Copyright 0 1985 by the Genetics Society of Aiiierira

ANIMAL MODEL STUDIES OF ALLELISM: CHARACTERIZATION O F ARYLSULFATASE B

MUTATIONS IN HOMOALLELIC AND HETEROALLELIC (GENETIC COMPOUND) HOMOZYGOTES WITH FELINE

MUCOPOLYSACCHARIDOSIS VI

MARGARET M. McGOVERN,* NADINE MANDELL,* MARK HASKINSt AND ROBERT J. DESNICK*

*The Division of Medical Genetics, Department of Pediatrics, Mount Sinai School of Medicine, New York, New York 10029; and 'The Section of Veterinary Pathology, University .f Pennsylvania

Veterinary School, Philadelphia, Pennsylvania 19104

Manuscript received October 15, 1984 Revised copy accepted April 8, 1985

ABSTRACT

The identification of a second structural gene mutation at the feline aryl- sulfatase B locus (MPS VIb) provided the opportunity to investigate the expres- sion of allelism by characterization of the residual enzymatic activity in feline mucopolysaccharidosis VI, an animal analogue of human Maroteaux-Lamy syn- drome. Matings were designed to produce affected homozygotes who were homoallelic for the MPS VI" and MPS V I b mutations or heteroallelic genetic compounds (MPS VI"/VIb). T h e physicokinetic and immunological properties of the partially purified residual hepatic arylsulfatase B isozymes from the affected homoallelic and heteroallelic cats were compared to those of the nor- mal feline enzyme. The residual hepatic arylsulfatase B activities from the inbred MPS VI" and MPS VIb homozygotes were distinguished by differences in physicokinetic and immunological properties. The newly identified mutant isozyme b had abnormal kinetic properties toward artificial and natural sub- strates, normal cryo- and thermostabilities, a normal molecular weight and an altered electrophoretic mobility. Polyacrylamide gel electrophoresis demon- strated that the mutant b subunits formed dimers with normal subunits in obligate heterozygotes (MPS VI'lb). In contrast, mutant isozyme a subunits from obligate MPS VI0/+ heterozygotes did not dimerize with the normal subunit, and the mutant and normal isozymes could be separated by anion exchange chromatography and polyacrylamide gel electrophoresis. Characterization of the partially purified residual hepatic arylsulfatase B activity from the heter- oallelic homozygotes revealed the presence of both mutant isozymes a and b. T h e demonstration of two allelic mutations in the feline arylsulfatase B gene documented the occurrence of genetic heterogeneity in feline mUCOpOlySdC- charidosis VI and permitted characterization of the enzymatic defect in ho- moallelic and heteroallelic (genetic compound) homozygotes, providing a model for the study of allelism in human genetic disorders.

D U R I N G the past decade, investigators have identified and characterized naturally occurring animal models of various human inborn errors of

metabolism (DESNICK, PATTERSON and SCARPELLI 1982; PATTERSON, HASKINS

Genetics 110: 733-749 August, 1985

734 M. M. MCGOVERN ETAL.

and JEZYK 1982), including animal analogues of more than ten human lyso- somal storage diseases (DESNICK et al . 1982). These animal analogues provide the unique opportunity to investigate and compare their molecular, enzymatic, pathophysiological and clinical defects with those of their human disease coun- terparts. In addition, the occurrence of different mutant alleles among the affected animal population provides a model system to study the expression of allelism. Such studies provide information concerning the pathophysiology of the metabolic disease, permitting the rational design and evaluation of various therapeutic strategies that could not be assessed adequately in clinical trials due to the limitations of human experimentation (DESNICK et al. 1982).

Previously, we described an animal analogue of the human lysosomal storage disease, mucopolysaccharidosis VI (MPS VI, Maroteaux-Lamy disease) in Sia- mese cats (JEZYK et al . 1977; HASKINS, JEZYK and PATTERSON 1979; HASKINS et al. 1980). The feline analogue and the human disease both are autosomal recessive disorders resulting from the deficient activity of arylsulfatase B (ASB) and the lysosomal accumulation of dermatan sulfate (JEZYK et al. 1977; MAR- OTEAUX and LAMY 1965; STUMPF et al. 1973; FLUHARTY et al. 1974; Mc- KUSICK, NEUFELD and KELLY 1978; BERATIS et al. 1975). More recently, we reported the physicokinetic properties of the residual mutant enzyme (isozyme a) from an inbred line of Siamese cats with MPS VI, designated MPS VI” (VINE et al . 1981). In contrast to the normal feline enzyme, the residual hepatic ASB activity from the affected cats had a markedly increased K , and was less stable to thermo-, cryo- and pH inactivation. In addition, the mutant isozyme a had approximately half the molecular weight of the normal feline enzyme, which was a homodimer (MCGOVERN et al. 1982). These findings suggested that the genetic mutation in feline MPS VI“ altered a subunit association site as well as the kinetic and stability properties of the mutant protein. Further studies re- vealed that the residual MPS VI” activity could be enhanced by treatment with thiol-reducing agents which caused the mutant subunits to dimerize (VINE et al. 1982). In nitro and in vivo experiments demonstrated that sulfhydryl re- duction of mutant isozyme a significantly increased its activity in mixed leu- kocytes and resulted in a concomitant decrease in dermatan sulfate levels (VINE e t al. 1982).

In this communication, we report the occurrence of a second structural gene mutation at the feline ASB locus which was discovered in a second inbred line of MPS VI cats, designated MPS VIb, and describe the physicokinetic properties of the residual ASB activity, mutant isozyme b. In addition, the properties of the highly purified hepatic ASB activities from homozygous and heterozygous members of the MPS VI” and MPS VIh kindreds were determined and com- pared. Furthermore, we describe the properties of the residual mutant ASB isoirymes from affected heteroallelic “compound” homozygotes, designated MPS VZ”’lb, who were the products of matings between heterozygotes (MPS VI”/ +; MPS VZCIb) from each of the MPS VI kindreds (Figure 1).

MATERIALS AND METHODS

Anzmal lznes: T h e two MI’S VI animal lines were established from geographically separate faiiiilies of Siamese m t s with no known history of conimon ancestors. The MPS VZ“ line was derived

ALLELISM IN FELINE MPS VI 735

MPS ma MPS molb MPS PTb

I t

Hwnozygous affected

Heterozygote +lo

Heterozygote t I b

0 S e x unknown

FIGURE 1.-Pedigree of the MPS VI" and MPS Vlb kindreds. Note that affected cats 220, 863, 865, and 1200 are heteroallelic genetic compounds.

by outcrossing the sire, 190 (see Figure l), of the propositus, 191, to a normal female, 735, and performing backcross father to daughter matings or matings among their offspring. The MPS VIb line was established by mating an affected male, 170, to a normal female, 569, and performing matings between half brothers and sisters. Continued intra-line matings expanded each colony. Matings between the two separate lines produced the affected MPS VI"" heteroallelic homozygous individuals, 220, 863, 865 and 1200. Affected homozygous and heterozygous carriers for MPS VI were identified by determination of their ASB activities in isolated leukocytes (MCGOVERN et al. 1981).

Specimen coZZection and preparation: Livers from normal Siamese cats and from affected homoal- lelic homozygous (191, 670, 745; 170, 577, 1071) and heterozygous (215, 744, 1131; 161, 218, 293) cats from the MPS VI" and MI'S VIb kindreds, respectively, and from affected heteroallelic (MPS homozygous cats (220, 863, 865) were removed and immediately frozen after the animals were sacrificed. The livers were stored at -55" until used. Mixed leukocytes were isolated from heparinized whole blood by dextran sedimentation as previously described (PERCY and BRADY 1968).

Materials: Dithiothreitol (DTT) was purchased from Calbiochem-Behring Corporation, San Diego, California. Diaminobenzidinetetrahydrochloride,4-methylumbelliferylsulfate(4-MUS),p-nitro- catechol sulfate (pNCS) and chondroitin sulfate B (dermatan sulfate) were from Sigma Chemical Company, St. Louis, Missouri. The Bio-Rad protein assay and polyacrylamide gel electrophoresis materials were from Bio-Rad Laboratories, Richmond, California. Diethylaminoethyl (DEAE)-cel- lulose was obtained from Whatman, Inc., Clifton, New Jersey. Concanavalin A-Sepharose (Con A- Sepharose) and molecular weight standards were purchased from Pharmacia Fine Chemcials, Pis- cataway, New Jersey. Amicon ultrafiltration apparatus and membranes were from Amicon Cor- poration, Lexington, Massachusetts. All other chemicals were the highest grade obtainable.

Enzyme Assays: Arylsulfatase A (ASA) and ASB activities in crude liver homogenates and in partially purified liver preparations were determined by the method of BAUM, DODGSON and SPENCER (1959) using pNCS as substate or by the procedure of KOLODNY and MUMFORD (1976) using 4-MUS as substrate. The ASB activity in mixed leukocytes was measured after separation of ASA and ASB by batch DEAE-cellulose chromatography as previously described (MCGOVERN et al. 1981). The ABS activities also were determined in the presence of 0 to 2.5 mM DTT according

736 M. M . MCCOVERN E T AL.

to the procedure of VINE et al. (1982). The effect of' dermatan sulfate on the pNCS activity of the partially purified enzyme preparations was determined over a range of 0 to 14 mg/ml of natural substrate using 10 and 20 n i ~ pNCS. K , values were determined from Dixon plots (DIXON 1953). One unit (U) of enzymatic activity was defined as that amount of enzyme required to hydrolyze 1 nmole of substrate per hour at 37". Protein was determined using the Bio-Rad assay according to the manufacturer's instructions (BRADFORD 1976).

Purijkation of ASB: Feline ASB was partially purified by batch and column DEAE-cellulose chromatography according to the method of MCGOVERN et al . (1982). These partially purified preparations were used as enzyme sources for all subsequent studies. Since the normal and mutant isozymes from MPS VIb heterozygotes were partially purified but not resolved by anion exchange chromatography, the partially purified normal and mutant activities from MPS VIa heterozygotes were pooled for comparative studies.

Molecular weight and electrophoretic studies: The molecular weights of the partially purified en- zymes were determined by sucrose density centrifugation performed by the method of MARTIN and AMES (1961). Vertical slab gel electrophoresis was performed in a Mini-Slab apparatus (Idea Scientific, Corvallis, Oregon) using 12% native polyacrylamide gels (7.0 X 9 cm) and tris(hydroxy- nlethyl)-aminomethane (Tris)-glycine buffer, pH 8.9, as previously described (MATSUDARA and BURGESS 1978). The gels were stained for protein using Coomassie blue or for ASB activity using pNCS as previously described (VINE et al. 198 1).

p H optima, Kinetic, stability and Con A-Sepharose-binding studies: Enzymatic activity us. pH curves, kinetic and pH optima studies and the cryo- and thermostability of each of the partially purified enzymes were determined according to the methods of MCGOVERN et al. (1982). The Con A- Sepharose-binding properties of each partially purified enzyme were assessed as previously de- scribed (VINE et al. 1981).

Immunological studies: New Zealand white rabbits were injected intradermally with 50 pg of honiogeneous normal feline or normal human hepatic ASB (MCGOVERN et al. 1982) in a 1:1 suspension of Freund's complete adjuvant. Booster injections (50 fig) were given at 2-wk intervals, and IgG from rabbit antisera was partially purified by ammonium sulfate precipitation as previously described (HARROE and INCELD 1973) and carbaniylated by the method of BJERRUM et al. (1973). The antibody was aliquoted and stored at -50". Each enzyme preparation was tested for immu- nological cross-reactivity with the rabbit antihuman or antifeline ASB antibodies by OUCHTERLONY (1970) double imniunodiffusion. Rocket immunoelectrophoresis was performed according to the method of ANDERSON et al . ( 1 98 1) with the following modifications. Agarose solutions ( 1 0%) were prepared in TEMED-acetate buffer (0.01 M h',N,N',N'-tetramethyl-l,2-diaminoethane and 0.029 M acetic acid), pH 5.0, and the center of the plates were filled with agarose containing 75 pl (approximately 0.25 mg of protein) of partially purified and carbamylated rabbit antifeline or antihuman ASB antibody.

RESULTS

Demonstration of residual ASB activity: Residual ASB activity was present in crude extracts of hepatic tissue and mixed leukocytes from cats affected with MPS VI. As shown in Table 1 , the MPS VI", MPS V I b and MPS VI"lb homo- zygotes had approximately 6% of normal ASB activity. T h e MPS VI" and MPS VI* heterozygotes had intermediate values of 42-48% of normal mean activity in these sources.

Partial purzfication of feline ASB: Table 2 summarizes the final specific activity and fold purification for each of the enzyme preparations from the normal feline and feline MPS VI homozygous and heterozygous livers. Batch DEAE- cellulose chromatography with 100 mM Tris-HCI buffer, pH 7 . 5 , completely separated the ASB from the ASA activity in each of the enzyme preparations. Subsequent analytical column chromatography on DEAE-cellulose equilibrated with 10 mM Tris-HCI buffer, pH 7.5, resulted in elution of the normal feline

ALLELISM IN FELINE MPS VI

TABLE 1

ASB activity in hepatic tissue and mixed leukocytes from normal and MPS VI", MPS VIb and MPS V1"" Siamese cats

737

ASB activity

Hepatic tissue Mixed leukocytes

% normal % normal Source U/" activity U/mg activity

Normal feline Mean Range ( n = 8)

Feline iMPS VI" Homozygotes

Mean Range ( n = 5)

Mean Range (n = 7)

Heterozygotes

Feline MPS VIb Homozygotes

Mean Range ( n = 3)

Mean Range (n = 4)

Heterozygotes

Feline MPS Vialb Mean ( n = 2)

13.8 10.5-18.4

0.83 0.69-0.92

5.9 5.2-8.7

0.75 0.51-0.84

6.1 5.5-7.3

0.79 0.66; 0.92

100 168 100 120-304

6.0 10.9 6.5 8.8-12.6

42.7 73 43.4 59-91

5.4 9.8 5.8 7.7-10.6

44.2 81 48.2 72-96

5.7 8.9 5.3 7.3; 10.8

enzyme at about 50 mM NaCl and differentiated the MPS VI", MPS VIb and MPS VIalb residual ASB activities by their respective elution profiles (Figure 2). Mutant isozyme a was recovered in the buffer wash (Figure 2A), whereas mutant isozyme b was bound to the resin and eluted with 50 mM NaCl (Figure 2B). The MPS VI"lb residual ASB activity was resolved into two activity peaks: one eluting in the buffer wash and a second activity peak eluting at 50 mM NaCI; these activity peaks eluted in the same position as the mutant a and b isozymes from the homozygous MPS VI" and MPS V I b enzyme preparations, respectively (Figure 2C). The partially purified enzymes were used to charac- terize their physical and kinetic properties as described below.

Molecular weight studies: The apparent molecular weights of the normal ASB enzyme and mutant isozymes a and b were determined by sucrose density centrifugation. The normal enzyme and the partially purified ASB activities from MPS VIb homozygotes and heterozygotes had molecular weights of 105,000. In contrast, mutant isozyme a from the MPS VI" homozygotes had a molecular weight of 52,000 and the MPS VI" heterozygotes had two enzyme forms with molecular weights of 52,000 and 105,000. Mutant isozymes a and

738 M. M. MCGOVERN E T AL.

TABLE 2

Comparative physicokinetic properties of partially pur$ed ASB from normal feline and feline MPS VI", MPS VIb and MPS VIaJb liver

Apparent Specific activity Purification Apparent K," V,,"

Source W / m d (-fold) pH optimum (mM) (U/%)

Normal feline Mean Range (n = 3)

Feline MPS VIa Homozygotes

Mean Range ( n = 3)

Mean Range (n = 3)

Heterozygotes

Feline MPS VIb Homozygotes

Mean Range ( n = 3)

Mean Range (n = 3)

Heterozygotes

Feline MPS VIaJb Isozyme a Isozyme b

4250 165 5.7 0.85 4170 3420-4930 138-193 0.80-0.89 4010-4310

870 145 5.7 25 880 8 10-930 130-155 870-895

2010 275 5.7 12 1970 1810-2160 265-280 10.5-1 3.0 1760-2100

630 180 5.2, 6.2 1 .3b 600 560-645 160-192 1.27-1.33 585-620

1420 190 5.4, 5.7 1 . I b 1,350 1380-1465 175-202 0.9-1.3 1240-1 460

460; 510 150; 160 5.7 27; 32 570 520; 580 175; 180 5.2, 6.2 0.8; 1.4 480

a Kinetic values determined with pNCS as substrate. K , determined at pH 5.2 and 5.4 for MPS VI* homozygotes and heterozygotes, respectively.

b from the MPS VZalb homozygotes had molecular weights identical with the mutant isozymes from the MPS VZa and MPS VZb homozygotes, respectively.

Electrophoretic properties: Charge differences between the normal enzyme and mutant isozymes a and b were revealed by polyacrylamide gel electrophoresis (Figure 3). The normal feline enzyme migrated most cathodally (lanes 1 and 9), whereas isozyme a migrated toward the anode (lane 2) and isozyme b was at an intermediate position (lane 4). When the residual ASB activity from the MPS Vlalb heteroallelic homozygote was electrophoresed, two bands were ob- served, corresponding to the mutant a and b isozymes, respectively (lane 6): mutant isozyme a and b subunits did not form a hybrid dimeric enzyme. Electrophoresis of the partially purified ASB activity from the MPS VI"" het- erozygote revealed that the mutant isozyme a subunits did not dimerize with the normal subunit and two protein bands were present at the positions of the normal enzyme and mutant isozyme a (lane 3). In contrast, electrophoresis of the partially purified ASB activity from the MPS VI+'b heterozygote revealed the presence of three bands, one at the position of the normal enzyme, one at the position of mutant isozyme b and a third, intermediate band that pre-

ALLELISM IN FELINE MPS VI 739

"0 20 40 60 80 100 120 140 160

\

3 500 B ' I I I I I I 1

L' a

200 -

Fraction Number FIGURE 2.-DEAE-cellulose chromatographic profiles of feline MPS VI" (A) (O), MPS VIb (B)

(C) (B) ASB isozymes. The columns were washed wtih 10 mM Tris-HCI buffer, (A) and MPS pH 7.5, and elution was with a NaCl gradient as described in MATERIALS AND METHODS.

sumably represented a hybrid molecule composed of one normal and one isozyme b subunit (lane 5).

pH optima and kinetic studies: The pH optimum of the partially purified normal feline and feline MPS VI" homozygous and heterozygous ASB activities was 5.7. In contrast, the MPS VIb homozygous and heterozygous ASB activities had biphasic curves with optima at pH 5.2 and 6.2 and pH 5.4 and 5.7, respectively. Kinetic studies at the pH optimum of each enzyme demonstrated

740 M. M. MCGOVERN ET AL.

1 2 3 4 5 6 7 8 9 FI(;URE 3.-Minislab gel e1r.c i r o p l ~ o r r ~ i ~ of Iionioiygous and hetero/\gous feline MPS VI'. M P S

V I b illid norinid feline hepatic ASH. Lane I , nornial feline ASB; lane 2. MPS VI'"; lane 3. MPS VIa/+; Iiiiir 4. MPS VIbJb; Line 5. MI'S Vib/+; lane ti, M P S VI'/b; lane 7. MPS VIa1' iso7ymr a; line N. MI'S VIaJb isoryiiie b; kine 9, normal feline ASB.

that the nornial enzyme and mutant isozyme b activities were saturated a t 5 and 7 mM pNCS, respectively. In contrast, mutant isozyme a could not be saturated at substrate concentrations up to 20 mM (the maximum solubility of the substrate). K,,, values calculated from Lineweaver-Burk plots were 0.85 mM (at pH 5.7) for the normal feline enzyme and greater than 25 mM (at pH 5.7) and 1.3 nlM (at pH 5.5) for mutant isozymes a and b, respectively. Kinetic studies a t pH 5.7, the optimum of the normal enzyme, revealed that mutant isozyme b was saturated a t 10 mM pNCS and had a K,, of 6.2 mM. T h e mutant a and b isozymes separated from the MPS VI"'h homozygote had K , values similar to those of the mutant isozymes isolated from the MPS VI" and MPS VIb homozygotes, respectively (Table 2).

Determination of enzymatic activity toward PNCS and 4-MUS: T h e activity of each partially purified enzyme preparation was determined using two artificial substrates, pNCS and 4-MUS (Table 3). Each enzyme displayed greater specific activity toward pNCS, with pNCS to 4-MUS activity ratios of 9.1, 39.5 and 85.1 for the normal enzyme and mutant isozymes a and b, respectively.

T h e effect of dermatan sulfate on the activity of the partially purified en- zymes was determined over an inhibitor range of 0 to 14 mg/ml using 10 and 20 mM pNCS. Dixon plots revealed that the natural substrate was a competitive inhibitor of pNCS. with Ki values of 0.4, 2.0 and 6.1 for the normal enzyme and mutant isozymes a and b, respectively (Table 3).

Con A-Sepharose-binding studies: Approximately 90% of the normal ASB ac- tivity was bound to Con A-Sepharose and elution resulted in recovery of 80% of the bound enzyme. In contrast, the MPS VI" isozyme was 75% bound with a 70% recovery and the ME'S VIb isozyme was only 55% bound with a recovery

ALLELISM IN FELINE MPS VI 741

T A B L E 3

Comparison ofpartially purajied normal feline and feline MPS VI", MPS VIb and VI"'b hepatic ASB activities toward various substrates

Source

Ki for der- matan sul-

4MU-S activ- pNCS activity fate" (me/ ity (U/w$ (U/mg) 4MU-S ml)

Normal feline 470 4,250 9.1 0.4

Feline MPS VIa Honiozygote 22 870 39.5 2.0 Heteroiygote 73 2,o 10 27.5

Feline MPS VIb Homozygote 7 630 85.1 6.1 Heterozygote 27 1,420 52.6

Feline MPS VIalb Isozyme a 11 510 46.4 Isozyme b 6 490 81.7

Final substrate concentrations were 20 mM pNCS and 5 mM 4MU-S. " K , values were determined from Dixon plots using 10 and 20 m M pNCS and

dermatan sulfate over a range of 0-14 mg/ml as inhibitor.

of 75%. The residual enzyme from the MPS VI"'b homozygotes was 60% bound with a recovery of 70%.

Stability studies: Thermal inactivation at 60" of the patially purified normal feline and feline MPS VI" and MPS VIb homozygous and heterozygous ASB activities revealed that the normal feline enzyme and mutant isozyme b from the feline MPS V I b homozygotes had mean half-lives of 55 and 60 min, re- spectively. In contrast, mutant isozyme a from the MPS VI" homozygotes had an average half-life of 10 min. Mutant isozyme a also was markedly more cryolabile than the normal feline activity. After 72 hr at -50", 85% of the initial normal activity was retained, whereas only 45% of mutant isozyme a activity was recovered. Mutant isozyme b had nearly normal stability and re- tained 85% of initial activity. The ASB activity in MPS VI" and MPS VZb heterozygotes had intermediate thermostabilities (half-lives of 28 and 65 min, respectively) and cryostabilities (68 and 88% of initial activity, respectively), and the values for isozymes a and b isolated from the homozygotes were similar to those from MPS VI" and MPS VIb homozygotes, respectively. The stability of the enzyme preparations also was determined in the presence of 0 to 100 mM pronase. The normal enzyme retained 100% of initial activity over the entire range tested. In contrast, mutant isozymes a and b retained only 50% of initial activity at a concentration of 10 mM pronase.

Effect of sulfiydryl reagents: Table 4 summarizes the effect of DTT on the partially purified hepatic enzymes and on the enzymatic activity in crude leu- kocyte extracts. The activity of mutant isozyme a in MPS VI" homozygotes was enhanced more than four-fold and the activity in MPS VI" heterozygotes was increased almost two-fold at a DTT concentration of 2.5 mM. The normal

742 M . M. MCGOVERN ET AL.

TABLE 4

Effect Of DTT on normalfeline and feline MPS VI", MPS VIb and MPS VIab ASB activity

Partially purified hepatic en7yme Leukocyte enzyme"

Source 0.25 l l lM D T T 2.5 m M D T T 0.25 mM D T T 2.5 mM D T T

Normal feline Mean 98 91 97 94 Range (n = 4) 95-99 87-93 95-99 93-95

Feline MPS VI" Homor ygotes

Mean 304 41 1 345 463 Range (n = 3) 297-3 12 408-4 I 5 3 3 0-35 7 450-474

Mean 156 178 169 184 Range (n = 3) 150- 162 174-18 1 158-1 75 180-188

Heterozygotes

Feline MPS VIb Homozygotes

Mean 102 104 99 101 Range (n = 3) 101-105 102-106 98-103 100-103

Mean 99 97 92 97 Range (n = 3) 98-100 96-97 88-97 93-101

Heterozygotes

Feline MPS Vlnlb Homozygotes

Mean Range ( n = 3)

Partially purified iso- zymesb

Isozyme a Isozyme b

31 1 454 97 95

234 303 219-246 296-3 10

Results are percentages of initial activities. ' Leukocytes were isolated from peripheral blood and crude extracts were prepared as described

The partially purified hepatic isozymes were separated by DEAE-cellulose chromatography as in MATERIALS AND METHODS.

described in MATERIALS AND METHODS.

enzyme and the activities from MPS VZb homozygotes and heterozygotes were not stimulated by the sulfhydryl-reducing agent. Similarly, mutant isozyrne a isolated from the MPS VZaIb homozygote was increased more than four-fold with 2.5 mM DTT, whereas mutant isozyme b was not enhanced at any D T T concentration tested.

Immunological properties: Ouchterlony double immunodiffusion revealed that the normal feline enzyme formed a single precipitation line with the rabbit antifeline ASB antibodies, whereas the MPS VI mutant isozymes did not (Fig- ure 4A). In contrast, the rabbit antihuman ASB antibodies cross-reacted with both the normal feline and feline MPS VI mutant ASB isozymes (Figure 4B).

ALLELISM IN FELINE MPS VI 743

FIGURE 4.--Ouchterlony double immunodiffusion of the partially purified hepatic ASB activities from normal Siamese cats (NF). MPS VI' (a), MPS VI' (b) and MPS VI'" (ab) homozygotes against antifeline (gel A) and antihuman (gel B) antibodies.

However, the precipitation line of the mutant isozymes had a spur indicating that there was partial nonidentity with the normal enzyme.

Rocket immunoelectrophoresis was used to estimate the amount of immu-

744 M. M. MCGOVERN ET AL.

1 2 3 4 5 6

FKXJRF. 5.-Rockrt iiniiiiinorlrrtr~~pli~~rrsis of normal fcline and frlinc Ml'.V VI' and MPS VI' Iiepiitic ASR. Gel A, Kabbit antifeline ASB antibody. Gel B, Rabbit antihuman ASB antibody. T h e MPS VI' and MPS VIb honio7ygote and heterozygote leukocyte extracts were diluted 15 for rocket ininiuiioelectrophoresis agiinst the antihuman ASB antibody. Lane I , normal feline ASB: lane 2. M f S VIaJa: lane. M f S VPJ+: lane 4. MPS VIbJb; lane 5. MPS VIbJ+: lane 6. MPS W J ' .

T A B L E 5

Korhet immunoelertrophoresk of normal feline and felint MPS VI'. MPS VIb and MPS ASR

CRI M/activicy mid

.Source Antifeline ASB Antihuman ASB

Normal feline 1 .o I .o

Feline M f A VI' Homozygote 3. I 7.2 Heterwzygote I .2 2.8

Homozygote 2.3 16.5

Feline M f S VP" 2.6 7.8

Feline MP.V VI'

Heterozygote 1 . 1 7.2

Rcxkct heights were measured under magnification (x4) and C R I M ratios determined using the standard curve for the normal enzyme.

ALLELISM IN FELINE MPS VI 745

noreactive ASB protein in leukocyte lysates from normal Siamese cats and affected homozygotes and heterozygotes with MPS VI (Figure 5). When 1.0 U of mutant isozyme a activity was immunoelectrophoresed, the amount of cross-reacting immunological material (GRIM) was 3.1 and 7.2 times that pres- ent for 1.0 U of normal feline enzyme using antifeline or antihuman ASB antibodies, respectively (Table 5). Similarly, for 1.0 U of mutant isozyme b activity, the amount of GRIM was 2.3 and 16.5 times greater than that ob- served for 1.0 U of normal activity with the antifeline and antihuman ASB antibodies, respectively.

Clinical features: The 19 affected cats currently living in the M P S VI colony were examined to determine whether the clinical severity of the disease varied among animals affected with MPS VI" or MPS VZb. Of these 19 homozygotes, 14 are from the MPS VI" line and five from the MPS VZb line. N o significant difference was observed between the two affected groups in the degree of facial dysmorphia, dysostosis multiplex, paralysis or corneal clouding. In addi- tion, the percentage of neonatal deaths and the age of death of affected ani- mals from the two lines were similar.

DISCUSSION

The discovery of two allelic mutations in the feline ASB structural gene provided the unique opportunity to investigate the physicokinetic and immu- nological properties of the residual ASB activities in affected homoallelic and heteroallelic cats with the feline analogue of human Maroteaux-Lamy syn- drome (MPS VI). Previous studies of the physicokinetic properties of the par- tially purified residual hepatic ASB (mutant isozyme a) from the MPS"'" homo- zygotes revealed that the mutation in the feline ASB structural gene altered the kinetic, stability and subunit association properties of the expressed mutant enzyme (VINE et al. 1981, 1982). Subsequently, a second unrelated cat with MPS VI was discovered (Figure 1; 170). The residual hepatic ASB activity from this affected homozygote had a distinctly different elution profile on anion exchange chromatography than that observed for mutant isozyme a (Figure 2), suggesting the presence of a second allelic mutation (MPS VZb) at the feline ASB locus. Thus, matings were undertaken to produce affected cats that were homoallelic for mutant alleles a or b in order to facilitate the char- acterization of each mutant isozyme.

Studies of the partially purified mutant isozyme b from MPS VZb homozy- gotes revealed distinct differences from those of mutant isozyme a. In contrast to mutant isozyme a, mutant isozyme b was a homodimer, which had a charge different from both the normal enzyme and mutant isozyme a (Figures 2 and 3). Mutant isozyme b had a slightly increased apparent K , and a markedly decreased apparent V,,, for the artificial substrate; however, mutant isozyme b had a markedly increased Ki for the natural substrate. These findings indi- cated that the binding of the natural substrate was altered and that, if radio- labeled dermatan sulfate was available, a markedly increased K , would be observed for the hydrolysis of the natural substrate by mutant isozyme b. In addition, mutant isozyme b had a normal or slightly increased cryostability and

746 M. M. MCGOVERN E T A L .

TABLE 6

Comparative physicokinetic and immunologzcal properties of partially purijed hepatic ASB from normal feline and feline MPS VI" and MPS VIb homoallelic homozygotes

Property

Molecular weight Subunit structure Effect of D T T Electrophoretic mobility (R,) Thermostability ( t l p at 60", min) Cryostability (% initial activity after

72 hr, -50") Protease stability CRIM/activity ratio" Apparent K , (mM for pNCS) Apparent V,,, (U/mg) Ki for dermatan sulfate (mg/ml)

Feline MPS Nornial feline v I'

105,000 52,000 Dimer Monomer None Dimerizes 0.62 0.15 55 10 87 45

Stable Unstable 1 .o 7.2

0.85 >25 4,170 880

0.4 2.0

Feline MPS Vlb

105,000 Dimer None 0.20 60 85

Unstable 16.5 1 . 3 600 6.1

ASB CRIM in isolated leukocytes was determined with polyclonal antihuman ASB antibodies.

thermostability. Immunological studies (using rabbit antihuman ASB antibod- ies) demonstrated that mutant isozyme b had a CRIM to activity ratio of 16.5 compared to a ratio of 1.0 for the normal enzyme protein; these findings indicated the full expression of a stable mutant protein. In addition, isozyme b was only 55% bound to Con A, compared to 90% for the normal enzyme, suggesting that the MPS VIb mutation altered the enzyme conformation re- quired for optimal lectin binding. These results were consistent with a struc- tural gene mutation at the ASB locus which produced a stable protein that could dimerize but was catalytically defective with a residual activity about 6% of normal. T h e finding of two distinct mutations in the ASB structural gene documents the presence of genetic heterogeneity in feline MPS VI (Table 6).

Studies of the partially purified hepatic ASB activity from obligate hetero- zygotes for the MPS VI" and MPS VZb mutations provided further information on the interaction of the mutant and normal enzyme subunits in vivo. Heter- ozygotes (MPS VZa'+) for the MPS VZ" mutation expressed both the normal dimeric enzyme and the mutant isozyme a , previously characterized as a mon- omer (VINE et al. 1981). T h e fact that the normal and mutant enzyme forms could be separated by anion exchange chromatography (data not shown) and that no evidence of association between the normal enzyme and mutant sub- units was found indicates that isozyme a was unable to dimerize with the normal subunit. In contrast, electrophoresis of the partially purified MPS VI+'b ASB activity revealed the presence of three bands, one at the position of the normal enzyme, one at the position of isozyme b and a third, intermediate band, which presumably represented a hybrid of normal and isozyme b sub- units. Thus, the isozyme b subunits were able to form dimers with each other as well as with normal enzyme subunits.

T h e immunological studies were notable since the mutant isozymes were recognized differentially by the antifeline and antihuman ASB antibodies. Nei-

ALLELISM IN FELINE MPS VI 747

ther mutant isozymes a or b formed a detectable precipitation line in Ouch- terlony double diffusion gels with polyclonal antibodies produced against the homogeneous normal feline enzyme (MCGOVERN et al. 1982). In contrast, the polyclonal antibodies raised against the homogeneous normal human enzyme (MCGOVERN et al. 1982) formed a precipitation line of identity with the normal feline enzyme and both mutant isozymes. However, the precipitation lines against the mutant isozymes were more reactive and both had spurs of noni- dentity with the normal feline enzyme. The differential recognition of the mutant isozymes and the normal feline enzyme may in part be due to the fact that the human enzyme is a monomer which presumably has additional and/ or unique epitopes that are not exposed by the normal feline dimeric enzyme (MCGOVERN et al. 1982; VINE et al. 1982). It appears likely that these epitopes are exposed on the mutant isozymes, particularly on the monomeric mutant isozyme a. These findings also relate to the amount of CRIM estimated for the mutant and normal enzymes using each antibody preparation (Figure 5 , Table 5) . Since the antifeline antibodies had a greater avidity toward the normal enzyme, and since the antihuman antibodies had a greater avidity toward the mutant isozymes, the estimated CRIM to activity ratios may be lower and higher, respectively, than the actual values. More accurate estimates could be obtained by the use of a polyclonal or monoclonal antibody prepa- ration that recognized the normal and mutant enzyme proteins equally,

The characterization of the feline ASB mutations and their expression in homoallelic and heteroallelic homozygotes are instructive as they relate to the phenotypic and biochemical expression of allelism in human recessive disor- ders. Except for mutations that have a high frequency in certain ethnic groups (i.e., sickle cell anemia, Tay-Sachs disease), most rare recessive traits presumably result from the inheritance of two different mutant alleles, i . e . , the affected homozygotes are heteroallelic at the disease gene locus. Usually heteroallelic homozygotes will have the same or similar phenotype to that expressed by individuals who are homoallelic for the same mutation, as exemplified by the allelism found in feline MPS VI. Although the occurrence of different mutant alleles usually results in the same phenotype, such allelism may provide the basis for the variable expressivity in disease severity in unrelated homozygotes. Occasionally, heteroallelic homozygotes will have a unique phenotype, inter- mediate to that of either homoallelic homozygote. A prime example of allelism leading to different phenotypes is at the a-L-iduronidase locus where different allelic mutations result in Hurler’s disease, characterized by severe mental retardation and dysostosis multiplex, or in Scheie disease, in which a-L-iduron- idase deficiency results in normal intellience and mild to moderate skeletal and connective tissue involvement (MCKUSICK et al. 1972). It has been suggested that the intermediate phenotype, the Hurler-Scheie disease, represents the homoallelic homozygote, with one Hurler and one Scheie allele (STEVENSON et al. 1976); however, the intermediate phenotype may also be the result of different mutant alleles (KAIBARA et al. 1979).

The studies of feline MPS VI also provide insight into the variability in response to specific therapy observed in certain human genetic diseases (MAT-

748 M . M. MCGOVERN E T AL.

SUI, MAHONEY and ROSENBERG 1983). The residual ASB activity in MPS VI"/" homoallelic homozygotes, and to a lesser extent in MPS VZ"lb heteroallelic homozygotes, can be enhanced several fold by the use of thiol-reducing agents which cause the mutant isozyme a to dimerize (VINE et al. 1982). The residual activity in affected cats that were homoallelic for the MPS VIb allele was not responsive to such enzyme manipulation therapy since the mutant isozyme b is a dimer with abnormal catalytic properties. Thus, the allelism in feline MPS VI provides a model for the increased understanding of genetic heterogeneity, as well as the variability in therapeutic response in human genetic disorders.

The authors thank LINDA LUCO and LINDA SITNICK for their expert clerical assistance. This research was supported in part by a grant (AM25759) from the National Institutes of Health. M. M. McG. was the recipient of a March of Dimes Birth Defects Foundation Medical Student Summer Fellowship. N. M . was supported by a National Institutes of Health predoctoral fellowship ( 5 T 3 5 AM07420).

LITERATURE CITED

ANDERSON, P. M., R. REDDY, C. E. ANDERSON and R. J. DESNICK, 1981 Characterization of the porphobilinogen deaminase deficiency in acute intermittent porphyria: immunologic evidence for heterogeneity of the genetic defect. J. Clin. Invest. 68: 1-12.

The assay of arylsulfatases A and B in human

Arylsulfatase B defxiency in Maroteaux-Lamy syndrome: cellular studies and carrier identification. Pediatr. Res. 9: 475- 480.

BAUM, H., €3. S. DODGSON and B. SPENCER, 1959 urine. Clin. Chim. Acta 4: 453-455.

BERATIS, N. G . , B. M . TURNER, R. WEISS and K. HIRSCHHORN, 1975

~ J E R R U M , 0. J., A. INCILD, H. LOWENSTEIN and B. WEETER, 1973

BRADFORD, M. M., 1976

Carbamylated antibodies used

A rapid and sensitive method for the quantitation of microgram quan-

Animal analogues of human inherited metabolic diseases: molecular pathology and therapeutic studies. pp. 27- 65. In: Animal Models of Inherited Metabolic Diseases, Edited by R. J. DESNICK, D. F. PATTERSON and D. G. SCARPEILI. Alan R. Liss, Inc., New York.

DESNICK, R. J., D. F. PATTERSON and D. G. SCARPELLI, (Editors), 1982 Animal Models ofznherited

DIXON, M., 1953

FLUHARIY, A. L., K. I*. STEVENS, D. 1.. SANDERS and H. KIHARA, 1974

for quantitation of human IgG. Scand. J. Inimunol. 2 (Suppl. 1): 145-148.

tities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 77: 248-254.

DESNICK, R. J., M. M . MCGOVERN, E. H. SCHUCHMAN arid M. E. HASKINS, 1982

Metabolic Diseases, pp. 1-519. Alan R. Liss, Inc., New York.

The determination of inhibitor constants. Biochem. J. 55: 170-171.

Arylsulfatase B deficiency in Maroteaux-Lamy syndrome cultured fibroblasts. Biochem. Biophys. Res. Commun. 5 9 455- 461.

Immunization, isolation of immunoglobulins, estimation of antibody titer. Scand. J. Immunol. 2 (Suppl. 1): 161-162.

HASKINS, M. E., 1'. F. JEZYK, R. J. DESNICK and D. F. PATTERSON, 1980 Feline models of mucopolysaccharidosis. pp. 393-414. In: Enzyme Therapy in Genetic Diseases:Z, Edited by R. J. DESNICK. Alan R. Liss, Inc., New York.

Mucopolysaccharide storage disease in three families of cats with arylsulfatase B deficiency: leukocyte studies and carrier identifica- tion. Pediatr. Res. 13: 1203- 12 10.

JEZYK, P. F., M. E. HASKINS, D. F. PATTERSON, U'. J. MEI.LMAN and M. GREENSTEIN,

HARROE, N. and A. INGELD, 1973

HASKINS, M. E., P. F. JEZYK and D. F. PATTERSON, 1979

ALLELISM IN FELINE MPS VI 749

1977 Mucopolysaccharidosis in a cat with arylsulfatase B deficiency: a model of Maroteaux- Lamy syndrome. Science 198: 834-836.

KAIBARA, N., M. EGUCHI, K. SHITARA and K. TAKAGISHI, 1979 Hurler-Scheie phenotype: a report of two pairs of inbred sibs. Hum. Genet. 53: 37-41

KOLODNY, E. H. and R. A. MUMFORD, 1976 Arylsulfatases A and B in metachromatic leukodys- trophy and Maroteaux-Lamy syndrome: studies with 4-methylumbelliferyl sulfate. Adv. Exp. Med. Biol. 2 3 9 239-251.

MAROTEAUX, P. and M. LAMY, 1965 Hurler’s disease, Morquio’s disease, and related muco- polysaccharidoses. J. Pediatr. 67: 313-31 6.

MARTIN, R. G. and B. N . AMES, 1961 A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J. Biol. Chem. 236: 1372-1379.

MATSUDARA, P. T. and D. R. BURGESS, 1978 Microslab linear gradient polyacrylamide gel elec- trophoresis. Anal. Biochem. 87: 387-396.

The natural history of the inherited methylmalonic acidemias. N. Engl. J Med. 308: 858-861.

An improved method for heterozygote identification in feline and human mucopolysaccharidosis VI, arylsulfatase B deficiency. Enzyme 26: 206-2 10.

MCGOVERN, M. M., D. T. VINE, M. E. HASKINS and R. J. DESNICK, 1982 Purification and properties of feline and human arylsulfatase B isozymes. J. Biol. Chem. 257: 12605-12610.

allelism and genetic compounds among the mucopolysaccharidoses. Lancet 1: 993-996.

The mucopolysaccharide storage diseases. pp. 1282-1 307. In: The Metabolic Basis of Inherited Disease, Edited by J. B. STANBURY, J. B. WYNGAARDEN and D. J. FREDRICKSON. McGraw-Hill Book Company, New York.

Handbook of immunodiffusion and immunoelectrophoresis. In: Hand- book of Experimental Immunology, Edited by D. M. WEIR. Blackwell Scientific Publications, Oxford.

Models of human genetic disease in domestic animals. pp. 263-340. In: Advances in Human Genetics, Vol. 12, Edited by H. HARRIS and K. HIRSCHHORN. Plenum Press, New York.

PERCY, A. K. and R. 0. BRADY, 1968 Metachromatic leukodystrophy: diagnosis wtih samples of venous blood. Science 161: 594-595.

STEVENSON, R., R. HOWELL, V. A. MCKUSICK, R. SUSKIND, J. W. HANSON, D. E. ELLIOT and E. F. NEUFELD, 1976 The iduronidase-deficient mucopolysaccharidoses: clinical and roentgen- ographic features. Pediatrics 57: 11 1-122.

Mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome). Am. J. Dis. Child. 126: 747-755.

Feline mucopolysac- charidosis VI: purification and characterization of the residual arylsulfatase B activity. Am. J. Hum. Gen. 33: 916-927.

VINE, D. T., M. M . MCGOVERN, E. H. SCHUCHMAN, M. E. HASKINS and R. J. DESNICK, Enhancement of residual arylsulfatase B activity in feline mucopolysaccharidosis VI by

Communicating editor: R. E. GANSCHOW

MATSUI, S. M., M. J. MAHONEY and L. E. ROSENBERG, 1983

MCGOVERN, M. M., D. T. VINE, M. E. HASKINS and R. J. DESNICK, 1981

MCKUSICK, V. A., R. HOWELL, I. HUSSELS, E. NEUFELD and R. STEVENSON, 1972 Allelism, non-

MCKUSICK, V. A., E. F. NEUFELD and T. E. KELLEY, 1978

OUCHTERLONY, 0, 1970

PATTERSON, D. F., M. E. HASKINS and P. F. JEZYK, 1982

STUMPF, D. A., J . H. AUSTIN, A. C. CROCKER and M. LAFRANCE, 1973

VINE, D. T., M. M. MCGOVERN, M. E. HASKINS and R. J. DESNICK, 1981

1982 thiol-induced subunit association. J. Clin. Invest. 6 9 294-302.