(Received for publication, April 21, 1995)
From the
The syntrophins are a biochemically heterogeneous group of
58-kDa intracellular membrane-associated dystrophin-binding proteins.
We have cloned and characterized human acidic (1-) syntrophin and
a second isoform of human basic (
2-) syntrophin. Comparison of the
deduced amino acid structure of the three human isoforms of syntrophin
(together with the previously reported human
1-syntrophin)
demonstrates their overall similarity. The deduced amino acid sequences
of human
1- and
2-syntrophin are nearly identical to their
homologues in mouse, suggesting a strong functional conservation among
the individual isoforms. Much like
1-syntrophin, human
2-syntrophin has multiple transcript classes and is expressed
widely, although in a distinct pattern of relative abundance. In
contrast, human
1-syntrophin is most abundant in heart and
skeletal muscle, and less so in other tissues. Somatic cell hybrids and
fluorescent in situ hybridization were both used to determine
their chromosomal locations:
2-syntrophin to chromosome
16q22-23 and
1-syntrophin to chromosome 20q11.2. Finally, we
used in vitro translated proteins in an immunoprecipitation
assay to show that, like
1-syntrophin, both
2- and
1-syntrophin interact with peptides encoding the
syntrophin-binding region of dystrophin, utrophin/dystrophin related
protein, and the Torpedo 87K protein.
Dystrophin, the protein product of the Duchenne muscular dystrophy locus, is a large membrane-associated cytoskeletal protein(1) . In order to understand the function of this protein in skeletal muscle, it is important to establish the molecular organization of dystrophin in the context of the membrane cytoskeleton. Dystrophin copurifies with a group of integral membrane glycoproteins and membrane-associated proteins called the dystrophin glycoprotein complex(2, 3, 4) . A number of these proteins have been further defined by their primary sequence and their biochemical properties.
A 58-kDa cytoplasmic peripheral membrane
protein was independently identified in the Torpedo electric
organ, and shown to localize to the postsynaptic neuromuscular junction
in mammals(5) . This 58-kDa synaptic protein also copurifies
with dystrophin and is now known as
syntrophin(6, 7, 8, 9) .
Dystrophin-associated syntrophin isolated from rabbit skeletal muscle
is heterogeneous; it appears as a triplet by one-dimensional
SDS-electrophoresis, and when separated by two-dimensional gel
electrophoresis, appears as two clusters of 58-kDa proteins with
different isoelectric points (pI), one which is slightly acidic (,
pI = 6.4) and the other which is quite basic (
, pI =
9) (10) . Phosphatase pretreatment of the isolated microsomes
results in some signal consolidation(10) , and phosphoamino
acid analysis of syntrophin isolated from Torpedo electric
organ shows that serine and tyrosine residues are
phosphorylated(11) .
The isolation of two distinct isoforms
of syntrophin in mouse(9) , and antibodies to a single cloned
isoform of rabbit syntrophin(12) , confirmed the biochemical
evidence that there are at least two distinct genes. Based upon partial
peptide sequences from purified rabbit muscle syntrophin, we
independently isolated human 1-syntrophin cDNA which was also used
to identify a distinct but related human muscle expressed sequence tag
(EST), (
)EST25263 (13) . The deduced amino acid
sequence of this human EST fragment was nearly identical to a portion
of mouse
2-syntrophin(9) . From all the available
sequences, we proposed that there are at least three syntrophin genes
in the mammalian genome. From their predicted amino acid sequences and
their calculated pI values, the acidic isoform was named
1-syntrophin, and the two basic isoforms
1-syntrophin and
2-syntrophin (see Table 1).
The widely expressed
C-terminal product of the DMD gene, dystrophin protein of 71 kDa
(Dp71), the dystrophin related protein (DRP or utrophin), and the 87K
relative of dystrophin also copurify with syntrophin when isolated by
immunoaffinity
techniques(6, 8, 14, 15) . The
suggestion that dystrophin interacts with syntrophin via its C terminus
was independently determined by blot overlay of dystrophin onto
isolated syntrophin(16, 17) . Recombinantly produced
1-syntrophin interacts with a small region within the C terminus
of dystrophin, revealing a strong binding site within exon 74 of
dystrophin(18) . This result was independently determined by
using bacterially expressed or in vitro translated portions of
dystrophin to overlay onto purified syntrophin bound to a solid support
and to show that syntrophin may have yet another binding site in a more
distal region on dystrophin(19, 20) . In addition,
1-syntrophin was shown to also interact with the homologous
regions of utrophin/DRP and the Torpedo 87K
protein(18) .
Despite their mRNA expression in a wide
variety of tissues(9, 12, 13) , the
syntrophin isoforms appear to have a remarkable specificity in their
submembranous localization in muscle. Isoform-specific antibodies
discriminate the localization of 1-syntrophin, which is expressed
throughout the sarcolemma, from
2-syntrophin, which localizes
specifically to the neuromuscular junction (NMJ)(21) . The
determinants of the isoform-specific localization are presumably due to
structural differences between these isoforms, but these are yet to be
determined.
Gibson and colleagues have noted that the syntrophins
contain two pleckstrin homology (PH) domains(22) , a small
100-residue domain originally found as an internally duplicated
motif in pleckstrin, the major substrate of protein kinase C in
erythrocytes(23, 24) . This domain has captured wider
attention because it is also found in a number of other intracellular
signaling and cytoskeletal proteins, such as
-spectrin,
phospholipase C
, the
-adrenergic receptor kinase, a number of
GTPases, and GTPase-activating proteins, many of which are
membrane-associated(22) . The deduced three-dimensional
structure of the N-terminal pleckstrin domain and the PH region of
-spectrin has been determined(25, 26) . A
hydrophobic lip of the pleckstrin
-barrel has been shown to bind
to phosphatidylinositol 4,5-bisphosphate, which may explain how many
PH-containing proteins are associated with the membrane without
containing classical membrane-anchoring groups(27) .
Adams and colleagues (28) have noted the homology between a conserved region in the middle of the syntrophin genes and a number of other membrane-associated proteins, the PDZ domain, named for the Post-synaptic density protein-95 (PSD-95,(29) ), the Drosophila discs large tumor suppresser protein(30) , and the Zonula occludens-1 protein (ZO-1(31) ). Since this motif is shared among these intracellular peripheral membrane proteins, it may also be the basis by which the syntrophins interact with another component of the membrane or membrane cytoskeleton.
We
report here the cloning and characterization of human 2-syntrophin
and
1-syntrophin. By comparing the amino acid sequences of human
1-,
2-, and
1-syntrophin, we have identified the
C-terminal 57 amino acids of syntrophin as a conserved
syntrophin-unique domain. The mRNA of
2-syntrophin is expressed in
a wide variety of tissues, whereas
1-syntrophin is predominantly
expressed in striated muscle. The human chromosomal sublocalization of
2-syntrophin is 16q23-24, and that of
1-syntrophin is
20q11.2. We have also verified the functional conservation of
1-,
2-, and
1-syntrophin in their ability to interact with
dystrophin and its relatives in an in vitro binding assay.
For 1-syntrophin, the
oligonucleotide pair 5`-TGG GAT CCA GGA CAT CAA GCA GAT TGG CT-3` and
5`-GTG AAT TCC CGT GCG CAG GGC AAA GGA GA-3`, amplified a
reverse-transcribed cDNA template from human adult muscle, using 100 ng
of template DNA and 100 pmol of each primer, with 30 cycles of the
following conditions: 55 °C for 1 min, 72 °C for 10 s, and 94
°C for 1 min. Amplified DNA was separated by 6% polyacrylamide gel
electrophoresis, and the 250-bp fragment was visualized by staining
with ethidium bromide(32) .
cDNA clones of
2-syntrophin were obtained by screening a human adult brain
library with the PCR probe that corresponded to EST25263 (above). The
phage 19-1 was isolated and the insert subcloned into the EcoRI site of Bluescript II
SK for
sequencing. The DNA of this partial cDNA clone was then isolated and
radiolabeled (OLB labeling kit, Boehringer Mannheim) to screen a human
adult muscle cDNA library. The clones HAM1 and HAM12 were subcloned
into Bluescript for sequencing.
cDNA clones of 1-syntrophin was
obtained by screening a human adult heart (left ventricle) cDNA library (33) with a PCR probe prepared from the primers described
above. Of the six resulting clones, all were subcloned into plasmid,
shown to map similarly, and partially sequenced for verification. The
clones LV31-1 and LV6-2 were sequenced entirely.
The entire sequence of both strands were obtained by the dideoxy nucleotide chain-termination method, with either Sequenase T7 polymerase (U. S. Biochemical Corp.) or an ABI automated sequencer with Taq DNA polymerase (Perkin-Elmer). Sequences were analyzed and aligned using the GCG software suite (Wisconsin University) in their default settings. Gaps determined pairwise identity scores, PileUp produced multiple alignments (Fig. 1Fig. 2Fig. 3), and ProteinStructure made local secondary structure predictions.
Figure 1:
A, restriction
map and schematic representation of isolated 2-syntrophin cDNA
clones. The open reading frame is indicated by an open box. The EST25263 sequence was used as the basis for cloning 19-1 from
a human adult brain cDNA library. The 19-1 clone was used as the probe
to isolate HAM1 and HAM12 from a human adult muscle cDNA library. The
region HHR is a chimeric sequence from an unrelated gene (see
``Results''). B, interspecies comparison of the
deduced amino acid sequence of the
2-syntrophins, between human
and mouse. Includes data of the N terminus of mouse
2-syntrophin
reported(28) . A dash indicates identity to the
residue at that position for the human homologue, and a space indicates gaps introduced by PileUp software to optimize the
alignment (see ``Materials and Methods''). The clone 19-1
contains an open reading frame from the amino acid under the symbol
``>.''
Figure 2:
A, restriction map and schematic
representation of isolated 1-syntrophin cDNA clones. The open
reading frame is indicated by an open box. The PCR product was
amplified from a human adult brain cDNA library, which was used to
isolate clones LV31-1 and LV6-2 from a human adult left ventricle
library. B, interspecies comparison of the deduced amino acid
sequence of the
1-syntrophins, between human, rabbit, and mouse. Dashes and gaps are the same as for Fig. 1.
The clone LV6-2 contains an open reading frame from the amino acid
under the symbol ``>.''
Figure 3:
Sequence comparison of the three human
syntrophins (A), 1-syntrophin (
1),
2-syntrophin (
2), and
1-syntrophin (
1). The sequence of
1-syntrophin contains a short
correction from our prior published sequence (13) discovered by
our colleagues (M. Adams and S Froehner, personal communication). A dash indicates identity to the residue at that position for
the
1-isoform, and a space indicates gaps introduced by
PileUp software to optimize the alignment (see ``Materials and
Methods''). Sequences in boxes indicates the region of
homology to the PH domain described in (22) , the black box indicates the PDZ domain described in (28) , and the gray region represents the syntrophin-unique domain (SU). B, schematic representation of the domain organization of the
syntrophins. The first PH domain is split (PH1a and PH1b) by the PDZ
domain; the second PH domain (PH2) follows tandemly; the
syntrophin-unique domain follows at the C
terminus.
The 2-syntrophin
candidate HAM1 contains a single large open reading frame (Fig. 1A), which begins with an ATG start codon in a
favorable context for the initiation of translation, and is flanked at
the 3` end with a polyadenylation signal at the appropriate distance
from a poly(A) tail (GenBank accession no. U40572). The ATG start codon
is 45 nucleotides upstream of another in-frame ATG start codon which is
the initiation codon in mouse
2-syntrophin(28) . In the
mouse
2-syntrophin gene, the codon corresponding to the first
human ATG is ATC, but the former ATG codon in human is in an extremely
favorable context for the initiation of translation(40) . The
deduced peptide is 540 amino acids in length, 58,000 in molecular
weight, and has a pI of 9.4. Its amino acid sequence is 96% identical
to its mouse homologue (Fig. 1B)(9, 28) .
The deduced peptide is 505 amino acids in length, predicted to be a molecular mass of 54 kDa, and have a pI of 6.4 (Fig. 2B). The open reading frame begins with the first ATG start codon in the cDNA, which is in a favorable context for the initiation of translation, and is flanked at the 3` end with a polyadenylation signal at the appropriate distance from a poly(A) tail. At the amino acid level, this human isoform is 94% identical to the published mouse sequence and 93% identical to the published rabbit sequence. All three sequences contain homologous start codons. In comparison to rabbit and human, the mouse cDNA bears an internal deletion of 6 amino acids near its N terminus (GAPREQ). The 4-amino acid internal insertion in mouse (SSAH) is considered to represent a rare splicing event to a nearby splice acceptor(28) .
The C-terminal 57 amino acids
( Fig. 3in gray) also forms a region of strong homology
among the three human syntrophins, but does not have homology to other
characterized proteins. This 57-amino acid sequence has been labeled
the syntrophin-unique domain, and is predicted by Chou-Fasman and
Garnier-Osguthorpe-Robson analysis to consist of from three to five
strands of -sheet separated by as many turns (see ``Materials
and Methods'').
Figure 4:
mRNA tissue distribution of the three
syntrophins. A, 1-syntrophin (reprinted from (13) ). B,
2-syntrophin. C,
1-syntrophin. The tissues represented in each lane are: 1, heart; 2, brain; 3, placenta; 4,
lung; 5, liver; 6, skeletal muscle; 7,
kidney; 8, pancreas.
A similar hybridization with 1-syntrophin probe reveals a
distinct pattern of expression from that of the
-syntrophins (Fig. 4C). A single 2.5-kilobase pair transcript is
expressed in relatively high levels in both skeletal muscle and heart,
with some low level expression in brain, pancreas, liver, kidney, and
lung, and none detected in placenta.
Figure 5:
PCR amplification of human
1-syntrophin from the NIGMS collection of genomic DNA of
monochromosomal somatic cell hybrids (see ``Materials and
Methods''). 1, 10 ng of cDNA clone LV31-1; 2, H
O, negative control; 3, NA10568, from
Chinese hamster cell line RJK88; 4, NA05862, from mouse cell
line 3T6; 5, NAIMR91, from human cell line IMR91; 6,
NA10791, hamster line with chromosome 7; 7, NA10115, hamster
line with chromosome 4; 8, NA10926B, hamster line with
chromosome 10; 9, NA10156B, hamster line with chromosome 8; 10, NA10478, mouse line with chromosome 20 and parts of
chromosomes 4, 8, and 10; 11, NA13140, a separate mouse line
with chromosome 20.
Human genomic clones of both 2-syntrophin and
1-syntrophin isolated from an EMBL3 human genomic library were
used for FISH analysis to independently confirm the mapping panel
results (see ``Materials and Methods''). The
2-syntrophin signal localized to the region between 16q23 and
16q24 (Fig. 6A), and
1-syntrophin uniquely
localized a signal to 20q11.2 (Fig. 6B). No secondary
hybridization signals were consistently seen to suggest other closely
related loci elsewhere in the genome.
Figure 6:
FISH localization of the syntrophins. A, more than 20 metaphase spreads were examined to localize
2-syntrophin to the long arm of chromosome 16, in the region
between 16q23 and 16q24. B, a probe to
1-syntrophin
uniquely identified a single pericentromeric locus on the long arm of
chromosome 20, localizing the signal to 20q11.2. See ``Materials
and Methods'' for details.
Figure 7:
2- and
1-syntrophin interact
with dystrophin and its relatives. A, translated peptides of
dystrophin (dys), utrophin/DRP (drp), and the Torpedo 87K protein (87K) are used to coprecipitate
2-syntrophin (lanes 1-5) and
1-syntrophin (lanes 6-10) peptides. The syntrophin peptides were also
combined with Dp71
110(18) , which lacks the syntrophin
binding region (lanes 4 and 9), or with d11 antibody
alone (lanes 5 and 10). B, FLAG fusion
proteins of the syntrophin binding domains of dystrophin (1 and 5), utrophin/DRP (2 and 6), and the Torpedo 87K protein (3 and 7) are used to
coprecipitate translated partial cDNAs of
2-syntrophin and
1-syntrophin. In the control lane (4 and 8) an
identical precipitation was performed in the absence of FLAG fusion
protein.
The
syntrophin proteins used in this assay consistently showed a low level
rate of aggregation that is also seen in the control lanes (Fig. 7, A, lanes 4, 5, 9, and 10, B,
lanes 4 and 8). To address this issue, the exon 74
homologous regions of dystrophin, utrophin, and 87K protein were
produced as FLAG fusion peptide as reported previously for
coprecipitation with 1-syntrophin(18) . All three proteins
were able to coprecipitate both
2- (Fig. 7B, lanes
1-3) and
1-syntrophin (Fig. 7B lanes
5-7). A background of nonspecific aggregation of
2- and
1-syntrophin was seen regardless of whether a specific antiserum
or the anti-FLAG monoclonal antibody was used. This back-ground is
variable from different experiments but is never higher than the
specific coprecipitation reactions.
In this report we conclusively confirm our previous hypothesis that there are three distinct but homologous human syntrophin genes. Their biochemical and genetic characteristics are summarized in Table 1. We have also shown that these homologous proteins are functionally conserved with respect to their in vitro binding properties to dystrophin, utrophin, and the 87K protein.
Comparisons of the three human syntrophins to each other, as well as
those sequences available in mouse and rabbit, demonstrate that for a
particular isoform of syntrophin there is a high degree of interspecies
conservation, with 96% identity for 2-syntrophin and at least 93%
for the three mammalian
1-syntrophins. In contrast, the three
human syntrophins are less strongly conserved with respect to each
other. The
1-syntrophin is 54 and 50% identical to its
1- and
2-syntrophin counterparts, respectively, and the
-syntrophins
are only 57% identical to each other.
The syntrophins contain two tandem PH domains (Fig. 3A, in plain box)(22) . The first PH domain is interrupted by a 162- to 182-amino acid region in which 80 amino acids are highly conserved among the three syntrophins (Fig. 3A, in black box). Adams and colleagues (28) have noted the homology between this conserved region and a number of other membrane-associated proteins, the PDZ domain. The PH and PDZ domains, either together or individually, may determine the specific membrane localization of syntrophin, either directly to a lipophilic membrane component (27) or via an integral membrane protein(41) .
In
comparing the three human syntrophins, we have found that the
C-terminal 57 amino acids of the three proteins are highly homologous
to each other (Fig. 3A, in gray). This region
is predicted to contain as many as five strands of -sheet, and
because it appears to be a unique motif among other known proteins, we
have called it the syntrophin-unique domain. It is possible that this
57-amino acid syntrophin-specific domain subserves syntrophin's
specific interaction with dystrophin and its relatives. To this point,
we have shown that the C-terminal two-thirds of the three translated
syntrophins can coprecipitate with the exon 74 region of dystrophin and
its relatives ( (18) and Fig. 7). These polypeptides do
not contain the domain PH1a and the PDZ region, but do contain PH1b,
all of PH2, and the syntrophin-unique domain (see Fig. 3).
Further functional analysis of syntrophin structure will be necessary.
Dystrophin and utrophin/DRP also have distinct localizations to the
muscle membrane, with dystrophin distributed throughout the sarcolemma
and utrophin/DRP found mainly at the neuromuscular
junction(42) . Since mouse 1-syntrophin is distributed
throughout the membrane and
2-syntrophin is found at the
neuromuscular junction(21) , we hypothesized that these two
isoforms of syntrophin had unique binding properties to the respective
dystrophin and utrophin/DRP proteins. The finding that all three
syntrophins can each bind to dystrophin and its relatives in vitro falls short in providing some clue as to how either the
syntrophins or dystrophins can localize to different specializations of
the sarcolemma. The coprecipitation procedure used here (Fig. 7)
does not quantitatively address this question. However, the differences
among the three syntrophins, which are especially marked in the
connecting loops to the PDZ domain (Fig. 3A), may
reflect the specialization of these individual genes to a particular
function, such as to interact with another protein, or as determinants
of their distinct subcellular localization.
At the level of the
mRNA, the -syntrophins share a common characteristic in that they
give rise to a set of transcript classes (Fig. 4). In contrast
to
1-syntrophin, whose five transcript classes are most abundant
in liver,
2-syntrophin transcripts are more homogeneously
expressed, most abundant in lung, and have three transcript classes (Fig. 4B). These results are similar to those found in
mouse
2-syntrophin, but the relative abundance of
2-syntrophin in human brain is much lower than that observed in
mouse(9) . In the cDNA clones that we have isolated so far, we
have not noticed any large differences in the sequences among the
clones that can account for these alternative forms, nor do the other
reported cDNAs from rabbit and
mouse(9, 12, 28) .
The 1-syntrophin
transcript is expressed as a single-sized transcript of 2.5 kilobase
pairs (Fig. 4C), and is strongly dominant in cardiac
and skeletal muscle. This representation of
1-syntrophin mRNA
expression shows somewhat more expression in extramuscular tissues than
that reported in mouse and rabbit tissues
previously(9, 12) , but may be attributable only to a
higher sensitivity in detection.
The tissue distribution of the
respective syntrophin mRNA also allow us to hypothesize what kinds of
inherited disorders may be caused by defects of these genes. The
sublocalization of 2-syntrophin to 16q23-24 (Fig. 6A), and its widespread tissue distribution
suggests that a defect of this gene would have consequences in multiple
organs. Because
1-syntrophin is so abundantly expressed in
striated muscle, we would predict that a defect of this gene would be
more inclined to result in a myopathic phenotype. The question of
whether this 20q11.2-encoded gene (Fig. 6A) is linked
to any autosomal neuromuscular diseases is currently under
investigation.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U40571 [GenBank]and U40572[GenBank].