©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Gain-of-Function Mutations Conferring Actin-severing Activity to Human Macrophage Cap G (*)

(Received for publication, August 25, 1994; and in revised form, October 17, 1994)

Frederick S. Southwick (§)

From the Department of Medicine, Infectious Disease Division, and the Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, Florida 32610

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Nonmuscle cell motility requires marked changes in the consistency and shape of the peripheral cytoplasm. These changes are regulated by a gel-sol transformation of the actin filament network, and actin filament-severing proteins are responsible for network solation. Macrophage Cap G, unlike all other proteins in the gelsolin family, caps but does not sever actin filaments. Two amino acid stretches in Cap G diverge markedly from the severing proteins: LNTLLGE and AFHKTS. Discrete mutations in Cap G have been generated to determine if these amino acid sequences are critical for actin filament severing. Conversion of LNTLLGE to the gelsolin actin-binding helix sequence (LDDYLGG) renders Cap G capable of severing actin filaments (half-maximal severing, 1-2 µM). Adding a second set of mutations, converting AFHKTS to GFKHV, enhances severing by 10-fold (half-maximal severing, 0.1-0.2 µM). These experiments support a critical role for these two regions in actin filament severing and showcase the power of gain-of-function mutations in clarifying structure-function relationships.


INTRODUCTION

Cell movement requires rapid remodeling of the peripheral cytoplasm, and remodeling is achieved by gel-sol transformations of the actin filament network. The peripheral cytoplasm of motile cells is organized into an extensive orthogonal network of actin filaments cross-linked by ABP-280. Dissolution or solation of this network is brought about by severing actin filaments. The protein thought to serve this function in many cell types is gelsolin. Understanding the mechanism by which gelsolin and other severing proteins break apart preformed filaments is critical to understanding how cells generate amoeboid movement and ingest particles by phagocytosis.

Gelsolin was the first member of a family of actin regulatory proteins to be described(1) . Other members include villin, adseverin, severin, and fragmin(2) . Like gelsolin, all of these members sever actin filaments. Macrophage Cap G, previously named macrophage-capping protein(3, 4, 5) , gCap39(6) , and Mbh1(7) , is a more recently discovered member of this family. To eliminate confusion, investigators have agreed to rename the protein Cap G: ``Cap'' to signify that this protein is the only member of the gelsolin family that caps the barbed ends of actin filaments, but cannot sever them(3) , and ``G'' to emphasize that Cap G shares highest sequence identity with gelsolin (49% identity)(4, 6, 7) . Like severin and fragmin, Cap G is one-half the size of gelsolin and villin, containing three rather than six repeat domains. Cap G, like all members of the gelsolin family, requires micromolar Ca to bind actin.

Cap G is the only member of the gelsolin family that does not sever actin filaments. I predicted that amino acid sequences shared by all of the severing proteins in this family, but divergent in Cap G, should identify those segments critical for severing function. Indeed, two sequences in Cap G proved to be divergent. To test my hypothesis, specific mutations were made to convert these divergent stretches to the amino acid sequences found in gelsolin. A gain of function was achieved, and Cap G was converted from a strictly capping to a capping and severing protein. My investigations provide functional support for the prediction of McLaughlin et al.(8) , based on crystallographic data of segment 1 of gelsolin, that the ``actin-binding helix'' (residues 100-117 of gelsolin) plays a critical role in actin filament severing.


EXPERIMENTAL PROCEDURES

Mutagenesis, Protein Expression, and Purification

Site-specific polymerase chain reaction mutagenesis (10) was used to place multiple codon substitutions as well as one deletion (His) in human macrophage Cap G cDNA (4) . To generate the GFKHV/LDDYLGG mutant, mutant GFKHV cDNA was used as the template for generation of the LDDYLGG mutation. Each mutant DNA was then subcloned into the pET12a expression vector(4) . In each instance following isopropyl-1-thio-beta-D-galactopyranoside exposure, transduced Escherichia coli BL21 cells readily expressed the protein of interest. Recombinant protein was purified by DEAE ion-exchange chromatography as described previously(4) . In all instances, the recombinant proteins were purified to homogeneity as assessed on Coomassie Blue-stained SDS-polyacrylamide gels. All mutant cDNAs used for the production of recombinant proteins were fully sequenced to confirm their construction. Gelsolin was purified from rabbit alveolar macrophages as described previously(1) .

Actin Filament Severing and Capping Assays

Skeletal muscle actin was purified and pyrene-conjugated using standard methods(5) . Gelsolin-actin filaments were generated by polymerizing 2 µM (final concentration) pyrenyl-actin overnight at 25 °C in S2 buffer (0.5 mM ATP, 1 mM dithiothreitol, 1 mM CaCl(2), 0.1 M KCl, 1 mM MgCl(2), and 10 mM imidizole HCl, pH 7.5) containing 10 nM (final concentration) gelsolin (purified from rabbit alveolar macrophages) (1) . These barbed-end capped filaments were diluted to a final concentration of 100 nM in S2 buffer containing varying concentrations of the mutant Cap G proteins, and depolymerization was monitored by a Perkin-Elmer LS5 fluorometer. For measurement of barbed-end capping, pyrenyl-actin (2 µM final concentration) was polymerized in buffer alone or in the presence of two different concentrations (40 and 20 nM final concentrations) of the native and mutant proteins to produce capped actin filaments. These filaments were then diluted 40-fold in S2 buffer, resulting in final concentrations of 50 nM for the actin filaments and 1 and 0.5 nM for the Cap G proteins.


RESULTS AND DISCUSSION

Primary Structural Comparisons of Cap G with Gelsolin, Villin, Severin, and Fragmin

I have taken advantage of Cap G's inability to sever filaments to identify amino acid sequences playing important roles in actin filament severing. I reasoned that any amino acid sequence shared by the four severing proteins, gelsolin, villin, severin, and fragmin, but not by Cap G, should identify sites most likely to be required for severing activity. Truncation mutants of gelsolin reveal that severing function is contained in the first 160 amino acids(11, 12, 13) . Therefore, my comparisons focused primarily on these regions. One region, previously suggested to be a putative severing domain(14) , shared substantial identity with Cap G (67%) (Fig. 1A), indicating that this region was unlikely to be responsible for severing. Two other regions, however, were found to have markedly divergent sequences in Cap G (Fig. 1, B and C). In one 14-amino acid region in domain 1, while there was a 65-70% identity among gelsolin, villin, severin, and fragmin, only a 25% identity was observed with Cap G (Fig. 1B). Comparisons with gelsolin using the PAM250 scoring index (15) also revealed a marked difference in the similarity score between Cap G and the three other barbed-end capping proteins (PAM250 matrix scores: villin, 57; severin, 47; fragmin, 56; and Cap G, 27). This region also shared a strong identity with the fourth domain of alpha- and beta-actins (Fig. 1B)(16) . The underlined sequence of actin in Fig. 1B demonstrated greater similarity to the severing proteins than to Cap G.


Figure 1: Sequence comparisons of Cap G, gelsolin, villin, fragmin, and severin. A, amino acid sequences were compared within the region previously suggested to be important for severing. The shaded areas show regions of high identity. Two regions (in B and C) revealed deviation of the Cap G sequence (boldfaceletters in unshadedblocks) from the consensus sequences shared by the severing proteins. Gaps are designated by dashes. The region of identity to the alpha- and beta-actin sequence is underlined in B. The region highly conserved among the severing proteins, but different in Cap G, is underlined in C.



The second region in which Cap G differed from the four other proteins was located at the end of the first domain and the beginning of the second domain of gelsolin (Fig. 1C)(7) . In a highly conserved 5-amino acid sequence, GFKHV, Cap G had one amino acid insertion as well as three amino acid substitutions. While Cap G had 2 polar uncharged amino acids at the COOH-terminal end of this consensus sequence (Thr and Ser), all severing proteins were characterized by a polar charged amino acid (His) followed by a hydrophobic residue (Val).

Generation of Gain-of-Function Mutations

To test my hypothesis that these regions are critical for actin filament severing, polymerase chain reaction site-directed mutagenesis was used to introduce these gelsolin amino acid sequences into Cap G cDNA. Using the pET12a vector, the mutant protein was then expressed in E. coli BL21 cells and purified to homogeneity using DEAE ion-exchange chromatography.

I first investigated the functional consequences of changing the sequence AFHKTS to GFKHV, thus making this amino acid stretch identical to gelsolin (Fig. 1C). As shown in Fig. 2and Fig. 5, like native Cap G, the GFKHV mutant failed to sever preformed actin filaments at final concentrations as high as 3 µM. The apparent K(D) for actin filament capping was also identical to that of native recombinant Cap G, 0.5 nM (data not shown).


Figure 2: Severing activity of GFKHV mutant Cap G protein. 2 µM (final concentration) pyrenyl-actin polymerized overnight in S2 buffer containing 10 nM (final concentration) gelsolin was diluted to a final concentration of 100 nM in S2 buffer containing final concentrations of 1.5 µM (solidcircles) and 3 µM (solidsquares) GFKHV mutant Cap G. Depolymerization was monitored by a Perkin-Elmer LS5 fluorometer. No acceleration in the depolymerization was observed, consistent with a lack of severing activity.




Figure 5: Bar graph comparing the dose dependence of the initial depolymerization rates (first 2 min) for the three mutant proteins.



Next, the sequence LNTLLGE was changed to LDDYLGG using the same methods (Fig. 1C). The resultant mutant protein possessed calcium-sensitive severing function ( Fig. 3and Fig. 5), although requiring high concentrations of protein to produce significant severing (1-2 µM final concentrations). The same concentrations of native recombinant Cap G failed to demonstrate severing activity (data not shown). Actin filament capping of the LDDYLGG mutant was similar to that of native Cap G (K(D) = 0.5 nM; see Fig. 6).


Figure 3: Severing activity of LDDYLGG mutant Cap G. Gelsolin-pyrenyl-actin filaments prepared as described in the legend to Fig. 2were diluted to 100 nM in S2 buffer alone (opencircles) or in S2 buffer containing the LDDYLGG mutant Cap G protein (0.5 (solidcircles), 1 (solid triangles), and 2 (solidsquares) µM final concentrations). Note the concentration-dependent increase in the depolymerization rate, indicating the production of increasing numbers of uncapped filament ends due to actin filament severing.




Figure 6: Measurement of barbed-end capping by native Cap G (open squares) and by the two severing mutants LDDYLGG (closed circles) and GFKHV/LDDYLGG (closed squares). The assay is described under ``Experimental Procedures.'' Pyrenyl-actin was polymerized in S2 buffer alone or in S2 buffer containing different concentrations of Cap G or the mutant Cap G protein. Actin filaments were then diluted 40-fold in S2 buffer to a final concentration of 50 nM. The resulting final concentrations of Cap G and the mutant Cap G proteins are depicted on the right side of the graph. The depolymerization rates were slowed to a similar extent by each of the proteins, indicating similar apparent affinities for the barbed filament ends. Initial fluorescence values were corrected to allow visual comparisons of the depolymerization rates of the different samples.



A third mutant protein was also generated containing both the GFKHV and LDDYLGG mutations. As shown in Fig. 4and 5, this mutant was capable of severing actin filaments at markedly lower concentrations than the LDDYLGG mutant (one-tenth the concentration), with significant severing being observed at final concentrations between 0.1 and 0.2 µM. The capping function of this mutant was also found to be similar to that of native Cap G (Fig. 6).


Figure 4: Severing activity of GFKHV/LDDYLGG mutant Cap G. Gelsolin-pyrenyl-actin filaments were diluted in buffer alone (control (opencircles)) or in buffer containing increasing concentrations of the GFKHV/LDDYLGG mutant (0.1 (solidcircles), 0.2 (solid triangles), 0.5 (solid squares) µM final concentrations) and were monitored as described in the legend to Fig. 2. Note that approximately one-tenth the concentration used in Fig. 3was required to generate a similar degree of severing.



The severing ability of cytoplasmic gelsolin purified from rabbit alveolar macrophages was also examined using the same assay system. As shown in Fig. 7, gelsolin was capable of severing at much lower concentrations than the GFKHV/LDDYLGG mutant (approximately one-fiftieth the concentration of gelsolin produced comparable severing).


Figure 7: Severing activity of full-length gelsolin. Gelsolin-pyrenyl-actin filaments were diluted in buffer alone (control (opencircles)) or in buffer containing increasing concentrations of gelsolin (1.2 (solid circles), 5 (solid triangles), 10 (solidsquares), and 20 (open triangles) nM final concentrations) and were monitored as described in the legend to Fig. 2. Note that approximately one-fiftieth the concentration of the mutant Cap G protein used in Fig. 5was required to generate a similar degree of severing.



Structure-Function Implications

To my knowledge, this is the first time that discrete mutations have resulted in the addition of a function to an actin regulatory protein. Gain of function is a far more powerful tool for inferring structure-function relationships than mutations that result in the loss of a function. There are a number of explanations for loss of function, including increased susceptibility to proteolysis or denaturation, changes in tertiary structure that serve to bury the functional domain, as well as direct changes in the functional domain of interest. Therefore, interpretation of a loss-of-function mutant is often difficult. Other investigators have used large cassette mutations to add a function to an actin regulatory protein(17) . Such experiments, however, fail to exactly localize structure-function relationships, narrowing functions only to large domains of 12-14 kDa.

My finding that the LDDYLGG region was capable of conferring severing function to a capping protein is consistent with the three-dimensional structure of the gelsolin-actin complex(8) . The LDDYLGG segment has been shown to be the central region of a long alpha-helix (extending from residues 100 to 117 in plasma gelsolin) that interacts within the cleft formed by actin subdomains 1 and 3. This actin-binding helix consists of a central region of apolar side chains flanked by polar hydrogen-bonding groups and is predicted to bring about severing by inducing steric clashes with subdomain 2 of the adjacent actin subunit in the same strand(8) . The present findings show that the LDDYLGG mutation alone in Cap G can sever actin filaments only at high concentrations, suggesting that other regions of the protein might also play a role in severing. Conversion of a second segment from the native sequence to that of gelsolin (i.e. addition of the GFKHV mutation) enhances severing activity by a factor of 10. This amino acid sequence is contained in the region of gelsolin (amino acids 150-160; see Fig. 1) known to confer severing activity to truncated gelsolin(11) . However, the gelsolin-(1-160) truncation mutant has recently been shown to have lower severing activity than full-length gelsolin(9) . Similarly, although the addition of the GFKHV mutation enhances Cap G's ability to sever, this change fails to increase severing activity to levels observed in full-length gelsolin. The GFKHV region in gelsolin is not required for side binding(9) ; therefore, the mechanism by which this amino acid sequence enhances severing in Cap G remains to be elucidated.

Significantly, none of the structural changes produced by my mutations affected Cap G's ability to cap the barbed ends of actin filaments, indicating that different structural determinants mediate the capping and severing of actin filaments. Further consideration of these gain-of-function mutant proteins is likely to divulge how other actin regulatory activities (e.g. actin nucleation, actin monomer binding, and actin filament side binding) relate to actin filament severing. Future investigations of these gain-of-function mutants promise to provide even further insight into how phagocytic cells sever actin filaments to generate the shape changes critical for amoeboid movement.


FOOTNOTES

*
This work was supported by Grant RO1 AI23262 from the National Institutes of Health. 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 Medicine, Infectious Disease Div., University of Florida College of Medicine, P. O. Box 100277, Gainesville, FL 32610. Tel.: 904-392-4058; Fax: 904-392-6481.


ACKNOWLEDGEMENTS

I thank Linda Erwin and Mylinh McClure for technical assistance. I also appreciate the helpful advice provided by Drs. David Helfman, Dan Purich, Brian Cain, and Paul McLaughlin.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.