(Received for publication, November 28, 1994; and in revised form, April 26, 1995)
From the
From a human retinal cDNA library, we have isolated cDNAs that
are homologs for the 2 and
subunits of chicken Cap Z. The
derived human
subunit shares 95% amino acid identity with the
chicken
2 subunit; the
subunit is 99% identical to the
chicken subunit residues 1-243. The remaining portion of the
human
subunit(244-272) diverges significantly with only 8
out of 29 C-terminal amino acids conserved between the two species.
This lack of conservation is of particular interest because the chicken
C terminus contains an actin-binding domain. Cosedimentation assays
with F-actin show that human Cap Z binds actin with an affinity equal
that of chicken Cap Z. These results point to the eight shared amino
acids as critical for actin binding, three of which are regularly
spaced leucines. These apolar residues and one outside the region of
divergence align well with those residues of the actin-binding
-helix proposed for gelsolin segment 1. The apolar residues as
well as three polar amino acids are also conserved in other capping,
capping and severing, and monomer-binding proteins. Amino acid
substitutions in the chicken
subunit of the two most highly
conserved leucines result in significant decreases in F-actin binding
activity. The human
2 gene (CAPZA2) has been mapped to
chromosome 7 position q31.2-q31.3 and the
gene (CAPZB)
to chromosome 1 region p36.1.
Cap Z has been identified in chicken as a nonsevering,
barbed-end actin-binding protein composed of and
subunits.
In chicken, two cDNAs have been isolated for the
subunit. The
1 and
2 isoforms, which share 85% identity, are the products
of two separate genes(1, 2) ; one gene is believed to
be responsible for
subunit expression(3) .
By capping
the barbed end of actin filaments, Cap Z regulates the growth of the
actin filament at the barbed end(4) . The actin-binding domains
of the chicken protein have been mapped by genetic manipulation of the
respective subunit genes. Deletion mutations have shown that the
C-terminal 55 amino acids of the 1 subunit(5) , and the
C-terminal 15 amino acids of the
subunit are necessary for
binding to F-actin in vitro(6) . When expressed as a
glutathione S-transferase fusion protein, the C-terminal 25
residues of the
subunit are sufficient to bind monomeric
actin(6) .
For comparative studies and as a step toward
identifying human mutations, we have isolated and sequenced cDNAs
encoding an and a
subunit for human Cap Z and mapped each
subunit gene to its chromosome. We also have expressed the subunits and
determined the binding affinity of the human heterodimer to F-actin.
Within the divergent
subunit C termini of the Cap Z and other
homologous capping proteins, we have identified several conserved
residues. We provide the first evidence that many of these conserved
amino acids from the Cap Z family of actin-capping proteins align with
similar amino acids in actin-capping/severing and monomer binding
families of proteins. To confirm the functional importance of these
conserved residues, we have made amino acid substitutions for two of
the most conserved residues in the
domain of chicken Cap Z and
studied the F-actin binding characteristics of the heterodimers
(
1/mutated
) in actin cosedimentation and binding assays.
and
cDNAs were transferred into pGEM
(Promega) in order to synthesize RNA for in vitro translation.
To enhance the level of expression from these constructs, an EcoRI/NcoI fragment containing the 5`-untranslated
region (UTR) from the immediate early gene, 475(10) , was
included. Using the forward primer
(5`-CCAGAAGGACCATGGCGGAT
-3`) and reverse primer
(5`-AAATCTAGATTGACAGTTCAGAATAC
-3`) for the
subunit and the forward primer
(5`-GAGACGCCATCATGAGTG
-3`) and the reverse primer
(5`-AAATCTAGATGTTATGTGACCTGTCG
-3`) for the
subunit and the templates pH
4-1 and pH
4-3, cDNAs for each
subunit were amplified. In addition, the sequences encoding the
initiating methionines for each subunit were modified to include an NcoI or the compatible site, BspHI; an XbaI
site was created at the 3` end of each cDNA (added restriction sites
are highlighted in the primers). By digesting the PCR fragment with
either NcoI or BspHI and XbaI, the cDNAs
were inserted into the pGEM vector behind the 475 5`-UTR. The cDNAs
were sequenced again to check for any nucleotide changes created by PCR
amplification.
Alteration
of Leu was achieved using a similar strategy. A forward
primer (5`-AGAGGGAGCTCTCTCAAGTGNNGACCCAG
-3`) and
reverse primer (5`-TGAGAGAGCTCCCTCT
-3`) were made
which changed the
subunit nucleotides 783 and 786, creating a SacI restriction site. These substitutions maintained
Glu
-Leu
. To alter the amino acid at
position 266, degenerate nucleotides were included at positions 796 and
797 (underlined) in the forward primer. Similar to the previous
constructs, a 5` PCR fragment was synthesized using the KpnI-containing forward primer and the SacI-containing reverse primer; a 3` PCR fragment was made
using the SacI forward primer (with degenerate nucleotides)
and T
primer. The fragments were then digested with the
appropriate restriction enzymes and ligated together at the SacI site. The resulting KpnI-XbaI fragment
was used to replace the wild type sequence in pGEM-
. The
nucleotide changes at positions 796 and 797 of these constructs
resulted in several amino acid substitutions at Leu
.
The localization of CAPZA2 to
chromosome 7 was further refined by Southern and PCR analysis of a
panel of 17 human-rodent somatic cell hybrids (12, 13) and a chromosome 7-specific yeast artificial
chromosome (YAC) library(14) . The human 2 cDNA was used
as a probe in the hybridization experiments; PCR products were
generated using the 3`-UTR primers to human
2 cDNA.
The CAPZB gene was regionally localized on chromosome 1 by fluorescence in situ hybridization (FISH) analysis according to established protocols(15, 16, 17) . A 2.2-kb CAPZB cDNA probe (containing the plasmid vector) was biotinylated with the Life Technologies, Inc. BioNick labeling kit (15 °C, 1 h)(15) . After overnight hybridization and washing, the signals were amplified and detected using published methods(17) . The FISH signals and the DAPI-banding patterns were photographed separately. Assignment of the FISH mapping data to a specific chromosomal band was then determined by superimposing the FISH signals on the DAPI-banded chromosomes(15) .
For quantitative binding assays
and to assess heterodimer formation, samples were gel-filtered on a 1
30-cm Superose 12 FPLC column (Pharmacia). The column was
equilibrated and run at a rate of 0.5 ml/min in 100 mM KCl,
0.2 mM dithiothreitol, 0.01% NaN
, 10 mM K
HPO
, pH 8.0. Samples were collected in
0.5-ml fractions. Ten microliters from each fraction were counted in
scintillation mixture to determine the column profile. Radiolabeled
products were electrophoresed on 10% SDS-polyacrylamide gels and
visualized by autoradiography. Computer images of the autoradiograms
were collected using the Eagle Eye II Still video system (Stratagene)
and analyzed by ImageQuant densitometry analysis software (Molecular
Dynamics) to determine the quantity of each subunit.
To calculate the binding of the intact and
mutated proteins to F-actin, the same assay was performed except that
gel-filtered heterodimer was used and the concentration of actin was
0.25 mg/ml. After washing the pellet three times in the above buffer
minus the 20% sucrose, the amount of labeled material in the pellet was
determined by scintillation counting. K values of the proteins were calculated using the LIGAND
program of Munson and Rodbard (18) as modified by
McPherson(19) .
Analysis of the human nucleotide sequence showed
that it shared 90% identity with the chicken
2 and 80% identity
with
1. The predicted amino acid sequence (Fig. 1A) revealed only 14 differences between the
human and chicken
2 subunits out of 286 residues.
Figure 1:
Amino acid sequences
of human Cap Z 2 and
subunits. The amino acid sequences of
the human Cap Z
2 subunit (A) and
subunit (B) are shown. Those amino acids that are different from
chicken
2 or
are shaded. Site in
subunit where
sequences diverge is indicated with
and C-terminal residues are
boldface.
The human
sequence from base 1-729 shared 87% nucleotide identity
with the chicken
nucleotide sequence resulting in 99% amino acid
identity between chicken and human. The remaining sequence of the human
subunit markedly differed from the chicken
(Fig. 1B). Sequence alignments revealed that a 113
nucleotide segment that corresponds to the C-terminal domain of the
chicken
subunit is absent in the human cDNA. As a substitute, 90
nucleotides that are similar (95% nucleotide identity) to a region
considered 3`-untranslated region in chicken have become coding region
for the C terminus of the human
subunit. The human and chicken C
termini differ in length and share only 27% identity.
Figure 2:
Gel filtration of radiolabeled human Cap Z
translation products. Human and
subunit RNAs were in
vitro translated individually or together in the presence of
[
S]methionine. The labeled translation products
were chromatographed on a FPLC Superose 12 column, and 0.5-ml fractions
were collected. Fractionation profiles of
(top panel)
and
(center panel) translation products show one major
peak; whereas, two peaks are seen when
2 and
subunits are
cotranslated (bottom panel). In the cotranslated
fractionation, one peak elutes at a position similar to the individual
subunits and the other elutes at a position identical to heterodimeric
native chicken Cap Z (not shown).
Figure 3:
Cosedimentation of Cap Z translation
products with actin. Pellets and supernatants are shown after
cosedimentation of in vitro translated human Cap Z subunits
and heterodimer with actin. Each product was assayed with and without
excess native chicken Cap Z. The S-labeled samples were
run on a 10% SDS-polyacrylamide gel and visualized by
autoradiography.
Figure 4:
Sequence comparison between the
subunit C-terminal domain of Cap Z and homologs and other capping,
capping and severing, and monomer-binding proteins. Upper portion of figure shows the alignment of the C-terminal domains of human
and chicken Cap Z
subunits(3) . Above the sequence, numbers indicate amino acid positions in the human and chicken
sequences and the arrow indicates the site of sequence
divergence between the two C termini. In the C-terminal region, those
amino acids that are conserved in human and chicken are indicated with
an asterisk. C-terminal regions from
subunits of other
known Cap Z homologs (35, 36, 37) are aligned
below and a consensus of those amino acids that are present in at least
three out of the five sequences is given. In the consensus, an array of
amino acids containing three regularly spaced leucines,
KXXXLXXE/DLXXALXXK/R, is
identified. In the lower portion of the figure, sequences from
functionally distinct actin-binding proteins (20, 26) are aligned with the Cap Z
sequences. Boxes indicate identical or conservatively substituted amino
acids among the different families of proteins. The numbers of the
first and last residues of the aligned sequence are given. Those apolar
amino acids that are believed to be involved in actin binding of
gelsolin S1 are shaded. The 5 amino acids thought to be
conserved in several actin-binding proteins are indicated in the
alignment by +.
When compared to other functionally distinct actin-binding proteins, such as the gelsolin(20) , gelsolin-related(21, 22, 23, 24, 25) , and monomer-binding (26) proteins, we found a similar pattern of apolar residues that aligned with the leucine-rich array of Cap Z (positions 258, 262, 266, and perhaps 265). In addition, an apolar amino acid immediately adjacent to the region of divergence in the human and chicken C termini (aligning with human position 243) was conserved. Polar amino acids that coincided with Cap Z positions 251, 261 and 269 were also present. Those residues that aligned with positions 251 and 261 were typically acidic; those with position 269 were basic.
Figure 5:
Actin cosedimentation with 1 and
mutated
subunits. Supernatants are shown after cosedimentation of in vitro translated chicken Cap Z
1 and mutated
subunits. Each product was assayed with and without excess native
chicken Cap Z. The
S-labeled samples were run on a 10%
SDS-polyacrylamide gel and visualized by
autoradiography.
Figure 6:
Heterodimers containing the mutated
subunit detected by gel filtration. Transcripts for chicken
1,
and several
subunits containing mutations coding for the
amino acid substitutions at positions Leu
or Leu
were synthesized in vitro. The
and
subunit
mRNAs were cotranslated and the radiolabeled translation products gel
filtered on a FPLC Superose 12 column. The top panel is an
elution profile of the Cap Z translation products containing the
mutations at
position 262; the bottom profiles products
which have mutations at
position 266. As in Fig. 2, two
peaks are observed for both sets of mutations in the
subunit, one
occurs at a position similar to native heterodimeric chicken Cap Z
(fraction 37) and the other at the position of the individual subunits
(fraction 41). Autoradiograms of either fraction 37 or 38 from each set
of heterodimers are shown in Fig. 7A, 2.
Figure 7:
Quantitation of the subunit composition of
heterodimers and binding affinities of the heterodimers to actin. A, autoradiograms of SDS-polyacrylamide gels showing the
relative amounts of radiolabeled subunits before (1) and after (2) gel filtration. B, the corresponding scans of the
gel-filtered heterodimers. The x axes represent arbitrary
distance units and are slightly different for each gel; the y axes represent the intensity of the S signal. C, the ratio of signals from the radiolabeled
and
subunits contained in the purified heterodimeric complex is given; the
ideal ratio for a 1:1 molar complex is
:
., 36:64%. D, the actin-binding affinities determinined by the binding
assays.
These complexes containing the
altered subunits were then tested for their ability to bind to
actin. The results of the cosedimentation experiments (Fig. 5)
correlated with calculated K
values
determined from the binding assays (Fig. 7D). Both show
that actin binding of Cap Z is more affected when the leucine at
position 262 is changed than the leucine at position 266. At each
position, some amino acid substitutions show greater alteration in
actin binding than others. A nonconservative substitution at either
leucine with a basic residue significantly reduced binding;
replacements with small apolar amino acids, such as Gly and Ala, also
showed marked reductions.
To refine the localization of CAPZA2, a panel of 17 human-rodent somatic cell hybrids containing defined regions of human chromosome 7 (12, 13) was analyzed by the described PCR assay. The results clearly indicated that the CAPZA2 gene mapped to the 7q31-q32 region (data not shown). The PCR primers were then used to screen a human chromosome 7-specific YAC library (14) and four YACs (HSC7E4, HSC7E1394, HSC7E1420, and HSC7E1440) were identified (Fig. 8). These YAC clones were previously localized to 7q31.2-q31.3 and shown to be part of a set of overlapping clones linking the MET proto-oncogene to the cystic fibrosis transmembrane regulator gene (CFTR)(12) . HSC7E4 (600 kb), HSC7E1394 (330 kb), and HSC7E1420 (360 kb) were found to contain both MET and CAPZA2; therefore, the two genes are separated by a maximum distance of 330 kb. The results of these mapping studies suggest a physical order of the following reference DNA markers for the 7q31.2-q31.3 region: cen-MET-CAPZA2-D7S122-WNT2-D7S633-(D7S677-CFTR)-tel.
Figure 8: A YAC contig surrounding the gene at 7q31.2-q31.3. Twelve YAC clones (A) spanning the MET, CAPZA2, WNT2, and CFTR genes (B) are shown. The location of the DNA markers used to define the YAC contig are shown (C); the shaded boxes represent the minimal region to which the DNA markers could be assigned. The exon specific primers (exons 1, 5, 10, 15, 20, and 24) are from the CFTR gene. The other DNA markers have been described(12) .
The CAPZB gene was regionally localized to chromosome 1 region p36.1 by FISH analysis (Fig. 9). The efficiency of hybridization of the cDNA clone to metaphase chromosome preparations was approximately 60%. DAPI banding was used to assign the signal specifically to chromosome 1, which was in agreement with the PCR mapping data. A total of 10 mitotic figures were photographed, and the results indicated that CAPZB mapped to 1p36.1 (Fig. 9).
Figure 9:
Regional localization CAPZB to
1p36.1. An idiogram of chromosome 1 summarizes the FISH analysis using
the subunit cDNA. Each dot represents the localization
of double fluorescent signals on banded chromosome
1.
Candidate
human 1 and
1 homologues were not isolated in this study.
Their lack of representation may be due to the source of the cDNA
libraries screened. Both may be absent or in low abundance in the
retina; alternatively, these isoforms may be absent in the human.
Similarly, the human heterodimer with its differing C-terminal
sequence binds actin with an affinity equal to or greater than the two
isoforms of chicken Cap Z(5) . As with the chicken protein, the
individual human subunits required one another for high affinity
binding. The similarity in binding yet lack of sequence identity with
the chicken actin-binding domain pointed to the eight conserved
residues as being potentially critical for actin binding.
A recent
structural study of gelsolin S1 fragment (30) complexed with
actin suggests that a patch of apolar residues inserts between
subdomains 1 and 3 of actin and is rimmed with hydrogen bonding
residues. From this model, several of the residues of the highly
conserved five amino acid sequence in gelsolin S1 as well as several
amino acids surrounding this sequence are involved in this interaction (30) . The leucine-rich motif (including position 243)
identified in the Cap Z family of proteins aligns well with the apolar
residues of gelsolin that are proposed to interact with actin. Like the
corresponding region of gelsolin, this sequence in Cap Z is predicted
to be helical.
Previous studies have failed to show strong
homologies between Cap Z and other actin-binding proteins. A weak
homology exists between chicken Cap Z subunit residues
113-152 and gCAP39 (Cap G) residues 116-160 and, to a
lesser extent, gelsolin residues 117-159(23) . Within
this segment of gelsolin, a short stretch of basic amino acids is
present which is thought to be important in phosphoinositide binding (31) . The findings of the present study provide the first
evidence of a potential functional link between the actin-binding
domains of Cap Z and of the gelsolin/severin and profilin families. The
fact that a common motif has been preserved among three distinct
families of actin-binding proteins is interesting, and suggests that
binding sites in all three families may have evolved from a common
source.
Note Added in Proof-Several cDNAs, including expressed sequence
tags (ESTs) have been identified recently which may represent partial
sequences of a human Cap Z 1 subunit.