-C Mascarelli F Vienet R Al-Mahmood S Courtois Y Labaree J (1999)

In vivo involvement of heparan sulfate proteoglycan in the bioavailability, internalization, and

catabolism of exogenous basic fibroblast growth factorMol Pharm 1 74-82

Comper WD (1995)Interplay Genet Phys

Dev Biol Forum. Workshop 199521

Ed Beysens D et al World Scientific Singapore CA 124 79513e

[De novo occurrence of sulphated polysaccharides of ECM and cell surface has been correlated with

the evolution of metazoa and differentiation in embryogensis]

Cox JF Saravia F Briones M Maria HS (1995)

Dose-dependent effect of heparin on fertilizing ability of goat spermatoza

Theriogenology 44 (3) 451-460 CA 123 246357v

{Cf Reyes R Rosado A Hernandez O Delgado NM (1989)Heparin and glutathione: physiological decondensing agents of human sperm nuclei


12/30

Gamete Research 23 39-47

and Valencia-Sanchez (1995) loc cit}

Castillo GM Templeton DM (1992)

Structure and metabolism of multiple heparan sulphate proteoglycans synthesized by the isolated rat

glomerulus

Biochem Biophys Acta 1136 119-128[In contrast to other matrix components HSPGs are in a state of dynamic turnover]

De Clercq E (1990)

Targets and strategies for the antiviral chemotherapy of AIDS

TiPS (11) 198-205

[Includes discussion of anti-HIV activity of sulphated polyanions including GAGs]

De Lisser HM Yan HC Newman PJ Muller WA Buck CA Albelda SM (1993)

Platelet/endothelial cell adhesion molecule-1 (CD31)-mediated cellular aggregation involves cell

surface glycosaminoglycans

J Biol Chem 268 (21) 16037-16046

Dhoot GK Gustafsson MK Ai Z Sun W Standiford DM Emerson CP Jr (2001)Regulation of Wnt singalling and embryo patterning by an extracellular sulfatase

Science 293 1663-1666

[HSPG regulator function in developmental signalling via HSPG sulfatase Qsulf1 (which has

homology with human lysosomal N-acetylglucosamine 6-sulfatase (HuG6Sulf)) expression of which is

induced by Sonic hedgehog in myogenic ]

Qsulf 1shares conserved C-Y, putativly stress-related, motifs with a human lysosomal 6 sulfatase). Stress factors are likely to influence such extracellular desulphation/desulphonation processes. The

specific G6OSO3-patterns for Wnt signalling in quail embryo development are achieved by an

extracellular sulphatase Qsulf1

Although Dhoot et al suggest that Qsulf1 (which has homology with Gln60sulphatas) regualates

localized responses to widely distributed developmental signals for embryo patterning by achieving

complete hydrolysis of GlcNAc6S in surface HSPG bound Wnts, perturbation via partial desulphation

might also disrupt of embryo development.

{Comparison of the amino acid sequences of Qsulf 1 and HsG6Sulf show conserved C-Y motifs}

cf Li et al (2000) HSPG expression of Nsulphotransferase (NST) during the transition of type I to type

II rat alveolal cells was dependent on cell density and matrix and was intense in conditions where cells

spread fully. 3-O sulphotransferase expression was however unchanged under these conditons)

Dietrich CP Nader HB Strauss AH (1983)

Structural difference of heparan sulfates according to the tissue and species of origin

Biochem Biophys Res Commun 111 865-871{cf Dietrich et al DATA (1996)

Arch Biochem Biophys 325 (1) 129

Nader HB Ferrerira TMPC Toma L Chavante SF Dietrich CP Casu B Torri G (1988)Maintenance of heparan sulfate throughout evolution: chemical and enzymic degradation, and 13-C-

n.m.r. spectral evidence

Carbohydr Res 184 292-300Cf Ferrerro et al (Dietrich CP) (1993)

J Biochem 25 (6) 1219 DATA CA 120 50475a

Ding K Sandgren S Mani K Belting M Fransson LA (2001)

Modulations of glycpican-1 sulfate structure by inhibition of endogenous polyamine synthesis.

Mapping of spermine-binding sites and heparanase, heparin lyase, and nitric oxide/nitrite cleavage sitesJ Biol Chem 276 50 46779-46791

Diringer H (1985) Funk Biol Med 4 129-141 cf Ehlers B Dringer HJ Gen Virol 65 1325-1330;

Diringer H Ehler B (1991) J Gen Virol 72 457-460;Cf also WuDunn D Spear PG (1989)


13/30

J Virol 63 (1) 52-58

{Cf Polyamines (cf Ding et al 2001 loc cit), platelet factor 4 and poly L-lysine are known to block viral

entry into cells presumably by a HS-dependent mechanism}

Dow KE Riopelle RJ (1992)

Influences of N-linked oligosaccharides in the presntation of neurite promoting activity of

proteoglycans released by neurones in vitro[Castenospermine CHECK and swainsoline inhibited [3H] glucosamine incorporation into heparan

sulphate and chondroitin sulphate proteoglycans]Cell Tisssue Res 268 (3) 553 DATA CA 117 45057z

Drzeniek Z Stocker G Siebertz B Just U Schroeder T Ostertag W Haubeck H-D (1999)

Heparan sulfate proteoglycan expression is induced during early erythroid differentiation of

multipotent hematopoietic stem cells

Blood 93 (9) 2884-2897

[HSPGs are involved including the binding and presentation of growth factors in cellular regulation via

the direct contact interactions of primitive hematopoetic progenitor cells with stromal cells.

A novel HSPG (not syndecan or glypican) which probably binds EPO growth factor was found on bone

marrow (BM) stem cells of the erythroid lineage but not of the myeloid lineage. The HSPG is

upregultedduring the early stages of erythroid differentation and downregulated when the cells matureinto erythrocytes. HSPG may also be involved in the adhesion and homing of early erythroid

precursors to the sites of erythropoiesis in the BM. Previous studies had shown HSPGs of bone

marrow stromal cells or their ECM bind IL3 and granulocyte-macrophage colony stimulating factor

GM-CSF) allowing biologically active presentation to hematopoietic cells]

Cf Brunner G et al (1994) An endogenous glycosylphosphatidyl inositol specific phospholipase D

releases b-FGF {FGF-2}-HSPG complexes from human bone marrow cells ibid 83(6) 2115 CA 120261727x

Dundas J et al DATA (2000)

Effect of heparin and liver heparan sulphate on interaction of HepG2-derived transcription factors and

their cis activating elements: altered potential of hepato cellular CHECK carcinoma heparan sulphate

Biochem J 350 (1) 245 CA 133 320328

[PG assembly in malignant tumours is greatly altered and may promote growth via depeletion in genecontrol activity. In vitro and in vivo experiments indicated that heparin and HS are capable of

inhibiting the induction of transcription factors (AP1, SO-1 ETS-1 and nuclear factor kappa B) wheras

FFIID was hardly inhibited. HS from DATA liver was five times more effective than from liver

carcinoma was less effective comparable to heparin in activity, indicating that altered HS

microstructure may prmotoe uncontrolled cell proliferation]

Edenber MJ Conrad HE Bizzo SV (1991)

Heparin oligosaccharides enhance tissue type plasminogen activator: a relation between

oligosaccharide length and stimulation of plasminogen activation

Biochemistry 30 (4) 10999 DATA CA 115 229145s[Enhancement of plasminogen activation by the smaller oligosachharides is mediated exclusively via

binding of tissue type plasminogen (Pg) activator (t-PA) whereas the larger oligosaccharides may

interact with both t-PA and Pg]

Edward M Oliver RF (1984)

Ascorbate increases the sulphation of glycosaminoglycans synthesized by human skin fibroblastsBiochem Soc Trans 12 304

Cf These authors (1983)

Changes in the synthesis and distribution of sulphated glycosaminoglycans in human skin fibroblasts

upon ascorbate feeding ibid 11 383

[Upon increase in ascorbate from zero to 50microg/ml, cell associated HS increased by a factor of 19.2;

Total GAGs in the pericellular/extracellular domain increased by 3.5 fold].

Elhadj S et al (2002)

Chronic pulsatile shear stress impacts synthesis of proteoglycans by endothelial cells: Effect on

platelet aggregation and coagulationJ Cell Biochem 86 (2) 239-250


14/30

Engelberg H (1999)

Actions of heparin that may affect the malignant processCancer 85 (2) 257-272

Esko JD Rostand KS Weinke JL (1988)

Tumor formation dependent on proteoglycan biosynthesisScience 241 1092-1096

Feyzi E Saldeen T Larsson E Lindahl U Salmivirta M (1998)

Age-dependent modulation of heparan sulfate structure and function

J Biol Chem 273 (22) 13395-13398

[HS from different species and cells differ in their pattern of sulphation in N-suphonate rich regions

including a specific microstructure thought specific for platelet derived growth factor (PDGF) AAL and

BBL binding which for a 76-year-old individual was 4-5 times greater than for a 21-year old individual

(whereas binding of FGF-2 was only marginally dfiferent between the two ages); this was suggested to

contribute to arteriosclerosis;

The authors noted that their results were the first time discovery of a characteristic systematic change

associated with the ageing process]

Fisher RE Blumenthal Y (1982)

J Biol Chem 257 (4) 1702-1704 CA 96 138847v

[Heparin inhibits RNA polymerase and modifies the trypsin cleavage of RNA polymerase both alone

and at a poly[dA.T.dA.P] template

Folkman J Klagsburn M (1987)Angiogenic factors

Science 235 442-447

[Heparin binding endothelial growth factorsheparin affinity allowed their separation and purification

FGF1 (acidic FGF) and FGF2 (basic FGF) originally studied 140 amino acid residues and 146 residue

had 53% absolute sequence homology gene sequence data showed both had 154 amino acid residues;

the receptors FGF1 mw 135-150,000, for FGF2 mw 125-145,000. Tyrosine phosphorylation was

known only for the FGF1 receptor}{Heparin binding growth factor (vascular endothelial growth factor related to FGF1) activities are also

implicated in glucose induced vascular disfunction (Stephan CC et al 1998, Diabetes 47 (11) 1771

CA130 64586f}.

Fransson L-A (1982)

Structural features of the contact zones for heparan sulphate self-association

Carbohydr Res 110 127-133

Cf Fransson LA Havsmark B (1982)

Correct secondary structure is required for self-association of heparan sulphate

Carbohydr Res 110 (1) 135 CA 98 16981e[Modification of heparan sulphate by N-desulphonation and N-acetyaltion abolished chain-chain

interaction]

Frei B (1991)

Ascorbic acid protects lipids in human plasma and low density lipoprotein against oxidative damage

Am J Clin Nutr 54 1113S-1118S[Only ascorbic acid is reactive enough to intercept oxidants in the aqueous phase before they cause

detectable oxidative damage to lipids being able to prevent the initiation process not achieved by other

investigated antioxidant molecules (bilirubin, uric acdi, protein thiols, alpha tocopherol, ubiquinol-10,

lycopene and beta carotene)]

Fritz TA et al (1994)J Biol Chem 269 (46) 28809 CA 121 249190e

[Differences in activation requires divalent cations: alpha-GlcNAcTI can use both Mn2+ and Ca2+ while

alphaGlcNAcTII can only use Mn2+

provides a mechanism by which Mn2+ status might influence HSPG biochemistry]


15/30

Fujiwara Y Kaji T (1999)

Lead inhibits the core protein synthesis of a large heparan sulfate proteolgycan perlecan by prolferating

vascular endothelial cells in cultureToxicology 133 (2,3) 159-169 CA 131 154569c

[Lead significantly decreased the accumulation of GAGs but more strongly affected HS than

chondroitin or dermatan sulphates. Lead seems to decrease perlecan core protein sytnthesis without

affecting the attached polysaccharide chain lengths. HSPGs are reduced but by a different mechansimsbetween growing and growth arrested cells]

Cf Kaji et al (1990) ibid 68 249-257

Furcht LT McCarthy JB (1995)

Identification of homologous heparin binding peptide sequence present in fibronectin and 70kDFa

family of heat-shock proteins

Biochim Biophys Acta 1251 (1) 135 DATA CA 123 249546j

Furuya S et al DATA (195)

Elimination of heparan sulphate by heparitinases induces abnormal mesodermal and neural formation

of Xenopus embryos

Dev Growth Differ 37 (3) 337 CA 123 165404f

[HS plays an indispensible role in establishing fundamental body building during early Xenopusdevelopment; heparanse deletion causes organism developments without CNS and mesodermal tissue

but chondroitinase produced no similar effects]

Gabizon R et al J Cell Physiol (1993CHECK)157 (2) 312-25 CA 120 28472a

Cf also Perez et al loc cit;

Gallagher JT Turnbull JE Lyon M (1992)

Patterns of sulphation of heparan sulphate

Int J Biochem 24 (4) 553 CA 117 42780u

[cf Galagher JT Lyon M Steward WP (1986)

Structure and function of heparan sulphate proteoglycans

Biochem J 236 313-325]

Gilat D et al (1995)

Molecular behaviour adapts to context : heparanase functions as an ECM degrading enzyme or as a T

cell adhesive molecule dependent on the local pH

J Exp Med 181 (5) 1929-1934 CA 288881u

[Migration of lymphocytes requires their adhesion. Penetration of the sub endothelial ECM is

associated with expression of ECM degrading enzymes such as endo beta D glucouronidase

(heparanase) which degrades HSPGs but only under relatively acidic conditions. At normal tissue pH

the enzyme attaches to HS without degrading it and creates an anchor for CD4+T cells]

Graham LD (1994)Tumour region antigens to the hsp90 family (gp96) closely resemble tumour associated heparanase

enzymes

Biochem J 301 (3) 917{cf Srivastava PK

Endo beta D glucuronidase (heparanase) activity of heat shock protein.tumour region active agent gp96

ibid 301 (3) 915 Review with 21 refs}[Cf also comments following this paper by Nicholson JL & Nakajima M and Srivastava PK]

The two most abundant HSPs (HSP70 and HSP90) are highly conserved among organism as diverse as

bacterial flies and man (cf Pelham HRB (1986) Speculation on the functions of the major heat shock

and glucose-regulated proteins, Cell 46 959-961).

Grant D (2000)http://web.ukonline.co.uk/dgrant/dg4/cf ibid dg/2

{Discussion sessions held with Prof KEL McColl and coworkers (Glasgow University)}

[Ascorbate and nitric oxide modulation of heparan sulphate biochemistry.

Tyrosine nitrate formation is indicative of the presence of NO metabolites DATA
http://web.ukonline.co.uk/dgrant/dg4/http://web.ukonline.co.uk/dgrant/dg4/http://web.ukonline.co.uk/dgrant/dg4/


16/30

Efffects of NO metabolites implicated in aetiology of rheumatic diseases (Stichentoch & Frolich 1998,

loc cit); pathological deaminative cleavage of heparan sulphate chains also implicated

Grant D Long WF Williamson FB (1987a)

Pericellular heparans may contribute to the protection of cells from free radicals

Med Hypoth 23 67-72 afforded by glycosaminoglycans, ibid 38 49-55]

Cf These authors(1987b)

Infrared spectroscopy of heparin-cation complexesBiochem J 244 143-149

(1989a)

A comparison of the antioxidant requirements of proteins with those of synthetic polymers suggests an

antioxidant function for clusters of aromatic and bivalent sulphur-containing amino aacid residues

Med Hypoth 28 245-253

[A hypothesis similar to part of the suggestion of Nakashima(1996) that evolutionary conserved C-Y

(and related serine and theonine as well as histidine) groupings in proteins are especially associated

with antioxidant protection and also with ligand binding to RNA and DNA protooncogene function]

(1990)

The dependence on counter-cation of the degree of hydration of heparin

Biochem Soc Trans 18 (6) 1283-1284(1991)

Heparin polypeptide interaction

Biochem J 277 569-571

(1989b)

Inhibiton by glycosaminoglycans of CaCO3 calcite) crystallization

Biochem J 259 41-55 CA 110 171240x(1992a)

Complexation of Fe2+ based ions by heparin is not a simple reversible thermodynamic process

Biochem Soc Trans 20 (4) 361s

Cf Biochem J 287 (3) 849-853 CA 117 226049u

[Zn2+ heparin binding; evidence for phase change mechanism]

(1992b)

Degenerative and inflammatory diseases may result from defects in antimineralization mechanismsafforded by glycosaminoglycans

Med Hypotheses 38 49-55

Grimm J Keller R de Groot PG (1988)

Lamellar flow induces cell polarity and leads to rearrangement of proteoglycan metabolism in

endothelial cells

Thromb Haemostasis 60(3) 437-441

Gupta P et al DATA (1998)

Structurally specific heparan sulphates support primitive haematopoiesis by forming multi molecularstem cell niches

Blood 92 (12) 4641 CA 130 137148b

Halpern J Carter BJ (1989)

Modification of human carcinoma SW13 cells by heparin and growth factors

J Cell Physiol 141 (16 DATA CA 111188341z[EGF and insulin like growth factor 1 (IGF 1) were not able to overcome heparin induced inhibition of

cell growth (due to strong complexing by heparin) but such inhibition was partly overcome by TGFe

and FGF-1 (weaker heparin complexes formed). Heparin-like substances present in the extracellular

matrix play an important role in the control of epithelial growth].

Hahnenberger R Jakobson AM Ansan A Wehler T Svahn CM Lindahl U (1993)Low sulphated oligosaccharides derived from heparan sulphate inhibited normal angiogenesis CHECK

Glycobiology 3 (6) 567-573 CA 120 235393r

[Attempts to define a saccharide structure responsible for the anti-angiogenic effects of HSPG

implicated the initial polymerisation product containing unsulphated and un-isomerized[GlcAbeta1,4-GlcNAcalpha 1,4]n sequences which also occur in Escerichia coli K5 capsular polysaccharide of


17/30

anti-angiogenic activity. However isomeric hyaluoronic acid with[GlcA beta 1,3-GlcNAc beta 1,4]n

structures was inactive]

Hansen LK OLeary JJ Skubitz APN Furcht LT McCarthy JB (1995)

Identification of a homologous heparin binding peptide sequence present in fibronectin and the 70kDa

family of heat shock proteins

Biochem Biophys Acta 1251 (1) 135-145 CA 123 249546s[LIGRK and LIGRR represent a common heparin binding motif in fibronectin , HSP70 and HSP70 and

are consistent with the proposed role of heparins and similar polyanionic structures in the functions of70kDa heat shock proteins]

Heller R CHECK Munsche-Pauley F Grabner R Till U (1999)

L-Ascorbic acid potentiates NO synthesis in endothelial cells

J Biol Chem 274 (12) 8254-8260

[Ascorbic acid enhances NO synthesis in endothelial cells this being postulated to be the origin of the

{Various linked signalling via HSPG systems suggested by this finding e.g. ascorbate (incr HSPG

biosynthesis?) whicle NO also may modulate HSPG biosynthesis and cause HS deaminative cleavage

(e.g. dependent on trace redox metal e.g. Cu availability) with resultant HS oligosaccharide or

exogenous heparin(oid) feedback signalling and modulation of HSPG biosynthesis}

Herbert J-M Maffrand J-P (1991)

Effect of pentosan polysulfate , standard heparin and related compounds on protein kinase C activity

Biochim Biophys Acta 1091 432-441

{Inhibiton of PKC by oligosaccharides is highly dependent on molecular weight}

Hu W-L Regoeczi E (1992)Hepatic heparan sulphate proteoglycan and the recycling of transferrin

Biochem Cell Biol 70 535-538

[Liver HSPG can some transferrin receptor functions; HSPG can facilitate the release of iron from

transferin]

Imai T et al (1993)

Heparin inhibits ET-1 and protooncogene cfos expression in cultured bovine endothelial cellsJ Cardiovasc Pharmacol 22 (suppl 8) 549 DATA CA 120 69189g

[Heparin probably inhibits proto ET-1 mRNA expression via a PTC dependent pathway]

Ishii K et al (1982)

Biochem Biophys Res Commun 104 (2) 541-547

[Heparin inhibited DNA topoisomerase ex mouse mammary carcinoma cells I50 = 0.20 microg/ml.

Other GAGs were much less effective]

Jarrett DRJ Grosset AB Rowland Payne CME (1989)

Ageing as a cause of raised serum ferritin in the absence of diseaseJ Clin Exp Gerontol 11 (3-4) 145-54

Jethmalan SM et al (1994)DATAHeat shock induced prompt glycosylation. Identification of P-SG67 as calreticulin CHECK

J Biol Chem 265 (38) 23602-23609 CA 121 224578z

[Acute heat shock initiates the phenomenon of prompt glycosylation which is characterized byselective glycosylation of specific cellular proteins]

Julian J Das SK Dey SK Baraniak D Ta V-T Carson DD (2001)

Expression of heparin/heparan sulfate interaction protein/ribosomal protein L29 during the estrous

cycle and early pregnancy in the mouse

Biol Reprod 64 1165-1175

Klevit RE (1991)

Recognition of DNA by Cys2His2 zinc fingers

Science 253 1367-1393[C2 containing zinc fingers are major DNA binding proteins]


18/30

[C2XXC2 also bind Cu ions as in metallothionenes]

Kaji T Yamamoto C Sakamoto M (1991)Effect of lead on the glycosaminoglycan metabolism of bovine aortic endothelial cells in culture

Toxicology 68 249-257

cf Fujiwara Y Kaji J (1999)

Toxicology 133 (2,3) 159 CA 131 154569

Kan M Wang F Kan M DATA Gabriel JL McKeehan WL (1996)Divalent cations and heparin/heparan sulfate cooperate to control assembly and activity of the

fibroblast growth factor receptor complex

J Biol Chem 271 (42) 26143-26148

(cf also Kan M Wu X Wang F McKeegan WL (1999)

Specificity of factors determined by heparan sulfate in a binary complex with receptor kinase

Ibid 274 15946-15952)

[cf also Colin S Jeanny JC Mascarelli F Vienet R Al-Makmood S Copurtois Y Labarre J (1999)

In vivo involvement of heparan sulfate proteoglycan in the bioavailability, internalization, and

catabolism of exogenous basic fibroblast growth factor

Molecular Pharmacology 1 74-82http://www.molpharm.org;

Danmon DH Halegoua S DAmore P Wagner JA (1992)Fibroblast growth factors, like nerve growth factor induce morphological differentiation of PC12 cells

regulated by glycosaminoglycans

Exp Cell Res 201 154 CA 117 40895m

David G Bernfield M (1998)

The emerging roles of cell surface heparan sulfate proteoglycans

Matrix biol 17 (7) 461-463 CA 130149916pKrufka A Guimond S RapraegerAC (1996)

Two hierarchies of FGF-2 signalling by heparin: mitogenic stimulation and high-affinity

binding/receptor transphosphorylation

Biochemistry 351 11131-11141 (CHECKp)

LaRochelle WJ Sakaguchi K Atabey N Cheon H-G Takagi Y Kinaia T Day RM Mike T Burgess

WH Bottaro DP (1999)

Heparan sulfate proteoglycan modulates keratinocyte growth factor signalling through interaction withboth ligand and receptor

DATA

Johnson GR Wange L (1994)

Heparan sulfate is essential to amphiregulin induced mitogenic signalling by the epidermal growth

factor receptor

J Biol Chem CHECK 269 (43) 27147 DATA CA 121 222877x

[Heparan sulphate is essential to amphiregulin induced mitogen signalling by the EGF receptor tyrosine

kinase]

Lind T Tufaro F McCormick C Lindahl U Lidholr K (1998)

The putative tumor suppressors EXT1 and EXT2 are glycosyltransferases required for the biosynthesisof heparan sulfate

J Biol Chem 273 (41) 26265-26268 CA 130 64007t

[McCormick C et al (1998) Nat Genet 19 152 had previously shown that EXT-1 rescues defectiveheparan sulphate biosynthesis and was now shown to elevate GlcA and GlcNAc transferase of mutant

cells; Lin X et al (2000) also demonstrated that EXT-deficient mice show disruption of gastrulation

and heparan sulphate biosynthesis (mutation in the human EXT-1 gene are responsible for multipleexotosis type. ]

Lyon M Gallagher JT (1998)

Biospecific sequences and domains of heparan sulphate and the regulation of cell growth and adhesion

Matrix Biol 17 (7) 485 DSATA

Fahem S Linhardt RJ Rees DC (1998)

Diversity does make a difference : fibroblast growth factor-heparin interactionCurr Opin Struct Biol 8(5) 578-586

Richard C Liuzzo JP Moscatelli D (1995)

Fibroblast growth factor2 can mediate cell attachment by linking receptors and heparan sulphate

proteoglycans in neighbouring cellsJ Biol Chem 270 (41) 24188-96


19/30

Kanner J Harel S Granit (1991)

Nitric oxide as an antioxidantArch Biochem Biophys 289 (1) 130-136

Cf Hinshelwood CN (1957)

The Structure of Physical Chemistry, Oxford, the Clarenden Press cf e.g. p395

Cf also Brown GC loc cit

Kao J Huey G Kao R Stern R (1990)Ascorbic acid stimulates production of glycosaminoglycans in cultured fibroblasts

Experimental Mol Pathol 53 1-10

[Heparan sulphate biosynthesis is differentially increased by ascorbate. The most pronounced effect of

altered GAGs upon ascorbate feeding at 25microg/ml (added 72 h prior to labelling and replendished

every 24 h) on cell cultured human foreskin fibroblasts was in boosting HSPG formation]

cf also Edward M Oliver RF (1994) loc cit who had previously published similar findings for human

embryonic endothelial fibroblast cells, and

Malemud CJ et al (1978) Connect Tissue Res 6(3) 171-9

who found that ascorbate boosts GAG biosynthesis in chondrocytes CA 9255968b

Karlinsky JB Rounds S Faraber HW (1992)Effects of hypoxia on heparan sulphate in bovine aortic and pulmonary artery endothelial cells

Circ Res 71 (4) 782-9 CA 118 78363v

[Hypoxia induced decreased sulphation of heparan sulphate but increased the AT(III) binding site

content]

Karlsson K Marklund SL (1988)Extracellular superoxide dismutase association with cell surface bound sulphated glycosaminoglycans

Basic Life Sci 49 647-650

Kishibe J Yamada S Okado Y Sato J Ito A Miyazaki K Sugahara K (2000)

Structural requirements of heparan sulfate for the binding to the tumor-derived adhesion

factor/angiomodulin that induces cord-like structures to ECV-304 human carcinoma cells

J Biol Chem 275 (20) 15321-15329

Knaus H-G Scheffauer F Romanin C Schindler H-G Glossmann H (1990)

Heparin binds with high affinity to voltage-dependent L-type Ca2+

channels. Evidence for an agonistic

action

J Biol Chem 265 11156-11166

Korengaga R Ando J Tsuboi H Yang W Sakumo I Toyoka T Kamiya A (1994)

Biochem Biophys Res Commun 198 (1) 213-19 CA 120 103328p

Lahiri B Lau PS Pousada M Stanton D Danishefsky I (1992)Arch Biochem Bioiphys 293 54-65

Linker A Hovingh P (1973)The hepartin sulfates (heparan sulfates)


[Amyloid heparan sulphates with high GlcNH2 contents]cf later work DATA( Lindahl et al)

Liu Z Perlin AS (1994)

Evidence of a selective free radical degradation of heparin mediated by cupric ion


Lahiri B Lai PS Pousada M Stanton D Danishefsky I DATA

Depolymerization of heparin by complexed ferrous ions

Arch Biochem Biophys 293 54-60

[Ascorbate promotes limited degradation of heparin


20/30

Previously heparin had been shown to be degraded by Fentons reagent and by activated phagocytes cfMetcalf DD et al (1990) loc cit

Horner AA (1971) loc cit found that multichain rat skin heparin was degraded by ascorbic acid tosingle chain structures possibly due to the action of trace metal impurities]

Li Z et al DATA (2000)

Expression of N-deacetylase/sulfotransferase and 3-O sulfotransferase in rat alveolal CHECK type IIcells

Am J Physiol 279 (2 part 2) L292-301 CA 133 250129w[HSPG sulphation enzyme RNAs were studied as influencing accumulation in basal lamina beneath

alveolal type I cells (e.g. during the transition from type II to type I cells); increased expression of N-

sulphotransferatse showed that GN-sulphonation was dependent on cell density and matrix and was

intense under condition where cells spread fully; the pattern of G 3 o sulphation was, however, was

not changed under these conditions as indicated by expression of the 3OST.

NaCl03 singificantl;y inhibited cultured type II cell spreading and the inhibition was reversed by

Na2SO4]

Libeu CO et al DATA (Linhardt RJ) (2001)

J Biol Chem 276 42 39138-DATA

[Defective binding of ApoE to HSPG associated with atherosclerosis via defective liver clearance alasorelevant to Alzheimers disease neural plaque formation]

Lind T Tufaro F McCormick C Lindahl U Lidholt K (1998)

The putative tumor suppressors EXT1 and EXT2 are glycosyltransferases required for the biosynthesis

of heparan sulfate

J Biol Chem 273 (41) 26265-26268 CA 130 64007t

Liu Z Perlin AS (1994)

Evidence of a selective free radical degradation of heparin mediated by cupric ion


Lindahl B Lindahl U (1997)

Amyloid-specific heparan sulfate from human liver and spleenJ Biol Chem 272 (42) 26091-26094

Long WF Williamson FB (1979)

Glycosaminoglycans, calcium ions and the control of cell proliferation

IRCS J Med Sci 7 429-434

Luck W (1965)

Ber Bunsen Phys Chem 69 (1) 69 (also ibid 626) DATA

Mandil AK et al DATA (1995)Kidney International 48 (5) 1508

[Heparin induced endothelial cell cytoskeletal reorganisation-possible mechanism for vascular

relaxation]

Mani K Jonsson M Edgren G Belting M Fransson L-A (2000)

A novel role for nitric oxide in the endogenous degradation of heparan sulfate during recycling ofglypican-1 in vascular endothelial cells

Glycobiology 10 (6) 577-586

[This process can be affected by Cu ions via catalysis of release from thiol storage sites]

cf Ishihari M (1998? DATA)

A new degradative pathway for heparan sulphate proteoglycans

Trends Glycosci Glycotechnol 10 (54) 331 DATA CA 130 63940e{NO and reactive oxygen species in regulation of the ECM metabolism

Cf Heller R Munsche-Pauley F Graham R Till U ()

L ascorbic acid potentiates nitric oxide synthesis in endothelial cells

J Biol Chem 274 (12) 8254-8260 CA 131 13677


21/30

McCarty MF (1997)

Glucosamine may retard atherosclerosis by promoting endothelial production of heparan sulphate PG

Med Hypoth 48 (3) 245 DATA CA 127 128534[Glucosamine dietary supplementation aids arteriosclerosis etc

cf also Theodosakis J Adderly B Fox B (1997) The Arthritis Cure Century Books ISBN 078123 7813

1]]

Metcalf DD Thompson HL Klebanoff SJ Henderson WR (1990)

Biochem J 272 51-57

Mislick KA Baldeschwieler JD (1996)

Proc Natl Acad Sci 93 12349-12354

Mishra G Castellot JJ (1999CHECK)

Heparin rapidly and selectively regulates protein tyrosine phosphorylation in vascular smooth muscle

cells

J Cell Physiol 187 (2) 205

Morano S Guidobaldi L Cipriani R Gabriele A Pantellini F Medici F DErme M DiMario U

(1999)High glucose modifies heparan sulfate biosynthesis by mouse glomerular epithelial cells

Diabetes.Metab Res Rev 15 (1) 13-20 CA 131 57278z

[Effect of 30mmol glucose produced a 50% decrease in total cellular proteoglycan biosynthesis

compared with physiological 5mmol; the effect was most centred on cell layer heparan sulphate

proteoglycans, three identified species being reduced in amount by 81-91%- this is the opposite effect

to what was found by Edward and Oliver (1984) loc cit for 50 mmol ascorbate on epithelial cellheparan sulphates where a 350% boost in total heparan sulphates but a 1950% increase in tyrpsin

resistant cell surface heparan sulphate proteoglycan was observed in this case as speculated by these

authors post synthetic degradation of heparan sulpahte might have occurred but this may have been

decreased by the effect of ascorbate. This suggests that ascorbate may reduce back to NO metabolites

such as nitrous acid known to efficiently deaminatively cleave heparan sulphate.

Heparin administration however enhances NO production by injured endothelia cf CA 120 182713m

CHECK as required for complex biofeedback heparan sulpahte processing/singalling]Cf Raats CJI et al DATA (2000)

Glomerular heparan sulfate alteration. Mechanism and relevance for proteinuria

Kidney Int 57 (2) 385 Ca 133 294275v

[Excess glucose in diabetes caused down regulation of kidney HSPG biosynthesis and reduction in

degree of sulphation of HSPG]

Mounkes LC Zhong W Cipres-Palacin G Heath TD Debs RJ (1998)

J Biol Chem 273 26164-26170

Murata K Yokoyama Y (1989)Acidic glycosaminoglycans in human atherosclerotic cerebral arterial tissues

Atherosclerosis (Shannon Irel) 78(1) 69-79 CA 111 151343a CHECK

[Data presented indicates a regular diminution of arterial wall heparan sulphate as a function of ageFeyzi et al 1998 (loc cit) found that the microstructure of arterial wall heparan sulphate showed a

systematic increase in the amount of trisulphated heparin-like sequence content;

perhaps this is evidence for a biofeedback related attempt by the organ to limit deleteriaouscalcification since more highly sulphated heparan sulphated would be predicted to provide such a

function cf Grant et al 1989b 1992b)]

Another age-dependent physiological marker is the reported human

age (in absence of disease) increase in the serum ferritin DATA

This has also been strongly implicated the aetiology of vascular damage linking Finnish mortality in

eastern Finnish men (Salonen et al (1990) loc cit).

Nader HB Ferreira TMPC Toma L Chavante SF Dietrich CP Casu B Torri G (1988)

Maintenance of heparan sulphate through evolution

Carbohydr Res 184 292


22/30

Nakashima I (1996)

Can cysteine direct tyrosine in signal transduction for environment-oriented gene control?

Nagoya J Med Sci 59 1-10[A hypothesis that cysteine-oriented signalling potentially directs the tyrosine-oriented signalling in a

mechanism of environmentally oriented control of internal signalling for gene control]

{cf Koch CA Anderson D Moran MF Elis C Pawson T (1991)

SH2 and SH3 domains: elements that control interactions of cytoplasimic signalling proteinsScience 252 668-674

These domains have a number of conserved tyrosines and another highly conserved cluster of basicamino acids (a potential heparin/HSPG binding site, previously proposed binding site for tyrosine

phosphates).

C-Y motifs (Grant et al 1989) occur in SH2 domain S (at conserved positions) in c-Src, c-Yes, Fgr,

Fyn, Lck, Lyn, Hck , Blk, at conserved Y but altered C in Vav, GAP-N (C deleted in GAP-C) PLC

gamma 1N and gamma 2N (but with possible methionine juxtapositions in Nck (Fer), PLCgamma 1C,

gamma 2C and GAP-C) and alternatively placed C-Y positions in p85 from same Y as above and from

a different Y in cFps; in tensin there is a replacement of Y by H at the conserved position in the IV

sub-domian)

(but apparently absent in c-Abl, Arg, Dab1 where the conserved Y is replaced, as is also the case with

p85 and in Nck with histidine;

v-Crk lacks C-Y and also a putative heparin binding site)}.

Nagasawa K Ogamo A Uchiyama H Matsuzaki K (1983)

Hydrophobicinteraction chromatography of glycosaminoglycuronans: the contribution of N-acetyl

groups in heparin and heparan sulfate to the affinity for hydrophobic gels, and variety of molecular

species in beef-kidney heparan sulfate


Nagasawa K Uchiyama H Sato N Hatano A (1992)

Chemcial change involved in the oxidative-reduction depolymerization of heparin

Carbohydr Res 236 165-180 CA 118 97491b

Nicholson JL Nakajima M (1994)

Similarity between heparanse enzyme and mammalian hsp90 like proteins[Hsp90 shares with heparanase occurrence at both cell surface and nuclear compartment. Attempts to

demonstrate heparanse activites with purified hsp90 were however not successful ]

cf Graham LD (1994)

Srivastava PK (1994)

Endo beta D glucouronidase (heparinase) activity of heat shock protein/tumour region antigen gp96

Ibid 301 (3) 915

{gp96 was suggested to be an evolutionary recent protein sharing multiple functions}

Cf Nakajima M Irimura T Nicolson GL (1988)

Heparanases and tumor metastasis

J Cell Biochem 36 157-167

Nishinage M et al DATA (1993)

J Clin Invest 192 (3) 1381 CA 119 24338u[Redox potential altered by H2O2 and cysteine which inhibit expression of anticoagulant heparan

sulphate by endothelial cells]

OBrien CA Ward NE Weinstein IB Bull AW Marnett LJ (1988)

Activation of rat brain protein kinase C by lipid oxidation products

Biochem Biophys Res Commun 155 (3) 1374 1380

[Activation of PKC by hydroperoxy fatty acids may be an early cellular response to oxidative stress]

Olin KL Potter-Perigo S barrett HR Wight TN Chair A (1999)Lipoprotein lipase enhances the binding of native and oxidized low density liproteins to versican and

biglycan synthesized by cultured arterial smooth muscle cells

J Biol Chem 274 (49) 34629-34636

[Other GAGs, chondroitins and dermatan sulphates, may be cofactors in HSPG dependent lipidprocessin, arterial walls and be involved in adverse effects of oxidised lipids]


23/30

Paka L Kako Y Obunike JC Pillarisetti S (1999)

Apolipprotein E containing high density lipoprotein stimulates endothelial production of heparansulfate rich in biologically active heparin-like domains. A potent mechanism for the anti-

atherosclerotic action of vascular apolipoprotein E

J Biol Chem 274 (8) 4816-4823

[Oxidised lipid adverse effect on heparan sulphate proteoglycans biosynthesis model of induction ofarterosclerosis]

cf Chang MY Potter-Perigo S Tsoi C Chair A Wight TN (DATA)Oxidised low density lipoproteins regulate synthesis of monkey aortic smooth muscle cell

proteoglycans that have enhanced native low density lipoprotein binding properties

J Biol Chem 275 (7) 4766-4773

Parrish RF Fair WR (1981)

Selective binding of zinc ions to heparin rather than to other glycosaminoglycans

Biochem J 193 407-41

[Copper ions have also been suggested to bind preferentially to heparin]

Paudel HK et al (1999)

Heparin induced conformational change in microtubule associated protein tau as detected by chemicaland phosphopeptide mapping

J Biol Chem 274 (12) 8029-8038 CA 131 13676y

Pauling L (1991)

In Block G Henson DE Levine M (Eds) (1991)

Ascorbic acid: biologic functions and relation to cancerProc Conf Natl Inst Health Bethesda MD Dept 112 1990

Am J Clin Nutr (1991) 54 1252S-1298S

[Pauling, while producing evidence supporting beneficial anti-viral and anti-cancer activities of a

therapeutic ascorbate dietary supplementation, lacked a convincing explanation for the observed effects

which can now be attributed to this major influence of ascorbate in HSPG biochemisty, but this can be

seen to be part of a wider dependence of HSPG biochemistry on redox status (e.g. as characterised by

the effects of hypoxia and glucose status on HSPG biosynthesis)]

Perez

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