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Department of Cell Biology, School of Medicine, University of Alabama, Birmingham, AL 35295
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Direct evidence which links PMN motility and cytoskeletal dynamics are derived in part from studies with pharmacological agents such as cytochalasins and phalloidins and studies of human cell motility disorders auch as the neutrophil actin dysfunction (NAD) (1, 2). In 1991, Coates and colleagues (3) described a unique PMN disorder, NAD with abnormal 47-kDa and 89-kDa proteins (NAD 47/89). The NAD 47/89 PMNs could not move and contained increased amounts of a 47-kDa and decreased amounts of an 89-kDa protein. In addition, the NAD 47/89 PMNs have a unique morphology and abnormal microfilamentous structure, resulting from defective actin polymerization in response to chemotactic factor stimulation, and this leads to a decreased ability of the cells to spread on glass (3, 4).
Cloning and sequencing of the 47-kDa-protein cDNA revealed a nucleotide sequence nearly identical to that of the lymphocyte-specific protein 1 (LSP1), a mouse cDNA sequence reported in GenBank (5) and subsequently identified as an F-actin binding protein (6). Human LSP1 is a phosphoprotein composed of 339 aa (7). The gene for this protein was cloned from mouse lymphocyte library using a subtractive hybridization technique (8), and studies showed that this gene is expressed in normal murine B and T lymphocytes and in transformed B cells but not in T lymphoma cell lines (5). Subsequent studies showed LSP1 is not limited to lymphocytes but is a pan-leukocyte protein (9, 10). Human LSP1 has an acidic N-terminal half which is 53% homologous to its mouse counterpart and a basic C-terminal half which is 85% homologous to its mouse counterpart (6). The basic C-terminal domain contains amino acid sequences homologous to two known F-actin binding protein caldesmon and the villin headpiece. Although it is known that LSP1 is an F-actin binding protein and is a very important regulator of microfilamentous cytoskeleton dynamics, it is not known which regions of the molecule are required for the F-actin binding activity. In the studies reported here, two methods were used to define the F-actin binding domains of this molecule. The results show that human LSP1 has at least three F-actin binding domains, all located in the basic C-terminal half, corresponding to the homologous regions of F-actin binding domains in two known actin-binding proteins: caldesmon and the villin headpiece.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Restriction enzymes BamHI and HindIII, T4 DNA ligase, and in vitro mutagenesis kit were purchased from Promega (Madison, WI). QIAEX II gel extract kit was from Qiagen (Santa Clarita, CA). Proband resin was obtained from Invitrogen (San Diego, CA). PCR primers were synthesized at the Department of Biochemistry and Molecular Genetics at the University of Alabama (Birmingham, AL), 125I-labeled G-actin was prepared in the University of Alabama cancer center. Prokaryotic expression plasmid pET21C was from Novagen (Madison, WI). Isopropyl ß-D-thiogalactopyranoside (IPTG), ampicillin, gelsolin and phalloidin were purchased from Sigma (St. Louis, MO). Taq DNA polymerase was purchased from Perkin-Elmer (Norwalk, CT). Bovine brain tubulin was purchased from Cytoskeleton (Denver, CO).
Rabbit skeletal muscle G-actin preparation
Rabbit skeletal muscle G-actin was prepared according to the method of Spudich and Watt (11). G-actin dialyzed against G-buffer (5 mM Tris-HCl (pH 8.0), 0.2 mM ATP, 0.2 mM DTT, 0.2 mM CaCl2, and 1 mM NaN3) was further purified by passage over a Sephadex G-75 column. Before use, the G-actin was centrifuged at 100,000 x g for 30 min in the Beckman (Palo Alto, CA) Optima TL Ultracentrifuge.
Construction of truncated LSP1 peptides
The strategy for creating truncated LSP1 peptides involved preparing PCR primers which place a BamHI restriction site in front of the 5' primer, a stop codon, and a HindIII restriction site behind the 3' primer (the LSP1 cDNA contains neither BamHI nor HindIII restriction sites). The PCR products were excised using BamHI and HindIII and purified by QIAEX II gel extract kit. The purified PCR products were ligated into prokaryotic expression vector pET21C, which had been cut with BamHI and HindIII and purified. When correctly ligated, this vector added six histidine residues to the C terminus of the peptide, which was then used to purify the peptides by nickel affinity chromatography.
Expression of truncated protein and protein purification
The constructed plasmids were transformed into Escherichia coli strain BL21 (DE3), the bacteria were induced with 1 mM IPTG for 3 h to express the proteins, the bacteria were lysed by three freeze-thaw cycles in liquid nitrogen and a 37°C water bath. After centrifugation at 100,000 x g for 1 h, the supernatants were passed through a nickel affinity column and the bound proteins were subsequently purified.
High-speed F-actin cosedimentation assay
Truncated and mutated LSP1 proteins in P-buffer (10 mM imidazole (pH 7.0), 75 mM KCl, 0.2 mM DTT, 0.2 mM EGTA, and 0.01% Nonidet P-40) were first spun at 100,000 x g at Beckman centrifuge for 45 min to remove aggregates. Assays of F-actin cosedimentation were performed by mixing actin with LSP1 in P-buffer containing 2 mM MgCl2 and 0.1 mM ATP. After incubation for 60 min at room temperature, the samples were spun at 100,000 x g for 45 min. The pellets were washed twice with P-buffer. Both supernatants and pellets were analyzed by SDS-PAGE, and then the bands were visualized by Coomassie blue staining and quantified by a Bio-Rad (Hercules, CA) densitometric scanner.
125I-labeled G-actin labeling
Column-purified G-actin was labeled with 125I-Bolton Hunter reagent (New England Nuclear, Boston, MA). The labeled G-actin was separated from the free 125I by passing over G-75 gel filtration column. The concentration of the G-actin was measured by BCA method.
F-actin ligand blotting
125I-labeled G-actin at 1 mg/ml was polymerized to F-actin in the presence of 0.2 µM gelsolin (100:1 mol actin to gelsolin) in polymerizing buffer (20 mM PIPES (pH 7.0), 50 mM KCl, 2 mM MgCl2, and 50 mM CaCl2) for 10 min on ice, then phalloidin was added to a final concentration of 40 µM and polymerization was continued at room temperature for an additional 15 min. 125I-labeled F-actin (125I-F-actin) was diluted to a final concentration of 50 µg/ml with blocking buffer (10 mM Tris-HCl (pH 7.5), 90 mM NaCl, 0.5% (v/v) Tween 20, 5% nonfat milk, 1% BSA, and 0.25% gelatin). Truncated LSP1 peptides were expressed in E. coli, and equal OD600 total cells were lysed with Laemmli buffer and E. coli proteins separated on SDS-PAGE and transferred to nitrocellulose (NC) membrane. The NC membrane was first treated with blocking buffer at room temperature for 2 h. After incubation with 50 µg/ml 125I-F-actin at room temperature for 6 h during constant agitation, the blots were then washed extensively with TBST (10 mM Tris-HCl (pH 7.5), 0.5% (v/v) Tween 20, and 90 mM NaCl) five times (15–30 min/wash). Blots were air-dried, and F-actin binding proteins were identified by autoradiography (12, 13). For cold probe competition assays, the blots were incubated with either cold F-actin or microtubule at room temperature for 2 h before the blots were air-dried.
In vitro site-directed mutagenesis
In vitro site-directed mutagenesis was done according to the protocol described for the Promega GeneEditor in vitro site-directed mutagenesis system. Synthesized oligonucleotides containing the mutated bases were phosphorylated by T4 polynucleotide kinase, then the phosphorylated oligonucleotides were annealed with the plasmid which contained the LSP1 cDNA. The primers were extended by T4 DNA polymerase, and the gap was ligated by T4 DNA ligase, the mutants were selected by DNA sequencing. The mutated proteins were purified by inserting the mutated gene into prokaryotic expression vector pET21C and subsequently induced with IPTG. The expressed proteins were purified.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Database searches of the LSP1 amino acid sequences to define
regions homologous to the actin-binding domains of known actin-binding
proteins revealed four regions of interest in the C-terminal half of
the protein. Two sequences separated by 23 aa were more than 60%
homologous to the F-actin binding sites of caldesmon (CI and CII, Fig. 1
) (14, 15, 16, 17). The CI region
contains a 33-aa sequence (aa 205–237), where 18 of 33 aa are similar;
the second region of homology, CII, spans 17 aa (aa 260–276), where 9
of 17 aa are similar. In addition, near the extreme C-terminal end of
LSP1, there are also two short stretches of amino acids (VI and VII)
that are almost identical to the KKEK sequences identified in the
headpiece of villin as an F-actin binding domain. This headpiece is
essential for the bundling activity and its morphologic effect(s) of
the villin molecule on cells (18, 19, 20) (see Fig. 1).
Furthermore, computer-based secondary structure analysis reveals a
series of 
-helices positioned between and following CI and CII. Also
three ß-pleated sheets that include the VI and VII domains follow the
helical structure and are similar to those found in other F-actin
binding proteins including spectrin and ABP280.
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Our initial results showed human LSP1 purified from Sf9 cells
infected with LSP1 cDNA recombinant baculovirus, similar to mouse LSP1,
can bind to F-actin. In addition, human LSP1 colocalizes with F-actin
in A7 melanoma cells that stably express LSP1 protein from the pCEP4
vector (21). Because the basic C-terminal half of LSP1
contains regions highly homologous to the F-actin binding proteins
caldesmon and villin headpiece, PCR was utilized to create an array of
truncated LSP1 peptide constructs and were analyzed for F-actin binding
activity. The constructs, designated LSP1 x-y where x is N-terminal and
y is C-terminal amino acid, were designed to include and exclude the V
and C homologous sequences in combinations and alone within unique
peptides. Truncated LSP1 cDNA was cloned into the prokaryotic
expression vector pET21C and transformed into E. coli strain
BL21 (DE3) and induced to express truncated peptides by IPTG.
Initially, the LSP1 truncates expressed in bacteria were screened for
F-actin binding domains by 125I-F-actin ligand
blot. Results of the 125I-F-actin ligand blot
screen for F-actin binding domains are shown in Fig. 2. In whole bacterial lysates of E.
coli expressing LSP1 peptides induced by IPTG,
125I-F-actin binds to several, but not all, LSP1
peptides, suggesting that LSP1 contain more than one F-actin domain. No
actin-binding peptides are present in control IPTG induced bacterial
lysates, and no binding was observed with the nonspecific control,
purified BSA protein. Gelsolin, a known F-actin binding protein on
ligand blots (4), is included as a positive control (Fig. 2C). Specific screening results show that the full-length
and C-terminal LSP1 (LSP1 181–339), but not the N-terminal LSP1 (LSP1
1–180), peptides bind F-actin in the ligand blot. Also, both the LSP1
1–305, which contains both CI and CII, and the LSP1 306–339, which
contains both VI and VII regions, bind to F-actin on ligand blots (Fig. 2A). F-actin binding by ligand blot via CI and CII
independently is also demonstrated with LSP1 181–245 and LSP1 246–295
peptides (Fig. 2B). The screening results suggest that LSP1
has at least three F-actin binding domains and that these binding
domains may differ in their apparent affinity for F-actin.
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A) but not the N-terminal
half (LSP1 1–180) (Fig. 3B) cosediment with F-actin. None
of these peptides alone sediment under these assay conditions. These
results are consistent with the overlay data that F-actin binding
domain(s) reside within the C-terminal half of the molecule.
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C). Thus, LSP1
has at least two F-actin binding domains. Because the VI and VII
domains are separated by only 7 aa, it was technically difficult to
express and purify VI and VII and analyze them as independent peptides.
In contrast, truncated peptides including CI or CII (LSP1 181–245 and
LSP1 246–295) were sufficiently large and sufficiently spaced to allow
independent analysis of CI and CII to further define F-actin binding
domains of LSP1. The results show these peptides each retain an F-actin
binding domain (Fig. 3D). In summary, these results show
LSP1 has at least three, and possibly four, F-actin binding domains,
CI, CII, and VI/VII. Given the sequence homologies, it is possible that
VI and VII can also independently bind F-actin. These results,
summarized in Fig. 4, support the
contention that LSP1 has at least three F-actin binding domains. Two
LSP1 peptides, LSP1 1–275 and LSP1 181–275, yielded unexpected
results. Although both contain the defined CI and CII sequences,
neither binds F-actin. Inclusion of 20 additional C-terminal amino
acids in these peptides (LSP1 1–295) restores F-actin binding,
suggesting that the 20 additional amino acids contribute important
secondary structure.
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Quantative analysis by Scatchard plot of murine LSP1 binding to
F-actin by Jongstra-Bilen et al. (6) suggested that murine
LSP1 might contain high- and low-affinity F-actin binding domain.
However, these domains were not clearly characterized. Our quantitative
analysis of F-actin binding by cosedimentation shows that LSP1
1–305 binding to F-actin is lower affinity than the binding of
full-length LSP1 (Fig. 5A).
This finding also suggests that LSP1 306–339 indeed contain another
F-actin binding domain. It should be noted that at equal molar ratio of
both LSP1 peptides, the quantity of LSP1 1–305 which cosediments with
F-actin is quantitatively less (<20% by cosedimentation) than that of
LSP1 1–339. The result indicates the CI, CII binding to F-actin is
weaker than full length and suggests the V regions may represent strong
binding domains while C regions may represent relatively weaker binding
domains. It is also possible that VI and VII domains may cooperate with
each other and with CI and CII to strengthen F-actin binding.
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B). VI and VII respectively contain the sequences KYEK and
KRYK that are similar to the villin headpiece sequence KKEK, the
F-actin binding domain of villin. Mutation of a single positively
charged amino acid to glutamic acid (E) in the VII domain (KYEK to
KYEE) weakens full-length LSP1 binding to F-actin (see Fig. 5B). Similar results were obtained with point mutations in
the VI region. Furthermore, quantitative assay of LSP1 wild-type and VI
or VII mutant binding to F-actin over a range of molar ratios of LSP1
to F-actin (Fig. 5C) shows LSP1 affinity for and saturation
binding to F-actin is dramatically altered by basic amino acid to
acidic amino acid changes in either VI or VII sequences. Similar
results are obtained on 125I-F-actin ligand blot
(data not shown). The results show both the VI and VII sequences are
independently important for F-actin binding. In combination with
analysis of LSP1 truncates these results demonstrate that LSP1 contains
at least three, and likely four, independent F-actin binding domains in
its C-terminal half. LSP1 binds to F-actin through electrostatic interaction
The actin-binding domains of many actin-binding proteins contain
clustered basic amino acids. This observation seems to be a repeating
theme in actin-binding proteins, which may reflect their evolutionary
origin. Actin-binding domains at clustered basic amino acids in
proteins include the KKGGKKKG sequence of myosin, DAIKKK sequence in
actin depolymerizing factor, cofilin and tropomyosin, and KSKLKKT
sequence in thymosin ß4 (22). Vandekerckhove
(22) has suggested that the extreme N-terminal peptide
Ac-DEDE of actin molecule is the binding site on actin that is the
ligand for these positively charged sequences on actin-binding
proteins. Several lines of evidences including chemical cross-linking
are consistent with this suggestion (22, 23). Such a
situation will inevitably lead to competition between different
actin-binding proteins for actin in cells. Because the F-actin binding
domains of LSP1 localize in the basic C-terminal half of the molecule,
we reasoned that the interaction of LSP1 with F-actin may also be
through electrostatic interaction and therefore that LSP1 may compete
with other actin-binding proteins. To test the possibility that the
LSP1 to actin interaction is controlled electrostatically, we increased
the concentration of KCl in the F-actin cosedimentation assay to
determine whether increased ionic strength would dissociate LSP1 from
F-actin. The increase in KCl concentration did not affect the
solubility of LSP1 protein (data not shown). Increasing KCl
concentration did not affect the polymerization of actin, as shown in
Fig. 6, which was consistent with the
fact that actin-actin interaction is mainly hydrophobic
(24). However, when the concentration of KCl was
increased, the binding of LSP1 to F-actin decreased. This observation
suggests that at least one of the three F-actin binding domains
associate with F-actin through electrostatic interaction. Such
electrostatic interactions may be nonspecific. However several lines of
evidence show the LSP1 interaction with actin is specific. As shown in
Fig. 7, 125I-F-actin ligand blot demonstrate cold F-actin
at >20-fold excess significantly dissociates
125I-F-actin from binding. This effect is
specific to F-actin polymer since 50- to 100-fold excess cold taxol
stabilized microtubule does not compete away F-actin binding.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we have mapped the F-actin binding domains of human LSP1, an F-actin binding protein. Overexpression of LSP1 modifies the cytoskeletal structure and motility of PMNs. The F-actin binding domains of LSP1 were mapped to three regions of the molecule: aa 181–245, aa 246–295, and aa 306–339. Each domain contains amino acid sequences homologous to those in the F-actin binding proteins caldesmon or the villin headpiece. Two complementary methods were utilized to define the F-actin binding domains. The high-speed F-actin cosedimentation assay utilizes purified native proteins and excludes specific or nonspecific interference from other proteins. The F-actin ligand blotting utilizes 125I-radioactive labeled F-actin. This method is more useful in screening for potential F-actin binding domains including those peptides that have weak binding to F-actin and those peptides which might form homologous aggregates or easily precipitate under the conditions of F-actin cosedimentation assay condition. With the F-actin cosedimentation assay, it is difficult to determine whether homologous aggregates might explain cosedimentation with F-actin. In contrast, 125I-F-actin ligand blot screens independent of peptide aggregates. One limitation of the F-actin ligand blotting is that this method uses denatured and renatured proteins, resulting in the uncertainty that some proteins may not be properly renatured. Combining these two methods provides the most accurate and easily interpreted results.
Although domain mapping through creation of actin-binding protein
truncates is a standard method for defining the functional domains of
actin-binding proteins, it has its limitation. For example, truncates
may have a significantly altered conformation resulting from deleting
adjunct sequences, and therefore may provide little insight into the
function of full-length actin-binding proteins. An example is the
microtubule associated protein 2 (MAP2) and tau molecules binding to
microtubule. It was shown that the extreme C-terminal hydrophobic
-helix in these proteins is responsible for the microtubule bundling
activity of MAP2 and tau by domain mapping (32). However,
in subsequent experiments exploiting site-directed mutagenesis and
deletion mutation experiments, it was shown that the regions identified
by truncation are not directly responsible for microtubule bundling
activity of these proteins (33). Therefore, caution should
be taken in interpreting data from truncated peptides alone to map the
functional domains of a molecule as the sole method for defining
actin-binding domains. The same situation has been encountered in this
study with analysis of LSP1 actin-binding domains. Clearly, the results
show the truncated peptide LSP1 181–245 and peptide LSP1 246–295,
which respectively contain CI and CII alone, can each bind to F-actin
independently, and LSP1 1–295, which include these two fragments, can
also bind to F-actin. However, LSP1 1–275, which include LSP1 181–245
and part of LSP1 246–295, does not bind to F-actin either in the
F-actin cosedimentation assay or the F-actin ligand-blotting assay.
This result suggests aa 275–295 may modify the conformation of the
truncates and therefore affect the F-actin binding. Thus the
three-dimensional structure of the molecule is as important as the
primary amino acid sequences. To fully confirm the findings reported
here, the crystal structure of the LSP1 would be required.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas H. Howard, Division of Hematology-Oncology, Department of Pediatrics, University of Alabama, 1600 7th Avenue South, Birmingham, AL 35233.
3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophil; NAD, neutrophil actin dysfunction; LSP1, lymphocyte-specific protein 1; IPTG, isopropyl ß-D-thiogalactopyranoside; 125I-F-actin, 125I-labeled F-actin.
Received for publication May 26, 1999. Accepted for publication May 30, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and two villin
headpiece-like (VI and VII) (
) amino acid sequences in the
C-terminal half of the molecule. These sequences are homologous to the
actin-binding domains of caldesmon and the villin headpiece. Shown in
single letter amino acid code are the location and sequences of the CI,
CII, VI, and VII homologous regions. Numbers reveal the amino acids in
N- to C-terminal direction for LSP1 (1–339), caldemon (597–667), and
villin (805–826). The boundary of truncated peptides are
labeled.
