The structure and metabolism of mucopolysaccharides (glycosaminoglycans) and the problem of the mucopolysaccharidoses

The Structure

and Metabolism

Mucopolysaccharides

of

(Glycosaminoglycans)

and the Problem

of the

Mucopolysaccharidoses*

‘l’he structures of the sulfated mucopolysaccharides are outlined and theit common and particular features pointed out. All are normally attached to specific proteins by covalent linkages to lorm large complex molecules. The biosynthesis of the sulfated mucopolysaccharides is considered in relation to the po5slble genetic defects of the mucopolysaccharidoses. The formation of the core protein is a prerequisite for the subsequent synthesis of the carbohydrate chains in both normal and abnormal cells. A linkage region consisting of atypical neutral sugars that joins the carbohydrate chain to protein is common to all the sulfated mucopolysaccharides apart from keratan sulfate. The formation of this region is strictly controlled by the specificities of the glycosyl transferases that form it. The synthesis of the main carbohydrate chain is dependent on prior

formation of the linkage region so that a defect in the formation of the linkage region or in glycosylation of the core protein cannot explain the abnormalities of the mucopolysaccharide diseases. Ocher possible explanations are considered, including defects in protein-pal!. sactharide structures that might produce inbalance in synthesis and degradaliorl. Faultv intracellular degradation of mucopolysaccharides has been shown in cultures of fibroblasts from patient5 with Hurler’s and Hunter’s syndromes, implying a deficiency of degradative enzymes. Deficiencies of certain lysosomal exo-glycoaidase5 found in some tissues of patients with mucopolysaccharidoses is unlikely to I)e the primary defect in view of the specificities of these enzymes and the strut. tures of the mucopolysaccharidoses. Some 01 the clinical features of the mucopolysaccharidoses may be explained by possible secondary effects ol’ the accumulation ol abnormal mucopolysacch;l. ritlr\ witllill the body.

which cannot be compreto mind some of the facts which are IIOW known about the structure and metabolism of mucopolysaccharides that apllear to have some bearing on the mucopolysac-cllaridoses, and which should be considered w!tell explanations of the genetic defect are adVanced. I-lie Iiomenclaturc of these compouncls has I)cen rex,isetl [I] as their structures have become

T

HLS KKIE:I~‘ review.

hensive.

aims

3 F~OIIItltc I)ivisiorl of Biochemistry, ~;~~lrns. I.ondon W.6., England. VOL.

4’,

established. The new and oltl syrlorlyn~a are listed in Table I. The tenn “glycosaminoglycan” is no~v generally used in the biochemical literatul-e in place of mucopolysaccharide, however since the latter ‘term is in common clinical lise it xvi11 be used in this article.

to bring

NOVEMBER

1969

THE

STRUCTI!RE

There

The Mathilda

and Terrncc

673

are

OF

several

MUCOPOI.YSACCHAI~I~~;S

excellent

Kennedy Institute

of

recent

reviews

Rhcum:~tolog~.

Rtrtt.

Structure

674

TABLE NEW

AN,,

OLD

NAMES

and Metabolism

of the Mucopolysaccharides-Muir

I

OF MUCOPOLYSACCHARIDES

New

Old

Chondroitin 4-sulfate Chondroitin 6-sulfate Dermatan sulfate Heparan sulfate Keratan sulfate Glycosaminoglycan

Chondroitin sulfate Chondroitin sulfate Chondroitin sulfate Heparitin sulfate Keratosulfate Mucopolysaccharide acid mucopolysaccharide Chondromucoprotein

Proteoglycan or Protein-polysaccharide

A C B

or

[2-51 which discuss how the chemical structures of the various mucopolysaccharides have been derived. These compounds consist of long unbranched chains made up of disaccharide repeating units (Fig. 1) of which one sugar is invariably an hexosamine and the other hexuronic acid with the exception of keratan sulfate which contains instead a galactose residue in place of hexuronic acid. All but hyaluronic acid contain sulfate groups which are attached to the hexosamines and in some cases also to the hexuronic acid moieties. These compounds are highly charged polyanions because of their uranic acid and sulfate groups and they occur normally in the matrix of connective tissue.

I :

H

They are manufactured locally by the appropriate connective tissue cells and extruded from them. They are also manufactured and stored within certain specialized circulating cells, such as platelets and leukocytes [6]. The anticoagulant heparin is also a mucopolysaccharide which is manufactured by, and stored within, mast cells [T], but it does not appear to be a structural component of extracellular connective tissue, although its chemical structure is closely related to that of heparan sulfate, which is a structural component [8]. From both structural evidence and work on the biosynthesis of mucopolysaccharides evidence is accumulating that probably none of the mucopolysaccharides occurs initially as free polysaccharide chains but are attached to protein by specific covalent linkages to form in most cases multichain complex molecules or proteoglycans, which are often of very high molecular weight. CHONDROITINSULFATES

Chondroitin 4-sulfate and chondroitin 6-sulfate differ only in the position of the sulfate group (Fig. 1A and 1B). They are susceptible to testicular hyaluronidase. They are the most abundant mucopolysaccharides in the body and occur both in skeletal and soft connective tissue. Apart from two apparently rare cases

OH

FIG. 1. Structure of disaccharide repeating units of mucopolysaccharides. A, chondroitin 4-sulfate. B, chondroitin B-sulfate. C, dermatan sulfate. D, hyaluronic acid. E, keratan sulfate. F. heparin. The sulfate group in brackets on C, of the uranic acid is not present in every disaccharide unit. AMERICAN

JOURNAL

OF

MEDICINE

(discussed later). they do not appear t0 be directly ilivolved in the mucopolysaccl~aritloses oilier t ban perlinps in type Iv [a] or Morquio’s disease. HoXvel.er muclt of the knowledge of t11c l)iosyntllesis and metabolism of mucopoly~accharitles and tlie way that proteoglycarlr are coiistrIi< ted has been derived from studies on c.llontll-oitin sulfate. From the more limited tlala obtained on other mucopolysaccharicles it app:~rs tllat to a large extent the chondroitin 4~ill;ites may be legartl~d as representative of tlie otliers. Frown the work mainly of Rod&n and his co~~Y~rkels [10-/q detailed information is now ii\ ailable about the chemical structure of the rc.,qion containiilg atypical neutral sugars that links chondroit in 4-sulfate to the protein nloict!. Trnlike the main part of the chain \\-llic h i\ digested by hyaluronidase, the linkage r(‘gion i\ resistant. .iftcr proteolysis of thontli-oitiit sulfate-proteins a peptide remains attached to the chondroitin sulfate through the linkage region. The structure of the linkage rqion is shown in Figure 2. It was clerived flom ;I study of the degradation products obt:linetl on hyaluronidase digestion followed by protense digestion of the proteoglycan. The resulting ,~lyr,opeptide on partial acid hydrolysis ga\~ oligosnccharides that were similar to those obtained from heparin after partial acid hytit-ol>sis. T1’Pth the use of specific enzymes and compariso~l lvith derivatives of known atruct:~re tlle complete structure of the linkage rcagion of both polysaccllarides was established uneclui\ocallp. The terminal amino acid, sc:riiie [ 141. is linked to xylose by its hydroxyl ~IXHI~)r/5]. This makes it labile to alkali, when the c-arl,ohydrate chains are detached from the pcptitlc or protein by a /3-carbonyl elimination of an alkoxide. This reaction is dia\gnostic for ( I-gly( osidic linkages to hydroxyamino acids, Tvllicli are destroyed in the process. It has heen used to demonstrate the existence of multichain proteoglycans, because single thains are released which are much smaller tllarl the complete molecule. These can then

peuetrate gels such as Sephadex@ G200 irom which the intact proteoglycan is excluded. The linkage of chondroitin &sulfate is exactly the same as that of chondroitin 4-sulfate rlh,17]. as are the linkage regions of heparin. licpar;ul sulfate and dermatnn sulfate. KERATAN

SI!I,FATE

Keratan sulfate appears to be exe ItGve to cartilage, nucleus pulposus and cornea. These tissues are particularly affected in Morquio’s disease (mucopolysaccliaridosis type 11’) ii1 which abnormal amounts of keratan sulfate ;~rt excreted in the urine. This mucopolysacc h;witle sllows considerable variations irk tietaiietl chemical composition [I81 and size [(.9], am1 an exact definition covering all the variables is not possible at ‘this time. .i\ major part of the chain is niatle up 01 III:disaccharide unit shown in Figure 1E. ‘l’hi:, differs from the disaccharides of the chondroitin sulfates and hyaluronic arid in having galactose in place of uranic acitl and in the positions of tile glycosidic linkages. The sulfate groups vary in number [20] and in prtsition [21,22]. Methylation studies show that keratan \ulfate is partially branched [21,X’], and there i+ an excess of galactose over hexosamine, wllicli are thought to be at the branch points. Sialic acid, f’ucose and mannose are present as minor constituents in most preparations 1211 and also xylosc identified by gas liquid cliromatography 123,241. The linkage to protein of cornea1 ant1 of cartilage keratan sulfates is different lor 1.11~ most part. The linkage in cornea1 keratan sulfate is stable to alkali [IR,%]. Tfte link appears to be a glycosylamine link between gluco+ amine and asparagine [18,20] and glulamine [IS]. The linkage to protein of cartilage keratan sulfate on the other hand involves threonine and serine [18,24]; in some instances threonine alone [25], these amino acids being destroyed during alkaline ,Celimination of the carbohydrate chain. The terminal stqar is

676

Structure

and Metabolism

of the Mucopolysaccharides-Muir

FIG. 3. Schematic diagram of the structure the possible variations. ”

to be galactosamin,e substituted in positions 3 and 6 [26], but there is evidence that some galactosamine is also situated elsewhere than at the point of linkage [27]. thought

CHONDROITIN SULFATE-KERATAN

SULFATE

PROTEOGLYCANS

The model for the structure of chondroitin proposed by originally sulfate-proteins Mathews and Lozaityte [28] and by Partridge and co-workers [29] consists of a protein core to which a number of polysaccharide chains are attached (Fig. 3). This model is still regarded as being essentially correct, although little is known about the number and variation in lengths of the chains or of their distribution along the protein core. Possible variations are shown schematically in Figure 3. The position is further complicated by the fact that chondroitin sulfate-proteins of cartilage and nucleus pulposus contain some keratan sulfate that cannot be separated from them [18,30--351. Attempts to do so without proteolysis merely succeed in segregating fractions richer in keratan sulfate from fractions richer in chondroitin sulfate [24,?1,32,?5$6]. A small proportion of the total chondroitin sulfate-protein of pig cartilage is free of keratan sulfate, however, and has been separated by zone electrophoresis from the remainder which was thereby enriched in keratan sulfate [37]. That keratan and chondroitin sulfates are attached to the same peptide has been shown by incomplete proteolytic digestion [38] when a peptide por-

of chondroitin

sulfate proteins

and

tion was isolated containing both types of mucopolysaccharide. A similar peptide has been obtained from normal urine which is present in greatly increased quantities in the urine of patients with Morquio’s disease (mucopolysaccharidosis type IV) [jr9]. Further evidence is obtained from hyaluronidase digestion of chondroitin sulfate-proteins, which resulted in a protein containing keratan sulfate together with the hyaluronidase-resistant linkage region of chondroitin sulfate and including some uranic acid 1331. The way that keratan sulfate is incorporated into the composite macromolecule is not known. The size of cartilage chondroitin sulfate-proteins bears some relationship to the keratan sulfate content, since from a given source, the larger that the proteoglycans are the more keratan sulfate they contain [40,41]. Nevertheless, those from immature cartilage contain less keratan sulfate than those of comparable size from mature cartilage [42]. The increase of keratan sulfate in proteoglycans with age [34] must therefore involve a complex process, which is reflected in the relative increase in cartilage with age of keratan sulfate over chondroitin sulfate [43]. The extent of this change varies with the site of the cartilage and is most marked during postnatal growth [44]. It is notable that the skeletal deformities of Morquio’s disease, such as those of the chest, become worse when rapid growth begins. The conditions of ,growth in tissue culture affect the amount of keratan sulfate produced by chon-

AMERICAN

JOURNAL

OF

MEDICINE

\tructure

and

Metahoiisln

of the Mucopolysaccharides-

of keratan suli-iii, ;rntl the ;unount I;~[c III aclult human articular cartilage shows ;I m;it Lccl 10~~~1topographical variation [4h,4/‘], tltoc~re

~vith depth from the articular surI.1ce rt~ore titan the chondroitin sulfate, where22 Ilunr;~n scmil11nar cartilage appears to conr;tin little if any keratart sulfate [48]. The forIrt;ILiott of collagen fibrils is affected by chontlroiLi1t sult’atc-lxoteitts [-/9,50]. P10teogl);cans are heterogeneous in other w:1)5 besitles silt and keratan sulfate content. ?Irvcr;~l n’-terminal amino acids have been IOLI~CI IF/\. Different fractions have different ;tmino ;I( id composition [?2,35,37,40,52] and N-terminal amino acid residues, as well as dificrenr antigenic tleterininants [do]. ‘ll~e sti~otig association of proteoglyrans with :I specific glycoprotein in cartilage to form aggtqatcs, as has recently been demonstrated / 531, Ittight account for some of the observed Itetero~c1teity but not for the considerable ~lillercnc (~5in kerat;m s11lfate content. illcw;rsing

tlYAT.I’KONIC

ACID

I i~;ilut~onic: acid is not conspicuously

impliand beCILIW of limited space consideration of it will Ix omitted. It is fully discussed in the reviews ;~lrc;itl> cited.

( ;~trd in the mucopolysaccharidoses,

I)eritt;1L;itt sulfate differs from the chonth-oitin sulfates in containing iduronic acid in place of glucuronic acid, its epimer at carbon ;1Lont 5 (Fig. I(:). It is present in soft connective ~iss~w ;tntl is abundant in skin, arterial walls ;~ntl lieart valves, organs that are affected in I It~rlcr’s and Hunter’s diseases (McKusick type I antI 1Ij. .4s far a\ 11x been ascertainecl it is I~OI-IIKI~~~ ;11~sc1tLfrom bone, cartilage, interverI rl)r;Il ciisc. c~ornea ;1nt1 the vitreous body. It ~otttl)ri\es the largest l’roportion of the ;~bnor1na1 Iti1r~~ol~ol~~accl1aritles in the urine of paI.iettt$ \viLlt Hurler’s and Hunter’s diseases and is tlic ;~lmoriri;il urinary mucopolysacchatide of 111~~Lar;11e;111s ant1 Iznmp syndrome (McKusick I\ 1’” VI). Recent work by Fransson and Rod& 154,551 ~11cl 11); Ftxnsson [5(i--581 has shown conclu\ively tliat altltouglt the greater part of the (Ii:1iIi consists of disaccharide units containing

itlirronic acid (Fig. IC) other regions contain tliaacc~liwitle units with glucuronic acid resi-

.bl/ri)

0 7 ‘“7

dues which are the same as the di~sac~l1;tI~Itlc units of the chondroitin sulfates. The latter .11x> susceptible to liyalLIronidase whereas th;it pit’t of the cllairl which contains id11roItic acitl is resistant to the en;lyme [ 5-!,55]. ‘1‘1~ pro~l~rcts of liy;iluronidase digestion range itt si/e from tlisaccharitle to polysaccharide ft2gttic.Itts, I lie latter predominating, whereas frag1ttrnts of iIitertttecliate size are scarce. As glucurotiic a( id is principally found iIt the stttaller Iragmrnts it would appear that the disaccharide [5513 units with glucuronic acid occur together iIt ~gro~~psor clusters, at variable intc~rvals along the chain. Dermatan sulfate can th11s IW regarded as a co-polymer with chontlroir in SLIIfate [SS]. Fransson [55] has shown further rhnt the dcrmatan sulfate of umbilical cord. urrlike that of skin, has sulfate groups attac.1tc.d to (i,; instead of Cl4 of the ,galactosamiIte residues in the regions containing glucuronic acid, 50 I hat the dermatnn sulfate of umbilical cord is ;I (‘I)poIytner with chondroitin (i-sulfate \vltcIeas that of skin is a co-polymer witlt cltotttlroi tin 4-sulfate. DermaLan sulfate with more I It;rtt otte sulfate group per disaccltaride uttil IIx l)een deccribed [j9] and found in the spleen of a patient with Hurler’s disease and in co~ne Iractiotis of tlermatan sulfate from thth riritie 01’ a patient with Mnratea11x and 1,x111\ \\rt(lt-c~titc (1ttttcol~o1~s;tc~cliaritlosis, type vi) [“‘)‘I. ‘-1s already mentioned, the sulfntc~tl strut 1ural Itt11co~~olysaccltarides of c-onnectivc li~suc are co~aletttly bo~mtl to specific 11onc.olla~etto1is proteins, from which they may l)e relt2sett by proteolytic digestion, when single l~o1~sacc:haride chaiIts are obtaiitrd wit11 ;I l~~)t itle 1r;ignient on the end. IVhen thmt~tn slllfate is prepared lvitlt hyal11rortidnse It-ci1ttttt~ttt to ixInovc cottL;imittating chondroitiIt ~11llaLc, it contains much less amino acid th;iIt Ivltert prcpared without 1541. (:l\~copeptitle~ cottt;rinittg scrInc 1tal.e been obtained from 5kitt dcrn1;1t;In sulfate 011 tligestion witlt Ii);;il11ritti~t;t~~~.FT‘liese coItLaittetl the same neutral sugars in tile 4;knie molar proportions [hl 1 as in the linkage region

of chordroitin sulfate. Idtnticnl oligm~tc-chrides Iiave been isolated jci?] Irotn Ix1cIcrial clioIttlroitinase digests of drrmaIa1t sulfate which were Glc.4$?( l-Y)&1 a ml Galp-( I-X)Gal/3(1-tl)xyl. Since the uranic ncitl t&dues in the vicinity of the linkage region arc almost

exclusively glucuronic acid [“I]. hvd~ironidase is iIl)le to cleave the linkage region and the

678

Structure

and Metabolism

of the Mucopolysaccharides-&IG

peptides to which it is attached from the rest of the chain. The xyIosyl-serine linkage makes dermatan sulfate susceptible to alkaline degradation [63], like chondroitin sulfate. Dermatan sulfate proteoglycans are much more difficult to extract than chondroitin sulfate proteoglycanis of cartilage. With the use of hydrogen bond-breaking reagents Lowther and co-workers have obtained dermatan sulfate proteoglycans from heart valves, skin and tendon [64-661. These contained more protein and were of lower molecular weight than those of chondroitin sulfate, and there were some differences in amino acid composition. Proteoglycans of dermatan sulfate possess a striking ability to precipitate soluble tropocollagen immediately at 4’~. at physiologic pH and ionic strength [67], a property that chondroitin sulfate proteoglycans do not have. It is suggested that its prime function is in the formation of collagen fibres. In the Hurler and Hunter syndromes the ‘tissues that contain much collagen, such as skin and heart valves, are affected, and cornea1 opacities are frequent in Hurler’s disease. In this connection it is of interest that Anseth and Fransson [68] have shown that dermatan sulfate, normally absent from corneas, appears during the formation of opaque scar tissue. HEPARAN

SULFATE

Heparan sulfate, like dermatan sulfate, is implicated in many of the mucopolysaccharidoses classified by McKusick [9] and is the principal mucopolysaccharide in the urine of patients with the Sanfillipo syndrome (mucopolysaccharidosis, type III). Like dermatan sulfate, it has not been identified in normal skeletal tissue or cornea. It is found mainly in blood vessel walls of various sizes [69] and appears to be a structural element, unlike heparin to which it is closely related. The generally accepted structure of heparin is shown in Figure 1F (for reviews see [2 and 3]), in which the glycosidic linkages differ from those of the chondroitin sulfates and dermatan sulfate in two respects, in having all 1-4 linkages, with the a-configuration. Heparin is thus resistant to the enzymes, both bacterial and animal, that degrade ,the chondroitin sulfates or dermatan sulfate. Adapted strains of Flavobacterium hepariniase [7] produce an enzyme that has been purified [70] which degrades heparin to

unsaturated disaccharide units by an elimination reaction. The close relationship between heparin and heparan sulfate is shown by the fact that both are degraded by this enzyme. The amino groups of heparin are mostly substituted by sulfate groups, whereas only a proportion are in heparan sulfate, the remainder being acetylated, as in all the other mucopolysaccharides. The flavobacterial enzyme, however, degrades both N-acetylated and N-sulfated portions [70,71]. In contrast the hexosaminide linkages with sulfamido groups can be selectively cleaved with nitrous acid at low temperatures [72]. Heparin obtained after proteolysis contains a residual peptide rich in serine and possessing the atypical sequence of neutral sugars of the linkage region (Fig. 2) [73-771 which occurs in the chondroitin sulfates and dermatan sulfate. Heparan sulfate obtained after proteolysis, like heparin, retains serine as the principal amino acid [7S], attached to a linkage region that appears to be the same as that of heparin [79]. Furthermore the N-acetylated portion of the polysaccharide chain is directly connected to the linkage region although some N-acetylated glucosamines are distributed further along the chain [72]. Similarly in heparin the N-acetylglucosamine residues are in the immediate vicinity of the linkage region [76] and are substituted at position 4 with uranic acid [80]. The glycosidic linkages of the N-acetylated and N-sulfated regions of the chain are therefore the same as would be expected from the mechanism of formation of the sulfamido groups whereby acetyl groups are displaced by sulfate from the N-acetylated polymer [SI]. The presence of some N-acetylglucosamine in heparin [72,82,83] and the variable amounts of sulfamido and N-acetyl groups in heparan sulfate [84], particularly that derived from the liver of patients with Hurler’s disease [79], suggest that heparin and heparan sulfate contain the same carbohydrate chain, differing only qualitatively in the relative proportions of N-acetyl and sulfamido groups [S]. The essential similarity of the carbohydrate chain is further shown by the presence of some iduronic acid in heparin [76,85,86] and heparan sulfate [84]. Heparan sulfate normally is not readily extracted from tissues, unlike heparin [8,87] or the heparan sulfate in the liver in Hurler’s AMERICAN

JOURNAL

OF

MEDICINE

S;tructure

and

Metabolisnl

of the Mucopolysaccharides-

tl:seas~ ii?\, IL occurs as a multichain IXoteo$i)c;~n that ma\ be degraded to a single chain poly~:tccharitlc ‘by protcolytic enzymes or by ;ill\aline cleavage [S] of the xylosyl-serine I,ond IIv p-cz~rbonyl elimination, in the same way as (lmndl.oitin sulfate proteoglycans [IS]. The Il~pKlll sulfate present in the liver of patients \vilh Hurler’s cliqease Teas easily extracted tvithout protcolysis and WAS deficient in amino a( id>, particularly serine, compared wit11 the hcpar;~n sulfate of aorta that was prepared aftei l)l-oteolysis of the tissue. It also had a very 11rucl1 lo\strr molecular weight than heparan s~ilf;~tca 01 aorta [Z], suggesting that it was partially degraded. This should be borne in nlintl lvhen the underlying defect of Hurler’s and Hunter‘s diseases is considered. BIOSYKTHESIS II-I recent years considerable advances have I~3l made in understanding how nlucopolysaccharides are synthesised and a large literature has acctunulated, much of which has I)cell lately reviewed [,W-911. There is space to discuss only some salient points here. 7-11~ uridine diphosphate (UDP) nucleotitle bk~g;rrj pl,ovide the monosaccharide intermediates Ilot only for transferase reactions that I)uiltl up the carbohydrate chains but also for the eri/ymes coriverting common precursors to sugar2 required for mucopolysaccliaride synthesis (Fig. 4). All these sugars are required for other purposes, however, with the probable ex(eptioll of IJDP-iduronic acid neerleti for cIerm;ltan sulfate and heparan sulfate synthesis. It sl~ould be noted that at two points feedback inhibition is exerted, namely, that of UDP~vlosr~ on the formation of UDP-glucuronic ;I( itl horn I!DP-glucosr [92] and that of UDPN-ac-etylglucosarnine on the formation of girlfrom fructose& phosc’os;~nline-fi-phosphate phate and glutamine [93]. The formation of 1 TDP-xylose from the decarboxylation of UDPglucuronic acid has been shown in cartila,ge “941 and hen oviduct [95]. Since xylose is the rcrminal sugar linking carbohydrate chains to jerin(a residues of core proteins in all the sulfated rnucopolysaccharides with the exception of keratan sulfate, the absence of the appropri‘lte acreptor protein would result in accumulation of IJDP-xylose which would in turn inhibit flu-ther synthesis of UDP-glucuronic .N itl. lrcquil-ctl for mucopolysaccharide manu-

” 0 t. :

47.

NOVEMBER

1969

!,?‘I

itlzb UDP--

UDP-Gal Glc-I-P

P Glucose

*

4f

) UDP-Glc

IdUA

4

I) UDP-GlcUA

Glc-6-P + F-6-P

GkN-6-P I

GlcNAc-6-P

+ GlcNAc-I-P

+ +4 UDP-GalNAc

FIG.

4.

Fornlation of uridine

the biosynthesis

nucleotides of mucopolysaccharides.

rvquiwci

for

facture. Thus puromycin inhibited synthesis oE mucopolysaccharide by cartilage cells 1961 and fibroblast cultures [97,98]. Cycloheximide, like puromycin, inhibits protein synthesis. Its effect on the synthesis of core protein by cartilage slices is immediate whereas inhibition of chontlroitin sulfate synthesis is delayed for thirty minutes [W]. Cl~ondroitin sulfate syntl~esis is thus controlled and initiated by the synthesis of acceptor protein, a process common 10 all glycoproteins. This is also true for the bynthesis of intracellular chondroitin sulfate-protein by human leukocytes, wl~ich is not exyported from the cell. Puromycin inhibited the incorporation of labelled serine. sulfate and glucosamirir: into the proteoglycan [ZOO]. The initial step in the synthesis of mucopolysaccllaricle chains is thus the transfer of IYDPxylose to a serine residue of the appropriate receptor protein. This transfer to entlogr~-lous protein acceptor has been demonstrated in cellfree preparations from embryonic chick cartilage [101] and mastocytoma cells (which manufacture heparin) [102] when “C-xylos;vl-serine was demonstrable after extensive proteolysis, as well as 14C-xylitol after alkaline borohydride reduction, showing that the newly formed xyloside could undergo ,&elimina t inn, iIS should that in the linkage region. It XV;ISt’llrther shown Clint incorporation ol labelled serine was inhibited by puromycin but not tile glycosylation, suggesting that preforrnctl protein was being utilised as the acceptor. In a series of elegant experiments Helting and Rod& [103,104] have shown recently how tile linkage region .is assembled to form the strictly defined structure shown in Figure 2. Previous work [loll had demonstrated that a

680

Structure

and Metabolism

of the Mucopolysaccharides-hiluir

cell-free preparation from embryonic chick cartilage incorporated 14C-galactose into protein from which galactosyl-xylitol could be obtained by alkaline borohydride reduction. Enzyme preparations catalysing the addition of each sugar of the linkage region have now been obtained from this source. The substrate specificity of each glycosyl transferase has been shown by the use of fragments of the linkage The region and their analogues [10?,104]. specificity of the reaction transferring the first galactose residue to xylose was not very exact since free xylose or various ,@xylosides were good acceptors. By using mixed substrates to test competitive effects, it was demomtrated that the transfer of the second galactose was catalysed independently of the first and had a different pH optimum. For the second transfer the sugar attached to the first galactose was decisive; the substrate had to be galactosyl ,81-4 xylose or the corresponding serine derivative. This requirement prevented the transfer of a third galactose. Using similar methods with mixed substrates Helting and Roden [104] showed that the enzyme catalysing the transfer of the first glucuronic acid to the linkage region (i.e., that of the first disaccharide repeating unit of the main chondroitin sulfate chain) had a substrate specificity that was different from the enzyme transferring glucuronic acid to substrates possessing N-acetyl galactosamine residues at their nonreducing ends, derived from the main chondroitin sulfate chain. Free galactose was an extremely poor substrate, but fragments of the linkage region containing galactosyl-galactose at the nonreducing end were good acceptors. Again the second sugar attached to the acceptor defined the specificity of the enzyme. In this case, however, although the P-configuration was essential the linkage between the two galactose residues was not decisive since PI-4 and /3126linked galactose disaccharides were acceptors. As Helting and RodCn point out, this lack of specificity is immaterial since the structure of the natural acceptor is defined by the specificities of the preceding reactions. A consequence of the specificity of the enzymes forming the linkage region is that a genetic defect resulting in the absence of any one of them would interrupt and so prevent the sequence of events leading to the formation

of all the mucopolysaccharides having this linkage. Puromycin inhibits the biosynthesis of mucopolysaccharides because the initial glycosylation cannot occur in the absence of acceptor core-protein to initiate chain synthesis. In the mucopolysaccharidoses a defect in enzymes synthesising the linkage region is therefore not a plausible hypothesis to explain the presence of free mucopolysaccharide uncombined with protein, as for example free heparan sulfate in Hurler livers [79]. Indeed puromycin inhibited mucopolysaccharide synthesis by Hurler fibroblasts [9S], indicating that a protein acceptor was necessary for chain initiation even in genetically abnormal cells. The way that alternate glucuronic acid and N-acetylgalactosamine residues are incorporated into the chondroitin sulfate chain has been studied by Telser, Robinson and Dorfman [96]. Enzyme preparations from chick embryo cartilage incorporate labelled UDP glucuronic acid and UDP-N-acetylgalactosamine into a polymer resembling chondroitin [105,106]. If chain growth takes place by sequential addition of each monosaccharide to the nonreducing end of the nascent chondroitin sulfate chain, ,then an oligosaccharide with one sugar at the nonreducing end should be an acceptor for the other sugar. Thus the hexasaccharide obtained by hyaluronidase digestion of chondroitin sulfate which had glucuronic acid at the nonreducing end was an acceptor for labelled UDP-N-acetylgalactosamine in the absence of UDP-glucuronic acid, but not vice versa, the product being a heptasaccharide. Similarly, a sulfate-free pentasaccharide with N-acetylgalactosamine at the nonreducing end could accept labelled UDP-glucuronic acid in the absence of UDP-N-acetylgalactosamine but not vice versa, the product being a hexasaccharide. The chick cartilage N-acetylgalactosamine transferase was specific for the group transferred, since no transfer took place with UDP-N-acetylglucosamine. Telser et al. [96] h ave suggested that the termination of chain elongation is the result of sulfation because the sulfated pentasaccharide from chondroitin 4-sulfate with a terminal N-acetylgalactosamine-4-sulfate group was not an acceptor for labelled UDP-glucuronic acid. All the enzymes involved in forming the linkage region, as well as the main chain, are present in the microsomal fraction. There AMER,CAN

JOURNAL

OF

MEDICINE

Structure

and

Metabolixll

Icl:II ion in dislribulion from nncrosomal fractions in 111~ c11q3ne~ wcjuiretl at the beginning and \ulfate synthesi’s [IO/“\. c,tttl 01 ~Iloiidioitin ‘I 11115Ilie ~)104)1 trallzferase was clriefly conI g1.i

1+..1s ;I I I ;lp[‘;l

I’(‘11

I 0qlr

~niootl~

to

frl~c~l t<) the

i~c~iiglr iileinbranes,

~vliereas

the

t’117\IIIC’\ lormiug

the main chontlroililt sull;itc cli;iiil UY~(: distributed more evenly l)cnmnbranes, and smooth i ‘1\‘(‘ClI I ough ~V~~CTC~;IS tlitl ~l~lfotr~in4fer~~se activity of the ~rl1001 II IIW

tractions

III~C-i~osoni;tl

1 iims

was

four

to

rough. From this it is ~~oarulatetl that the Iiascent proteoglycan passes .~lOlH+ ;(I1 ~~cwlling iroin wme~

111x1 of

assembly the

Golgi

die

line

I he ~11. .\ Gmilar to storage

>yntllcsis

of

apparatus

ATanules

of intracellular

enzymes, prior

transfer takes

finally to export

from place

chondroitin

microafter

the

sulfate

i)y IIIIWI~ m~~eloid leukocytes [108] and ~uniably of heparin in mast cells [109].

prc-

ISiological sulfation reactions require S-phosphoadenos: I-5-l)llosphosulfate (PAPS) as an ilrlcrniediale [IlO]. The enzymes transferring mllar~ from P.\PS to c,hondroitin sulfate [207, I /I !.,ind Lo heparin (1091 are also present in a par1 I(-ulate fraction. In the absence of PAPS a polymer

identified a, c-llondroitin whicll is particle-bound is for-rnccI Irom labelled monosaccharide precur5orj itsing a cell-free preparation from cartilage //Oh.lZ-7]. In the presence of PAPS, howas chronever, a >nlfated polymer identified clroitill hIdfate [I”] is formed, showing that sul fat ion aud polymerisation are independent both processes orcur in proc”s~‘s altlioL# C~OSCI)roximity in the cell. A similar situation ill the synthesis of heparin [IZ?], and Llic. alwnce ol time lag in sulfation during l,ol!~~l(‘i.i\atioll sl~)ws that both processes take plan-c, at the same time [IOS]. There is no cvi-

oI)taill+

it

tleiitc, Iio-~\ever, that sulfated polymer5 c-an be loi mctl lrom \ulfatetl monosaccharide nucleo1itlcs >\I,(11 as I!l)P-N-~~cctylgalactosalni~i~ -&sulI;IIV, \vllic h WI\ not utilized

for polymer formarat epiphyses [I 261. Hc*parin tontains N-sulfate groups, with a small rrtinibel. of N-acetyl Xqoups, whereas I~ef);t~~an >rlllare contains a more equal but variable prol)or(ion of both, as already pointed out. The bios),nthesis of the N-sulfate groups has I)cen s11own by Silbert [82] to take place by tliaplacement of N-acctyl ~groups. Using UDPN-~i(.(~tyl,~lli(os;iinirie labelled with tritium

I ion [/I-/j and embryonic

\‘(,1..

47 , N,,VEMIDI..R

1969

0x1

.WNII

of the Muc~opol~saccllarides

in the acelyl group, and 1JI)l’-~lucui.c~liic: ac.irl. it w;i4 sliown thal in thr nbsencc 01 1’ \PS all N-acetylatetl polymer \vas formecl 1)) ii Iilic rosomal preparation from m:Is~ocylotrl:~ t (*IIs. III

Llie pi’eseiic’e of P.IPS, Iiowc\~cr, tlrcl~c~ \2’:152 loss of as 1nucl1 ;Is half tile lalIw1lC~1 ;I( CL!1 groups and ;I corresponding formalioir 01 Nsulfate well

gronps. durilig

sulfated that

7‘1Gs reaction or

after

product

tlis~~lacenie~il_

took

place

l)olynleris;itioli.

was the most

anionic,

of N-acetyl

grout)\

q11a11) l’l1(,

N-

slio\virig \vas j)r<)-

gressive, which is further e\,iclence lor lllc 1 iell that tlie difference between hcpatxii sullalrc and liepaiiii is mainly ;I qudilativc ollc [Xl. It is therefore remarkable that there arc no (iinical signs of abnormal hcparin nictabolisirl iri the rntlco~,olysaccharitloses in wliic II liq);iran sulfate is implicated (types 1, n and III). Mewan and Davidson [117] consitler that tll( sulfotransferase in chick embryo cartil;tge lacks specificit), since several exogenous mti~pol\saccharides can act as acceptors, including heparan and dermatan sulfates, \vliicIi are’ noL present in this tissue. The) also suggc5t 1I/S] that sulfation of chontlroitin sulfate t;ikc.s place on CA of the galactosamine resitlties lvheri the polysaccharide is attached to protein. but on (:,; if it is free. As opposed to this l
Dorfman [I 191 find that the proporliorls of cliondroiti~i 4-sulfate and 6-sulfate s~iitlie~isctl by minced embryonic chick cartilage v;tries with the stage of development of rile eml)rvo, in agreement with the changing pi.oport iolis previously found [I-30], so that it apprars utilikely that tile position of sulfntioli is dccitlctl by the state of the receptor. The proportions of the IWO isomers were established by the use of Proteus vulgaris chondroitinasc, whit Ii gives /Ior f-sulfated unsaturated disacchal~icles. Kobinsori [121] has shown further that the pi‘oportion of tlie isomeric chontlroitin sulfates formed by cartilage in uitro is similar to that irl 0710 at the same stage of developiiieiit. .\ crllfree enzyme system from embryonic chick cal.tilage likewise produced both isomcrj of e~~dogenons cliontlroitin sulfate from P.\PS. However. the relative proportion of clioii~ll~oit in Isulfale formed was affected by pF1 antI 1)r llle time of incubation, being grcatcr ;I( e;lrlicl times. These results suggest that t1iei.c \\‘erc two sulfolransferases in this tissue. On tlie otlirr

hand, the line structure influence the position

of the receptor inigllt of sulfa&n, bclcause

682

Structure

and Metabolism

of the Mucopolysaccharides-Muir

Fransson [56] found that dermatan sulfate from umbilical cord contained 4- and 6-sulfate groups, in which 6-sulfated galactosamine residues were preferentially located next to glucuronic acid residues whereas 4-sulfated galactosamines were prevalent in regions containing iduronic acid. An octasaccharide was isolated that contained sulfate groups in both positions, in support of these conclusions. That umbilical cord contains chondroitin 6-sulfate [122] may be of some significance. Relatively little is known about the formation of the complete proteoglycan. Interpretatations of results meet with two difficulties when a tissue like cartilage is used; the first is the structural heterogeneity of the chondroitin sulfate-proteins, and the second is the relatively small amount of material synthesised during the experiment compared with pre-existing extracellular material. Thus for example different fractions of cartilage chondroitin sulfateproteins showed differences in the rates of incorporation in vitro of labelled precursors [152]. Several investigators [106,123-1251 find that chondroitin sulfate chains with the highest specific activity are invariably those that are most anionic and/or largest. This appears to be so whether the labelled precursor is sulfate or glucosamine, or whether the material is entirely intracellular as with human leukocytes or derived from various proteoglycan fractions of cartilage that are mainly extracellular. Leukocytes contain a chondroitin sulfate-protein whose synthesis is inhibited by puromycin [IOO]. On gel-chromatography the separated chondroitin sulfate chains with the highest specific activity are those of largest molecular size [124], but these represent only a small proportion of the total. It is suggested that the differences in molecular size and nonuniformity of labelling result from synthesis and partial degradation taking place in different parts of the cell at the same time [I@‘]. Kleine, Kirsig and Hilz [126], on the other hand, found that the fraction of highest specific activity from cartilage, which like that from leukocytes also had the highest charge density, was inhibited less than any other fraction by puromycin. DEGRADATION

Since mucopolysaccharides are normally covalently bound to protein, degradation can affect the carbohydrate or protein moieties. The structural importance of the protein, at

least of the proteoglycans of cartilage, is evident from the loss of matrix that follows intravenous injection of papain [127]. Proteolytic enzymes of lysosomes when released produce the same result (for reviews see [5,90,128]). Breakdown of the core protein is the initial step which allows the fragments to diffuse out from the tissue. In contrast, degradation of the mucopolysaccharide chain requires enzymes of greater specificity. Although hyaluronic acid and both the chondroitin sulfates are effcctively degraded by enzymes closely similar to testicular hyaluronidase that are present in lysosomes [129,130], the existence of analogous endoglycosidases that degrade dermatan, heparan and keratan sulfates is less certain; the amounts or activities appear to be too low for them to be demonstrated unequivocally as yet. The normal animal is thus capable of breaking down considerable amounts of injected chondroitin sulfate [I311 but not the other sulfated mucopolysaccharides, much of which may be recovered in the urine [232]. In the mucopolysaccharidoses it is these mucopolysaccharitles which are present in large amounts in the organs and urine. This situation could arise if the normal degradative mechanism is limited and there were an overproduction. The small amount of dermatan sulfate that is present in normal urine is of comparable molecular size with that in the urine of patients with Hurler’s disease [I%] so that the capacity to degrade dermatan sulfate may not be impaired. Matalon and Dorfman [98] have isolated the dermatan sulfate from cultured Hurler fibroblasts after digestion with papain and found it to be of comparable molecular weight as that of normal tissue dermatan sulfate and also to retain one residue of serine per chain. On the other hand the dermatnn sulfate in the urine of patients with Hurler’s disease is deficient in amino acids [I331 and resembles in molecular weight normal tissue dermatan Sulfate that has been de
JOURNAL

OF

MEDICINE

Structure

and Metabolism

of the Mucopolysaccharides-;M1/1’1

II!, injectetl labelled dermatan or chondroitin sr!lf;tte tltcy cannot depade dermatan sulfate ;I> the) do exogenous chondroitin sulfate. V;I~ Hoof and Hers [2?7] suggest that. the defect in the mucopolysaccharidoses is clue to a tlciic icnc), in 01113or another of the lysosomal cll/.vlne4. TI~LIS. for example, ,8-galactosidase ac,ti’\ itI was frequently low in certain organs such a5 the liver, brain and skin although norn1a1 in other organs such as the kidney and spleen. :!t thr s;nne time other lysosomai glycosidascs were greatly increased, which they consider ma) be a secondary effect of the mucopol)s;rct~l~aritle accumulation in the lysosomes I\ lien lhey are filled Xzith material that they cannot degrade [138]. The enzyme deficiencies that lvere observed by Van Hoof and Hers, I\-hich included in some cases an absence of c+ f:lco5i~lasr [I 391, did not correlate well with McKusick’s classification, and they suggest a new classification based on the enzyme deficiencies. Somewllat similar results have been obtained by others, the most common being a tleficic.licy in /Lgalactosiclase [140-1421, which ITas been separated on gel chromato
47,

NOVEMBER

1969

083

in complete mucopolytact linkage region saccharides. In view of the specificity of enzymes forming the linkage region (as alread\ discussed) it is just conceivable that were some’ /3-galactositlase to be outside the l~sosomes in the vicinity of the biosynthetic machinerv it could interrupt chain growth on( e this 1~s been initiated by the first glycosylation of the acceptor protein with xylose. Since a pl~ogressive localization of enzymes synthe5ising thr linkage region and the mucopolysacc.haritle chain has been demonstrated [107], the nascent chains whose galactose of the linkage region had been cleaved by ,@galactositlasc might not wholly regain their galactose before pasGng on to the next position of the biosynthetic machinery. This would have to assume that in tile normal situation some degradation takes pl;~e at the same time as synthesis and at the same locus in the cell. A deficiency of /3-galactositlnse would then allow more chains than normal to be formed; more than would be required for export from the cell for structural needs. ‘Flie difficulty of accounting for an abnormality of one or another rather than all the mucopolysaccharides remains, however, unless the intracellular enzyme deficiency is localised to teltain tissue5 or organs (as the work of Van Hoof and Hers and iickerman shows) whose cells might make one rather than another mucopolysaccharide. On the other hand tllc mucopolysaccharidoses are generalised diseases, not confined to connective tissue or to a particular organ. Another suggestion is that the abilitv to form proteoglycans of normal structure is in some way impaired [39,1?,f], the defective compound being removed more rapidly than tht> normal one and appearing in the urine in ;t partially cle~gratletl form. This might explain the illcreased serum levels of mucopolysaccliarides in patients with Hurler’s disease [149]; the low molecular weight and amino acid content of heparan sulfate [79] and dermatan sulfate [Z?3] in the livers of such patients: the higher t.han normal degree of sulfation of dermaLan sulfate in the spleen [60]; the lower than normal sulfation of urinary chondroitin sulfate-peptide of patients with Morquio’s disease (mucopolysaccharidosis, type IV) whose keratan sulfate-chonclroitin sulfate peptide was deficient in amino acids [?9]. It is suggested [97] that the absence of the correct structural components outside the cell might impair normal feedback

684

Structure

and Metabolism

of the Mucopolysaccharides-Muir

inhibition and lead to overproduction, analogous to the replacement of matrix of cartilage in organ culture that has been partially removed by digestion with enzymes 17150,154. Material that stains with metachromatic dyes is present in skin cells of patients with Hurler’s disease [I531 and in a proportion of the circulating lymphocytes [154], which persists in culture [155,156] for several weeks. Net synthesis of mucopolysaccharide by fibroblasts in the presence and absence of ascorbic acid shows that although the amount secreted into the medium was similar, Hurler cells retained within them much more than normal cells, a difference that was greatly accentuated in the presence of ascorbic acid [157]. Matalon and Dorfman [97] f ound similarly that the uptake of labelled precursors into mucopolysaccharides within Hurler cells in culture increased greatly with time compared to normal cells, whereas pulse-chase experiments showed that the secretion of newly formed mucopolysaccharide into the medium was the same. Using labelled sulfate Fratantoni, Hall and Neufeld [f44] have concluded that the abnormal cells synthesise and secrete mucopolysaccharides at the same rate as normal fibroblasts but that the intracellular pool continuously increases and never reaches a steady state, implying faulty degradation of mucopolysaccharides. The kinetics of secretion suggest that the bulk of the intracellular material is not the precursor of the extracellular material in Hurler cells. Chase experiments showed that most of the prelabelled intracellular material is not subsequently ,secreted as macromolecular compounds. It is suggested that there are two intracellular pools, a secretory pool of small size and rapid turnover and a storage pool in which the rate of degradation is slower than that of normal cells but the rate of synthesis is not. The cytoplasm thus becomes continuously filled with partially degraded mucopolysaccharide as the size of the intracellular pool increases. The presence of partially degraded material in the livers of patients with Hurler’s disease [79,1?3] would thus be explained, but this implies that the disease involves the lack of as yet unidentified degradative enzymes for heparan and dermatan sulfates. It is notable, however, that the dermatan sulfate chains themselves of the proteoglycan within Hurler fibroblasts do not appear to be degraded [98]. Chase experiments with normal cells show that much of the pre-

labelled intracellular material enters the medium as low molecular weight products [244]. Fratantoni, Hall and Neufeld [145,146] have now found that cells from a different genotype, or the culture medium therefrom, correct the storage abnormality of Hurler or Hunter cells, whereas the medium or cells from different subjects having the same genetic defect do not. Thus Hunter and Hurler cells can mutually correct each other’s storage defect, even though both diseases involve the same mucopolysaccharides. This finding also explains why mothers who are heterozygotes do not show any mucopolysaccharide abnormality although when their cells are separately cloned approximately half develop metachromatic inclusion bodies [158]. The normal cells of the heterozygote mosaic will correct the Hunter cells’ abnormality. Fratantoni, Hall and Neufeld [146] have shown that the corrective factor is heat labile and macromolecular. It accelerates the degradation of intracellular mucopolysaccharides in recipient cells, the size of the intracellular pool is diminished and its half life approaches that of normal cells. The prelabelled intracellular material which enters the medium under the influence of the corrective factor is of low molecular weight. Serum is not necessary for activity, which is present in a soluble fraction of disrupted cells. The factor was not destroyed by digestion with nucleases or trypsin and had no ,@galactosidase activity. It appears that there is a lack of a factor specific for Hunter or for Hurler cells, because the factor which corrects Hurler cells is produced by all genotypes other than Hurler and that which corrects Hunter cells by all genotypes other than Hunter cells. These results do not support the possibility of a mutation of a regulator gene [97]. The necessary degradative enzymes which are presumably lacking may either be induced or their loss from the cell prevented by the corrective factor. The deficiency of P-galactosidase in liver and increased levels in serum and urine of patients [142] might suggest the latter possibility. The continuous synthesis and storage of chondroitin sulfate in the granules of leukemic as opposed to normal leukocytes is somewhat analogous to the situation in Hurler fibroblasts [108] in which the stored material is likewise partially degraded. The chondroitin sulfateprotein of guinea pig Kurloff cells induced by estrogens is not degraded, however, [159], and

Structure t 11e pt aces\ tilelv

leatling

and Metabolism

to storage

may

be en-

here

FI alantoni

and

lrom

kin&c

associates

amniotic

Illetllotl

for

Hut-ler is

the

and

IOO

;I

period

fllrtlter

but

1nint1 lvhen

brought

a biochemical

;tnotl~r

01 explaining to explain

lvith

disease

(lie janie

explana-

why one

rather

than

is involved,

why different

escrete

of tnllc-ol”)l!s;lccllaridc.

to

L4s MeI1 as

il\rlco~)ol~saccl~~~ride

.~Iso ~~rc~s\ar)

as

need

displaying

be

whose

it is

subjects

differing relative

propor-

I ;II~. Jntle~l two siblings with the Hunter >yn(l~ome [I61 j cxcretctl different amounts and rclati\-c

proportions (Table

Sin<-<, tlrcxv UYIY netic sion

not

tliff&nccs of tilcir

rclls

tall sulfatr tall

sulfate

ones

arc: 5tron,g [I621

r11a1 tlc’~ratlation rail

the

sulfate

in

I)‘ttiellrs raisetl

11met1 11) 111~ (Table bc rxpla

lielit\

with

ined

wllnt

of III)

of

the

dermakera-

the

nor-

1 (,G.E;.) 1

1 Now: ” o I.

NOVEMBER

with

tlroitin hel)aran results).

This

droitin

sulfate

excretion

the

need

explaining

as much

urine large

output

ot c-lion-

to a more

mutation

whicll

any

and

unpultlished

be due in

chon-

as tlermatan

III, Muir,

extremely may

ok mucopoly-

almost

(Table

of tlip genetic

and

It is not tural

primary too t\ould

comprclteilsi\~~

tural

complexity

tilage

and

vessel

hepa-

very

sulfate

others

[I631

In two pa-

Hurler’s

are of

the

IIV-

which

syn-

that

the

may

be

earlier there

might

view

with

also

with

is very

its

the much

might

and in

of

keratan

Tile

reason disease

i:, conlplcted skeleton,

sulfate

lIefore

in llle

body.

III

Type

Total output

Dermatan Sulfate

I I I III VI 1

1,260 576 170-615 504 220

625

of the mucopolysaccharides

I-leparan Sulfate

18 180

(hnlciroitirt

h~llfarr~

119

346 150-565

i 16 730 l’-so 50 37

430 Approx. 2-3

were determined

as in Table

I)e

effects,

Morquio’s

the

[l64_l, not

the consequent

tissues.

development

rest

as blood age

cumulative

growth

car-

the vat-iety

tissues

which

strucof

change

is spared

because than

in the

the

in such

of connective skull

of

age and

produce

during

tile struc-

normal

proteoglycsns

abnormality

great

whether

entirely In

the changes

walls,

remodelling

way.

at present

mucopolysaccharidoses.

pal-titularly

chondroitin

known

proteoglycans

fluids

1969

in

effect

some-

to be

was

sulfate

The

by

large

there

sulfate

tissue

and

a very

saccharides,

and

The amounts and proportions 47.

20 11

disease.

No. of

1 I’K.P.1

P.P. T.P.

a structural

is impaired

TABLE

Paricnt.5

54 45

of mucopolysaccharides

lysosomal

that

acid

in the same

appeared

into and

of dertnatan

Hurler’s

excretion

coultl

inhibitors

lysosomes

with

Sulfate* su1ratc* Sull;lie*

(yr.)

* The mucopolysaccharides were isolated and their composition estimated by thp amount digrsted hy hyaluronidase and the molar ratios of glucosaminc to galactosamine before and after digestion [ /h?l].

in

whose

Since

sulfate

of hyaluronic presence

ge-

is raised

acid

[157].

it is possible

bet ;III~~ of tile

\vh;lt

acid

Ilyaluronic

droitm

pothesis.

syndrome

as 1~11 as heparan

hy:rlrrronitla~e

their

the expres-

gene.

Hurler’s

rmrmal

results).

twins,

of hyaluronic Inore

than

tlermatan

modified

mutant

lz.ith

pt’oduced

metliilni

have

common

I)atierlts

and

unpublished

itlentical

may

‘1‘11~ excretion sonle

ol heparan Muir,

JI:

(.:ho!l-

Heparan

matan

P.P. T.P.

dromr

amounts

tiotls

~u11;11~

ii

other

such

may

I ~OII 01 tile mucopol~saccharidoses. IIW 111ohl~n

Patient

for dia%gnosis.

should

considering

cells

before

Lo be useful

I’\\‘, i SlHI.lNI

Ikr.\q

This

to the

some

OF

SYNDROME

HlJNTER’S

storage diagno-

syndrome

points

used

syndromes.

in culture

.fI)no~ 1rla1 bchasionr Sonlc~

and

tile Sanfillipo

1I~II long

uptake

WITH

similar

intrauterine

it1 principle

11~uc-o~~ol~~accl~:11-idoses. those

out

IIunter

applicable

have

to carry

on the

sulfate

\i\ $)I’ the

[160]

fluid

experiments

III l;~l~lletl

of

TABLE. II URINARY MUCOPOLYSACCHARIDES

ttifferent.

cells

OXi

of the Mucopolysaccharides--Muir

II.

686

Structure and Metabolism of the Mucopolysaccharides-AJzkr

The presence of sulfated mucopolysaccharides in the normal grey as opposed to white matter of the brain [165] may be a factor in mental deterioration of patients if the compounds are structurally abnormal or if dermatan and heparan sulfates which are found in the patients’ brains [266] are preferentially deposited in the grey matter. Other secondary effects may result from the presence in the tissues of sulfated mucopolysaccharides that cannot be degraded. Nonspecific increase in lysosomal enzymes, particularly protease, could have widespread effects on connective tissue and increase the turnover of its constituents, which might contribute to the appearance of more than normal amounts of chondroitin sulfate in the urine (Table III). The abnormal presence of dermatan sulfate in Hurler cartilage [167] might cause some of the skeletal abnormalities of patients since repeated administration of dextran sulfate to weanling rats produced a disorder of endochondral msification [16S]. The presence of labelled dex!tran sulfate at the sites of deposition was shown by autoradiography. It is possible that ‘the severity of such secondary effects may depend upon the type as well as the amounts of abnormal mucopolysaccharides in the tissues, and might vary with the age and stage of development of the patient. REFERENCES I.

2.

3.

4.

5.

6.

7.

8.

JEANLOZ, R. W. The nomenclature of mucopolysaccharides. Arthritis & Rheumat., 3: 233, 1960. BRIMACOMBE, J. S. and WEBBER, J. M. Mucopolysaccharides: Chemical Structure, Distribution and Isolation. Amsterdam, 1964. Elsevier Press, Inc. MUIR, H. Chemistry and metabolism of connective tissue glycosaminoglycans (mucopolysaccharidoses). In: International Review on Connective Tissue Research, vol. 2, p. 101. Edited by Hall, D. A., New York, 1964. Academic Press, Inc. SCHILLER, S. Connective and supportive tissues: mucopolysaccharides of connective tissue. Ann. Rev. Physiol., 28: 137, 1966. BARRETT, A. J. Cartilage. In: Comprehensive Biochemistry, vol. 26B, p. 425. Edited by Florkin, M. and Stotz, E. H. Amsterdam, 1968. Elsevier Press, Inc. OLSSON, I. and GARDELL, S. The isolation and characterisation of glycosaminoglycans from human leukocytes and platelets. Biochim. et biophys. acta, 141: 348, 1967. KORN, E. D. The synthesis of heparin by slices of nlouse mast cell tumour. J. Biol. Chem., 234: 1321, 1967. LINDAHL, U. Proceedings of the North Atlantic

Treaty Organisation Advance Study Institute. Edited by Balazs, E. A. New York, 1969. Academic Press, Inc. In press. 9. MCKUSICK, V. A. Heritable Disorders of Connective Tissue. St. Louis, 1963. C. V. Mosby Co. 10. GREGORY, J. D., LAURENT, T. C. and RODI?N, L. Enzymatic degradation of chondromucoprotein. J. Biol. Chem., 239: 3312, 1964. 11. ROD&N, L. and ARMAND, G. Structure of chondroitin+sulfate-protein linkage region: isolation and characterisation of the disaccharide. J. Biol. Chem., 241: 65, 1966. 12. LINDAHL, U. and RODBN, L. The chondroitin 4-sulfate protein linkage. J. Biol. Chem., 241: 2113, 1966. 13. ROD~N, L. and SMITH, R. Structure of the neutral disaccharide of the chondroitin-4.sulfate protein linkage region. J. Biol. Chem., 241: 5949, 1966. 14. MUIR, H. The nature of the link between protein and carbohydrate of a chondroitin sulphate complex from hyaline cartilage. Biochem. J., 69: 195, 1958. 15. ANDERSON, B., HOFFMAN, P. and MEYER, K. The O-serine linkage in peptides of chondroitin 4 or B-sulfates. J. Biol. Chem., 240: 156, 1965. 16. SCHMIDT, M., DMOCHOSKI, A. and WIERZBOWSKA, B. Galactose xylose as a structural component of vertebrate chondroitin sulphate protein complex. Biochim. et biophys. acta, 117: 258, 1966. 17. HELTING, T. and ROD~N, L. The carbohydrate protein linkage region of chondroitin g-sulfate. Biochim. et biophys. acta, 170: 301, 1968. 18. SENO, N., MEYER, K., ANDERSON, B. and HOFFMAN, P. Variations in keratosulfates. J. Biol. Chem., 240: 1005, 1965. 19. LAURENT, T. A. and ANSETH, A. Studies on corncal polysaccharides. II. Characterisation, chemistry and metabolism of connectit-e tissue glycosaminoglycans (mucopolysaccharides). &per. Eye Res., 1: 99, 1961. 20. MATHEWS, M. B. and CIFONELLI, J. A. Comparative biochemistry of keratosulfate. J. Biol. Chem., 240: 4140, 1965. 21. BHAVANDAN, V. P. and MEYER, K. Studies on keratosulfates: methylation, desulfation and acid hydrolysis studies in old human cartilage keratosulfate. J. Biol. Chem. 243: 1052, 1968. 22. BHAVANAN, V. P. and MEYER, K. Studies on keratosulfates: methylation and partial acid hydrolysis of bovine cornea1 keratosulfate. J. Biol. Chem.,

243:

4352,

1968.

23.

BALDINI, G., BROVELLI, A. and CASTELLANI, A. A. Gas chromatography identification of neutral sugars in keratan sulphates from different sources. Ital. Biochem. J., 17: 4, 1968. 24. GREILING, H. and STAHLSATZ, H. W. Glykosaminoglykan-peptide aus dem humanen Kniegelellkknorpel. Ztschr. physiol. Chem., 350: 449, 1969. 25. TsIc14Nos, C. P. and MUIR, H. Structural studies on a keratan sulphate-chondroitin sulphate-protein hybrid. (Abstract 490.) Presented at the 4th Mreting of the Federal European Biochemical Sociery, p. 123. 1968. 26. BKAY, B., LIEBERMAN, R. and MEYER, K. Structure of human skeletal keratosulfate. The linkage region. 1. Riol. Chem., 242: 3373, 1967. AMERICAN

JOURNAL

OF

MEDICINE

M. I$. anti (.I.wo\, 5. ,tcid 111u<0p0!\saccharide patterns in ageing human carlila;:<,. I. Clin. Invert., 45: 1103, 196F. 45. sHUI,hlAN, H. J. and MF~ER, I(.Cellular tlilicrc1lti;1tion and the agcir1g process in ca1.1ilageno11? tissues. J. Exijer. Aled., I!%: 1!!53, IOGH. 44.

I.O~AITYTE, I. Sodium chon,l1oitin sllllate-protein tomplexes of cartilage. I. \lolcc ula~, \\t,igilt atltl shape. .4rclc. Bioche~?z., 7-t: 158, 195x.

2% x.1xI mvs, AL. II. and

MA’THE\YS,

46. STOC~WLL.L,R. A. and SCOTT, J. E. l~istrii1ution of acid glycosaminoglycans in I~un,an :,I tic 11I:rr I all i lage. Nafure, 215: 1376, 19A7. 47. MAROUI)AS,A., MUIR, H. and \~‘INClU\I, J. ‘I‘llC CO1relation of fixed negative charge will1 $yCO\aminoglycan content of human articular cartilage. B~orlzim. et biol>hys. acln, I ii: .1!P.1969. 48.

SOLHEM,

I<.

sen1ilunar I9G5. 31. I~OIPM\X,

I’.. MAIHWRN, T. I~R\V. 1% l’lotein-polvsaccharides

A., TGEYER, K. and of bovine cartilage_ Extraction and clectrophoretic stndies. .I. Iliol. Cl/e,,/.. “42: 3799, 1967. 32. RIISNRLRC, I.. SCHURFIIT, M. and SANDSON,J. Proctin~l~01~sactliaricles of bovine nutlcus pulposus. I. Iliol. C:hpw., 21’1: .1691, 1967.

33. ‘I \,(,.,!XC~S. (:, I’. and h[UIR, H. I\ hyhritl protcin~~I\~:Icc~I~I 1~1e of kc>ratan sulpllate and chontlroitin wlpllate flotll pig laryngeal cartilage. /i/ociic*!1i. /.. 101: 26C, 19F7. 34. liI(cj/., \\‘. an11 ~5ikI11Y:hE, E. Chenlischc und makrolnolekuliirr Altei,,cr~rlderungen van Poly-sactllar-itl-ploreinen aus menschlichen Rippenknor1~1. Zbzhr. physiol. Chem., 348: 665, 1967. 35. ROWIUERG, I..,JOHNSON, B. and SCI&BERT, XI. 7‘11~ l~rotcir~-l~~)I~sacc~~ari~les of human costal cartilage. 1. Clin. I?~w.<\t., 48: 5.13, 1969. 36. PI.wwI, V. ;intl PEUKINI-MILLE, A. Keratan sulfate protein colnplexcs fl-om human costal cartilage. In: The Ctiemical Physiology of Mucopolyhacct1arides. p 139. Etlited by Quiniarclli, (;. Boston. 1965. I.ittle, I%1own & Co. 37. hl1 1R, IT. an<1 JACOBS. S. Protein-polysaccharidea of pig laryng:cal cartilage. Bioclfem. I., 103: 367, IY(i7. 38.

E L.. ;rt~tl CIFONELLI, J, A. Linkage of tho11tiroilin sulfate and keratan sulfate to a
KOWNRL~!hl,

39. KAPLAN, I)., McKvslcK, \‘., TRLBACH, S. and I.iU4RI.S. 13. Keratnsulfate-cllolltlroitin sulfate pcptidc fr(lm urine, of patients with Morquio \~ntlr0nlc ~XIucopolysaccharitlo~is. Iv). J. Lab. ti Clin. Med., ;1: 48, 1968. 40. ~SICANOS, C. P. and hfuIR, H. Studies on protcirrpolvsaccharitles from pig laryngeal cartilage. Heterogeneity, fractionation and characterisation. Blocifr:rn. J., 113:885, 1969. 41. kANI).I; K. II. and MUIR, H. Differences in COINposition and size of protein-polysaccharidcs exLracted from pig articular cartilage. I;ERS Letter.r. 4: 16, 1969. 42. b.ANI)T, K. 13.and >ItiIR,H. Characterisation of protein-polysaccharides of articular cartilage from mature and immature pigs. ~ioc/1ev1. J, in press. 43. KWl.AS, I). :1nd I\IFYER, K. Ageing tartilagc. .\rtflr?r. 183: 1267, 1959,

of

hunlat1

‘I‘he gl\to~;11llinogl~c~ll1~ 01 Iru11~11 cartilage. 1. 0.<10 Cilv fff~\p. 15: 127.

B. P. and LowrHEK, D. A. l’hr cllect of chondroitin sulphatc pfotcirrs on ll1r Ir~nn;1ricm of collagen fibrils ill vilro. Hiocllej?!. I., 109: 857. 1968. 50. MATHEWS, M. B. and I~IXKCR, L. ‘1’11~c~ff~~r ol acid mucopolysaccharide proteins on fih1 il lor111ation fro1n collagen solutions. Biochem. 1.. 109: 517, 1968. 51. SERAFISI-FRACASSIM, (;.. I'E'ILR\, .I‘.1. $111<1 1,I.OKEANI. I,.The pr~‘tein-poIysacciI;tricie cotl1plex of bovi11e nasal cartilage: studies on tl11, p1o1ein core. Rioclrr~. J., 105: 569, IYFi. 52. PAL., S., I)OC.ANGES, P. and SCHURERI., .\I. 1‘11c separation of new forms of protein-I)olysaccll;1rides of bovine nasal cartilage. I Iliol. Cilvn,., “41: 4261, 1966. 53. HAXAI.~., ‘i’. C. and SAJDERA, S. \\’ l’totcin-polysaccl1aritlc complex f1om bovine nasal cartilage. .The function of glycoprotein in fhc formation of aggrcgatcs. 1. Rio!. Chcm., 244: 238 I, 196!l.

49.

I‘OOLE,

54. FRAXWN;, I.. A. and ROD~N, L. Srrucrurr 01 tlc1I11i1. lan sulfate. I. Degradation b) testicular- tz\nluro11itlase. I. Viol. Chem., 242: 4161, 1967. 55. FKANSSO~, L. A. and ROD&N, L. Structure of &1111;1tan sulfate. II.Characterisation of protluclh 01). rained by hyaluronidase digestion of dermatan sulfate. 1. Bid. Chem., 242: 4170. 1967.

*

56. I~II.zsso~\~,1.. A. Structure of dcrmaran slllfatc. 111. ‘1%~ hybrid structure of der111atan sl1lfrcte fro111 ultil1ilical cord. /. Biol. Chenz., 243: 150 I. 1968. D

L. .\. Structure of dermatan \rllfate. 1’. ‘I’he hybrid sttucture of dernlatan sulfate fron1 hog intcslinal 1nucosa. Ark. Kemi, 29: !)5. 1968.

57. kR\NSWu,

58. FRANSSOS, L. ;\. St1-ucture and metabolism 01 111~ protcoglycans of dermatan sulfate. Proceedings of the Advanced Study Institute of tile Piorrh Atlantic Treaty 0rganization. Editor Ilalat, E. .I. Academic P1,ess, Inc. In press. 59. SUZUKI, S., SAIIO, H., YA.WMAGATA, I., ANNO, I(.. SENO, N., KAWAI, Y. and FIJKUHASHI, T. Fomnation 0f three types of disulfatctl disaccharides from chondroitin sulfates hy chondroitinase digestion. .I. Riol. Chern., 243: 1543, 1968. GO. CALATRONI, A. and I)1 i'ERKANrE, N. Oversulfated dermatan sulfate extrartcd from E-lurler spleen. /fnnl. Biochem., “5: 370, 1968. 61.

FKNSSOY,

L.

(I:.II(. [“olein

A.

Clycopcptide linkage lcgio;l

from the carbohyof pig skin derma.

688

62. 63.

64.

65.

66.

67.

Structure

and Metabolism

of the Mucopolysaccharides-Muir

tan sulfate. Biochim. et biophys. acta, 156: 311, 1968. STERN, E. I. The linkage of dermatan sulfate to protein. Fed. Proc., 27: 596, 1968. BELLA, A. and DANISHEFSKY,I. The dermatan sulfate-protein linkage region. J. Biol. Chem., 243: 2660, 1968. TOOLE, B. P. and LOWTHER, D. The isolation of dermatan sulphate protein complex from bovine heart valves. Biochim. et biophys. acta, 101: 364, 1965. PRESTON, B. N. Physical characterisation of dermatan sulphate protein. Arch. Biochem., 126: 974, 1968. TOOLE, B. P. and LOWTHER, D. The organisation of hexosamine-containing compounds in bovine skin. Biochim. et biophys. acta, 121: 315, 1966. TOOLE, B. P. and LOWTHER, D. Dermatan sulphate isolation from and interaction with protein: collagen. Arch. Biochem., 128: 567, 1968.

68. ANSETH, A. and FRANSSON,L. A. Unpublished results, 1969. 69. MANLEY, G. Changes in vascular mucopolysaccharides with age and blood pressure. Brit. J. Exper. Path., 46: 125, 1965. 70. LINKER, A. and HOVINCH, P. The enzymic degradation of heparin and heparitin sulfate. I. The fractionation of crude heparinase from Flavobacteria. /. Biol. Chem., 240: 3724, 1965. 71. YOZIZAWA, Z. Enzymic degradation of w-heparin (whale heparin). Biochim. et biophys. acta, 141: 600, 1967. 72. CIFONELLI, A. Reaction of heparitin sulfate with nitrous acid. Carbohydrate Res., 8: 233, 1968. 73. LINDAHL, U., CIFONELLI, J. A., LINDAHL, B. and ROD~N, L. The role of serine in the linkage of heparin to protein. J. Biol. Chem., 240: 2817,1965. 74. LINDAHL, U. and ROD~N, L. The role of galactose and xylose in the linkage of heparin to protein. J. Biol. Chem., 240: 2821, 1966. 75. LINDAHL, U. The structure of xylosylserine and gaIactosyl-xylosyl-serine from heparin. Biochim. et biophys. acta, 130: 361, 1966. 76. LINDAHL, U. Further characterisation of heparinprotein linkage region. Biochim. et biophys. acta, 130: 368, 1966. 77. LINDAHL, U. Structure of the heparin-protein linkage region. Ark. Kemi, 26: 101, 1966. 78. JACOB, S. and MUIR, H. A heparin sulphate peptide from aorta. Biochem. J., 87: 38P, 1963. 79. KNECHT, J., CIFONELLI, J. A. and DORFMAN, A. Structural studies on heparitin sulfate of normal and Hurler tissues. J. Biol. Chem., 242: 4652, 1967. 80. LINDAHL, U. Glucuronic acid and glucosamine-containing oligosaccharides from the heparin-protein linkage region. Biochim. et biophys. acta, 156: 203, 1968. 81. SILBERT, 1. E. Biosynthesis of heparin. IV. N-deacetylatlion of a precursor glycosaminoglycan. I. Biol. Chem., 242: 5153. 1967. 82. JA&JES, L. B., ~AVANACH,.L. and LAVALLEE, A. A comparison of biological activities and chemical analyses for various heparin preparations. k’rzneimittel-Forsch., 17: 774, 1967. 83. KOTOKO, T., YOZIZAWA,Z. and YAMUCHI, F. Com-

84.

85.

86.

87.

88.

89.

90.

91.

92.

93.

94.

95.

96.

97.

98.

99.

100.

parison of heparin specimens isolated from bovine, porcine and whale organs. Arch. Biochem., 120: 553, 1967. CIFONELLI, A. and DORFMAN, A. The uranic acid of heparin. Biochem. 6 Biophys. Res. Commun., 7: 41, 1962. RADNAKRISHNAMURTHY, B. and BERENSON, G. Identification of uranic acids in mucopolysaccharides. Arch. Biochem., 101: 360, 1963. WOLFSON, M. L., HONDA, S. and WANG, P. Y. The isolation of L-iduronic acid from the crystalline barium acid salt of heparin. Carbohydrate Res., 10: 259, 1969. SERAFINI-FRACASSINI,A., DUR~AND, J. J. and FLOREANI, L. Heparin protein complex of ox liver capsule. Isolation and chemical characterisation. Biochem. J., 112: 167, 1969. BOSTROM, H. and RODBN, L. Metabolism of glycosaminoglycans. In: The Amino Sugars, vol. IIB, p. 45. Edited by Jeanloz, R. W. andBalazs, E. A., New York, Academic Press, Inc. 1966. SILBERT, J. The metabolism of polysaccharides containing glucuronic acid. In: Glucuronic Acid. Free and Combined, p. 385. Edited by Dalton. New York, 1966. Academic Press, Inc. DODGSON,K. S. and LLOYD, A. G. Metabolism of acidic glycosaminoglycans (mucopolysaccharides). In: Carbohydrate Metabolism and its Disorders, vol. 1, p. 169. Edited by Dickens, E., Fandle, P. J. and Whelan, IV. J. New York, 1968. Academic Press, Inc. STOOLMILLER,A. and DORFMAN,A. The metabolism of glycosaminoglycans. In: Comprehensive Biochemistry. Edited by Florkin, M. and STOTZ, E. H. Ansterdam. Elsevier Press, Inc. In press. NEUFELD, E. F. and HALL, C. W. The inhibition of UDP-D-glucose dehydrogenase by UDP-D-xylose. A possible regulatory mechanism. Biochem. Q Biophys. Res. Commun., 19: 456, 1965. KORNFELD, S., KORNFELD, R., NEUFELD, E. F. and O’BRIEN, P. J. The feedback control of sugar nucleotide biosynthesis in the liver. Proc. Nat. Acad. SC., 52: 371, 1964. CASTELLANI,A. A., CALATRONI,A. and REGHETTI, P. G. UDP-xylose formation in cartilage. Ital. J. Biochem., 16: 5, 1967. BDOLAH,A. and FEINGOLD,D. S. Decarboxylation of uridine diphosphate-D-glucuronic acid by an enzyme preparation from hen oviduct. Biochem. Q Biophys. Res. Commun., 21: 543, 1965. TELSER, A., RORINSON,H. C. and DORFMAN, A. The biosynthesis of chondroitin sulfate-protein complex. Proc. Nut. Acad. SC., 54: 371, 1965. MATALON,R. and DoRFMAN, A. Hurler’s syndrome. Biosynthesis of acid mucopolysaccharides in tissue culture. Proc. Nat. Acad. SC., 56: 1310, 1966. MATALON, R. and DORFMAN, A. The structure of acid mucopolysaccharides produced by Hurler fibroblasts in tissue culture. Proc. Nat. Acad. SC., 60: 179, 1968. COLE, N. N. and LOWTHER, D. A. The inhibition of chondroitin sulphate protein synthesis by cycloheximide. FEBS Letters, 2: 351, 1969. OLSSEN, I. Chondroitin sulphate proteoglycan of human leukocytes. Biochim. et biophys. acta, 177: 24, 1969. AMERICAN

JOURNAL

OF

MEDICINE

Structul‘e

I(IZ. (,HI UWI-K. E. F: , HALL.

and Metabolisln

of the Mucopolysac~l-laricies

<:. IV.

ant1 I\‘IXJFFL~, E. F. t Il\co\~l:~~io~l oi scrinc residue hy a l~l~P-~~lose ~,roic.it~ s\l~,\~lruansf(,rase from mouse lllastOcy,o,,,;,. Ivrl,. Wiwl~c!ll.. 116: 391, 1966. of chonII):<. III I I,\(,. I .III~ lZo~)i;u, I,. Biosynthesis ~lloitill \ul1;11~. I. (.alactosyl transfer iu the Iortnarion of th<, (;rrhohyclrate-protein linkage Iqion. ./_ m/c>/. Cl~rnz., 244: 2790, 1969. .I‘.;111dKo~kv, L. Biosynthesis of chon101. Ill 1.1.lh(., tlloilin .sull;tt(~. 11. (~lucuronosyl transfer in the !OI n1ation of the carlwhydrate linkage region. J. /Rio/. C/rem., “44: “79’1, 1969. IO,;. 1’1N.M.\N, R. I... erLl.bl.:K, A. Zinc1 DORFMAN, i\. The i~~os~rrrlwsis of chontlroitin sulfate by a cell-free j)tq;ar~~tion. /. niol. (:lrrrra., 239: 3623, 1964. IlKi. %1!
DAVIDSON, E. A. Mucopolysaccha. in chiLk embryo cartilage. I. Prop. colic\ of the sulfation system. 1. Bio2. C:he,rf., 2%“: 1685. 1967. 118. XII-U\N. K:.. and D.~\‘Ds~N, E. A. MucopolysacSl~b~41.

E. and

1ide sulfation

!‘o,.

47.

NOX’EMREK

1969

.\/l/rir

OX’)

119. ROHINSON, H. C. and DOKFM.\N, \ I’llc ~llllxtlon chick of chondroitin sulphate in wrlhtvonic tarrilagc t:piphysc\, J. Biol. (.lfr,r,r.. 2 I 1: 348. 1!)69. 120. M .\~H~.Ns, hl. B. Molecular e\olut ion 01 con~~t~c~iw: tissue. In: Strllcturc ant1 Function 01 Connccli\c~ and .Sk
125.

126.

127.

128.

129.

130.

131.

IV. ~..

133. 154. CONSTANTOPOUHIS, G. Hurler-1 lun1(,r a~ntlrotn~. Ccl Illtmtion and dialysis of u1 inar\ 111ucopo1vsaccharides. Sn turc, 220: 583, 196X. 135. lj’4N Hoop, F. and 111%. II. G. I.‘ult~a\tluc LIIYC tic:. cellules hPpatiqucs dan la nralatlic tl(, Hurler (gargo>lisme). Cornpt. 1.c72
690

Structure

and Metabolism

of the Mucopolysaccharides-Muir

137. VAN HOOF, F. and HERS, H. G. The abnormalities of lysosomal enzymes in mucopolysaccharidoses. European Biochem. J., 7: 34, 1968. 138. V’AN DUIJN, P., WILLIGHAGEN, R. G. J. and MEIJER, A. E. F. H. Increase of acid phosphatase activity in mouse liver after dextran storage. Biochem. Pharmacol., 2: 177, 1959. 139. VAN HOOF, F. and HERS, H. G. Mucopolysaccharidosis by absence of o-fucosidase. Lance& 1: 1198, 1968. 140. ACKERMAN, P. A. Lysosomal acid hydrolases in the liver in gargoylism, deficiency of 4-methylumbelliferyl-p-galactosidase. Scandinav. J. CZin. Q Lab. Invest., 22: 142, 1968. 141. GERICH, J. E. Hunter’s syndrome: ,+galactosidase deficiency in skin. New England J. Med., 280: 799, 1969. 142. ACKERMAN, P. A., HULTRERG, B. and ERIKSON, 0. Enzyme patterns in tissue and body fluids in the mucopolysaccharidoses. Clin. chim. acta, 25: 97, 1969. 143. OCKERMAN, P. A. and HULTBERG, B. Fractionation of 4-methylumbelliferyl-,+galactosidase activities in liver in gargoylism. Scandinav. .I. Clin. Q Lab. Invest., 22: 199, 1968. 144. FRATANTONI, J. C., HALL, C. W. and NEUFELD, E. F. The defect in Hurler’s and Hunter’s syndromes. Faulty degradation of mucopolysaccharides. Proc. Nat. Acad. SC., 60: 699, 1968. 145. FRATANTONI, J. C., HALL, C. W. and NEUFELD, E. F. Hurler and Hunter syndromes. I. Mutual correction of the defect in cultured fibroblasts. Science, 162: 570, 1968. 146. FRATANTONI, J. C., HALL, C. W. and NEUFELD, E. F. The defect in Hurler aand Hunter syndromes. II. Deficiency of specific factors involved in mucopolysaccharide degradation. Proc. Nat. Acad. SC., in press. 147. SPRANCER, J., TELLER, W., KOSENOW, W., MURKEN, J. and HUSEMANN, E. Die HS-MucopolysacchariSanphilipo (Polydystrophe oligodose von phrenie). Ztschr. Kinderheilk., 101: 71, 1967. 148. KAPLAN, D. A heparitin-serine compound from human urine. Biochim. et biophys. acta, 136: 394, 1967. 149. CALATRONI, A., DONELLY, P. V. and DI FERRANTE, N. The glycosaminoglycans of human plasma. J. Clin. Invest., 48: 332, 1969. 150. BOSMAN, H. R. Cellular control of macromolecular synthesis: rates of synthesis of extracellular macromolecules during and after depletion by papain. Proc. Roy. Sot. London, s.B, 169: 399, 1968. 151. FIT~ON-JACKSON, S. Control of macromolecular synthesis by environmental factors. Proc. Roy. Sot. London, s.B, 1969. 152. KRESSE, H. and BUDDECKE, E. Stoffwechsel Heterogenitlt von Chondroitin 4-sulfat Proteinen und Kollagenstoffwechsel in Rindernasenknorpel. Ztschr. Physiol. Chem., 349: 1497, 1968. 153. HAMBRICK, G. 1V. and SCHEIE, H. G. Studies in the

154.

155.

156.

157.

158.

159.

160.

161. 162.

skin in Hurler’s syndrome. Mucopolysaccharidoses. Arclz. Dermat., 85: 455, 1962. MUIR, H., MITTWOCH, U. and BITTER, T. The diagnostic value of isolated mucopolysaccharides and of lymphocyte inclusions in gargoylism. Arch. Dis. Childhood, 38: 358, 1962. BOWMAN, J. E., MIT~WOCH, U. and SCHNEIDERMAN, L. J. Persistence of mucopolysaccharide inclusions in culture of lymphocytes from patients with gargoylism. Nature, 195: 612, 1962. DANES, B. S. and BEARN, A. G. Hurler syndrome and genetic study in cell culture. J. Exper. Med., 123: 1, 1966. SCHAFER, I. A., SULLIVAN, J. C., SVEJCAR, J., KOFAED, J, FAN B. and ROBERTSON, W. Study of the Hurler syndrome using cell culture definition of the biochemical phenotype and the effect of ascorbic acid on the mutant cell. J. Clin. Invest., 47: 321, 1968. DANES, B. S. and BEARN, A. C. Hurler’s syndrome. A genetic study of clones in cell culture with particular reference to Lyon hypothesis. J. Exper. hfed., 126: 509, 1967. DEAN, M. F. and MUIR, H. A chondroitin sulphateprotein in Kurloff cells from guinea pig spleens. FEBS Letters, in press. FRATANTONI, J. C., NEUFELD, E. F., UHLENDORF, W. and JACOBSON, C. B. Intra-uterine diagnosis of Hunter and Hurler syndromes. New England J. Med., 280: 686, 1969. EMANUEL, R. W. Saint Cyr Lecture. London. To be published. ARONSON, N. N. and DAVIDSON, E. A. Lysosomal hyaluronidase from rat liver. II. Properties. J. Biol. Chem., 242: 441, 1967.

163. BERGG~RD, I. and BEARN, A. G. The Hurler syndrome. A biochemical and clinical study. Am. J. Med., 39: 221, 1965. 164. KUMAR, V., BERIXNSON,G. S., R~Iz, H., DALFERES, E. R. and STRONG, J. P. Acid mucopolysaccharides of human aorta. I. Variation and maturation. J. Atherosclerosis Res., 7: 573, 1967. 165. SINCH, M. and BACHAWAT, B. K. The distribution and variation with age of different uranic acid containing mucopolysaccharides in brain. J. Neurochem, 12: 519, 1965. 166. MEYER, K., HOFFMAN, P., LINKER, A., GRUMBACH, M. M. and SAMPSON, P. Sulfated mucopolysaccharides of urine and organs in gargoylism (Hurler’s syndrome). Additional studies. Proc. Sot. Exper. Biol. 6 Med., 102: 587, 1959. 167. MEYER, K. Studies on Hurler’s syndrome. Canad. M. A. J., 84: 851, 1961. 168. TOURTELLOT~E, C. D. and DZIAWIATKOWSKI, D. D. A disorder of endochondral ossification induced by dermatan sulfate. J. Bone d7 Joint Surg., 47A: 1185, 1965. 169. BITTER, T. and MUIR, H. Mucopolysaccharides of whole human spleens in generalized amyloidosis. 1. C[in. Invest., 45: 963, 1966.

AMERICAN

JOURNAL

OF

MEDICINE