(Received for publication, October 31, 1994)
From the
We have characterized a new ankyrin gene, expressed in brain and
other tissues, that is subject to extensive tissue-specific alternative
mRNA processing. The full-length polypeptide has a molecular mass of
480 kDa and includes a predicted globular head domain, with membrane-
and spectrin-binding activities, as well as an extended
``tail'' domain. We term this gene ankyrin based
on its giant size and general expression. Two brain-specific isoforms
of 480 kDa and 270 kDa were identified that contain a unique stretch of
sequence highly enriched in serine and threonine residues immediately
following the globular head domain. Antibodies against the serine-rich
domain and spectrin-binding domain revealed labeling of nodes of
Ranvier and axonal initial segments. Ankyrin-binding proteins also
known to be localized in these specialized membrane domains include the
voltage-dependent sodium channel, the sodium/potassium ATPase,
sodium/calcium exchanger, and members of the neurofascin/L1 family of
cell adhesion molecules. The neural-specific ankyrin
polypeptides are candidates to participate in
maintenance/targeting of ion channels and cell adhesion molecules to
nodes of Ranvier and axonal initial segments.
Ankyrins are peripheral membrane proteins believed to
interconnect integral proteins with the spectrin-based membrane
skeleton (reviewed in (1) and (2) ). Two ankyrin genes
have been characterized so far: ankyrin and
ankyrin
. Ankyrin
was originally characterized
as a component of the erythrocyte membrane skeleton. In the rat brain,
ankyrin
is localized to the plasma membrane of a
subpopulation of postmitotic neurons(3) . The second gene,
ankyrin
, exists as two developmentally regulated
alternatively spliced variants with molecular sizes of 220 kDa and 440
kDa. The 440-kDa isoform contains a predicted extended
``tail'' domain in addition to the membrane and
spectrin-binding domains and is targeted to the plasma membranes of
unmyelinated and premyelinated
axons(4, 5, 6) .
Ankyrin has been shown to
associate with the voltagedependent sodium channel in vitro and to co-localize with this molecule at nodes of Ranvier, axonal
initial segments, and the neuromuscular
junction(7, 8, 9, 10) . It is
generally believed that the maintenance of highly localized
concentrations of the voltage-dependent sodium channel at the axonal
initial segments and nodes of Ranvier is important to the initiation
and propagation of the saltatory action potential. An ankyrin isoform
at the node of Ranvier was initially identified by immunofluorescence
using an antibody raised against erythrocyte ankyrin which
showed cross-reactivity with other members of the ankyrin gene
family(8) . As the nodal isoform of ankyrin was still present
in ankyrin
-deficient mice (carrying the nb mutation) and
was not recognized by antibodies specific for ankyrin
or
ankyrin
, it was concluded that the ankyrin isoform present
at the node of Ranvier was the product of an unidentified ankyrin
gene(9) .
In this study, we describe the cDNA sequence of a third ankyrin gene with alternatively spliced isoforms expressed in brain as well as a variety of other tissues. The two largest protein isoforms, which contain an unusual serine-rich sequence, are expressed only in nervous tissue. Specific antibodies raised against this serine-rich sequence stain axonal initial segments and nodes of Ranvier in cryosections from the rat brain. The two novel ankyrin isoforms represent the first specialized cytoplasmic protein components of these physiologically important membrane domains.
Figure 1:
cDNA sequence of
ankyrin 480 kDa. The putative polyadenylation sequence is boxed. The derived amino acid sequence is also shown, using
the single letter code, numbered in italics. The ANK repeats
of the membrane-binding domain are shown in bold and underlined. The serine-rich domain is also shown in bold and boxed. The peptide sequences used in the production
of antibodies to the spectrin-binding domain are shown in bold
italics.
Figure 2:
Comparison of ankyrin 480
kDa with ankyrin
440 kDa and predicted structure. A, dot matrix alignment of the derived amino acid sequence of
ankyrin
480 kDa against that of ankyrin
440
kDa. Each dot represents a minimum identity of 60% over a
window of 8 residues. Also shown is the relative position of three rat
cDNAs (a, b, and c). B,
hydrophilicity profile and predicted model for the ankyrin
480 kDa structure.
To raise antibodies against the serine-rich
domain (Fig. 2A), rat ankyrin cDNA c
(nucleotides 5028-6042) was subcloned into pGEMEX, and the
recombinant fusion protein was purified and injected into rabbits. The
resulting antiserum against the rat ankyrin
serine-rich
domain was affinity-purified using immobilized recombinant protein
after initially depleting the serum of gene 10 antibodies using
recombinant gene 10 protein coupled to Sepharose CL-6B.
Immunoblot
analysis of crude membrane samples was carried out as described
previously(3) , with bound antibodies detected using I-labeled protein A and autoradiography.
Ankyrin is more closely related
to ankyrin
than to ankyrin
with an overall
homology of 71% amino acid identity when compared with ankyrin
and 57% amino acid identity when compared with
ankyrin
. Fig. 2A shows a dot matrix alignment of
the derived amino acid sequence of ankyrin
480 kDa against
that of ankyrin
440 kDa. As with all members of the ankyrin
gene family, the two proteins show extensive homology in their
membrane- (74% amino acid identity) and spectrin-binding (67% amino
acid identity) domains and only small areas of homology in their
carboxyl-terminal domains.
Unlike ankyrin, ankyrin
and ankyrin
have a predicted ``tail''
domain inserted between their spectrin and carboxyl-terminal domains.
Biophysical studies of recombinant polypeptides derived from
ankyrin
440 kDa suggest that much of this domain is largely
unstructured and has the configuration of an extended random coil.
Comparisons of this domain with other random coil polypeptides of
similar sizes such as MAP2 suggest that this domain may be
approximately 200 nm in length (6) . Although the
``tail'' domain for ankyrin
shows only 20% amino
acid identity with that of ankyrin
, the sequence of this
domain also shows features consistent with an extended structure and
has a hydrophilicity profile (Fig. 2B) and amino acid
composition similar to that of the ankyrin
440-kDa tail
domain(6) . As shown in the dot matrix alignment (Fig. 2A), the ankyrin
tail domain contains
multiple small stretches of homology with the ankyrin
440-kDa tail domain. These homologies and the strong identity
between the membrane- and spectrin-binding domains of these molecules
suggest the evolution of ankyrin
or ankyrin
as
the result of a gene duplication event.
The ankyrin 440-kDa tail domain contains a large number of predicted sites
for phosphorylation, particularly by the enzymes casein kinase 2 and
protein kinase C(6) . Although the tail domain of ankyrin
also contains a large number of sites for casein kinase 2, it is
missing many of the potential sites for protein kinase C, which are
located in a group of fifteen 12-amino acid repeats present at the
amino-terminal end of the ankyrin
440-kDa tail domain.
These repeats are not found in ankyrin
.
The 480-kDa
ankyrin also contains a novel domain of approximately 40
kDa (Fig. 1, residues 1478-1908), which causes a
displacement in the dot matrix alignment (Fig. 2A).
This domain is serine-rich (35% serine and threonine residues) and
extremely conserved between rat and human (87% identity). A data base
search indicates that the serine-rich domain shares sequence homology
with a number of glycosylated proteins such as agglutinin and mucin.
Features of the ankyrin 480-kDa sequence are summarized
in the model shown in Fig. 2B. This model predicts a
globular head domain consisting of the membrane- and spectrin-binding
domains and a long extended unstructured tail domain. This model is
based on previous biophysical studies of the membrane- and
spectrin-binding domains of ankyrin
(12) and on
similar studies of the tail region of ankyrin
440
kDa(6) , as mentioned earlier.
As all further studies were
carried out using rat tissues, three cDNAs representing 4.2 kb of the
rat ankyrin sequence were obtained from a rat brain cDNA
library using human cDNAs as probes. The three rat cDNAs covered
nucleotides 789-1881 (a), 2913-5094 (b), and
5028-6042 (c) of the human ankyrin
sequence.
Homologies between rat and human ankyrin
over the 4.2 kb
were typically of the order of 70% identity at the nucleic acid level.
The relative positions of the three rat cDNAs in the ankyrin
molecule are underlined in Fig. 2A.
Figure 3:
Northern blot analysis of ankyrin transcripts. Poly(A
) RNA (20 µg) from the
rat testis (lane 1), lung (lane 2), kidney (lane
3), and brain (lane 4) was fractionated in 1%
formaldehyde/agarose gels and transferred to nitrocellulose. Filters
were hybridized with
P-labeled rat ankyrin
cDNAs from the membrane-binding domain (A),
spectrin-binding domain (B), and serine-rich domain (C).
The cDNA probe from the spectrin-binding
domain also hybridizes under stringent conditions with two smaller
transcripts of 5.5 kb and 4.2 kb (Fig. 3B). As
transcripts of this size were not observed in kidney mRNA, hybridized
with probes from ankyrin or
ankyrin
(4) , we conclude that these smaller
transcripts are genuine products of the ankyrin
gene
produced by alternative mRNA processing. In contrast, the probe
encompassing ANK repeats 5-15 does not hybridize to the 5.5-kb
transcript and hybridizes only weakly to the 4.2-kb transcript (Fig. 3A). This suggests that the smaller isoforms may
be missing some or all of their membrane-binding domain. Multiple
transcripts of ankyrin
(ankyrin 3) have also been noted by
Peters et al. (13).
To further elucidate the expression of
ankyrin isoforms, antibodies were raised against the
ankyrin
spectrin-binding (@SpBd) and serine-rich
(@SRd) domains. The use of these antibodies in immunoblot
analysis of multiple rat tissues is shown in Fig. 4. In crude
membrane fractions from the rat brain (Fig. 4A, lane 1), antibody against the serine-rich domain recognizes
two polypeptides of 480 kDa and 270 kDa (Fig. 4A, lane 1), which presumably are encoded by the 15-kb and 10-kb
transcripts. These polypeptides are not seen in the kidney (lane
2), lung (lane 3), testes (lane 4), spleen (lane 5), liver (lane 6), or heart (lane 7).
Antibody against the serine-rich domain also recognizes a doublet of
bands of approximately 90 kDa in size, present in all tissues studied
(data not shown). These bands are not recognized by the
spectrin-binding domain antibody, however, and immunofluorescence with
antibody against the serine-rich domain in rat tissues other than the
brain shows no discernible staining.
Figure 4:
Immunoblot analysis of ankyrin expression in adult rat tissues. Tissue homogenates and crude
membrane samples were prepared for SDS-polyacrylamide gel
electrophoresis from the rat brain (lane 1), kidney (lane
2), lung (lane 3), testes (lane 4), spleen (lane 5), liver (lane 6), and heart (lane
7). Equivalent loadings of membrane proteins were subject to
immunoblot analysis using antibodies raised against the serine-rich
(@SRd) (A) and spectrin-binding (@SpBd) (B) domains. Lane 1` shows a 5
longer exposure
of lane 1 blotted with
@SpBd.
In contrast to immunoblot
analysis with antibody against the serine-rich domain, analysis of the
same tissue samples with antibody against the spectrin-binding domain
identified a number of different polypeptides (Fig. 4B). A range of major polypeptides from 190 kDa
to 72 kDa are seen in multiple tissues, supporting the results from
Northern blot analysis that suggest tissue-specific alternative mRNA
processing. Fig. 4B, lane 1`, shows that the
antibody also recognizes the 480-kDa and 270-kDa ankyrin proteins in the brain upon longer exposure of the autoradiograph.
These large proteins were not seen in any of the other tissues (lanes 2-7) when using longer autoradiograph exposures,
nor were any previously undetected immunoreactive peptides observed
with these exposure times.
Figure 5:
Immunofluorescence localization of
ankyrin isoforms at the node of Ranvier. 4-µm
cryosections of the rat sciatic nerve (A, B, and D) or spinal cord white matter (C) were stained with
either @SRd (A and C) or @SpBd (B and D). A` and D` are DIC micrographs
of the corresponding immunofluorescence. Arrows indicate the
nodes of Ranvier. Arrowheads (B) indicate bundles of
unmyelinated axons in the sciatic nerve. Bars represent 10
µm.
Figure 6:
Immunofluorescence localization of
ankyrin isoforms at the axonal initial segment. 4-µm
cryosections from the CA3 region of the hippocampus (A),
cerebellum (B), and cortex (D) were stained with
@SRd. Arrows indicate staining of the initial segments
of neurons. Large arrowheads indicate Ranvier nodes observed
in the corpus callosum (cc) and white matter (wm) of
the cerebellum. Small arrowheads (B) appear to
represent staining of the unmyelinated axons of granule cells (gc). C represents the insert area of A in
greater detail. E shows an individual Purkinje cell (pc) from another area of the cerebellum, in greater detail.
The unstained axon is indicated by open arrowheads. Bars represent 10 µm.
This study presents the complete cDNA sequence of a new
ankyrin gene (ankyrin), which has 480-kDa and 270-kDa
neural-specific isoforms localized at axonal initial segments and nodes
of Ranvier. An unusual feature of the neural-specific ankyrins is a
40-kDa serine-rich domain with sequence similarity to mucins and
glycoproteins. 480-kDa ankyrin
is closely related to
440-kDa ankyrin
, an isoform of the major ankyrin gene in
the brain. Both molecules possess predicted extended tail domains
subject to alternative splicing(6) . Currently, we are not able
to distinguish whether the two 480-kDa and 270-kDa isoforms detected by
antibodies against the serine-rich domain are both present at the node
and/or axonal initial segments. However, both polypeptides are expected
to contain membrane-binding and spectrin-binding domains based on
Northern and immunoblot analysis. Ankyrin-binding proteins also known
to be localized in these specialized membrane domains include the
voltage-dependent sodium channel(7, 15) , the
Na/K-ATPase(15, 16) , Na/Ca
exchanger(15, 17) , and members of the neurofascin/L1
family of cell adhesion molecules(18) . The neural-specific
ankyrin
polypeptides are candidates to participate in the
maintenance/targeting of ion channels and cell adhesion molecules to
nodes of Ranvier and axonal initial segments. It is of interest that
initial visualization of nodes of Ranvier by electron microscopy
revealed a submembrane specialization(15, 19) .
Neural-specific forms of ankyrin
may represent a component
of this structure(20) .
The role of ankyrin and
ankyrin
440-kDa isoforms in myelination and the
establishment of nodes of Ranvier remains to be determined.
Unmyelinated axons contain 440-kDa ankyrin
, which is
down-regulated as myelination takes place(6) . Development of
membrane specializations at the node of Ranvier appear early in
myelination(21) , suggesting that a number of ankyrin isoforms
may be present in the axolemma during myelination. Joe and
Angelides(22) , using an antibody raised against erythrocyte
ankyrin, have observed that clustering of the voltage-dependent sodium
channel occurs independently of ankyrin. The development of
isoform-specific antibodies will permit evaluation of the role of
ankyrin in axonal differentiation and clustering of ion channels at the
initial segment and node of Ranvier.
Alternatively spliced isoforms
of ankyrin are expressed in a number of tissues ( Fig. 2and Fig. 3)(13) . Some of these isoforms
are missing areas of their membrane-binding domain based on Northern
blot analysis, although they all contain a spectrin-binding domain (Fig. 3) (13) . A similar feature has been noted in mRNA
transcripts of ankyrin
(5) . Smaller isoforms
missing the membrane-binding domain might be involved in functions
different from that proposed for ankyrin as a membranecytoskeleton
linker and may even localize to subcellular compartments other than the
plasma membrane.
Ankyrin has been implicated in the establishment of
cellular polarity, particularly in cultured kidney epithelial
cells(23) . The tissue-specific expression of ankyrin isoforms suggests that this ankyrin gene is able to use a number
of different combinations of functional domains, that may have a role
to play in the establishment of specialized membrane domains as well as
currently unanticipated functions.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U13616[GenBank].