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Oversulfated dermatan sulfate extracted from Hurler spleen
ANALYTICAL
25, 370-378
BIOCHEMIBTRY
Oversulfated
ALBERT0 Department
(1868)
Dermatan Sulfate from Hurler Spleen
CALATRONI
NICOLA
AND
Extracted
DI
of Biochemistry, Baylor University Houston, Texas VOdG
FERRANTE’
College
of Medicine,
Received January 8, 1968 The presence of heparan sulfate and dermatan sulfate in the urine of a patient affected by “polydystrophic dwarfism” (Maroteaux-Lamy syndrome or mucopolysaccharidosis VI) was recently demonstrated in this laboratory (1). The urinary glycosaminoglycans were fractionated by chromatography on Dowex l-X2 (2). Heparan sulfate was eluted as a single peak by 0.70M NaCl but dermatan sulfate was eluted as a series of peaks by NaCl of increasing molarity (from 1.20 to 2.0 M) . Analyses of the various fractions of dermatan sulfate indicated that they differed in degree of sulfation, the molar ratio sulfur/hexosamine varying progressively from 1.20 to 1.82 (Table 1). TABLE 1 Fractionation on Dowex l-X2 of Urinary Glycosaminoglycans from a Case of Polydystrophic Dwarfism HtXOSrtlIkE. Molarity eluting
0.80
of N&I whtion
0.7 to 1.0 1.2 1.4 1.6 1.6 2.0
Hexuronic borate)
0.95 0 0.84 0 9F 1.0 0.72 0.64
acid
Glucoaamine
Galactossmine
0.83
0.17
Sulfur
1 .o 1.0
1 .02 0 I ‘20 1 X7
1.0
1.44
1.0 1.0
1.75 1.82
0
-
Values are molar or atomic ratio per mole of total hexosamine. A search of the literature indicated that a moderate oversulfation of glycosaminoglycans obtained from urine of patients with various forms of mucopolysaccharidoses had already been noticed in other laboratories, although no particular attention had been given to the observation (3,4). 1Established investigator of the American Heart Association. 370
OVERSULFATEiD
DER’MATAN
371
SULFATE
These findings prompted us to analyze the glycosaminoglycans of Hurler spleen in order to investigate the possible presence of dermatan sulfate fractions with different degrees of sulfation. MATERIALS
AND
METHODS
Part of the spleen of a patient who died with the autosomal recessive form of Hurler’s syndrome (mucopolysaccharidosis I) was obtained through the kindness of Drs. Harvey S. Rosenberg and Don B. Singer of the Department of Pediatrics at Baylor University College of Medicine and the Department of Pathology of Texas Children’s Hospital. A fragment of the spleen (3.8 gm, wet weight) had been frozen; the remnant (16.9 gm, wet weight) had been preserved in 4% formalin. Dermatan sulfate (preparation RO 12232/715), extracted from beef lungs was generously donated by Dr. Kurt von Berlepsch, HoffmannLa Roche & Co., Basel, Switzerland. Two preparations of heparan sulfate (1.25B and 1.5A) were a gift of Dr. J. Anthony Cifonelli, La RabidaUniversity of Chicago Institute, Chicago, Illinois. Heparin was purchased from Calbiochem (B grade, lot 63525,162 U/mg). Uranic acid was determined (1) by the carbazole method of Dische (5), (6) by the borate modification described by Bitter and Muir (6), and (3) by the naphthoresorcinol method of Pelzer and Staib (7). Total amino sugars and the ratio glucosamine/galactosamine were measured, after hydrolysis in 4 N HCl for 8 hr at lOO”C, with the Technicon amino acid analyzer, using a 0.6 X 66 cm cation-exchange column and the ninhydrin reagent.2 Total sulfur was measured after hydrolysis of the material in a sealed tube with 25% formic acid for 24 hr at lOO”C, with the following modification of the method described by Antonopoulos (8). After addition of the benzidine reagent, ethyl alcohol, and amyl alcohol to an aliquot of the hydrolyzate, the tubes are shaken and allowed to stand for 3 hr at -20”. Then 0.2 ml of a suspension of Adsorbosil-G23 (75 mg in 95% ethanol/amyl alcohol/25% formic acid 7/5/3) is added to each tube before centrifugation at 0” for 10 min. After removal of the supernatant with a Pasteur pipet, the tubes are inverted on a filter paper for 10-15 min. The mouths of the tubes are wiped with filter paper wet with ethanol, and 1 ml of ice-cold absolute ethanol/ether 2/l is added to each tube. After resuspension of the residue by vigorous shaking, the walls of the tubes are rinsed with additional 0.5 ml of ethanol/ether solution, and t’he tubes are centrifuged again for 10 min at 0”. The supernatant is dis‘We wish to thank Drs. ’ Adsorbosil-G2, without Pennsylvania.
A. and binder,
V. Pedrini for details of the Appljecj Science Laboratory,
method. Inc., State
College,
~~;tr(Ii~(l, :t11(1the ~esiclue antI tl~c walls of the> tubes :+I’(’ UX&~ twice with 1.*5 1111of ether at room tcmperaturc. After ccntrifugation, the final resitlue is ~usl~n(lecl in 0.5 ml 1.0 i\: HCl. After 30 min at room tempcraturc, tlicl hydrochlo: ic acid is collected by centrifugation, and the residue is extracted twice more, once with 0.5 ml 1 N HCl and once with 0.5 ml tlistillcd water. The three extracts are combined, am1 the optical density is reacl against, an internal blank :\t 248 111,~in ;t 1 cm ~11. A (~:tlibratio~~ lilacs, ranging from 0.5 to 2 ;LK of S, is usc~tl for the ralculwtiolls. Rccovcr? st.:~ndard solut.ion of K,S;o, rnngcd tcsls Ijcrformed on dilutionh of :i IwIn-ret1 96 and 1035%. InfrarC(l analyses were l~for1~1c~1 with t.hc Heckman TR9 spectrophoto~~rrtr~~, using :L inicrol)cllc~t of KBr (2, 3 ing) colrt:Lillillg 50-100 jr,g of s:mplr.
‘l’l!o frozen fragment of spleen n~ltl the one fixed in fornlaliu were l~o~~~ogenizec-1repeatedly in acetone at 4”C, using a Willems Polytrolr ~lltra~ollic tlisintegrator, Iliodel PT 20, 0D type (Brinknlunn InstrumenLs, 111~2..Westbury, N. Y.) for 15 FCC each time. The two air-dried residues (0.730 gm for the frozen sample and 3.45 gm for the formalin-fixed samp1~‘1 were homogenizccl repeatedly in water with the Polytron at 4°C. ‘1’1:~~watw extracts of the same residue were combined, reduccl in volume, :111clclcsalte(l hy ultrafiltration through a IXaflo ~ntmbran~, t,yljcl ITXI 1 (,Jinicon C’orporation. (‘ilI1~l~l~iCl~;c~,Llwss.). ‘rho Ilonfiltrabl(~ mwtrrials wc~re precipitated at 4” with 3 ml of 9.5(;/ tthitllol containing 10% pot assinm acetate, washed wit!) 95Fj anal ahsolutc ethanol, at~d dried with ct)hcr. Since various anal\~ces of the two crude extracts did not rcavcxl any substantial difference, fu!,ther work was performed on the 65.4 mg of crude material extracted from the formalin-fixed sample. A 40 ms wliquot of this crude extract, cont,aining 13..5’$ hexuronic acid (horate), W-IS applied to a 0.9 X 10 cm rolumn of Dowex 1-X2, 200-403 mesh. Clform, which was washed with 250 ml H,O and then elut,ccI with NaCl solutions of gradually increasing niolaritp, thrw 10 ml frwrtions hcinrr collected for each NaCl increment. The fraction!: rorrcsponding to each cluting solution were combined, desalted and concentrated by ultrafiltration, cleared by filtration through a fine sinterrrl-glass rliqc, and lyoI’hilizc?l. 8crrntccn milligrams of dcrmatan slllfatc RO 12232 /715 ancl Xl urr: of lwpwl~in wcv prnrrsscrl with tllcs s;:jmp tw)lrlicllj(>.
I”iKure I itlclirates the patterns of elut.ioll from Uowrx 1-X2 ob18~illrcl \\,it II the spleen extract and the standard rlermwtan sulfwtcb. Eightv-nine
Values
are molar
0.4 - 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 3.0 4.0
Molsrity of NaCl eluting solution
or atomic
ratio
1.73 (2.3) 1.61(15.0) 1.14(20.0) 0.90 (20.8) 0.87 (25.7) 0.82 (26.0) 0.89 (26.5) 0.87 (10.5) 0.94 (8.0) 0.84 (7.5)
l-X2
hexosamine.
0.42 0.44
4.26 4.25
per mole of total
0.44
4.28
Figures
RO
12232/715
indicate
1.41 1.72 1.65
1.17 1.04
2.05 1.27 1.21
2.2
-
Sulfur
per cent of hexuronic
0.89 0.89 0.83
0.11 0.11 0.17
0.40 0.73 0.95
0.60 0.27 0.05
0.98 0.97
0.45
0.55
0.02 0.03
0.38
0.62
Hexosamine Galsctosamine Glucosamine
Sulfate
in parentheses
p
TABLE 3 of Dermatan
Carbazole/naphthoresorcinol
Ratio OD/OD
on Dowex
Borate/ carbazole
Fractionation
acid in each fraction
7.5 3.9 0.3 0.3 0.3
0.3 0.6 3.0
0.2
0.8
Dry weight, mg
E d
1
E
5
lCI
B 5
OVERSULFATED
DERMATAN
SULFATE
375
24002200-
2ooo
-
Dsrmatan
-----
Hurler
ROl2237/715
sulfate
spleen
g 1600e cz 1400.I! s.g 1200$ IOOOI” so0s 600400200
c-x4
v-n-1
r--
:-=-:
.2 .3 .4 .5 .6 .7 .B .9
I
1.2 1.4 1.6 1.6 2 3 4
NaCI,M
FIQ. 1. Chromatography on Dowex l-X2 (200400 mesh), Cl- form, of drrmatan sulfate RO 12232/715 and of an aqueous extract of Hurler spleen.
per cent of the hexuronic acid present in the former and 91% of that present in the latter were recovered in the various fractions, whose analyses and dry weights are presented in Tables 2 and 3. DISCUSSION
The analytical data of the spleen fractions eluted from the Dowex l-X2 column with 0.5 to l.OM NaCl are not sufficient for the identification of the compounds present therein. However, on the basis of the analytical results obtained for similar fractions extracted from Hurler liver, it is possible to assume that the glucosamine-containing compound is heparan sulfate. The fractions eluted with 1.4 to 3.0 M NaCl contain essentially galactosamine and produce color ratios borate/carbazole and carbazole/ naphthoresorcinol similar to those found for dermatan sulfate in various laboratories (6, 9). Their infrared spectra are similar to those obtained with fractions 1.2, 1.4, 1.6, and 1.8 M derived from standard dermatan sulfate and are characterized by absorption bands at 925 and 850 cm-*
(as found ill clioiiclroitiu 4-sulfate) but iiot :~t 8120 cln-’ (as fou!:tl ill chondroitin 6-sulfate). Also evident is a strong absorption band at 1640 to the presence of cm-l and a weaker one at 1550 cm*, attributable N-acetyl residues. The spectra of standard heparan sulfate and various heparin fraction:: differ because of absorption at 820 CII~-~ but not at 928 and 850 cm-l and for absence of absorption at 1550 cm-l. The var:ous heparin fractions also lack the absorption band at 1640 cm’. 011 the basis of these data, the compouncl containing galactosaminc 4-sulfate that is present in these fractions may be identified as clt~rmatan sulfate. The ratio sulEur/l~esosa-lmi~~e of these fractions is above unit,y, :tnd this oversulfation cannot he accounted for with the assumpt~ion tllnl the gluco~alnine-colltairliilg contaminant allay rcprcscnt hcpari~l with .‘, S atoms per tctrasaccharitlc unit. Fransson and Rod&l i 10) have isolated, from l)orcine skin, fractions of dcrlllatnn sulfate wit,11 somcl dcgrc~~ of ov(‘rsulfation at111 have ascribctl this finding, at least in part, to pos;“ihlr tlestruction of hexoeamine during acid hydrolysis. However, our repentc~d observations of orersulfated fractions of dermatan sulfate from differrtll sources and wit’h different degrees of protein contamination make cluestionable such an int’crpretation and seem to indicate that this glpco$aminoglpcan may exist in Hurler tissues and urine at various 1~~~~1s of sulfation. The data of Franrson and Rod&i (10’1 and the clemonstr:lt ion of a S/hcxosaniilic ratio abovcb unity in two minor fritrtiotls isolatclcl front stanclard tlermatan sulfate 12.0 alid 3.0 M NaClJ indicate, Iiowcvc~i,. that oversulfation of this glycosaminoglycan should not 1)~ conA~lr~~ef1 :th sprcific of Hurler’s spndromc or other Il~llropolp~acc~l~:~]~i~los~~s.T~I f:ic*t , Suzuki ha:: clcmonstrnted, in prcl>arations of clrrmat:~t1 sulfittcs fro111 hccf liing~, t#lic preseucc of fractious possessing t,wo sulfate residtlcbs part’ disaccharide unit’, one in position 4 of the ncctylg:llactosaminc at~l t.ll(b other in position 2 or 3 of the hexuronic acid Cl 1) It is of partJiculat, interest to mention that no new bands of infrared absorption wrre noticctl which could be assignedto the latter residues. What remains to be seen, then, is whether oversulfated dermatan sulfate is more abundant ill Hurler tissues and urine than in normal org:lus and tissues. To this extent! it is interesting to mention that, Knecht cf (II. ( 12) haye isolated frolrl Hurler liver fractions of heparan sulfattl with ratios S/llexosamine varying between 1.8 and 3.0. While these authors were in the position to compare these fractions of heparan Fulfate wit11 Pimilar ones obtained from normal human aortas, we have been unal)lc. SO far, to prepare sufficient amounts of dermatan sulfate from normal human organs :ttd, therefore, liar-c, hcrl~ forcac,dto rely for cwmp:brison nn preparations clcrived from other species. I)orfmnn :1t1(1Itis co-workers. having demonst,rn,ted t,h:lt, the gly-
cosaminoglycans which accumulate in Hurler’s ayuclroiuc UC rcltlti\ cl>. free of protein, suggested originally that these protein-clcficieiit, unbc~~~ I glycosaminoglycans diffuse readily to the blood stream and may 1)~ excreted in the urine and deposited in various organs. Moreover, tile clefective protein-glycosaminoglycan complex would fail to exerciac :t 11orma1 fredback inhibition on the synthetic processes, thus leading 1-o :L continuous synthesis of the carbohydrate moiety (13 1. Recent expt+ mcnk performed in various laboratories on cultures of skin fihrol&lsts from Hurler’s patient,s have confirmed the occurrence of exccssivcl SJ.IIt,hesis and storage of glycoeaminoglycans in these cells (I 4-l 6). Ilo~Cver, their synthesis is strictly Ilepentlent, as in embryonic chick chontl~ ocytes (17)) on the availability of a presynthesized protein acceptor ( Ifii. glycosaminoglycans of Hurler’s orgalls .slr011l~l Thus, the protein-scarce he considered as the product either of quantitatively deficientj syn thcl ic mechanisms or of qualitatively inadequate l)rocesses of itrtrarcliul:lr degradation (12). Bc as it may, this either primary or secondary pyotc,ilr deficiency does not explain satisfactorily the ubiquitous accumulatio!l 01 glycosaminoglycans seen in various mucopolysaccharidoses, as one wo111~1 expert t,he protein-poor glycosaminoglycans to be more soluble and mo!‘cs rapidly excreted in the urine than the normal protein-glyrosnt~~i~~o~l~~.~~~ complexes. In fact, experimental conditions which cause degradation of the protein part of normal complexes (like the administration of p:lp.lilt or Inrgc closes of vitamin a) result in an increased blood IWCI :III~I urinary excretion of glycosaminoglycans as a consequence of their nlol)ilizatioll from the tissues (18, 19). Thus, it seems improbable tllat thcb :tccumulation of glycosaminoglycans is a direct consequence of the cleficicncy of their protein moiety. The recent observations of I1Ircz:~n :mtl Davidson (201 seem to provide the missing pathogenetic link. Those> authors have demonstrated that the protein portion of the complex, although not necessary for sulfate incorporation, may play a regulator? role in the process by determining, through steric influences, which site. :~IY+ to be sulfated. While under normal conditions only specific site,c ~V~~tllrlbe ncrrSsit)le to t,he sulfotransferase (i.e., the 4 position of :icetp!lr:Il:lrtosaltliltc iI1 XII wtmct of chick embryo cartilage), in absence of t]lcJ ?t’“otpiu moiety other sites would become exposecl and possibly s:lllf:tt(l(l, <~i(*h :IS the 6 IJositioll of thtb amino sugars :inrl the hy(lro.xyl gro,llj,< of tjlc8 Ilrouie acid. On tltc hasi? of these dat,n, one would like to Fpcxculat(l thal the deficiency of the protein part in Hurler complexes may allow over.wlfation of the carbohydrate moieticy. The rceulting hipher charge density would increase the possibility of electrostatic linkages between anionic sites :lnd tissue proteins, thus contributing to the progressivcb deposition of ~l~cos~minorrlpcnllr; in vnrioii9 tjss11(3,<.
378
CALATRONI
AND
DI
PERRANTE
SUMMARY
Oversulfated dermatan sulfate has been found in an aqueous extract of Hurler spleen. It is possible that the deficiency of the protein moiety of the glycosaminoglycan complexes in this disease may allow oversulfation of the carbohydrate polymer, thus contributing to its deposition in various organs. This investigation and by grants-in-aid Association.
ACKNOWLEDGMENTS was supported by United States Public Health Grant AM-16311 from the American Heart Association and the Texas Heart REFERENCES
1. FILLS, N., BARNES, F. L., AND DI FERRANTE, N., J. Clin. Endocrin. Metab. 28, 26 (1968). 2. SCHILLER, S., SLOVER, G. A., AND DORFMAN, A., J. Biol. Chem. 236, 983 (1961). 3. MEYER, K., CRUMBACH, M. M., LINKER, A., AND HOFFMAN, P., Proc. Sot. Exptl. Biol. Med. 97, 275 (1953). 4. BERGGARD, T., AND BRZRN, A. G., Am. J. Med. 39, 221 (1965). 5. DISCHE, L., J. Biol. Chem. 167, 189 (1947). 6. BITTER, T., AND MUIR, H. M., Anal. Biochem. 4, 330 (1962). 7. PELZER, H., AND STAIB, W., Clin. Chim. Acta 2, 407 (1957). 8. ANTONOPOULOS, C. A., Acta Chem. Scnnd. 16, 1521 (1962). 9. TELLER, W. M., ROSEVEAR, J. W., AND BURKE, E. C., Proc. Sot. Exptl. Biol. Med. 108,276(1961). 10. FRANSSON, L. A., AND ROD~N, L.. J. Biol. Chem. 242,417O (1967). 11. SUZUKI, S., J. Bial. Chem. 235,358O (1960). 12. KNECHT, J., CIFONELLI, J. A., AND DORFMAN, A., J. Biol. Chem. 242, 4652 (1967). 13. DORFMAN, A., Biophys. J. 4, 155 (1964). 14. DANES, B. S., AND BEARN, A. G., J. Exptl. Med. 123, 1 (1966). 15.DANES, B. S., AND BEARN, A. G., J. Ezptl. Med. 124, 1181 (1966). 16. MATAL~N, R., AND DORFMAN, A., Proc. Natl. Acad. Sci. U. S. 56, 1310 (1966). 17.TELSER, A., ROBINSON, H. C., AND DORFMAN, A., Proc. Natl. Acad. Sci. U. S. 54, 912 (1965). 18. THOMAS, L., J. Exptl. Med. 104,245(1956). 19. THOMAS, L., MCCLUSKEY, R. T., POTTER, J. L., AND WEISSMANN, G., J. Exptl. Med. 111, 705 (1960). 20. MEEZAN, E., AND DAVIDSON, E. A., J. Biol. Chem. 202, 6956 (1967).
25, 370-378
BIOCHEMIBTRY
Oversulfated
ALBERT0 Department
(1868)
Dermatan Sulfate from Hurler Spleen
CALATRONI
NICOLA
AND
Extracted
DI
of Biochemistry, Baylor University Houston, Texas VOdG
FERRANTE’
College
of Medicine,
Received January 8, 1968 The presence of heparan sulfate and dermatan sulfate in the urine of a patient affected by “polydystrophic dwarfism” (Maroteaux-Lamy syndrome or mucopolysaccharidosis VI) was recently demonstrated in this laboratory (1). The urinary glycosaminoglycans were fractionated by chromatography on Dowex l-X2 (2). Heparan sulfate was eluted as a single peak by 0.70M NaCl but dermatan sulfate was eluted as a series of peaks by NaCl of increasing molarity (from 1.20 to 2.0 M) . Analyses of the various fractions of dermatan sulfate indicated that they differed in degree of sulfation, the molar ratio sulfur/hexosamine varying progressively from 1.20 to 1.82 (Table 1). TABLE 1 Fractionation on Dowex l-X2 of Urinary Glycosaminoglycans from a Case of Polydystrophic Dwarfism HtXOSrtlIkE. Molarity eluting
0.80
of N&I whtion
0.7 to 1.0 1.2 1.4 1.6 1.6 2.0
Hexuronic borate)
0.95 0 0.84 0 9F 1.0 0.72 0.64
acid
Glucoaamine
Galactossmine
0.83
0.17
Sulfur
1 .o 1.0
1 .02 0 I ‘20 1 X7
1.0
1.44
1.0 1.0
1.75 1.82
0
-
Values are molar or atomic ratio per mole of total hexosamine. A search of the literature indicated that a moderate oversulfation of glycosaminoglycans obtained from urine of patients with various forms of mucopolysaccharidoses had already been noticed in other laboratories, although no particular attention had been given to the observation (3,4). 1Established investigator of the American Heart Association. 370
OVERSULFATEiD
DER’MATAN
371
SULFATE
These findings prompted us to analyze the glycosaminoglycans of Hurler spleen in order to investigate the possible presence of dermatan sulfate fractions with different degrees of sulfation. MATERIALS
AND
METHODS
Part of the spleen of a patient who died with the autosomal recessive form of Hurler’s syndrome (mucopolysaccharidosis I) was obtained through the kindness of Drs. Harvey S. Rosenberg and Don B. Singer of the Department of Pediatrics at Baylor University College of Medicine and the Department of Pathology of Texas Children’s Hospital. A fragment of the spleen (3.8 gm, wet weight) had been frozen; the remnant (16.9 gm, wet weight) had been preserved in 4% formalin. Dermatan sulfate (preparation RO 12232/715), extracted from beef lungs was generously donated by Dr. Kurt von Berlepsch, HoffmannLa Roche & Co., Basel, Switzerland. Two preparations of heparan sulfate (1.25B and 1.5A) were a gift of Dr. J. Anthony Cifonelli, La RabidaUniversity of Chicago Institute, Chicago, Illinois. Heparin was purchased from Calbiochem (B grade, lot 63525,162 U/mg). Uranic acid was determined (1) by the carbazole method of Dische (5), (6) by the borate modification described by Bitter and Muir (6), and (3) by the naphthoresorcinol method of Pelzer and Staib (7). Total amino sugars and the ratio glucosamine/galactosamine were measured, after hydrolysis in 4 N HCl for 8 hr at lOO”C, with the Technicon amino acid analyzer, using a 0.6 X 66 cm cation-exchange column and the ninhydrin reagent.2 Total sulfur was measured after hydrolysis of the material in a sealed tube with 25% formic acid for 24 hr at lOO”C, with the following modification of the method described by Antonopoulos (8). After addition of the benzidine reagent, ethyl alcohol, and amyl alcohol to an aliquot of the hydrolyzate, the tubes are shaken and allowed to stand for 3 hr at -20”. Then 0.2 ml of a suspension of Adsorbosil-G23 (75 mg in 95% ethanol/amyl alcohol/25% formic acid 7/5/3) is added to each tube before centrifugation at 0” for 10 min. After removal of the supernatant with a Pasteur pipet, the tubes are inverted on a filter paper for 10-15 min. The mouths of the tubes are wiped with filter paper wet with ethanol, and 1 ml of ice-cold absolute ethanol/ether 2/l is added to each tube. After resuspension of the residue by vigorous shaking, the walls of the tubes are rinsed with additional 0.5 ml of ethanol/ether solution, and t’he tubes are centrifuged again for 10 min at 0”. The supernatant is dis‘We wish to thank Drs. ’ Adsorbosil-G2, without Pennsylvania.
A. and binder,
V. Pedrini for details of the Appljecj Science Laboratory,
method. Inc., State
College,
~~;tr(Ii~(l, :t11(1the ~esiclue antI tl~c walls of the> tubes :+I’(’ UX&~ twice with 1.*5 1111of ether at room tcmperaturc. After ccntrifugation, the final resitlue is ~usl~n(lecl in 0.5 ml 1.0 i\: HCl. After 30 min at room tempcraturc, tlicl hydrochlo: ic acid is collected by centrifugation, and the residue is extracted twice more, once with 0.5 ml 1 N HCl and once with 0.5 ml tlistillcd water. The three extracts are combined, am1 the optical density is reacl against, an internal blank :\t 248 111,~in ;t 1 cm ~11. A (~:tlibratio~~ lilacs, ranging from 0.5 to 2 ;LK of S, is usc~tl for the ralculwtiolls. Rccovcr? st.:~ndard solut.ion of K,S;o, rnngcd tcsls Ijcrformed on dilutionh of :i IwIn-ret1 96 and 1035%. InfrarC(l analyses were l~for1~1c~1 with t.hc Heckman TR9 spectrophoto~~rrtr~~, using :L inicrol)cllc~t of KBr (2, 3 ing) colrt:Lillillg 50-100 jr,g of s:mplr.
‘l’l!o frozen fragment of spleen n~ltl the one fixed in fornlaliu were l~o~~~ogenizec-1repeatedly in acetone at 4”C, using a Willems Polytrolr ~lltra~ollic tlisintegrator, Iliodel PT 20, 0D type (Brinknlunn InstrumenLs, 111~2..Westbury, N. Y.) for 15 FCC each time. The two air-dried residues (0.730 gm for the frozen sample and 3.45 gm for the formalin-fixed samp1~‘1 were homogenizccl repeatedly in water with the Polytron at 4°C. ‘1’1:~~watw extracts of the same residue were combined, reduccl in volume, :111clclcsalte(l hy ultrafiltration through a IXaflo ~ntmbran~, t,yljcl ITXI 1 (,Jinicon C’orporation. (‘ilI1~l~l~iCl~;c~,Llwss.). ‘rho Ilonfiltrabl(~ mwtrrials wc~re precipitated at 4” with 3 ml of 9.5(;/ tthitllol containing 10% pot assinm acetate, washed wit!) 95Fj anal ahsolutc ethanol, at~d dried with ct)hcr. Since various anal\~ces of the two crude extracts did not rcavcxl any substantial difference, fu!,ther work was performed on the 65.4 mg of crude material extracted from the formalin-fixed sample. A 40 ms wliquot of this crude extract, cont,aining 13..5’$ hexuronic acid (horate), W-IS applied to a 0.9 X 10 cm rolumn of Dowex 1-X2, 200-403 mesh. Clform, which was washed with 250 ml H,O and then elut,ccI with NaCl solutions of gradually increasing niolaritp, thrw 10 ml frwrtions hcinrr collected for each NaCl increment. The fraction!: rorrcsponding to each cluting solution were combined, desalted and concentrated by ultrafiltration, cleared by filtration through a fine sinterrrl-glass rliqc, and lyoI’hilizc?l. 8crrntccn milligrams of dcrmatan slllfatc RO 12232 /715 ancl Xl urr: of lwpwl~in wcv prnrrsscrl with tllcs s;:jmp tw)lrlicllj(>.
I”iKure I itlclirates the patterns of elut.ioll from Uowrx 1-X2 ob18~illrcl \\,it II the spleen extract and the standard rlermwtan sulfwtcb. Eightv-nine
Values
are molar
0.4 - 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 3.0 4.0
Molsrity of NaCl eluting solution
or atomic
ratio
1.73 (2.3) 1.61(15.0) 1.14(20.0) 0.90 (20.8) 0.87 (25.7) 0.82 (26.0) 0.89 (26.5) 0.87 (10.5) 0.94 (8.0) 0.84 (7.5)
l-X2
hexosamine.
0.42 0.44
4.26 4.25
per mole of total
0.44
4.28
Figures
RO
12232/715
indicate
1.41 1.72 1.65
1.17 1.04
2.05 1.27 1.21
2.2
-
Sulfur
per cent of hexuronic
0.89 0.89 0.83
0.11 0.11 0.17
0.40 0.73 0.95
0.60 0.27 0.05
0.98 0.97
0.45
0.55
0.02 0.03
0.38
0.62
Hexosamine Galsctosamine Glucosamine
Sulfate
in parentheses
p
TABLE 3 of Dermatan
Carbazole/naphthoresorcinol
Ratio OD/OD
on Dowex
Borate/ carbazole
Fractionation
acid in each fraction
7.5 3.9 0.3 0.3 0.3
0.3 0.6 3.0
0.2
0.8
Dry weight, mg
E d
1
E
5
lCI
B 5
OVERSULFATED
DERMATAN
SULFATE
375
24002200-
2ooo
-
Dsrmatan
-----
Hurler
ROl2237/715
sulfate
spleen
g 1600e cz 1400.I! s.g 1200$ IOOOI” so0s 600400200
c-x4
v-n-1
r--
:-=-:
.2 .3 .4 .5 .6 .7 .B .9
I
1.2 1.4 1.6 1.6 2 3 4
NaCI,M
FIQ. 1. Chromatography on Dowex l-X2 (200400 mesh), Cl- form, of drrmatan sulfate RO 12232/715 and of an aqueous extract of Hurler spleen.
per cent of the hexuronic acid present in the former and 91% of that present in the latter were recovered in the various fractions, whose analyses and dry weights are presented in Tables 2 and 3. DISCUSSION
The analytical data of the spleen fractions eluted from the Dowex l-X2 column with 0.5 to l.OM NaCl are not sufficient for the identification of the compounds present therein. However, on the basis of the analytical results obtained for similar fractions extracted from Hurler liver, it is possible to assume that the glucosamine-containing compound is heparan sulfate. The fractions eluted with 1.4 to 3.0 M NaCl contain essentially galactosamine and produce color ratios borate/carbazole and carbazole/ naphthoresorcinol similar to those found for dermatan sulfate in various laboratories (6, 9). Their infrared spectra are similar to those obtained with fractions 1.2, 1.4, 1.6, and 1.8 M derived from standard dermatan sulfate and are characterized by absorption bands at 925 and 850 cm-*
(as found ill clioiiclroitiu 4-sulfate) but iiot :~t 8120 cln-’ (as fou!:tl ill chondroitin 6-sulfate). Also evident is a strong absorption band at 1640 to the presence of cm-l and a weaker one at 1550 cm*, attributable N-acetyl residues. The spectra of standard heparan sulfate and various heparin fraction:: differ because of absorption at 820 CII~-~ but not at 928 and 850 cm-l and for absence of absorption at 1550 cm-l. The var:ous heparin fractions also lack the absorption band at 1640 cm’. 011 the basis of these data, the compouncl containing galactosaminc 4-sulfate that is present in these fractions may be identified as clt~rmatan sulfate. The ratio sulEur/l~esosa-lmi~~e of these fractions is above unit,y, :tnd this oversulfation cannot he accounted for with the assumpt~ion tllnl the gluco~alnine-colltairliilg contaminant allay rcprcscnt hcpari~l with .‘, S atoms per tctrasaccharitlc unit. Fransson and Rod&l i 10) have isolated, from l)orcine skin, fractions of dcrlllatnn sulfate wit,11 somcl dcgrc~~ of ov(‘rsulfation at111 have ascribctl this finding, at least in part, to pos;“ihlr tlestruction of hexoeamine during acid hydrolysis. However, our repentc~d observations of orersulfated fractions of dermatan sulfate from differrtll sources and wit’h different degrees of protein contamination make cluestionable such an int’crpretation and seem to indicate that this glpco$aminoglpcan may exist in Hurler tissues and urine at various 1~~~~1s of sulfation. The data of Franrson and Rod&i (10’1 and the clemonstr:lt ion of a S/hcxosaniilic ratio abovcb unity in two minor fritrtiotls isolatclcl front stanclard tlermatan sulfate 12.0 alid 3.0 M NaClJ indicate, Iiowcvc~i,. that oversulfation of this glycosaminoglycan should not 1)~ conA~lr~~ef1 :th sprcific of Hurler’s spndromc or other Il~llropolp~acc~l~:~]~i~los~~s.T~I f:ic*t , Suzuki ha:: clcmonstrnted, in prcl>arations of clrrmat:~t1 sulfittcs fro111 hccf liing~, t#lic preseucc of fractious possessing t,wo sulfate residtlcbs part’ disaccharide unit’, one in position 4 of the ncctylg:llactosaminc at~l t.ll(b other in position 2 or 3 of the hexuronic acid Cl 1) It is of partJiculat, interest to mention that no new bands of infrared absorption wrre noticctl which could be assignedto the latter residues. What remains to be seen, then, is whether oversulfated dermatan sulfate is more abundant ill Hurler tissues and urine than in normal org:lus and tissues. To this extent! it is interesting to mention that, Knecht cf (II. ( 12) haye isolated frolrl Hurler liver fractions of heparan sulfattl with ratios S/llexosamine varying between 1.8 and 3.0. While these authors were in the position to compare these fractions of heparan Fulfate wit11 Pimilar ones obtained from normal human aortas, we have been unal)lc. SO far, to prepare sufficient amounts of dermatan sulfate from normal human organs :ttd, therefore, liar-c, hcrl~ forcac,dto rely for cwmp:brison nn preparations clcrived from other species. I)orfmnn :1t1(1Itis co-workers. having demonst,rn,ted t,h:lt, the gly-
cosaminoglycans which accumulate in Hurler’s ayuclroiuc UC rcltlti\ cl>. free of protein, suggested originally that these protein-clcficieiit, unbc~~~ I glycosaminoglycans diffuse readily to the blood stream and may 1)~ excreted in the urine and deposited in various organs. Moreover, tile clefective protein-glycosaminoglycan complex would fail to exerciac :t 11orma1 fredback inhibition on the synthetic processes, thus leading 1-o :L continuous synthesis of the carbohydrate moiety (13 1. Recent expt+ mcnk performed in various laboratories on cultures of skin fihrol&lsts from Hurler’s patient,s have confirmed the occurrence of exccssivcl SJ.IIt,hesis and storage of glycoeaminoglycans in these cells (I 4-l 6). Ilo~Cver, their synthesis is strictly Ilepentlent, as in embryonic chick chontl~ ocytes (17)) on the availability of a presynthesized protein acceptor ( Ifii. glycosaminoglycans of Hurler’s orgalls .slr011l~l Thus, the protein-scarce he considered as the product either of quantitatively deficientj syn thcl ic mechanisms or of qualitatively inadequate l)rocesses of itrtrarcliul:lr degradation (12). Bc as it may, this either primary or secondary pyotc,ilr deficiency does not explain satisfactorily the ubiquitous accumulatio!l 01 glycosaminoglycans seen in various mucopolysaccharidoses, as one wo111~1 expert t,he protein-poor glycosaminoglycans to be more soluble and mo!‘cs rapidly excreted in the urine than the normal protein-glyrosnt~~i~~o~l~~.~~~ complexes. In fact, experimental conditions which cause degradation of the protein part of normal complexes (like the administration of p:lp.lilt or Inrgc closes of vitamin a) result in an increased blood IWCI :III~I urinary excretion of glycosaminoglycans as a consequence of their nlol)ilizatioll from the tissues (18, 19). Thus, it seems improbable tllat thcb :tccumulation of glycosaminoglycans is a direct consequence of the cleficicncy of their protein moiety. The recent observations of I1Ircz:~n :mtl Davidson (201 seem to provide the missing pathogenetic link. Those> authors have demonstrated that the protein portion of the complex, although not necessary for sulfate incorporation, may play a regulator? role in the process by determining, through steric influences, which site. :~IY+ to be sulfated. While under normal conditions only specific site,c ~V~~tllrlbe ncrrSsit)le to t,he sulfotransferase (i.e., the 4 position of :icetp!lr:Il:lrtosaltliltc iI1 XII wtmct of chick embryo cartilage), in absence of t]lcJ ?t’“otpiu moiety other sites would become exposecl and possibly s:lllf:tt(l(l, <~i(*h :IS the 6 IJositioll of thtb amino sugars :inrl the hy(lro.xyl gro,llj,< of tjlc8 Ilrouie acid. On tltc hasi? of these dat,n, one would like to Fpcxculat(l thal the deficiency of the protein part in Hurler complexes may allow over.wlfation of the carbohydrate moieticy. The rceulting hipher charge density would increase the possibility of electrostatic linkages between anionic sites :lnd tissue proteins, thus contributing to the progressivcb deposition of ~l~cos~minorrlpcnllr; in vnrioii9 tjss11(3,<.
378
CALATRONI
AND
DI
PERRANTE
SUMMARY
Oversulfated dermatan sulfate has been found in an aqueous extract of Hurler spleen. It is possible that the deficiency of the protein moiety of the glycosaminoglycan complexes in this disease may allow oversulfation of the carbohydrate polymer, thus contributing to its deposition in various organs. This investigation and by grants-in-aid Association.
ACKNOWLEDGMENTS was supported by United States Public Health Grant AM-16311 from the American Heart Association and the Texas Heart REFERENCES
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