- Email: [email protected]
Two small deletion mutations of the HEXB gene are present in DNA from a patient with infantile Sandhoff disease
Biochimica et Biophysica Acta, 1138 (1992) 315-317 © 1992 Elsevier Science Publishers B.V. All rights reserved 0925-4439/92/$05.00
315
BBADIS 61135
Two small deletion mutations of the HEXB gene are present in D N A from a patient with infantile Sandhoff disease B e t h M c I n n e s 1, Charlotte A. B r o w n 1,2 a n d D o n J. M a h u r a n 1,2 1 Research Institute, Hmpitalfor Sick Children, Toronto (Canada) and 2 Department of Clinical Biochemistry, University of Toronto, Toronto (Canada)
(Received 30 August 1991)
Key words: Sandhoff disease; Hexosaminidase; Tay-Sachs disease; Gene mutation; (Infantile)
Lysosomal /~-hexosaminidase (EC 3.2.1.52) occurs as two major isozymes hexosaminidase A (a/3) and B (/~/~). The a subunit is encoded by the HEXA gene and the /3 subunit by HEXB gene. Defects in the ot o r / ~ subunits lead to Tay-Sachs or Sandhoff disease, respectively. While many HEXA gene mutations have been reported only three HEXB gene mutations are known. We report the characterization of two rare HEXB mutations present in genomic DNA from a single fibroblast cell line, GM203, taken from a patient with the infantile form of Sandhoff disease. The first is a single base pair deletion in exon 7 changing the codon for Gly-258, GGA, to GA and the second, a two base pair deletion in exon 11 changes the codons for Arg-435/Val-436, A G A / G T C , to AGTC. Each mutation produces a frame shift in the affected allele that results in a premature stop codon 17 or 20 codons downstream, respectively. These mutations also result in the inability to detect/3-mRNA by Northern blot analysis of total mRNA. These data are consistent with the idea that the severe infantile form of Tay-Sachs or Sandhoff disease is associated with a total lack of residual hexosaminidase A activity. Introduction
/3-hexosaminidase A (a/3) is a lysosomal enzyme responsible for the hydrolysis of G~2 ganglioside. Interest in the hexosaminidase system has been generated by the occurrence of a group of autosomal recessive neurodegenerative disorders known as the GM2 gangliosidoses. Three major categories of the disease are known depending on which of three different genes involved in the degradation of GM2 ganglioside is defective. The first gene product is the G~z-activator protein which serves as a transport protein for the ganglioside. The other two genes encode the subunits of heterodimeric hexosaminidase A. Defects in the a subunit (encoded by the H E X A gene on chromosome 15) result in Tay-Sachs disease and defects in the /3-subunit (encoded by the H E X B gene on chromosome 5) result in Sandhoff disease. The lack of functional hexosaminidase A leads to the lysosomal accumulation of GM2 ganglioside in brain and peripheral nervous tissue. These disorders displays a spectrum of clinical severity, ranging from the most severe infantile
forms to juvenile, chronic and adult-onset variants (reviewed in Ref. 1). Extensive homology in both the H E X A and H E X B gene structures demonstrate their common evolutionary origin [2]. While more than a dozen mutations in the H E X A gene have been described (reviewed in Ref. 3), only three mutation have been characterized in the H E X B gene [4-6]. Surprisingly one of these, a 16 kb deletion of the 5' end, accounted for 27% of the Sandhoff alleles analyzed [4]. This mutation is probably the same as that described by Bikker et al. using pluse-field gel electrophoresis [7]. In this report we describe a patient with the infantile form of Sandhoff disease who was a compound heterozygote for two rare H E X B gene mutations. One is a 1 bp deletion in exon 7, and the other is a 2 bp deletion in exon 11. We have previously shown that neither allele produces detectable /3-mRNA by Northern blotting [8]. This is presumably due to the mutations generation of premature stop codons in the resuiting reading frame shifts. Materials and Methods
Correspondence: D.J. Mahuran, Research Institute, The Hospital For Sick Children, 555 University Avenue, Toronto, Ontario, Canada, M5G 1X8.
Cell line GM203 The cell line was obtained from the Human Genetic Mutant Cell Line Repository, Camden NJ (U.S.A.).
316 The patient was of C a u c a s i a n / I n d i a n ancestry and was diagnosed at 1 year of age. Both the clinical and biochemical/enzymatic data available are indicative of a classical infantile Sandhoff phenotype [8-10].
PCR PCR was performed on genomic D N A diluted to (1.5 /xg/4 #1 which had been sheared by vortexing. The conditions used were those recommended by PerkinElmer Cetus Corporation except for the addition of 1U of Perfect Match (Stratagene). AmpliTaq polymerase was used in a 50 /.tl reaction volume. To amplify sequences from intron 6 through to intron 7, 250 ng each of oligonucleotides 525 (sense), 5 ' - A T C A A A T G C A A G C A C A A T T G T - 3 ' and 524 (antisense), 5'-GTG A C A G A A C A A G A C T C C A - 3 ' , were used as primers for 3(I cycles of 94°C for 30 s, 56-58°C for 30 s, and 72°C for 90 s. This resulted in a 394 bp fragment which was sequenced directly (below). To amplify sequences from intron 9 to intron 11 oligonucleotides 528 (sense), 5 ' - C A C C T C T C A A A A T G C A A G A A 3 ' , and 529 (antisense), 5 ' - A A T G G T T G C T T C A C T T A C C A - 3 ' , were used as primers for 30 cycles of 93°C for 1 min, 60°C for 1 min and 72°C for 3 min. This resulted in a 1058 bp fragment which was sequenced directly (below). Direct sequencing To avoid detection of Taq artifacts in cloned PCR fragments, we directly sequenced the products of each PCR. PCR fragments were isolated by agarose gel electrophoresis and purified with Geneclean Kit (Bio 101). The gel-purified fragments were sequenced with 500 ng of oligonucleotide 524 or 525 (above) for the
exon 7 mutation ~r oligonucleotidc 52 ( 5 ' - ( q ( - C A A T C T T G T C C A T A G C T - 3 ' ) for the mutation in exon 11. The method of direct sequencing used was adapted from the Sequenase protocol (United Stales Biochemical Corp.), as previously reported [11]. Results
Direct nuclcotide sequencing of PCR fragments was performed on all 14 exons and exon/intron junctions with the exception of the extreme 5' end of the H E X B gene (data not shown) from fibroblast line GM203, Two mutations were detected. The first, a 1 bp (G) deletion in exon 7, changes the codon for Gly-258, GGA, to G A (Fig. 1), and results in a frame shift which introduces a stop codon 17 codons downstream. The second, a 2bp (AG) deletion in exon 11 changes the codons for Arg-435/Val-436, A G A / G T C , to AGTC. This also results in a frame shift with a stop codon produced 20 codons downstream (Fig. 2). Analysis of our collection of fibroblasts from 19 Sandhoff patients [4,8] demonstrated that both mutations were unique to this cell line. Thus, the patient was a compound heterozygote for two rare Sandhoff mutations. Discussion
The identification of two allelic d e l e t i o n / f l a m e shift mutations in the cell line GM203 from a patient with the infantile form of Sandhoff disease is consistent with the hypothesis that the infantile phenotype of Tay-Sachs or Sandhoff disease is associated with a complete absence of residual hexosaminidasc A activity.
GM203
Normal Exon 7 T Ser263 C G Leu262 T
T.
T Set261 C T Tyr260 A
1 C Ser259 G
A Gly258 G
G g a c
G
A
T
C
G
A
T
C T C C
T Leu263
T G
c T
T
G Cys262
T T C
T T Leu261
T
_~
T A
T T lie260
T
.~.
C G
T C Ala259
A A A G~A Glu258 GMG
g
g
a c
a c
Intron
Fig. 1. Autoradiogram of a 6% polyacrylamide/urea gel showing sense sequence obtained for genomic DNA from patient GM203 and from a normal individual in the intron 6/exon 7 region of the HEXB gene. Patient GM203 is heterozygousfor 1 bp (G) deletion in exon 7 which changes the codon for Gly-258, GGA, to GA (Fig. 1). This results in a frame shift with a stop codon produced 17 codons downstream.
317 GM203
G A
T T Leu 433 Ser 434 Ser 435 His 436 Ser 437
Ile 438
normal T
C~G
G A G T C A *T C A G T G T C fi T A
"
A G T C A T C I C A G T G T ¢
Leu 433
G
Ala 438
Ser 434 Arg 435 Va1436 Thr 437
T
A
.~
oj--
A
A
Ser 439
Fig. 2. Autoradiogram of a 6% polyacrylamide/urea gel showing antisense sequence obtained for genomic DNA from patient GM203 and from a normal individual in exon 11. Patient GM203 is heterozygous for a 2 base pair deletion (AG) at Arginine-435/Valine-436 in exon 11. This results in a frame shift with a stop codon produced 20 codons downstream.
We had previously found that this cell line lacked residual hexosaminidase A activity and detectable /3m R N A by Northern blot analysis [8]. The mechanism by which a mutation causing a premature stop codon results in a low steady-state level of affected m R N A is unclear. One possible explanation is that the 3' end of the m R N A is no longer protected by ribosomes and is therefore more susceptible to RNAse digestion. Such a theory would predict that the length of the 3' untranslated end of an m R N A would be inversely proportional to its stability. Alternatively, a defect in /3-globin m R N A metabolism has been shown to be unrelated to cytoplasmic instability, suggesting that the premature stop codon in this case alters intranuclear stability a n d / o r cytoplasmic transport [12,13]. The major mutation responsible for Tay-Sachs disease in the Jewish population is a 4 bp insertion in exon 11 of the HEXA gene. This produces a stop codon 6 codons after a-Ile-425 (aligns with /3-Ile-454) and like the two /3 mutations we report here, no detectable ce-mRNA by Northern blot analysis [14]. Interestingly, in the aligned sequences of the a and /3 cDNAs the stop codon generated by this HEXA gene mutation occurs within 6 codons of that generated by the 2 bp deletion mutation in the HEXB gene described here. However, another such HEXA mutation in exon 13 produces normal steady-state levels of am R N A a n d a n a polypeptide that is shortened by 22 r e s i d u e s a t its c a r b o x y t e r m i n u s [15]. T h u s , the present d a t a suggests that the length of the 3 ' u n t r a n s l a t e d e n d o f t h e a - a n d / 3 - m R N A is inversely proportional to m R N A stability.
Acknowledgments T h i s w o r k w a s supported through a M e d i c a l R e search Council of Canada operation grant No. MA10435 t o D . M . F u n d i n g f o r B . M . w a s t h r o u g h a fellowship awarded by The Hospital F o r S i c k C h i l d r e n F o u n dation.
References 1 Sandhoff, K., Conzelmann, E., Neufeld, E.F., Kaback, M.M. and Suzuki, K. (1989) in The Metabolic Basis of Inherited Disease (Scriver, C.V., Beaudet, A.L., Sly, W.S. and Valle, D., eds.), pp. 1807-1839, McGraw-Hill, New York. 2 Proia, R.L. (1988) Proc. Natl. Acad. Sci. USA 85, 1883-1887. 3 Mahuran, D.J. (1991) Biochim. Biophys. Acta 1096, 87-94. 4 Neote, K., Mclnnes, B., Mahuran, D.J. and Gravel, R.A. (1990) J. Clin. Invest. 86, 1524-1531. 5 Mitsuo, K., Nakano, T., Goto, I., Taniike, M. and Suzuki, K. (1990) J. Neuro. Sci. 98, 277-286. 6 Dlott, B., D'Azzo, A., Quon, D.V.K. and Neufeld, E.F. (1990) J. Biol. Chem. 265, 17921-17927. 7 Bikker, H., Van den Berg, F.M., Wolterman, R.A., Kleijer, W.J., De Vijlder, J.I.M. and Bolhuis, P.A. (1990) Hum. Genet. 85, 327-329. 8 0 ' D o w d , B.F., Klavins, M.H., Willard, H.F., Gravel, R., Lowden, J.A. and Mahuran, D.J. (1986) J. Biol. Chem. 261, 12680-12685. 9 Rattazzi, M.C., Brown, J.A., Davidson, R.G. and Shows, T.B. (1976) Am. J. Hum. Genet. 28, 143-154. 10 Wood, S. (1978) Hum. Genet. 41, 325-329. 11 Winship, P.R. (1989) Nucleic Acids Res. 17, 1266. 12 Humphries, R.K., Ley, T.L., Anagnou, A.W. and Nienhuis, A.W. (1984) Blood 64, 23-32. 13 Takeshita, K., Forget, B.G., Scarpa, A. and Benz Jr, E.J. (1984) Blood 64, 13-22. 14 Myerowitz, R. and Costigan, F.C. (1988) J. Biol. Chem. 263, 18587-18589. 15 Lau, M.M.H. and Neufeld, E.F. (1989) J. Biol. Chem. 264, 21376-21380.
315
BBADIS 61135
Two small deletion mutations of the HEXB gene are present in D N A from a patient with infantile Sandhoff disease B e t h M c I n n e s 1, Charlotte A. B r o w n 1,2 a n d D o n J. M a h u r a n 1,2 1 Research Institute, Hmpitalfor Sick Children, Toronto (Canada) and 2 Department of Clinical Biochemistry, University of Toronto, Toronto (Canada)
(Received 30 August 1991)
Key words: Sandhoff disease; Hexosaminidase; Tay-Sachs disease; Gene mutation; (Infantile)
Lysosomal /~-hexosaminidase (EC 3.2.1.52) occurs as two major isozymes hexosaminidase A (a/3) and B (/~/~). The a subunit is encoded by the HEXA gene and the /3 subunit by HEXB gene. Defects in the ot o r / ~ subunits lead to Tay-Sachs or Sandhoff disease, respectively. While many HEXA gene mutations have been reported only three HEXB gene mutations are known. We report the characterization of two rare HEXB mutations present in genomic DNA from a single fibroblast cell line, GM203, taken from a patient with the infantile form of Sandhoff disease. The first is a single base pair deletion in exon 7 changing the codon for Gly-258, GGA, to GA and the second, a two base pair deletion in exon 11 changes the codons for Arg-435/Val-436, A G A / G T C , to AGTC. Each mutation produces a frame shift in the affected allele that results in a premature stop codon 17 or 20 codons downstream, respectively. These mutations also result in the inability to detect/3-mRNA by Northern blot analysis of total mRNA. These data are consistent with the idea that the severe infantile form of Tay-Sachs or Sandhoff disease is associated with a total lack of residual hexosaminidase A activity. Introduction
/3-hexosaminidase A (a/3) is a lysosomal enzyme responsible for the hydrolysis of G~2 ganglioside. Interest in the hexosaminidase system has been generated by the occurrence of a group of autosomal recessive neurodegenerative disorders known as the GM2 gangliosidoses. Three major categories of the disease are known depending on which of three different genes involved in the degradation of GM2 ganglioside is defective. The first gene product is the G~z-activator protein which serves as a transport protein for the ganglioside. The other two genes encode the subunits of heterodimeric hexosaminidase A. Defects in the a subunit (encoded by the H E X A gene on chromosome 15) result in Tay-Sachs disease and defects in the /3-subunit (encoded by the H E X B gene on chromosome 5) result in Sandhoff disease. The lack of functional hexosaminidase A leads to the lysosomal accumulation of GM2 ganglioside in brain and peripheral nervous tissue. These disorders displays a spectrum of clinical severity, ranging from the most severe infantile
forms to juvenile, chronic and adult-onset variants (reviewed in Ref. 1). Extensive homology in both the H E X A and H E X B gene structures demonstrate their common evolutionary origin [2]. While more than a dozen mutations in the H E X A gene have been described (reviewed in Ref. 3), only three mutation have been characterized in the H E X B gene [4-6]. Surprisingly one of these, a 16 kb deletion of the 5' end, accounted for 27% of the Sandhoff alleles analyzed [4]. This mutation is probably the same as that described by Bikker et al. using pluse-field gel electrophoresis [7]. In this report we describe a patient with the infantile form of Sandhoff disease who was a compound heterozygote for two rare H E X B gene mutations. One is a 1 bp deletion in exon 7, and the other is a 2 bp deletion in exon 11. We have previously shown that neither allele produces detectable /3-mRNA by Northern blotting [8]. This is presumably due to the mutations generation of premature stop codons in the resuiting reading frame shifts. Materials and Methods
Correspondence: D.J. Mahuran, Research Institute, The Hospital For Sick Children, 555 University Avenue, Toronto, Ontario, Canada, M5G 1X8.
Cell line GM203 The cell line was obtained from the Human Genetic Mutant Cell Line Repository, Camden NJ (U.S.A.).
316 The patient was of C a u c a s i a n / I n d i a n ancestry and was diagnosed at 1 year of age. Both the clinical and biochemical/enzymatic data available are indicative of a classical infantile Sandhoff phenotype [8-10].
PCR PCR was performed on genomic D N A diluted to (1.5 /xg/4 #1 which had been sheared by vortexing. The conditions used were those recommended by PerkinElmer Cetus Corporation except for the addition of 1U of Perfect Match (Stratagene). AmpliTaq polymerase was used in a 50 /.tl reaction volume. To amplify sequences from intron 6 through to intron 7, 250 ng each of oligonucleotides 525 (sense), 5 ' - A T C A A A T G C A A G C A C A A T T G T - 3 ' and 524 (antisense), 5'-GTG A C A G A A C A A G A C T C C A - 3 ' , were used as primers for 3(I cycles of 94°C for 30 s, 56-58°C for 30 s, and 72°C for 90 s. This resulted in a 394 bp fragment which was sequenced directly (below). To amplify sequences from intron 9 to intron 11 oligonucleotides 528 (sense), 5 ' - C A C C T C T C A A A A T G C A A G A A 3 ' , and 529 (antisense), 5 ' - A A T G G T T G C T T C A C T T A C C A - 3 ' , were used as primers for 30 cycles of 93°C for 1 min, 60°C for 1 min and 72°C for 3 min. This resulted in a 1058 bp fragment which was sequenced directly (below). Direct sequencing To avoid detection of Taq artifacts in cloned PCR fragments, we directly sequenced the products of each PCR. PCR fragments were isolated by agarose gel electrophoresis and purified with Geneclean Kit (Bio 101). The gel-purified fragments were sequenced with 500 ng of oligonucleotide 524 or 525 (above) for the
exon 7 mutation ~r oligonucleotidc 52 ( 5 ' - ( q ( - C A A T C T T G T C C A T A G C T - 3 ' ) for the mutation in exon 11. The method of direct sequencing used was adapted from the Sequenase protocol (United Stales Biochemical Corp.), as previously reported [11]. Results
Direct nuclcotide sequencing of PCR fragments was performed on all 14 exons and exon/intron junctions with the exception of the extreme 5' end of the H E X B gene (data not shown) from fibroblast line GM203, Two mutations were detected. The first, a 1 bp (G) deletion in exon 7, changes the codon for Gly-258, GGA, to G A (Fig. 1), and results in a frame shift which introduces a stop codon 17 codons downstream. The second, a 2bp (AG) deletion in exon 11 changes the codons for Arg-435/Val-436, A G A / G T C , to AGTC. This also results in a frame shift with a stop codon produced 20 codons downstream (Fig. 2). Analysis of our collection of fibroblasts from 19 Sandhoff patients [4,8] demonstrated that both mutations were unique to this cell line. Thus, the patient was a compound heterozygote for two rare Sandhoff mutations. Discussion
The identification of two allelic d e l e t i o n / f l a m e shift mutations in the cell line GM203 from a patient with the infantile form of Sandhoff disease is consistent with the hypothesis that the infantile phenotype of Tay-Sachs or Sandhoff disease is associated with a complete absence of residual hexosaminidasc A activity.
GM203
Normal Exon 7 T Ser263 C G Leu262 T
T.
T Set261 C T Tyr260 A
1 C Ser259 G
A Gly258 G
G g a c
G
A
T
C
G
A
T
C T C C
T Leu263
T G
c T
T
G Cys262
T T C
T T Leu261
T
_~
T A
T T lie260
T
.~.
C G
T C Ala259
A A A G~A Glu258 GMG
g
g
a c
a c
Intron
Fig. 1. Autoradiogram of a 6% polyacrylamide/urea gel showing sense sequence obtained for genomic DNA from patient GM203 and from a normal individual in the intron 6/exon 7 region of the HEXB gene. Patient GM203 is heterozygousfor 1 bp (G) deletion in exon 7 which changes the codon for Gly-258, GGA, to GA (Fig. 1). This results in a frame shift with a stop codon produced 17 codons downstream.
317 GM203
G A
T T Leu 433 Ser 434 Ser 435 His 436 Ser 437
Ile 438
normal T
C~G
G A G T C A *T C A G T G T C fi T A
"
A G T C A T C I C A G T G T ¢
Leu 433
G
Ala 438
Ser 434 Arg 435 Va1436 Thr 437
T
A
.~
oj--
A
A
Ser 439
Fig. 2. Autoradiogram of a 6% polyacrylamide/urea gel showing antisense sequence obtained for genomic DNA from patient GM203 and from a normal individual in exon 11. Patient GM203 is heterozygous for a 2 base pair deletion (AG) at Arginine-435/Valine-436 in exon 11. This results in a frame shift with a stop codon produced 20 codons downstream.
We had previously found that this cell line lacked residual hexosaminidase A activity and detectable /3m R N A by Northern blot analysis [8]. The mechanism by which a mutation causing a premature stop codon results in a low steady-state level of affected m R N A is unclear. One possible explanation is that the 3' end of the m R N A is no longer protected by ribosomes and is therefore more susceptible to RNAse digestion. Such a theory would predict that the length of the 3' untranslated end of an m R N A would be inversely proportional to its stability. Alternatively, a defect in /3-globin m R N A metabolism has been shown to be unrelated to cytoplasmic instability, suggesting that the premature stop codon in this case alters intranuclear stability a n d / o r cytoplasmic transport [12,13]. The major mutation responsible for Tay-Sachs disease in the Jewish population is a 4 bp insertion in exon 11 of the HEXA gene. This produces a stop codon 6 codons after a-Ile-425 (aligns with /3-Ile-454) and like the two /3 mutations we report here, no detectable ce-mRNA by Northern blot analysis [14]. Interestingly, in the aligned sequences of the a and /3 cDNAs the stop codon generated by this HEXA gene mutation occurs within 6 codons of that generated by the 2 bp deletion mutation in the HEXB gene described here. However, another such HEXA mutation in exon 13 produces normal steady-state levels of am R N A a n d a n a polypeptide that is shortened by 22 r e s i d u e s a t its c a r b o x y t e r m i n u s [15]. T h u s , the present d a t a suggests that the length of the 3 ' u n t r a n s l a t e d e n d o f t h e a - a n d / 3 - m R N A is inversely proportional to m R N A stability.
Acknowledgments T h i s w o r k w a s supported through a M e d i c a l R e search Council of Canada operation grant No. MA10435 t o D . M . F u n d i n g f o r B . M . w a s t h r o u g h a fellowship awarded by The Hospital F o r S i c k C h i l d r e n F o u n dation.
References 1 Sandhoff, K., Conzelmann, E., Neufeld, E.F., Kaback, M.M. and Suzuki, K. (1989) in The Metabolic Basis of Inherited Disease (Scriver, C.V., Beaudet, A.L., Sly, W.S. and Valle, D., eds.), pp. 1807-1839, McGraw-Hill, New York. 2 Proia, R.L. (1988) Proc. Natl. Acad. Sci. USA 85, 1883-1887. 3 Mahuran, D.J. (1991) Biochim. Biophys. Acta 1096, 87-94. 4 Neote, K., Mclnnes, B., Mahuran, D.J. and Gravel, R.A. (1990) J. Clin. Invest. 86, 1524-1531. 5 Mitsuo, K., Nakano, T., Goto, I., Taniike, M. and Suzuki, K. (1990) J. Neuro. Sci. 98, 277-286. 6 Dlott, B., D'Azzo, A., Quon, D.V.K. and Neufeld, E.F. (1990) J. Biol. Chem. 265, 17921-17927. 7 Bikker, H., Van den Berg, F.M., Wolterman, R.A., Kleijer, W.J., De Vijlder, J.I.M. and Bolhuis, P.A. (1990) Hum. Genet. 85, 327-329. 8 0 ' D o w d , B.F., Klavins, M.H., Willard, H.F., Gravel, R., Lowden, J.A. and Mahuran, D.J. (1986) J. Biol. Chem. 261, 12680-12685. 9 Rattazzi, M.C., Brown, J.A., Davidson, R.G. and Shows, T.B. (1976) Am. J. Hum. Genet. 28, 143-154. 10 Wood, S. (1978) Hum. Genet. 41, 325-329. 11 Winship, P.R. (1989) Nucleic Acids Res. 17, 1266. 12 Humphries, R.K., Ley, T.L., Anagnou, A.W. and Nienhuis, A.W. (1984) Blood 64, 23-32. 13 Takeshita, K., Forget, B.G., Scarpa, A. and Benz Jr, E.J. (1984) Blood 64, 13-22. 14 Myerowitz, R. and Costigan, F.C. (1988) J. Biol. Chem. 263, 18587-18589. 15 Lau, M.M.H. and Neufeld, E.F. (1989) J. Biol. Chem. 264, 21376-21380.