©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Sequence Analysis and Chromosomal Localization of Human Cap Z
CONSERVED RESIDUES WITHIN THE ACTIN-BINDING DOMAIN MAY LINK CAP Z TO GELSOLIN/SEVERIN AND PROFILIN PROTEIN FAMILIES (*)

(Received for publication, November 28, 1994; and in revised form, April 26, 1995)

Emily A. Barron-Casella (1)(§) Michelle A. Torres (1) Stephen W. Scherer (2) (3) Henry H. Q. Heng (2) (3) Lap-Chee Tsui (2) (3) James F. Casella (1)

From the  (1)Department of Pediatrics, Division of Hematology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, the (2)Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, M5S 1A8 Canada, and the (3)Department of Genetics, Research Institute, The Hospital for Sick Children, Toronto, Ontario, M5G 1X8 Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

From a human retinal cDNA library, we have isolated cDNAs that are homologs for the alpha2 and beta subunits of chicken Cap Z. The derived human alpha subunit shares 95% amino acid identity with the chicken alpha2 subunit; the beta subunit is 99% identical to the chicken subunit residues 1-243. The remaining portion of the human beta 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 alpha-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 beta subunit of the two most highly conserved leucines result in significant decreases in F-actin binding activity. The human alpha2 gene (CAPZA2) has been mapped to chromosome 7 position q31.2-q31.3 and the beta gene (CAPZB) to chromosome 1 region p36.1.


INTRODUCTION

Cap Z has been identified in chicken as a nonsevering, barbed-end actin-binding protein composed of alpha and beta subunits. In chicken, two cDNAs have been isolated for the alpha subunit. The alpha1 and alpha2 isoforms, which share 85% identity, are the products of two separate genes(1, 2) ; one gene is believed to be responsible for beta 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 alpha1 subunit(5) , and the C-terminal 15 amino acids of the beta 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 beta 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 alpha and a beta 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 beta 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 beta domain of chicken Cap Z and studied the F-actin binding characteristics of the heterodimers (alpha1/mutated beta) in actin cosedimentation and binding assays.


MATERIALS AND METHODS

Screening of Retinal cDNA Library

A human retinal cDNA library constructed in gt10 (kindly provided by Dr. Jeremy Nathans) (7) was screened with cDNAs of the chicken alpha and beta subunits. For each of the two screens, approximately 180,000 plaques were transferred onto nitrocellulose, and the filters were prepared for hybridization using standard procedures(8) . Hybridizing plaques were isolated and purified by limiting dilution and rescreening.

Subcloning into pBS and pGEM

Phage DNA was isolated from plate lysates using standard procedures(8) . After digesting the purified phage DNA with EcoRI, the cDNA insert was gel purified. The resulting cDNAs, which ranged in size from 1-3 kb, (^1)were subcloned into pBluescript KS+ (pBS, Stratagene) and sequenced using the dideoxy chain termination method(9) . One clone containing the full-length alpha subunit cDNA (pHalpha4-1, 1.6 kb) and two clones with the complete beta subunit cDNA (pHbeta4-2, 1.0 kb and pHbeta4-3, 2.2 kb) were sequenced on both strands.

alpha and beta 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 alpha subunit and the forward primer (5`-GAGACGCCATCATGAGTG^7-3`) and the reverse primer (5`-AAATCTAGATGTTATGTGACCTGTCG-3`) for the beta subunit and the templates pHalpha4-1 and pHbeta4-3, 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.

Construction of Substitution Mutations

Substitution mutations of Leu and Leu were created in the chicken beta subunit by PCR using the template pGEM-beta. In the synthesis of two Leu mutations, restriction enzyme sites were introduced into the nucleotide sequence coding for Glu-Leu-Ser. To accomplish the modifications, two C-terminal PCR fragments were generated for each restriction site. The 5` PCR fragments were synthesized using a forward primer containing a KpnI site (5`-GTTCTGGTACCATGAA-3`) and a reverse primer with either the restriction site Eco47III (5`-TTGAGAGCGCTCCCTCTG-3`) or HindIII (5`-TTGAGAAGCTTCCCTCTG-3`). The forward primers used for the 3` PCR fragments were the complement of the reverse primers used in synthesis of the 5` PCR fragments; the reverse primer was the T(7) primer (sequence found in pGEM 3` to the multicloning site). After synthesis, the 5` PCR products were digested with KpnI and either Eco47III or HindIII and the 3` PCR fragments were digested with either Eco47III or HindIII and XbaI. The 5` and 3` fragments were ligated together at the new restriction site and substituted for the wild type KpnI-XbaI fragment containing the beta C-terminal domain in pGEM-beta. When the Leu encoding sequence was mutated to include an Eco47III site, Leu was changed to Arg; when altered to a HindIII site, Ala was generated.

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 beta 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(7) 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-beta. The nucleotide changes at positions 796 and 797 of these constructs resulted in several amino acid substitutions at Leu.

Analysis of Sequence

Nonredundant nucleotide sequence data bases were searched for sequences homologous to human Cap Z using the BLASTN program from the National Center for Biotechnology Information (Bethesda, MD) using the BLAST network service.

Chromosomal Localization

Using primers designed from the 3`-UTR of the cDNAs, the human alpha2 and beta subunit genes were localized to two different chromosomes by PCR. The primers for the alpha2 screening were 5`-GTATTCTGAACTGTCAAG-3` (forward) and 5`-CTAGAATGTCCATTATTGACCCC-3` (reverse); those for the beta were 5`-CCTCTGTTTCATGCTAACC-3` (forward) and 5`-CTCTATGGAAATGTGGAGAGACTG-3` (reverse). Using the recommended conditions for AmpliTaq (Perkin-Elmer), 100 ng of DNA from each sample of the NIGMS human-rodent somatic cell hybrid mapping panel 2 (11) were assayed. Thirty cycles of amplification were performed under the following conditions: 1-min denaturation at 94 °C, 2-min annealing at the primer-specific temperature (52-55 °C for alpha primers and 60 °C for beta primers), and 2-min extension at 72 °C.

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 alpha2 cDNA was used as a probe in the hybridization experiments; PCR products were generated using the 3`-UTR primers to human alpha2 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) .

In Vitro Transcription/Translation and Chromatography

S-Labeled protein was synthesized in vitro using transcription and translation kits (Promega) as described previously(5) . For transcription, 1 µg of each plasmid was used per every 10 µl of reaction mixture and 0.3-0.5 mM mCAP analogue 5`7MeGppp5`G (Stratagene) was added. Synthesis was initiated from the SP6 promoter. For each subunit, approximately 8 µl of RNA were translated in a 100-µl reaction solution containing 70 µl of rabbit reticulocyte lysate. A total of 16 µl of RNA (8 µl for each subunit) was used in the translation of the heterodimer. 100 µCi of methionine (DuPont NEN) were included in these reactions to label the resulting protein. An estimate of the amount of protein synthesized was calculated based on the number of methionines/subunit and trichloroacetic acid-precipitable counts as described previously(5) .

For quantitative binding assays and to assess heterodimer formation, samples were gel-filtered on a 1 times 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(3), 10 mM K(2)HPO(4), 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.

Cosedimentation and Binding

The cosedimentation of the individual subunits and various heterodimers with chicken F-actin was tested by incubating equimolar amounts of the in vitro translated product with 0.5 mg/ml F-actin for 15 min in the presence or absence of excess Cap Z (3.8 µM), as described elsewhere(5) . After centrifugation through a 20% sucrose cushion (100,000 rpm for 20 min in a Beckman Airfuge centrifuge), the pellets or supernatants were electrophoresed on a 10% SDS-polyacrylamide gel.

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) .


RESULTS

Nucleotide and Predicted Amino Acid Sequence

Several human cDNAs were isolated from a human retinal library for both the Cap Z alpha and beta subunits. In the alpha screening, 38 hybridizing plaques were identified; 15 plaques hybridized with the beta probe. For each subunit, the cDNA inserts from 5 plaques chosen randomly were isolated, cloned and sequenced. All of the cloned cDNAs contained varying amounts of the same alpha or beta subunit sequence. GenBank accession numbers for the human alpha2 and beta cDNAs are UO3269 and UO3271, respectively. At the time of submission to the databank, sequence identity was detected with three databank entries (nos. T11326, D12250, and M26658), none of which contained complete subunit cDNA sequence nor had been identified as Cap Z.

Analysis of the human alpha nucleotide sequence showed that it shared 90% identity with the chicken alpha2 and 80% identity with alpha1. The predicted amino acid sequence (Fig. 1A) revealed only 14 differences between the human and chicken alpha2 subunits out of 286 residues.


Figure 1: Amino acid sequences of human Cap Z alpha2 and beta subunits. The amino acid sequences of the human Cap Z alpha2 subunit (A) and beta subunit (B) are shown. Those amino acids that are different from chicken alpha2 or beta are shaded. Site in beta subunit where sequences diverge is indicated with and C-terminal residues are boldface.



The human beta sequence from base 1-729 shared 87% nucleotide identity with the chicken beta nucleotide sequence resulting in 99% amino acid identity between chicken and human. The remaining sequence of the human beta subunit markedly differed from the chicken beta (Fig. 1B). Sequence alignments revealed that a 113 nucleotide segment that corresponds to the C-terminal domain of the chicken beta 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 beta subunit. The human and chicken C termini differ in length and share only 27% identity.

Actin Cosedimentation and Binding Studies with Human Cap Z

The divergence of the human beta sequence in a putative actin-binding domain of the chicken subunit raised the question of whether the human protein retained actin-binding activity. To address this question, radiolabeled human alpha and beta subunits produced by in vitro translation were examined for their ability to form a heterodimeric protein and bind to actin. In vitro translated, S-labeled human alpha (Fig. 2, top) and beta (center) subunits chromatographed on a Superose 12 FPLC column showed a major peak at fraction 41. When both alpha and beta were translated together, two peaks were seen (bottom). One peak eluted from the column at a position similar to that of the individual subunits; the other eluted at fraction 37, in a position identical to heterodimeric native chicken Cap Z. An aliquot of fraction 37 electrophoresed on a SDS-polyacrylamide gel and autoradiographed showed approximately equimolar amounts of each subunit when analyzed by densitometry and adjusted for S incorporation. Upon incubation and sedimentation of the individual subunits and heterodimer with F-actin, only the heterodimer pelleted appreciably with actin (Fig. 3). Both sedimentations could be reduced by the addition of excess native chicken Cap Z. Binding studies performed with the gel-filtered heterodimers (repeated >10 times) showed a K of approximately 7 times 10M.


Figure 2: Gel filtration of radiolabeled human Cap Z translation products. Human alpha and beta 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 alpha (top panel) and beta (center panel) translation products show one major peak; whereas, two peaks are seen when alpha2 and beta 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.



Sequence Comparison between the C-terminal Domain of Human and Chicken Cap Z beta Subunits and Other Barbed End, Actin-binding Proteins

The ability of the human protein to bind actin despite its weak similarity to the presumed actin-binding domain in the chicken protein prompted further examination of the C-terminal amino acid sequence. Aligned C-terminal domains of human (29 amino acids) and chicken (34 amino acids) revealed 8 amino acids that were identical (Fig. 4). Within the Cap Z family of actin-binding proteins, these 8 residues are conserved with the exception of the glutamine (Q) at position 270. A few of the intervening amino acids also appear to be maintained or conservatively substituted as shown in the consensus sequence for Cap Z and homologs. The most obvious feature of the comparison was an array of three, regularly spaced leucines that aligned with positions 258, 262, and 266 of human and chicken.


Figure 4: Sequence comparison between the beta 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 beta 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 beta 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 beta 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.

Substitution Mutations Affect Actin Binding of Cap Z

To determine whether the two most highly conserved leucines in the actin-binding domain were critical for binding, amino acid substitutions at positions 262 and 266 of the chicken beta subunit were created. These proteins were first studied by actin cosedimentation assays (Fig. 5). The cosedimentation experiments with alpha1 and the mutated beta subunits suggested the following order of actin-binding affinities of the altered proteins, Trp > Ala > Arg > Gly > Ala > Arg. Because these cosedimentation results could be confounded by incomplete heterodimer formation, the radiolabeled products were also purified by gel filtration. All of the mutated beta subunits formed heterodimers suggesting that a global disruption of protein folding was not responsible for the loss in cosedimentation. These complexes eluted in fractions 37 and 38 (Fig. 6), similar to wild type Cap Z. To quantitate the relative amounts of alpha and beta subunits, the purified complexes were electrophoresed on SDS-polyacrylamide gels and autoradiographed. Densitometry scans of the autoradiograms (Fig. 7, A-C) revealed ratios of alpha and beta subunits (Fig. 7C) which were very close to the idealized ratio of the labeled subunits (alpha:beta, 36:64% ) determined by the number of [S]methionines incorporated into each subunit.


Figure 5: Actin cosedimentation with alpha1 and mutated beta subunits. Supernatants are shown after cosedimentation of in vitro translated chicken Cap Z alpha1 and mutated beta 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 beta subunit detected by gel filtration. Transcripts for chicken alpha1, beta and several beta subunits containing mutations coding for the amino acid substitutions at positions Leu or Leu were synthesized in vitro. The alpha and beta 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 beta position 262; the bottom profiles products which have mutations at beta position 266. As in Fig. 2, two peaks are observed for both sets of mutations in the beta 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 alpha and beta subunits contained in the purified heterodimeric complex is given; the ideal ratio for a 1:1 molar complex is alpha:beta., 36:64%. D, the actin-binding affinities determinined by the binding assays.



These complexes containing the altered beta subunits were then tested for their ability to bind to actin. The results of the cosedimentation experiments (Fig. 5) correlated with calculated Kvalues 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.

Chromosomal Localization of the Human Cap Z alpha and beta Subunit Genes

Chromosomal localizations of CAPZA2 and CAPZB were determined by PCR screening of NIGMS human-rodent somatic cell hybrid mapping panel 2(11) . PCR products of the expected size for the alpha2 (343 bp) and beta (231 bp) subunit genes were only amplified from the hybrid cell lines containing human chromosome 7 and human chromosome 1, respectively (data not shown).

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 beta subunit cDNA. Each dot represents the localization of double fluorescent signals on banded chromosome 1.




DISCUSSION

Sequence Comparison between Human and Chicken Cap Z

Our results show that the majority of human Cap Z is over 95% identical to the alpha2beta heterodimer of chicken Cap Z. One of the striking findings in this study was the decrease in conservation to 27% in the C-terminal domain of the beta subunit. The nucleotide sequence shows that a 113-nucleotide segment present in the chicken beta cDNA is not represented in the human cDNA. This results in a region considered 3`-UTR in chicken becoming the coding sequence for the human beta subunit C terminus. A chicken beta subunit cDNA with a C terminus similar to our human clone has been identified recently and given the name beta2(27) . Although the genomic structure is not yet known for either the chicken or human beta genes, the most probable explanation is that the missing portion of coding sequence has been alternatively spliced from the beta subunit RNA.

Candidate human alpha1 and beta1 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.

Lack of Homology in an Actin Binding Domain

Previous sequence comparisons between capping proteins of lower eukaryotes and chicken Cap Z have demonstrated a strong conservation of primary structure except in the C-terminal region of the beta subunit(6) . It was therefore surprising that an actin- binding domain was mapped to this region of the chicken beta subunit. When expressed as a fusion protein, the C-terminal region alone did not cap actin filaments but instead bound monomeric actin with a K of 325 nM. Data base searches between this sequence and monomer binding or barbed-end capping proteins revealed no significant similarities(6) .

Similarly, the human heterodimer with its differing beta 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.

Conserved Amino Acids in the C-terminal Domain

Contained in those few conserved amino acids is a leucine-rich array that is present in variable form in other Cap Z homologs, KXXXLXXE/DLXXXLXXK/R. This sequence is found within the C-terminal 25 amino acids of the chicken betasubunit presumed to contain an actin-binding site. Our experiments show that amino acid substitutions at Leuand Leuwith either polar or small apolar residues greatly reduced the capping protein's ability to bind actin without impairing the ability of the subunit to interact with the heterologous subunit. The leucines at positions 262 and 266 of Cap Z align with leucines contained within a highly conserved five amino acid peptide described in the gelsolin and profilin protein families and in a wide range of actin-binding proteins(28, 29) .

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 alpha 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 beta 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.

Chromosomal Localization of Human Genes

At this time, it is unclear if any genetic disease might result from mutations in the human Cap Z genes. Interstitial deletions involving the q21-32 region of chromosome 7 are commonly observed chromosome abnormalities(32) . The clinical presentation of these patients do not define a definite clinical phenotype, but the common features of delayed development, ear malformations, feeding difficulties, low birth weight, simian creases, and an unusual ``catlike'' cry are often observed(32) . As additional genes are placed on the physical map of chromosome 7, it should be possible to correlate the clinical features with the genotypes of patients with deletions. The MET and CFTR genes have already been used in several allelic loss studies(33, 34) , so the precise mapping of CAPZA2 to this interval should contribute in these and other analyses. Likewise, the localization of CAPZB to chromosome 1p36.1 will also provide a valuable DNA marker for similar physical mapping studies of chromosome 1.


FOOTNOTES

*
This work was supported in part by funds from National Instiutes of Health Grant R01 AR40697 (to J. F. C.) and a CGAT Grant from the Medical Research Council of Canada (to L.-C. T.). This study was done during the tenure of an Established Investigator Award from the American Heart Association (to J. F. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pediatrics, Johns Hopkins University School of Medicine, Ross Research Bldg., Rm. 1125, 720 Rutland Ave., Baltimore, MD 21205. Tel.: 410-955-6132; Fax: 410-955-8208.

(^1)
The abbreviations used are: kb, kilobase(s); UTR, untranslated region; PCR, polymerase chain reaction; FISH, fluorescence in situ hybridization; DAPI, 4,6-diamidino-2-phenylindole; YAC, yeast artificial chromosome; bp, base pair; 5`7MeGppp5`G, p^1,5`-(7-methyl)guanosine-P^3-5`-guanosine triphosphate.


ACKNOWLEDGEMENTS

We thank Linda Thomas for helpful assistance with the manuscript.

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 alpha1 subunit.


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