Acta neurol. scandinav. 56, 389-396, 1977

1The Neurochcmical Institute, Copenhagen, *Institute for Life Science and Chemistry, Roskilde IJnivcrsity, Roskilde, Wnivcrsity Hospital, Department of Pcdiatrics,

Rigshospitalet, Copenhagen, and 4The State Institute of Mentally Retarded.

LYSOSOMAL (LEUCOCYTE) PROTEINASE AND SULFATASE LEVELS IN DYGGVE-MELCHIOR-CLAUSEN (DhlC) SYKDROME

s. c. RASTOGI,~ J. CLAUSEN,~ J. c. MELCHIOR,3 H. V. DYGGVE‘ and G. E. JENSEN~

ABSTRACT

Patients with the DhIC syndrome have been suggested to possess a specific sulfatase abnormality and/or to be deficient i n a proteinase cleaving glycoprotein-acid mucopolysaccharide (AMP) linkage. We have previously found in DMC patients an abnormal excretion of urinary AMPS of which hyaluronic acid and chondroitin sulfate (A+C) were oversulfated and keratosulfatc and heparan sulfate wcre undersulfated. Lysosomal acid protcinase, i.e. cathepsin D (EC 3.4.23.5) and neutral proteinase: elastase (EC3.4.21.11) and cathepsin G wcre found to be normal in DMC patients. However, a,-macroglobulin in serum was raised. This increase may be associated with a complex formation of a,-macroglobulin with a neutral proteinase released from the cells. Increased levels of chondroitin sulfate N-acetylgalactosamine-6-sulfate sulfatase and sulfamidase and decreased enzymic levels of arylsulfatase A and B (EC3.1.6.1) were found in leucocytes of DMC patients. The sulfatase activities assayed in the present study that a specific sulfatase abnormality may exist in

support our theory the DMC syndrome.

Since Dgggve, Melchior and Clausen (1962) described three siblings from Greenland with a uniform symptomatology consisting of retarded growth, mental retardation, skeletal abnormalities and with abnormal excretion of acid mucopolysaccharides (AMP) in the urine, this syn- drome has been described several times. The clinical appearance of these children has much in common with Morquiro’s or Hurler’s syn- drome. Until now 19 definite cases including the three original patients have been described (Dyggve et al. 1977). All 19 patients were short statured, had the typical radiological abnormalities, and mental re- tardation was always traced.

Our original observation of a slightly increased urinary AMP excre- tion has repeatedly been confirmed by us over the years (Clausen e f al. 1970, Rastogi et al. 1977). The urinary AMPs of these patients were rich in protein, reducing sugar and sialic acid. Furthermore urinary hyal- uronic acid and chondroitin sulfate ( A + C) were oversulfated, but kerato- and heparan sulfate fractions were undersulfated. Our recent studies also suggested the syndrome to be an inborn error of Glyco- protein-AMP metabolism (Rastogi et al. 1977) with the lack of de- gradation of these substances when assayed in uitro using radioactive precursors.

The purpose of the present study was to investigati in DMC patients, the levels of lysosomal enzymes cleaving protein-AMP complexes, e.g. acid proteinase (cathepsin D) and neutral proteinases (elastase and cathepsin G) . Inhibitors of neutral proteinase, e.g. a,-antitrypsin and a,-rnacroglobulin (Janoff 1975, OhZsson 1975) were also determined in the serum of the patients. Furthermorc, since we found changes in degree of sulfation of urinary AMPs of DMC patients, lysosomal sul- fatase activities were also assayed in DMC patients.

MATERIALS AND METHODS MATERIALS

Chemicals Azocascin and shark cartilage condroitin-6-sulfate were purchased from Calbio-

chcm., USA, p-Nitrocatecholsulfate and hyaluronidave (3000 U.S.P. units/mg) were obtained from Sigma. [ N-sulfonate-35Sl heparin (30 mCi/g) and Nuclear Chicago solubilizer (NCS) were purchased from The Radiochemical Center, Amersham. Immunodiffusion plates and standard serum were purchased from Behringwerke AG, Germany. All other chemicals were of highest purity available from E. Merck or BDH, England.

Control subjects ,

chosen as control subjects. Patient material

A bricf account of the three patients studied has reently been reported (Rastogi et 01. 1977) while an up-to-date history of these patients has been described else- where (DUggve et al. 1977).

Laboratory personnel without any history of inborn error of metabolism were

METHODS

Preparation of leucocutes fo r enzyme a s s a ~ s

8 ml venous blood was collected i n 2 ml dextran-heparin-sodium chloride (6: U.05: 0.75 w/w) in 100 ml redistilled water) and was allowed to stand at room temperature for 90 min. After this time, plasma was removed and diluted with 0.87 per cent (w/v) aqueous ammonium chloride (1: 4). The diluted plasma was

391

centrifuged at 600 g for 10 min and the pellet was washed two times with 10 ml of 0.15 M cold sodium chloride. The washed pellet (95 per cent leucocytes) was suspended in 0.6 ml redistilled water and sonicated for 1 min (0’) a t 50 watts using Sonifier B-12 (Branson Conic Power Co., Danbury, Connecticut). The sonicated material was dialysed for 4 h against 0.15 M sodium chloride at 4” for the assay of chondroitin sulfate N-acetylgalactosamine-6-sulfate sulfatase (6-sulfatase), while for the assay of other enzymes the sonicated material was dialysed against redistilled water. Tre dialysed material was termed as “leucocyte preparation”.

Assall of cathepsin I) Using denatured hemoglobin as substrate, cathepsin D was assayed a t pII 3.8

(Anson 1939). In a totalvolume of 1 ml, 100 p1 leucocyte preparation (200-300 pg protein) was incuhated at 37“ with 5 mg substrate in 0.1 M sodium acetate buffer, pH 3.8. 800 p1 10 per cent trichloroacetic acid were added to thc mixture after a 30 rnin incubation period and centrifuged at 3000 Q for 10 min. To 1000 pl supcr- natant, 1600 pl 1 N NaOH and 600 41 1 N Folin Ciocalteau reagent were added and absorbance was measured a t 750 nm after 1 h. A blank was run in each ease and double assays were carried out for cach sample. Tyrosine (0.25-1 nmole) treated in the same way as incuhation mixtures served as standard. Enzyme activity was expressed as colour produced equivalent to nmole of tyrosine/min/mg protein.

Assay of neutral proteinases (elastase and cathepsin G ) Using 3 per cent azocasein as substrate, cathepsin G was assayed at pH 7.7 and

elastase a t pH 8.7 (S tarkey & Barrett , 1976 a, b). To 50 +I leucocyte preparation (150-200 pg protein), 400 p1 redistilled water, 50 p1 substrate and 1.5 ml tris-HC1 (0.1 M ) containing 1 M sodium chloride were added and mixture incubated at 37”. After 60 min, 500 p1 10 per cent trichloroacetie acid was added to the incubation mixture, shook for 30 scc and centrifuged a t 3000 g for 10 min. Optical density (OD) of the supernatant was measured at 366 nm. A blank was run in each case in the same way except that the leucocyte preparation was added a t the end of incubation period. The enzyme activity was expressed as A OD of the supernatant/h/mg protein.

AsaaU of arylsulfatase A & B Arylsulfatase A and B were assayed using p-nitrocatechol sulfate as substrate

(Daum et aZ. 1959). Both the enzymes were assayed in 50 p1 leucocyte preparation (150-200 pg protein). Arylsulfatase A was assayed a t pH 5.5 and arylsulfatase B was assayed at pH 6.0. The enzyme activity was expressed as nmoles of nitro- catechol released/h/mg protein.

Assay of “6-sulfatase” The enzyme was assayed as described by Singh et al. (1976) a t pH 4.8 in 0.1 ml

leucocyte preparation (300-400 pg protein). The substrate, 6-sulfated tetrasaccharide, was prepared as described by these authors by the hyaluronidase degradation of shark cartilage-chondroitin-6-sulfate. The enzyme activity was expressed as nmoles of sulfate released/h/mg protein.

All the enzymes were assayed as changes i n OD in a Zeiss Spectrophotometer (Type P M ) using cuvetts with 1 cm light path.

AssaU of sulfamidase

Lysosornal sulfamidase activity was assayed at pH 4.5 by estimation of 35SO,--

liberated from (N-sulfonate-3~S) heparin. Leucocytes were sonicated (0') for 1 min (1000 eycles/second) in 0.05 M sodium acetate buffer containing 0.15 m sodium chloride, pH 4.5 and centrifuged for 20 min at 13.500 g. The supernatant thus obtained was used for sulfamidase assay. The assay mixture (optimal conditions), total volume 0.65 ml consisted of leucoeyte extract (300 pg protein), 2 mg heparin and labclled substrate (approx. 200,000 cpm) dissolved in the acetate buffer. 10 pl toluene was added as an antimicrobiological agent and the mixture was incubated at 37". After incubation for 18 h, 0.1 ml 0.01 hl non-radioactive sodium sulfate was added and the incubation mixture was allowed to equilibrate a t 37" for 2 h. 8 ml redistilled H,O and 2 ml 2.5 per cent aqueous cetyltrimethylammonium bromide were then added in order to precipitate heparin. The mixture b a s allowed to stand at 4" for 20 h. It was then centrifuged at 10,000 g for 2 h. The supernatant was transferred in new tubes and allowed to stand at 4' for further 24 h. After thiq period, both original and new tubes were centrifuged and the supernatant discarded. Pellets were dried a t 110" for 24 h and dissolved in 1 ml NCS whirling mixer and mixed. The solubilized material was then counted in 15 ml toluenc (containing 75 mg 2,2-p-phenylene-bis (5-phenyl) oxazole and 6 g 2,5-diphenyl oxazole/l). A blank was run in the same way except that the labelled substrate was added after the addition of sodium sulfate. Corrrctions for background and decay were made and the enzyme activity was expressed as epm released/h/mg protein. Initial studies revealed linearity of the assay for total protein in the assay mixture above 160 fig. Due to the radioactive decay of 3% the assay of sulfarnidase was made simultane- ously on pathological and control leucocytes.

Quantitation of serum proteins

According to standard procedure of Behringwerke AG (Germany), a,-antitrypsin and a,-macroglobulin in serum of patients were determined using respective M-partigen plates and a standard serum. Protein concentration was expressed a s mg/100 ml serum.

RESULTS

The reaction kinetics of all the assayed enzymes were found similar to those described in original methods. Enzyme activities of proteinases assayed are presented in Table 1. Lysosomal acid proteinase (cathepsin D) and neutral proteinases (elastase and cathepsin G) in DMC syn- drome were normal except that cathepsin D activity which was de- creased in the female patient (H.J.).

It is evident from Table 2 that all three patients had decreased aryl sulfatase B activity. Aryl sulfatase A activity in male patients was at the lower range of control subjects, but it was significantly decreased in the other patient. In contrast, lysosomal 6-sulfatase activity in male patients was at the upper range of normal subjects while that was more than double in the case of female patient. The sulfamidase activity in

393

one of the patients (case L.J.) was more than double that of the control subjects. In case M.J. a normal activity was traced.

The serum proteinase inhibitors assayed are described in Table 3. a,-antitrgpsin was at normal level in two male patients studied. Serum a,-macroglobulin concentration was raised in both patients.

Table 1. Lgsosomal enzymic activities of proteinase in DMC syndrome and control subjects.

Name Cathepsin D Cathepsin G Elastase units units units Sex - Age

Patients J.J. H.J. L.J.

M 30 0.64 1.32 F 26 0.42 0.50 M 19 0.70 2.0

0.89 0.45 1.03

Control Subjects Mean +. S.D. Range

6M 15-45 5 F 16-41

0.83 1.17 0.07 0.16

0.56-1.12 0.45-2.0

0.97 0.13

0.38-1.58

For units see the text. Normal distribution was documented.

Table 2. Arglsulfatase A and 0, 6-sulfatase and sulfamidase enzyme activities in normal subjects and DMC patients.

Sulfamidase Xrylsulfatase A Arylsulfatase B 6-sulfatase per cent of

units units units control Name Sex Age units

Patients J.J. H.J. L.J.

Control subjects Mean k S.D. Range

M 30 50.4 27.5 15.0 100 740 F 26 28.5 12.8 43.0 - M 19 58.2 26.3 16.0 262 %

6 M 15-45 5 F 16-41

73.6 98.7 11.8 25.9 4.1 7.7 1 .o 5.9

57.4-88.6 57.1-1 19.7 7.5-16.9

For units see the text. Normal distribution was documented.

394

Table 3. Concentration of serum proteins in DMC patients.

Name a,-antitrypsin a,-macroglobulin mg/100 ml mg/100 ml

J.J. L.J.

228.8 428.0 317.5 457.0

Normal subjects Range 200-400 150-350

Indicated by Behringwerke.

DISCUSSION

On the basis of our findings of an unbalanced incorporation of radio- active leucine, galactosamine and fucose, but a normal sulfate incorpo- ration in lymphocytes of DMC patients, a proteinase deficiency giving rise to a decrcased cleavage of glycoprotein-AMP complex has been sug- gested (Rastogi et al. 1977). Acid proteinase, cathepsin D (Held et al. 1968) and neutral proteinases : collagenase, elastase and cathepsin G (Janoff 1975, Malmud h Janoff 1976) have been shown to break proteo- glycans into protcin and AMP by acting on serine residues. Results of the present study have shown a normal level of the acid proteinase and of the two neutral proteinases assayed. Thus a probable proteinase deficiency in DMC syndrome may not involve serine proteinases. I t is also known that a,-macroglobulin inhibits the action of lysosomal collagenase and elastase (Janoff 1975, Ohlsson 1975). Thus, a high concentration of a,-macroglobulin found in two of the patients may be associated with a complex formation of a,-macroglobulin with a lysosomal proteinase released from the cells.

Our prcvions studies on pathochemical abnormalities in DMC syn- drome have revealed normal lysosomal enzymic activities of enzymes known to be involved in lysosomal storage diseases, i.e. a-L-fucosidase, a-D-mannosidase, /3-D-galactosidase, /3-D-galactosaminidase, /I-D-gluc- uronidase and acid phosphatase (Clausen et al. 1970). A normal or the raised activity found in the present communication of 6-sulfatase (H.J.) clearly distinguishes the DMC syndrome from the Morquio syndrome (MataZon et al. 1974, Singh et al. 1976). The raised level of 6-sulfatase found in leucocytes may explain undersulfated keratan sulfate excreted by DMC patients (Rastogi et al. 1977). The raised level of lysosomal sulfamidase activity is in agreement with the undersulfated heparan sulfate found in the patients. The significantly decreased aryl sulfatase

395

( A ) and B activity may chemically relate the DMC syndrome to Maroteoux-Lamy’s syndrome (Sturnpf et al. 1973, Fluharty et al. 1974, Beratis e t al. 1975), but the clinical findings in the latter are different from those of the DhIC syndrome. Arylsulfatase B assayed with nitro- catechol sulfate as a substrate may cover over several sulfatases, thus explaining the clinical differences. Though we have not been able to assay various sulfatases involved in mucopolysaccharidoses, the results of the present study support our view that a multiple sulfatase ab- normality and possibly a specific sulfatase deficiency may, in part, explain the DMC syndrome. The increased or decreased activities of dif- ferent sulfatases assayed with artificial substrate may fit with a normal sulfate incorporation in lymphocytes (Rastogi et al. 1977) and fibro- blasts (Spranger et al. 1975) as well as with undersulfated or over- sulfated AMP found in urine of DMC patients. Furthermore a multiple sulfatase abnormality and oversulfated or undersulfated urinary AMPS found in DMC patients are not in agreement with McKusick’s (1972) view that DMC syndrome does not possess abnormality in AMP metabolism.

REFERENCES

Anson, M. L. (1939) : The estimation of pepsin, trypsin, papain and cathepsin with haemoglohin. J. Gen. Physiol. 22. 7945 .

Baum, II., K. S. Dodgson & B. Spencer (1959) : The assay of arylsulfatase A and I3 in human urine. Clin. Chim. Acta 4, 453-455.

Beratis, N. G., B. M. Turner, H. Wciss & K. Hirschorn (1975): Arylsulfatase B deficiency in Maroteaux-Lamy syndrome : Cellular studies and carrier iden- tification. Pediat. Hes. 9, 475-480.

Clausen, J., H. V. Dyggve & J. C. Melchior (1970) : Chemical and enzymic studies of a family with skeletal abnormalities associated with mental retardation. Clin. Chim. Acta 29, 197-207.

Dyggve, H. V., J. C. Melchior & J. Clausen (1962) : Morquio-Ullrich’s diseasc. Arch. Dis. Childh. 37, 525-534.

Dyggve,H.V., J. C. Mclchior, J. Clausen & S. C. Rastogi (1977) : The Dyggve-Melehior- Clausen (DMC) syndrome. A 15 year follow-up and a survey of the present clinical and chemical findings. Neuropadiatrie 8, I n press.

Fluharty, A. L., R. L. Stevens, D. L. Sanders & H. Kihara (1974) Arylsulfatase B deficiency in Maroteaux-Lamy syndrome cultured fibroblasts. Biochem. Biophys. Res. Commun. 59, 455-461.

Held, V. E., 0. Hoefele, G. Reich, U. Stein, E. Werries & E. Buddecke (1968): Wirlrungssynergismus chondroitin-4-sulfat protein abbauender enzyme des arteriengewebes. Z. Klin. Chem. u. Klin. Biochem. 6, 244-250.

Janoff, A. (1975) : A t least three human neutrophil lysosomal proteinases are capihle of degrading joint connective tissues. Ann. N.Y. Aead. Sci. 256, 402-408.

Matalon, R., B. Arhogast, P. Justice, I. K. Brandt & A. Dorfman (1974) : Morquio

syndrome : deficiency of a chondroitin sulfate K-acetylhexosamine sulfate sulfatase. Biochcm. Biophys. Res. Commun. 61, 709-715.

Mckuscik, V. A. (1972): Heritable disorders of connective tissues, 4th ed., p. 629. Mosby, St. Louis.

Melmud, C. J. & A. Janoff (1976): Human polymorphonuclear lcucocyte elastase and cathepsin G mediate the degradation of lapine articular cartilage proteo- glycan. Proc. Acad. Sci. (Wash.) 256, 254-262.

Ohlsson, K. (1975) : a,-antitrypsin and a,-macroglobulin interactions with human neutrophil collagenase and elastase. Ann. N.Y. Acad. Sci. 256, 409-419.

Rastogi, S. C., J. Clausen, J. C. Melchior & H. V. Dyggvc (1977): The Dyggve- Mclchior-Clausen syndrome. Clin. Chim. Acta. In press.

Singh, J., N. DiPerrante, P. Niebcs & D. Tabella (1976) : N-acetylgalactosamine-6- sulfate sulfatase in man: Absence of enzyme in Morqtrio syndrome. J. Clin. Invest. 57, 10361040.

Spranger, J., P. Maroteaux 8 V. M. Derkaloustian (1975) : The Dyggve-Melchior- Clausen syndrome. Radiology 1 14, 415-421.

Starkey, P. #. & A. J. Barret (1976a): Human cathepsin G: Catalytic and im- munological properties. Biochem. J. 155, 273-278.

Starkey, P. M. & A. J. Barret (1976b): Human lysosomal elastase: Catalytic and immunological properties. Biochem. J. 155, 265-271.

Stumpf, D. A., J. H. Austin, A. C. Crocker & hi. LaFrance (1973) : Mucopolysaccharid- osis Type VI (Marotcaux-Larny syndrome). Am. J. Dis. Child. 126, 747-755.

Received May 21, accepted May 24, 1977

S. Rastogi, Ph.D. Neurochemical Institute RAdmandsgade 58 DK-2200 Copenhagen N Denmark