cDNA comprising the entire length of the human muscle glycogen debranching enzyme was cloned and its nucleotide sequence determined. The debrancher.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Swiety for Biochemistry and Molecular Biology, Inc.
VOl. 267, No. 13, Issue of May 6, pp. 9294-9299 1992 Printed in ~.S.A.
Molecular Cloning and Nucleotide Sequence of cDNA EncodingHuman Muscle Glycogen Debranching Enzyme” (Received for publication, December 31, 1991)
Bing-Zhi Yang$, Jia-Huan Ding$, Jan J. EnghildQ, YongBao$, and Yuan-Tsong Chen$V From the Department of $Pediatrics and §Pathology, Duke University Medical Center, Durham, North Carolina27710
cDNA comprisingthe entire length ofthehuman brancher glucosidase hydrolyzes the remaining a-1,6 branch was cloned and point glucose residue. Full debranching enzyme activity remuscle glycogen debranching enzyme its nucleotide sequence determined. The debrancher quires bothcatalytic activities. The presence of glycogen mRNA includes a 4545-base pair coding region and a debranching enzyme has been demonstrated in many species, 2371-base pair3”nontranslatedregion.Thecalcuincluding yeast (6),parasite (7), fish (8),and mammal (1, 2, lated molecular mass of the debrancher protein derived 9); the debrancher cDNA, however, has not yet been cloned fromcDNAsequence is 172,614 daltons, consistent nor has the primary structure of this multifunctional enzyme (M, 165,000 been determined in any species. with the estimatedsize of purified protein f 600). A partialaminoacidsequence (13 internal Genetic deficiency of glycogen debranching enzyme in man tryptic peptides with a total of 213 residues) deter(Type I11 glycogen storage disease, GSD-111)’ causes hepatomined on peptides derived from purified porcine musmegaly, hypoglycemia, short stature, and variable myopathy cle debrancher protein confirmed the identity of the (10). Some features of GSD-I11 suggest that molecular analycDNA clone. Comparison of the amino acid sequence predicted from the humanglycogen debrancher cDNA sis of this disease and gene coding of debranching enzyme may contribute significantly to our knowledge of the mechawith the partial protein sequence of the porcine debrancher revealed a high degree (88%)of interspecies nisms involved in theregulation of tissue-specific gene expressequence identity.RNA blot analysis showed that de- sion. In GSD-111, therearenaturally occurring mutations affecting the expression of the debranching enzyme in virbranchermRNA in humanmuscle,lymphoblastoid cells, and in porcine muscleare all similar in size (-7 tually all tissues, and there are mutations affecting the dekilobases). Twopatients with inheriteddebrancher branching enzyme limited to certain type of tissues; yet the deficiency had a reduced level of debrancher mRNA, enzyme is a monomeric protein and appears to be identical in whereas two other patients had no detectable abnor- all the tissues (9-12). Cloning and sequencing of debrancher mality in RNA blots. The isolation of the debrancher cDNA are prerequisite for studying the molecular basis of this cDNA and determination of its primary structureis an disease and understanding the controlling mechanisms inimportant step toward defining the structure-functionvolved in tissue-specific gene expression and the structurerelationship of this multifunctional enzyme and in un- function relationship of this interesting multifunctional enderstanding the molecular basis of the111type glycogen zyme. storage disease. We reported previously on the purification of glycogen debranching enzyme, characteristics of antibody, and immunoblot analysis of the enzyme in different subtypes of GSDGlycogen debranching enzyme is a large monomeric protein I11 (9, 13). Using this antibody to screen the human muscle (Mr= 165,000 +. 500) with two independent catalytic activi- cDNA expression library, we now report the isolation and ties, 1,4-a-D-g1ucan:1,4-cY-D-glucan 4-a-~-glycosyltransferase nucleotide sequences of cDNA encoding the complete human (EC 2.4.1.25) activity and amylo-1,6-glucosidase (EC 3.2.1.33) muscle debrancher protein. activity, occurring at separate sites on a single polypeptide EXPERIMENTALPROCEDURES chain (1-5). The debranching enzyme together with phosphorylase are responsible for complete degradation of glycogen. Construction of the Human Muscle cDNA Library in Agtll ExpresAfter phosphorylase has hydrolyzed the outer glucose resi- sion Vector-Total RNA was prepared from human muscle obtained dues, the glycogen chains contain short outer branches. De- from Duke University Medical Center Tissue Bank by the quanibrancher transferaseactivity transfers 3 glucose residues from dinum chloride extraction method (14). Poly(A)+ RNA was isolated oligo(dT)-cellulose column chromatography. Double-stranded one short branch to the end of another and then the de- by cDNA was synthesized from poly(A)+ RNA with either oligo(dT) or
* This work wassupported by National Institutes of Health Grants DK 39078 and Mol-RR30, National Center for Research Resources, General Clinical Research Centers Program, a grant from the Muscular Dystrophy Association, and the Duke GSD fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s)reported in this paper hos been submitted to the GenBankTM/EMBL DataBank withaccession numbeds) M85168. ll To whom correspondence and reprints should be addressed Dept. of Pediatrics, Duke University Medical Center, P. 0.Box 3028, Durham, NC 27710. Tel.: 919-684-2036; Fax.: 919-684-8944.
random hexanucleotides as primers, using a cDNA synthesis system kit (Amersham International). The system is based on the method described by Gubler and Hoffman (15). Methylation of internal EcoRI sites, addition of linkers, digestion of linked cDNA, separation of cDNA from excess linker molecules, insertion of cDNA into Xgtll arms, and in vitro packaging of recombinant phages were performed, using the Amersham cDNA cloning system kit according to manufacturer’s instructions. The yield of recombinants was 4 X 106/pg of cDNA. Immunoscreening of Agtll cDNA Expression Library with Anti-
’
The abbreviations used are: GSD, glycogen storage disease; PCR, polymerase chain reaction; HPLC, high performance liquid chromatography; kb, kilobase pair(s).
9294
Nucleotide Sequence of Human Glycogen Debrancher cDNA body-The library was screened with antibody, as described by Huynh et al. (16). A polyclonal antiserum raised in rabbits against purified porcine muscle debranching enzyme was prepared as described previously (9,13). Thisantiserum was further affinity-purified by adding antiserum (0.5 ml) to a nitrocellulose disk containing 20 pg ofpurified debranching enzyme. The specific antibody eluted was used as the probe for immunoscreening. Positive clones were identified using an alkaline phosphatase-linked immunodetection system with anti-rabbit IgG as a second antibody (Protoblot immunoscreening system, Promega Biotech). Positive phages were purified to homogeneity, and their cDNA inserts were isolated and subcloned into the plasmid vector pGEM-3zf(+) (Promega Biotech). Screening of the library with 32P-labelednucleic acid probes was as described by Benton and Davis (17). Cloningof 3’ and 5’Ends cDNA-The RACE (“rapid amplification of cDNAends”) protocol was used to synthesize cDNA corresponding t o the 5’ and 3’ end of the transcript (18). For the 5’ end clones, poly(A)+ RNA from human muscle was subjected to reverse-transcription with the use of 5’ gene-specific primers. Two primers were used, one span from debrancher cDNA position 641 to 661 and another from 868 to 885 (See Fig. 2). After removal of excess dNTP and primers, the cDNA wastailed with dATP and terminal deoxynucleotidyl transferase (Bethesda Research Laboratories). Second strand synthesis was performed with AmpLiTag DNA polymerase (Perkin-Elmer Cetus), using an oligonucleotide primer containing (dT)17and an adaptor sequence with endonuclease recognition sites. This adaptor, together with another primer (cDNA position from 280 t o 302), was then used for PCR. The PCR products were directly inserted into aplasmid vector PCR” 1000 (Invitrogen Corp.). The 3’ end clones were generated with the use of similar procedures. The first strand was synthesized with an oligonucleotide containing a (dT),7-adapter sequence. PCR amplification was carried out with the linkers sequence and a 3’ end gene specific primer (cDNA position from 5368 to 5386). All PCRs were performed for 35 cycles. DNA Sequencing-The nucleotide sequence was determined directly from double-stranded DNA using the dideoxy-chain termination method (19) with 35S-dATPas thelabeling component. T7or Sps promotor oligonucleotide primers and a Sequenase DNA sequencing kit (United Stated Biochemical Corp.) were employed. The strategy used for cDNA sequencing is outlined in Fig. 1. When no appropriate fragments could be obtained, an oligonucleotide complementary to the debrancher cDNA was used as a primer. In the phage cDNA inserts containing internal EcoRI sites, the region flanking the restriction enzyme sites was sequenced to confirm that EcoRI does not cut out an additional fragment. The data base of the nucleotide sequence represents an average nucleotide reduncancy of approximately four, both strands being covered throughout the entire sequence. Trypsin Digestion, Peptide Separation, and Amino Acid Sequence Analysis-Due to the difficulty in obtaining sufficient amounts of human tissue, porcine muscle debranching enzyme was purified as described previously (9). Approximately 2.5 mgof purified porcine muscle debranching enzyme was reduced with 5 mM dithiothreitol and alkylated with 15 mM iodoacetic acid in 6 M guanidine hydrochloride, 200 mM Tris-HC1,pH 8.3. The alkylated sample was dialyzed into 50 mM NH,HCOs and dried in a Speed Vac (Savant). The dry protein was dissolved in 5 ml of 50 mM Tris-HC1, pH 8.3, and digested with 50 pgof bovine trypsin (sequencing grade, Sigma) at 25 “C overnight. Following the enzymatic digestion, the peptides were separated by reverse-phase HPLC by using various columns supplied by Applied Biosystems (Aquapore RP-300, 4.6 X 250 mm and 2.1 X 220 mm), Vydac (Vydac Cia, 2.1 X 250 mm) and Beckman (Ultrasphere Octyl, 2 X 250 mm). The initial peptide separation was performed on the Aquapore RP-300 4.6-mm column employing an LKB 2152 controller and two LKB 2150 pumps to form a gradient from (a) 0.1% trifluoroacetic acid to ( b ) 90% acetonitrile, 9.9% HzO, 0.1% trifluoroacetic acid. The peptides were further purified in an Applied Biosystems 130A HPLC, using the narrow-bore columns and the solvent system described above. Automated Edman degradation was carried outin an Applied Biosystems 477A sequencer with on-line analysis of the phenylthiohydantoins, using an Applied Biosystems 120A HPLC. Samples were applied to Portonprotein or peptide sample pupport disks and Edman degradation was carried out employing the modified cycles Pl-BGN and P1-1 recommended by Porton Instruments. Northern Blot Analysis-Poly(A)+ RNA was fractionated on 1% agarose formaldehyde denaturing gel, transferred to a nylon mem-
9295
brane, and hybridized to DNA probes, radiolabeled with 32P,by the random primer DNA-labeling method (20). To study lymphoblastoid cells, RNA probes were synthesized from cDNA cloned in PGEM vector (Promega Biotech) with T7 or Sps RNA polymerase (21). Computer Analyses of Sequences-Nucleic acid and protein sequence and sequence homology analyses were performed using the software packages compiled by the Mac Vector Sequence Analysis Programs (International Biotechnologies, Inc.) and theUniversity of Wisconsin Genetics Computer Group Computer Programs.
RESULTS ANDDISCUSSION
Isolation of Glycogen Debrancher cDNA Clones-Approxi-
mately lo6 plaques of a human muscle cDNA library primed with random hexanucleotides andconstructed in a Xgtll expression vector were screened using the polyclonal antiporcine debrancher antibody asthe probe. Fifty position clones were obtained. Southern hybridization showed these clones could be grouped into four major groups, each with the presence of overlapping cDNA inserts. Group 1contained five overlapping clones (inserts ranged from 0.3 to 0.9 kb) and hybridized to a mRNA species of 2.8 kb in size. Group 2 contained three overlapping clones (inserts ranged from 0.6 to 0.9 kb) and hybridized to a mRNA species of 1.5 kb. These clones were not further studied because they hybridized to a mRNA species smaller than theminimum size of the mRNA coding unit for debranching enzyme. Group 3 contained nine overlapping clones with inserts ranging from 1.4 to 2.8 kb. Group 4contained seven overlapping clones with inserts ranging from 0.3 to 0.8 kb. Both groups hybridized to a mRNA of -7.0 kb (see below). The largest clone in each group (D-71 and D-35, Fig. 1)was used as the probe to rescreen the same library and also a library constructed with oligo(dT) as the primer. Eleven positive clones were isolated. Three of these 11 clones bridge D-71 and D-35, indicating that the original groups represented portions of a single large cDNA species. The immunoreactive clones D-71, D-35, and the threelargest clones obtained through rescreening (D-155, D-157, D-159), were subjected to nucleotide sequencing analysis, according to the strategy shown in Fig. 1. The inserts of the different clones sequenced derived, in all likelihood, from a common transcript, since all of the overlapping regions sequenced were identical. None of the cDNA clones contained the poly(A) tract. Further cDNA clones corresponding to the 5’ and 3’
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FIG. 1. Partial restriction mapand sequence strategy of glycogen debrancher cDNA. D-35 and D-71 are cDNA inserts contained in phage isolated by immunoscreening. D-157, D-159,and D-155 are cDNA inserts obtained by screening with DNA probe D35 and D-71. Further 5‘ and 3’ ends of cDNAwere obtained by RACE protocol (see “Experimental Procedures” for details). The arrows indicate the direction and extent of sequencing. Blackened region denotes the coding region of the debrancher mRNA.
Nucleotide Sequence of Human Glycogen Debrancher cDNA
9296
ends of the transcriptwere derived from experiments with the "RACE" procedure (18). Nucleotide Sequenceof Human Muscle Glycogen Debrancher cDNA-The nucleotide sequence constructed from the overlapping cDNA clones and the5' and 3' ends of clones together contain 6961 bases, plus a tract of poly(A) (Fig. 2). The size of the cDNA corresponds to thesize of the debrancher mRNA (-7 kb), estimated by Northern blot analysis from both human and porcine muscle poly(A)+ RNA (Fig. 3). The coding region of debrancher cDNA contains 4545 nucleotides, starting with the putative initiation codon contained within the sequence ACGATGA which could be part of the consensus initiation sequence described by Kozak (22). The sequence encodes a polypeptide of 1515 residues with a calculated molecular mass of 172,614 daltons, consistent with the molecular weight (Mr = 165,000 f 500) of the purified protein estimated by gel filtration, gel electrophoresis, and sedimentation equilibrium methods (1-3). The 3'-nontranslated region is 2371 bases long, which begins with the stop codon at
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N
0 1876 AGTACTACMTTGTTTCTATGGCATGTTGTGCTAGTGGMGTACMGAGGCTATGATGM
376 TGGGIGCAGACTTAGGGTTGCAAAAGMTCAGGCTACMCATGATTCATTTTACCCCA W
P
1816 GATATTACGCATGATMTGAGTGTCCTATTGTGCATAGATCAGC~TATGATGCTCTTCCA
3 1 6 CCCTTGGACTGTGTTACTCTTCAGACATTTTTAGCTMGTGTTTGGGACCTTTTGATGM P
C
Q
GCATATMTAGTCATGMGAGGGCAGATTAGTTTACCGATATGGAGGA~CCTGTTG~
S
85
S
N
N
L
CTGGACMTGTCTTTGTTACTAGACTGGGCATTAGTTCCTTMTMGAGAGGCMTGAGT
A
GGTGGAGGTTACATAGTTGTGGACCCCATTTTACGTGTTGGTGCT~TMTCATGTGCTA G
D
N
1756 TCCTTTGTTCAGCCCTGTTTGAGGCCTTTMTGCCAGCTATTGCACATGCCCTGTTTATG
196 CTTMTCTGCMCMTCTGGTTCATTTCAGTATTATTTCCTTCMGGITGAGAAAAGT 256
I696
L
R
L
65
K
R
GCTAGGMTTTGCMCCCMTTTATATGTAGTAGCT~MCTGGAT A
p
C
u
r
o
A
1
L
r
c
A
r
T
T
L
L
D
3016 TGGMGCACATGTCMGCTTTGTTCAGMTGGTTCMCCTTTGTG~CACCTTT~TTG G
~
~
~
~
n
p
n
s
s
r
v
~
~
~
s
FIG. 2. Nucleotide sequence of human muscle glycogen debrancher cDNA and its deduced amino acid sequence. The positive numbers of nucleotides start at thefirst residue of the coding sequence. Amino acids are designated by the single-letter code. Underlined regions represent the matching porcine muscle debrancher sequence obtained by automated Edman degradation of the tryptic peptides with substitutions as indicated. Parentheses indicate equivocal amino acid assignments. Boxes contain the initiation coding, stop codon, and polyadenylation signal sequences.
~
Y
~
r
L
~
v
~
s
~
~
of Human Glycogen Debrancher cDNA
Nucleotide Sequence
9297
4696 ATTTTTTTTAATCTACAGAGGTAGATTTCAATTTGAATCAGAAAGAAATATCATTACCAA 3076 GGTTCAGTTCMCTCTGTGGAGTAGG~TTCCCTTCCCTGCCMTTCTTTCACCTGCC G
~
V
~
L
C
G
V
G
K
F
P
~
L
P
I
3136
CTMTGGATGTACCTTATAGGTTITGAGATCAC~G~GGAGCMTGTTGTGTT
3196
TCTCTACCTGCAGGCTTACCTCATTTTTCTTCTGGTATTTTCCGCTGCTGGGGMGGGAT
3316
ATTATTTTAGCATTTGCGGGTACCCTGAGGCATGGTCTCATTCCTMTCTACTGGGTGM
L
~
D
~
P
Y
R
L
N
E
I
T
K
E
K
~ L A A G L P H F ~ ~ G I F R C W C 3256 ACTTTTATTGCACTTACACGTATACTGCTGATTACTGGACGCTATGTAGAAGCCAGGMT T I
F
I
I
A
L
L
A
R
F
C
A
I
G
L
T
L
L
I
R
T
H
G
G
R
L
Y
I
V
P
E
N
A
L
G
I
Y
A
R
Y
N
C
R
D
A
V
W
W
~
D
I
O4936~ AATACCCTCTGGTTCATTAATATTARCACCACCACGTACTATAGTATTCTTAGMTACAGTGC ~
N
~
~
~
G
L
4816 ATARRAATCATGTCATCTTCTATTTGTACAGAAATGMAATAAAATATGMAATAATGAA ~ ~ ~ I O ~ ~ 4876 AGAAATGMAAGATAGCTTTTMTTGTGTGGTATATATAATCTTCAGTAACAATACATACTG 4996 TCACTGCATTTAATARRTTATTTAATAAATGATGMTGATAGAAGTTTCCATCTACAATAT
E
O
~
C
~
I
~
~ 5116 ATTTTTCTGTAATCAATGCCAACAGTATATTTTATATGATTTACTTATGTGAGGAAACAT
I 5176 I ~ GCARRTGCATTAGGAAATTTTTTCCTARRAACAGTTTTGTMAATTAGTATTGAGTTCTA ~
P
IC)-
5236 TTCAGTATTATAAGATAGCTTACRTTTTTCMAATGGAAATTGTCGGTCATATTTCTAGAA
3436 G A T T A C T C T ~ T G G T T C C I T G G T C T A G A C A T T C T C M G T G C C C A G T T T C C A G M T G D
Y
;
,
K
B
V
P
N
G
L
D
I
L
K
C
P
V
S
R
M
l
l
6
5
5296 CTTTAAAGAAAAMGAATGTTATATTAGTTTTCTMAACTCAACTATCTTTAGTCATGTT
3496 TATCCTACAGATGATTCTGCTCCTTTGCCTGCTGGCACACTGGATCAGCCATTGTTTG~ Y
3556
T
D
D
S
A
P
L
P
A
G
T
L
D
O
P
L
F
E
1
)
8
N
A
5356 C~TCTATTGCTAGATCATAGTAGATACTGGTTTTCTATTAACTCMAACCTACATT
5
5416 GACAAGTTTAACATTGAGAGAATCTTAAC~TATGGATATGAATTCAGTAGATATC
GTCATACAGGMGCMTCC~CACATGCAGGGCATACAGTTCCGAGIGGMTGCT
V
3616
P
I
Q
E
A
~
Q
K
H
~
O
G
I
O
F
R
E
R
l
2
0
5
5476 TTAAATTCAATMAATCACTGGAAGTTTTTCATGATAACTTATTTTAAGATGCCTTMAA
GGTCCCCAGATAGATCGICATGMGGACGMGGTTTTMTATMCTGCAGGAGTTGAT G P Q I D R N ~ K D E G F N I T A G
V
D
~
~ ~ ATCTTARRTGTCACMAAGGAAARRGGTTTTTMCATTTACATGAGTTAACATTTTTTCAT ~ 5536
D
K
~
5596 ~ ~ ACAACTTATTTCCTAGATAGAATTTTTTACTGTTTTTTACTGTTTTCTTMG~CAGT ~
3676 C~ICAGGATTTGTTTATGGAGGITCGTTTCMTTGTGGCACATGGATGGATI E
3736
T
W
ATGGGAG~AAGTCACAGAGCTAGICAGAGGMTCCCAGCCACACCMGAGATGGGTCT ~ G ~ S D R A ~ N ~ G
E
T
G
F
V
Y
G
G
N
R
F
N
C
G
~
~
5656 TAAATCATTATCCATTCAGTTGGAAGAAAGTAGTGGCAAGAATTCTTTCATTGCTATATA P
A
T
3796 GCTGTGG~AATTCTGGGCCTGAGT~AATCTGCTGTTCGCTGGTTGCTGGMTTATCCI A
V
E
K
N
I
I
V
G
L
~
K
~
A
V
R
K
R
H
W
L
L
E
L
03856~TATTTTCCCTTATCATGMGTCACAGThGACATGGIGGCTATIGGTC
3916
F
P
Y
H
E
V
T
V
G
K
A
I
S
K
Y
D
E
W
N
R
K
I
O
D
N
F
E
K
L
F
V
~
V
~
~
H
~
3976 G M G A C C C T T C A G A T T T ~ A A T G ~ G C A T C C ~ A A T C T G G T T C A C ~ A A C G T G G C A T A T A C E
D
P
~
D
L
N
E
K
H
P
N
L
V
H
K
R
G
I
Y
~
D
S
Y
G
A
S
S
P
W
C
D
Y
Q
L
R
P
N
F
T
l
3
6
5
A
~
V
V
A
P
E
L
F
T
T
E
K
A
W
K
A
L
E
A
E
K
K
L
L
G
P
L
C
B
K
T
L
D
P
Y
C
G
I
Y
D
N
A
L
D
N
D
N
Y
N
D
L
D
B
~
~
N
Y
H
P
G
P
E
W
L
W
P
I
G
~
A
F
K
G
~
~
L
R
A
K
Y
V
F
L
~
S
R
R
H
L
Y
~
V
I
H
G
L
P
E
R
E
S
T
P
W
T
K
A
G
K
L
T
P
I
E
I
V
4
4456 CTGACCMTGAGMTGCCCAGTACTGTCCTTTCAGCTGTG~AACACMGCCTGGTCMTT L T N E N A ~ Y ~ P F ~ ~ E 4516
L
T
I
L
L
T
L
Y
D
L
V
4576 ACTGTATTATAGGATGCMGGTCATCATATGTITGCTATATGCACAGGCTCMGTTGT 4636 TTT-TCTCATTTATTATMTATTGATGCTCMTTAGGTMGATTGT~G~TTG
8
V
R
~
~
~
5 8 9 6 TCAGATGTCAAATACCCATGCTTGAAAGCTCGTGTMTTTACTTTAAGATTATCTGCCTG 59~ 56 ~CTCTTCTTCAAAGCTGACCTTGCTTTAGAAATAGTTTTAACTAGCTTAGTTTTCTGGTTT ~ 6 0 1 6 CCMAACT-TAGATTAAATCCTACAAATTTAAGGACAGTTGTGACAGTAATCTGACC ~
~
~
~
6~3 7 6~ GTTATCTCTAGTAAGGCAGATACCCACGTTGGTARRTTTTTTAGGATATTGTGTTGCACTA 6 4 3 6 GMAACTAAGTGGTTCATATTTCTAATGAGGMGATTAATGAAAGAACATTGTTAT~TTC ~
~
~
1515
6~ 5 5~6
~ ~ MAATACAGMTTAGATTTTTCAGGTGTCATTTGACTAAACGTTTCGGTAGAATGCTT
6 61 6 CATACTTGAGTCATGCTGGATAAGGTATTGTATTTCAACAATGGACTATGCCTTGGTTTT
5
T
TTATTACAGATATTMGTATGCMTT
A
GCTACTATTCTTGAGACACTTTATGATTT
A
~
6 4 9 6 TGCGTGGTATATTTTAAAGTTTAAGAAGGCATGTTAAACATTATTTCCTCTATGGTAGTT
4396 MTGTTCTTTCCCGACATTATGTTCATCTTGAGAGATCCCCTTGG~AAGGACTTCCAGM N
~
5776 ~ ~ARRTGTACCATTTTTTTAGTTAGTTTACAGGTTACATACCCMAACCTTAACTATGACTAA ~ ~
~
~
4336 TTATATTTTTCCAGATTGATGGGCCCGGAGACTACTGCIGACTATAGTTTTGGTTI L
~
6 3 1 6 ACAGTTAAATGTGACCAMAMATTMAAGTTCACAATTTTTTTAATGTAGCCATTTGGG
4276 TTCMTTATCACCMCGACCTGAGTGGCTGTGGCCTATTGGGTATTTTCTTCGTGC~ F
~
6 2 5 6 GCGAGCTAGGAAAGGTAACAGI\AAACCAGCATATTTAATCAAAGCAAGAAGTAATCGCTG
42 16 GTTTACTGTGGMTTTATGACMTGCATTAGACMTGACMCTACMTCTTGCT~AAGGT V
G
6~1 9 6~ AACCATTAGTTTATCAAAGGTTTATGTAGTAGTTTTGTTGCTGTACCCTAACTTTGATATTCA ~
~
4156 A T T G C A G ~ T T G C T T G G T C C C C T T G G C A T G ~ C T T T A G A T C C A G A T G A T A T G I
D
6 1 3 6 TTGTTCMTATATACCCTTCTCTAAACTGTGCGGGTMAAGGAATGACTGTCCTTGAGAG
4096 ATAGCMTGGTTGTGGCCCCTGAGCTCTTTACTACAG~GCATGG~AAGCTTTGGAG I
~
6076 ACTATCTATAAATACATTGGACATTGGTTTCCARRTTCTCCCTTTCTCTTCAGTTCCTTCC
4036 IGATAGTTATGGAGCTTCMGTCCTTGGTGTGACTATCAGCTCAGGCCTMTTTTACC K
P
5716 ATATTCAGTGCCTCATTTATACCTAATMAATAATGGTATTTTMAATAATGCTACTTTC 5836 GAAATTAAAGAACMAACCAGCARRTCTMAACTTCTGGGCAGC~TATATAAATGCT
R
TCATATGATGACTGGMCAGhTACMGACMCTTTG~GCTATTTCATGTTTCC S
~
5056 ATGTTCCTAAATCGAGCACAGATGTTCARRTCTATGCTTTCATTTTTTCACTGATATATTA
L
W
E
4756 TGAAATGTGTTTGAGTTCAGTAAGAATTATTCAAATGCCTAGAAATCCATAGTTTGGAAA P A ~ ~ ~ ~
~
R
R
3376 GGCATTTATGCCAGATACMTTGTCGGGATGCTGTGTGGTGGTGGCTGCAGTGTATCCAG
L
Q
A
6 6 7 6 TCACTAATC~~RAATCMAATTACTCTCTTTMCATGATAAATGAATTTACCAGTTTAGTATG ~ ~ I ~ ~ O ~ 6 7 3 6 CTGTGGTATTTTAAT~GTTTTCAAAGATAA~TGGGMAACA~G~GACTGGTCATATTGA 6 7 9 6TGAATATTGTAACATGTGAATTGTGTGATCCATTTCTGATATGTCTTGAACTACTGTGTCTA ~~~~GTAGCCAAATGTCATTGTTACCTCTGTGTGTTAAG~TATTTTC~~GGTC
691 6
P G
FIG.2.-continued
were matched with the amino acid sequence predicted by the human muscle debrancher cDNA. In thiscomparison only 25 A B differences were observed, resulting in 88%sequence identity 1 2 1 2 kb on the amino acid level. The differences consist mainly of -23.1 single amino acid substitution, with the notable exception - 9.4 that a proline is inserted between residue 171 and 172. In -0 - n r - 6 . 6 those amino acids which differ between species, the majority 28s * - 4.4 of the substitutions are conservative. The marked similarity is present throughout the available protein sequence, suggest-- 2.3 2.0 ing a selective pressure to maintain the structure. 18sNorthern Blot Analysis of Type 111Glycogen Storage Disease-To investigate the levels of mRNA encoding debranching enzyme in patientswith type I11 glycogen storage disease, - 0.6 we studied lymphoblastoid cells from these patients.Because the debrancher mRNA is present in relatively low amounts FIG. 3. Northern blot analysis of poly(A)+ RNA from muscle in the lymphoblastoid cells as compared with the debrancher of porcine (lane I ) and human (lune 2),probed with human debranching enzyme immunoreactive cDNA clones D-71 (A) mRNA inthe muscle (data notshown), we used a radiolabeled and D-35 ( B ) . 5 fig of poly(A)' RNA was separatedon a 1% antisense RNA derived from clone D-71 as the probe in our denaturing agarose gel, transferred to a nylon membrane, and hybrid- RNA blot study. As shown in Fig. 4,the lymphoblastoid cell ized with :'2P-labeled cDNA probes. HindIII-digested phage XDNA debrancher mRNA is about 7 kb, similar in size to the defragments were included as molecular weight markers (fragment size brancher mRNA in the muscle. Two mutant cell lines from in kilobase pairs). GSD-I11 patients showed reduced amounts of debrancher mRNA (lunes 1 and 3), whereas the debrancher mRNA of
Nucleotide Sequence of Human Glycogen Debrancher cDNA
9298 I
Debrancher mRNA
2
3
4
5
6
7
+
-28s
FIG. 4. Northern blot analysis of poly(A)+RNA from human lymphoblastoid cell lines. 5 pg of poly(A)+RNA was separated on a 1% denaturing agarose gel, transferred to a nylon membrane, and hybridized with "P-labeled antisense RNA probe synthesized from debrancher cDNA clone D-71. Lanes I , 3, 6,and 7 contained RNA from patients with type 111 glycogen storage disease. Lanes 2, 4, and 5 are control samples from patients with other metabolic diseases. The same filter was further hybridized with "P-labeled y-actin cDNA probe. Debranching (1331) enzyme Phosphorylase(33S)
FIG. 5. Sequence homology between human muscle debranching enzyme and glycogen binding region of rabbit muscle phosphorylase. The first amino acid residue in each sequence is numbered in parentheses on the left, and the last amino acid residue is numbered on the right. Dashes represent gaps introduced to optimize the alignment. Residues which are identical between the debranching enzyme and phosphorylase are boxed.
two other patients (lanes 6 and 7) were similar in size and amount to the control cells (lanes 2,4, and 5 ) .The same blot was further hybridized with y-actin DNA probe. No striking difference in the y-actin mRNA levels was observed among these cell lines. The reduced amount of a specific mRNA species, detected with cloned debrancher cDNA in patients deficient in debranching enzyme activity, provides genetic evidence that the cloned cDNA is an authentic copy of the debrancher mRNA. The data that someGSD-I11 patients have reduced amounts of debrancher mRNA, whereas others have no detectable abnormalities at themRNA level,suggests there is a genetic heterogeneity causing defective debranching enzyme activity in these patients. Search of Sequence Homology between GlycogenDebrancher and Other Proteins-A computer search of the protein and nucleotide sequence databank did not reveal any significant sequence identity to other proteins. The following sequences were directly compared with the debrancher sequence because of functional similarity: human lysosomal a-glucosidase (23, 24). amylase (251, and rabbitsucrase-isomaltase complex (26). None of them showed significant sequence homology to the debrancher protein. A remarkable sequence homology has been reported between lysosomal a-glucosidase and sucraseisomaltase complex, suggestingthat these enzymes are derived from the same ancestral gene (23,26). Our data indicates that
glycogen debranching enzyme does not belong to this gene family. It has been speculated that in eukaryotic glycogen debranching enzyme, the association of glucosidase and transferase activities into a single polypeptide may be an evolutionary development (27, 28). An analogy is the fatty acid synthesizing system, which in yeast and mammal is multienzyme and in bacteria consists of separate enzymes (29). Since there is no similar type of glycogen debranching enzyme in bacteria, we searched for sequence homology in bacteria pullulanase (30), isoamylase (31), and amylomaltase (32). Pullulanase and isoamylase hydrolyze 1,6-a-~-glucosidiclinkage of certain branched a-D-glycan, whereas amylomaltase is a 4a-glucantransferase which will transfer maltose onto a growing dextrin chain. No significant sequence identity was observed between human glycogen debranching enzyme and these bacteria proteins. It is possible that a comparison of the amino acid sequence of yeast debranching enzyme with these bacterial proteins may reveal conserved regions of protein sequence; this will await cloning and sequencing of yeast debranching enzyme. Functional Domains-Several functional domains of glycogen debranching enzyme have been proposed from enzymological studies. Nelson and colleagues (5, 33) used reversible inhibitors and a catalytic site-directed irreversible inhibitor to analyze the two activities and envisioned that the enzyme has a single overlapping or interacting polymer binding site(s),flanked by a glucosidase on one side and a transferase on the other. As stated above, we found no specific region in the sequence of glycogen debranching enzyme which bears clear homology to the catalytic sites identified or proposed in lysosomal a-glucosidase and sucrase-isomaltase complex (23, 26). Molecular dissection of GSD-I11 patients with isolated transferase or glucosidasedeficiencywill help to identify regions important in these catalytic activities. In our attempts toidentify the polymer binding site of the debranching enzyme, we first searched for sequence homology to a common starch-binding domain in starch-degrading enzymes (34). The starch-binding domain is located at the COOH-terminal of these enzymes. No clear homology was identified. We then compared sequence data generated by crystallographic analysis of muscle phosphorylase. The glycogen-binding domain of phosphorylase is from amino acid residues 322 to 485, with residues 396-416 providing most of the contact with glycogen (35, 36). Computer analysis of the debranching enzyme sequence to theglycogen binding domain of phosphorylase revealed a sequence of limited homology in two potential segments at the COOH-terminal of the debranching enzyme, separated by a gap of 31 residues (Fig. 5). Although this region may be a candidate, the glycogen-binding site in debranching enzyme could not be clearly identified. When x-ray crystallographic structures of the debranching enzyme or sequences of other glycogen binding proteins become available, comparison with them may reveala definitive domain for polymer binding. REFERENCES 1. Gordon, R. B., Brown, D. H., and Brown, B. I. (1972)Biochem.
Biophys. Acta 289.97-107 2. White, R.C., and Nelson, T. E. (1974)Biochem. Biophys. Acta 365,274-280 3. Taylor, C., Cox, A. J., Kernohan, J. C., and Cohen, P. (1975)Eur. J. Biochem. 51,105-115 4. Bates, E. J., Heaton, G. M., Taylor, C., Kernohan, J. C., and Cohen, P. (1975)FEBS Lett. 58,181-185 5. Gillard, B. K., and Nelson, T. E. (1977)Biochemistry 16, 29783987 6. Lee, E. Y. C., Carter, J. H., Nielsen, L. D., and Fischer, E. H.
Nucleotide Sequence of Human Glycogen Debrancher cDNA (1970) Biochemistry 9, 2347-2355 7. Werries, E., Franz, A., and Geiqemeyer, S. (1990) J. Protozool. 37(6), 576-580 8. Becker, J. U., Long, T. J., and Fischer, E. H. (1977) Biochemistry 16,291-297 9. Chen, Y.-T., He, J.-K., Ding, J.-H., and Brown, B. I. (1987) Am. J. Hum.Genet. 41,1002-1015 10. Howell, R. R., and Williams, J. C. (1983) in The Metabolic Basis ofznherited Disease (Stanbury, J. B., Wyngaarden, J. B., Fredrickson, D. S., Goldstein, J. L., and Brown, M. S., eds) 5th Ed., pp. 141-166, McGraw-Hill, New York 11. Van Hoof, F., and Hers, H. G. (1967) Eur. J. Biochem. 2, 265270 12. Brown, B. I., and Brown, D. H. (1968) in Carbohydrate Metabolism and Its Disorders (Dickens, F., Randle, P. J., Whelan, W. J., eds) Vol. 2, pp. 123-160, Academic Press, New York 13. Ding, J. H., de Barsy, Th., Brown, B. I., Coleman, R.A., and Chen, Y.-T. (1990) J. Pediatr. 1 1 6 , 95-100 14. Cox, R. A. (1968) Methods Enzymol. 12, 120-129 15. Gubler, U.,and Hoffman, B. J. (1983) Gene (Amst.) 25,263-269 16. Huynh, T. V., Young, R. A., and Davis, R.W. (1985) in DNA Cloning: A Practical Approach (Glover, D. M., ed) Vol. 1, pp. 49-78, IRL Press, Washington, D. C. 17. Benton, W. D., and David, R. W. (1977) Science 196,180-182 18. Frohman, M. A,, Dush, M. K., and Martin, G.R. (1988) Proc. Natl. Acad. Sci. U. S. A . 85,8998-9002 19. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc.Natl. Acad. Sci. U. S. A . 74,5463-5467 20. Feinberg, A. P., and Vogelstein, B. (1983) Anal. Biochem. 1 3 2 , 6-13 21. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn,
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K., and Green, M. R. (1984) Nucleic Acid Res. 1 2 , 7035-7056 22. Kozak, M. (1986) Cell 4 4 , 283-292 23. Martiniuk, F., Mehler, M., Tzall, S., Meredith, G., and Hirschhorn, R. (1990) DNA Cell Biol. 9 , 85-96 24. Hoefsloot, L. H., Hoogeven-Westerveld, M., Kroos, M.A., van Beeumen, J., Reuser, A. J. J., and Oostra, B. A. (1988) EMBO J. 7,1697-1704 25. Nakamura, Y.,Ogawa,M., Nishide, T., Emi, M., Kosaki, G., Himeno, S., and Matsubara, K. (1984) Gene (Amst.) 28, 263270 26. Hunziker, W., Spiess, M., Semenza, G., and Lodish, H. F. (1986) Cell 4 6 , 227-234 27. Walker, G. J., and Builder, J. G. (1967) Biochem. J. 1 0 5 , 937942 28. Lee, E. Y . C., Smith, E. E., and Whelan, W. J. (1970) Miami Winter Symp. 1 , 139-150 29. Amy, C. M., Witkowski, A., Naggert, J., Williams, B., Randhawa, Z., and Smith, S. (1989) Proc.Natl.Acad. Sci. U. S. A . 8 6 , 3114-3118 30. Katsuragi, N., Takizawa, N., and Murooka, Y. (1987) J. Bacteriol. 169,2301-2306 31. Amemura, A,, Chakraborty, R., Fujita, M., Noumi, T., and Futai, M. (1988) J. Biol. Chem. 263,9271-9275 32. Pugsley, P., and Dubreuil, C. (1988) Mol. Microbiol. 2 , 473-479 33. Nelson, T. E., White, R. C., and Gillard, B. K. (1979) ACS Symp. Ser. 88,131-162 34. Svensson, B., Jespersen, H., Sierks, M. R., and MacGregor, E. A. (1989) Biochem. J. 264,309-311 35. Weber, I. T., Johnson, L. N., Wilson, K. S., Yeates, D.G. R., Wild, D. L., and Jenkins, J. A. (1978) Nature 274,433-437 36. Sprang, S., and Fletterick, R. J. (1979) J. Mol. Biol. 131, 523551