AAMP, a Newly Identified Protein, Shares a Common Epitope with α-Actinin and a Fast Skeletal Muscle Fiber Protein

EXPERIMENTAL CELL RESEARCH ARTICLE NO.

225, 306–314 (1996)

0181

AAMP, a Newly Identified Protein, Shares a Common Epitope with a-Actinin and a Fast Skeletal Muscle Fiber Protein M. E. BECKNER,*,1 H. C. KRUTZSCH,* S. KLIPSTEIN,* S. T. WILLIAMS,* J. E. MAGUIRE,* M. DOVAL,† AND L. A. LIOTTA* *Laboratory of Pathology, National Cancer Institute, NIH, Bethesda, Maryland 20892; and †The Washington Hospital Center, Washington, DC 20010

AAMP (angio-associated migratory cell protein) shares a common epitope with a-actinin and a fasttwitch skeletal muscle fiber protein. An antigenic peptide, P189, derived from the sequence of AAMP was synthesized. Polyclonal antibodies generated to P189 readily react with AAMP (52 kDa) in brain and activated T lymphocyte lysates, a-actinin (100 kDa) in all tissues tested, and a 23-kDa protein in skeletal muscle lysates. The antibody’s reactivity for a-actinin can be competed with the purified protein. Activation of T lymphocytes does not alter the degree of a-actinin reactivity with anti-P189 as it does for AAMP’s reactivity in these lysates. Competition studies with peptide variants show that six amino acid residues, ESESES, constitute a common epitope in all three proteins in human tissues. The antigenic determinant is continuous in AAMP but discontinuous (or assembled) in aactinin. a-Actinin does not contain this epitope in its linear sequence so reactivity is attributed to an epitope formed by its secondary structure. Limited digestion of the reactive proteins with thermolysin destroys anti-P189’s reactivity for a-actinin while reactivity for recombinant AAMP is retained. Specificity of antiP189 for human skeletal muscle fast fibers seen on immunoperoxidase staining may be explained by antiP189’s reactivity with a 23-kDa protein found only in skeletal muscle lysates. Its pattern of reactivity is the same as that obtained using monoclonal anti-skeletal muscle myosin heavy chain in type II (fast-twitch) fibers. q 1996 Academic Press, Inc.

INTRODUCTION

The study of a newly discovered protein, AAMP (angio-associated migratory cell protein), has led to the identification of an epitope that is shared with other proteins, a-actinin in muscle and non-muscular tis1 To whom correspondence and reprint requests should be addressed at Laboratory of Pathology, Bldg. 10, Rm. 2A-33, NCI, NIH, 9000 Rockville Pike, Bethesda, MD 20892. Fax: 301-402-0043.

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0014-4827/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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sues, and an unknown skeletal muscle restricted protein. AAMP has been found in migratory type cells such as endothelial cells, trophoblasts, tumor cells in the circulation, activated T lymphocytes, etc. By immunoperoxidase staining it has been seen intracellularly in multiple cell types [1]. It is also seen extracellularly in cultures of endothelial cells (unpublished). It has been conserved through evolution in that a closely related protein (YCR072c) in Saccharomyces cerevisiae has been found. Sequence motifs in AAMP (two immunoglobulin type domains and six WD40 repeats) warrant its inclusion in both the immunoglobulin superfamily and the WD40 repeat family of proteins [1]. This is unique and may imply a multifunctional role for AAMP in migratory cells. Many immunoglobulin superfamily members are binding proteins. They are usually extracellular and function as adhesion proteins, but some are intracellular [2, 3]. The WD40 repeat proteins belong to a more recently described family. Many are part of intracellular, multimeric complexes that participate in regulatory events [4 –12]. A growing consensus about the function of these proteins proposes that their tandem repeats bind to other proteins in stable, structural relationships, while their nonrepeat portions, which contain variable sequence motifs, participate in dynamic, reversible interactions that may account for a variety of specific functions [8, 11, 13–15]. The WD40 repeats contain approximately 40 amino acids and commonly end with tryptophan and aspartic acid residues. bTransducin, a well-known signal transduction protein that was first found to contain these repeats [16], serves as a prototype of the family and is often mentioned as a relative of newly identified proteins with these repeats. It’s signaling activities are shared by some but not all of the family members [7, 14, 17, 18]. Non-skeletal muscle a-actinin has been found in diverse cell types (smooth muscle, brain, kidney, liver, platelets, fibroblasts, etc.) and has been detected in plasma membrane-associated structures. Skeletal muscle or myofibrillar type a-actinin is found only in the Z lines. Both classes of a-actinin bind F-actin but usually

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differ in their sensitivity to calcium for this activity. In regard to actin, a-actinin is thought to function in two roles: (1) anchoring actin filaments to a rigid structure and (2) regulating dynamic changes of the filament structure [19–21]. a-Actinin may be involved in cell motility due to its ability to structure the actin network in retraction fibers and in less adherent pseudopods [22]. Others have also found cross-reacting antibodies between a-actinin and other proteins that are dependent on secondary structure using peptides derived from cross-reacting proteins as immunogens [23–25]. The epitope shared by AAMP, a-actinin (muscle and nonmuscle), and a skeletal muscle protein was found in a peptide, P189, that was derived from AAMP’s sequence. P189 was synthesized to generate antibodies and for use in functional studies. Anti-P189 showed specific reactivity for AAMP in tissue and to recombinant AAMP by competition on immunoblots [1]. P189 is a synthetic, bipolar peptide (RRLRRMESESES) with positive charges in its amino-terminal region and negative charges in its carboxyl-terminal half. The immunoreactivity of the carboxyl half, ESESES, is the focus of this study. MATERIALS AND METHODS Production of peptides. Peptides, P189 and its variants (P350, P357-P360, and P369), were synthesized on a Biosearch Model 9600 peptide synthesizer using standard Merrifield solid-phase synthesis protocols and t-butoxycarbonyl chemistry. The peptides were analyzed by reverse-phase high-performance liquid chromatography. The identification of P189 as a sequence likely to elicit significant antibody production was obtained using the method of Hopp and Woods [26, 27]. Production of polyclonal antibodies. Polyclonal anti-peptide antibodies specific for P189 were generated in rabbits using P189 conjugated to bovine albumin. Peptide was injected every other week in 1-mg protein aliquots. The rabbits were bled (10 ml serum) during the intervening weeks. Antibodies from inactivated sera were affinity purified on columns of peptide covalently attached to Affi-Gel 10 beads (Bio-Rad Lab, Richmond, CA). Polyclonal anti-recombinant AAMP (anti-rAAMP) was prepared as previously described [1]. Lysate preparations. Human tissues were homogenized in 3% sodium dodecyl sulfate and 4% b-mercaptoethanol buffer at 1007C. Whole cell lysates of A2058 melanoma cells (passaged 15–20 times) were prepared in 0.5% Nonidet P-40 buffer. T lymphocytes from the peripheral blood of a normal donor were isolated by elutriation (95% pure) (gift of Dr. Larry Wahl, National Institute of Dental Research, NIH). Cultures of cells were stimulated with phytohemagglutinin, 1 mg/ml, and harvested at timed intervals. Cell lysates (1.5 ml buffer/ 60 million cells) were prepared with 3% sodium dodecyl sulfate (SDS), 4% b-mercaptoethanol, shearing, and boiling. Thermolysin digestions. Rabbit a-actinin (0.9 mg/ml) (Sigma, St. Louis, MO) was dialyzed in digestion buffer (40 mM ammonium acetate, 1 mM calcium chloride) to remove storage buffer and protease inhibitors. a-Actinin and recombinant AAMP (0.22 mg/ml) were partially digested with thermolysin (Protease Type X from Bacillus thermoproteolyticus) (Sigma) [28]. Thermolysin concentration for both digestions was 0.01 mg/mg substrate protein. Limited proteolysis occurred in 15-min incubations at 377C. Controls without thermolysin were treated similarly. Anti-a-actinin (polyclonal rabbit) (Sigma)

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was used to identify the protease-resistant products of a-actinin on immunoblots. Immunoblot preparation. Lysates and protein molecular weight standards were reduced, electrophoresed in 10% polyacrylamide – SDS gels, and transferred to blots. Blots were reacted with affinitypurified anti-P189 (1 –2 mg/ml), anti-rAAMP (0.1 or 1 mg/ml), or antia-actinin (1:1000) as the primary antibodies and goat anti-rabbit (H/L) horseradish peroxidase conjugate (0.5 mg/ml) (Kirkegaard & Perry, Gaithersburg, MD) as secondary antibody. Blots were developed with diaminobenzidene. Epitope localization with peptide variants of P189. The amino acid sequence in a-actinin, AAMP, and the 23-kDa skeletal muscle protein that reacted with anti-P189 was identified. Immunoblot reactions were performed on lysates of brain and skeletal muscle and purified recombinant AAMP using peptide variants of P189 for competition of anti-P189’s reactivity. Competing peptides were used at 50 1 the concentration of anti-P189 (1 mg/ml) on a weight basis. The degree of peptide competition of antibody reactivity is indicated by grades: 4/ Å complete or ú95%, 80% õ 3/ õ 95%, 30% õ 2/ õ 80%, 1/ Å detectable and õ30%, and 0 indicates no competition. The peptide variants of P189 (RRLRRMESESES) included P350 (MSRERESRERSL), P357 (QQLQQMESESES), P358 (RRLRRMQSQSQS), P359 (RRGRRGESESES), P360 (RRLRRMEAEAEA), and P369 (RLRRMESESE). Immunohistochemical staining. The ABC (avidin biotinylated horseradish peroxidase macromolecular complex) method of immunohistochemical staining (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA) was used to stain skeletal muscle. For comparison of anti-P189 and anti-skeletal myosin (type II, fast-twitch fiber), sections of human pectoralis muscle (autopsy material, 62year male who died of an intracranial hemorrhage), fixed in formalin and paraffin embedded, were deparaffinized and reacted separately with the primary antibodies. Monoclonal (mouse IgG1 isotype, clone MY-32) anti-skeletal myosin (fast) from ascites fluid (Sigma), monoclonal (mouse IgG1 isotype, clone EA-53) anti-a-actinin (sarcomeric) from ascites fluid (Sigma), and affinity-purified, polyclonal anti-P189 from rabbit sera were used at dilutions of 1:400, 1:800, and 1.5 –3 mg/ml, respectively. Anti-a-actinin (sarcomeric) required proteolytic digestion before staining. MOPC at 1:333 (Cappel/Organon, Inc.), rabbit immunoglobulin (Sigma) at concentrations comparable to anti-P189, and second antibodies only were used as negative control antibodies. Any endogenous peroxidases were previously quenced with hydrogen peroxide. The slides had been blocked (diluted serum in which the secondary antibody had been made) prior to primary antibody exposure. Appropriate mouse and rabbit second antibodies were used. Diaminobenzidine was used for stain development. For double labeling, it was used with and without nickel chloride. Slides were counterstained with either methyl green or Mayer’s Hematoxylin Solution (Sigma).

RESULTS

Detection of proteins in mammalian cells and tissues with polyclonal anti-P189. A survey of human cells and tissues using immunoblots shows that the antipeptide antibody, anti-P189, reacts with several proteins of different molecular weights (Fig. 1A). The 52kDa protein, AAMP, which is seen in brain (human and calf), has been previously described [1]. It is known to contain the linear sequence of P189 near its amino terminus. The 100-kDa protein is present in all human cells and tissues tested: nonmalignant (brain, skin, kidney, lung, lymph node, liver, skeletal muscle, heart, small bowel mucosa, and lymphocytes) and malignant

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FIG. 1. Immunoblots of human tissues reacted with polyclonal anti-P189. (A) Lanes 1–9 contain reduced tissue lysates of human brain, skin, kidney, metastatic melanoma, lung, lymph node, liver, skeletal muscle, and heart, 40 mg protein per lane. Lane 10 contains prestained protein molecular weight standards that are 215, 100, 69, 43, and 28 kDa, in vertical descending order. AAMP is present in many tissues [1] but the anti-P189 epitope is most available in brain (shown here) and activated T lymphocytes as shown in Fig. 3. a-Actinin has been seen in all tissues tested (normal and malignant). The 23-kDa band has been seen consistently and exclusively in skeletal muscle. The unlabeled bands do not react as consistently or as strongly as the labeled bands. Anti-P189 was 1 mg/ml. (B) Lanes 1 –8 contain human brain lysate in decreasing amounts, 50, 26.5, 13, 6.6, 3.3, 1.6, 0.8, and 0.4 mg protein per lane, respectively. Lane 9 contains prestained protein molecular weight standards that were loaded as described above. Anti-P189 was 2 mg/ml. Samples were reduced, electrophoresed in 10% polyacrylamide gels with SDS present, and transferred to nylon membranes.

(metastatic melanoma and small bowel adenocarcinoma). A 23-kDa protein is seen only in skeletal muscle. Other immunoreactive bands react with less intensity and/or are inconsistently present on repeated immunoblots. Anti-P189’s reactivities (2 mg/ml) for the 52- and 100-kDa proteins in brain were compared by reacting it with a blot in which decreasing amounts of brain tissue lysate were loaded in each lane. The 52kDa band, AAMP, was the predominant band at low concentrations (Fig. 1B). Identification of the 100-kDa protein as a-actinin. Known proteins with a molecular weight similar to that of the largest anti-P189 reactive band (seen in all tissues) include a-actinin (a dimer of 100-kDa subunits). It is also widely distributed, available in pure form, and was therefore tested. The reactivity of pure a-actinin (0.5 to 8 mg per lane) from human placenta corresponds with that of the 100-kDa band seen in skeletal muscle (54 mg protein per lane) (Fig. 2A). The reactivity of the 100-kDa band with anti-P189 (2 mg/ml) on an immunoblot of human A2058 melanoma cells (80 mg protein/lane) was successfully competed with pure aactinin (189 mg/ml) (Fig. 2B). Other purified intracellular proteins, myosin regulatory light chain (DTNB chain), and myosin essential light chains (alkali chains 1 and 2), 18 mg each, failed to show reactivity with antiP189 (2 mg/ml) on immunoblots (not shown). Expression of AAMP and a-actinin in a time course of T lymphocyte activation using anti-P189. The reactivity of AAMP’s ESESES epitope in activated T cells is shown. The stability of a-actinin’s expression in a time course of T lymphocyte activation is contrasted

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with the increase of AAMP’s expression seen on an immunoblot reacted with anti-P189 (Fig. 3). a-Actinin serves as an internal standard in the demonstration of phytohemagglutinin’s (1 mg/ml) stimulation of AAMP’s expression in normal T lymphocytes (31.5 mg protein/ lane) in tissue culture. Immunoblots of gels loaded with equal numbers of cells rather than protein showed similar results. Anti-rAAMP showed a similar pattern of reactivity for AAMP in T cell activation compared with anti-P189. However, it did not cross-react with a-actinin (not shown). Loss of a-actinin’s reactivity with anti-P189 after thermolysin digestion. Lack of linear sequence in aactinin’s primary structure that corresponds with the sequence of P189 suggested that reactivity with antiP189 is due to a reactive epitope formed by a-actinin’s secondary structure. Thermolysin shows specificity for only a few digestion sites (two major and two minor) in a-actinin [29]. This led to its use as an agent that would destroy a-actinin’s secondary structure without significantly damaging its primary structure. Thermolysin’s (100 mg/ml) digestion of a-actinin (900 mg/ml) completely eliminated the reactivity of anti-P189 (2 mg/ ml) for it and its digestion products after a 15-min incubation at 377C, whereas the digestion products still reacted with anti-a-actinin (Fig. 4). Reactivity of antiP189 for recombinant AAMP, treated with thermolysin also, was still present. Determination of the anti-P189 epitope in recombinant AAMP and three reactive human tissue proteins. Peptide variants of P189 (RRLRRMESESES) were used for competition studies of anti-P189’s reactivity

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FIG. 2. Confirmation of a-actinin reactivity with anti-P189 on immunoblots. (A) Purified a-actinin (100 kDa) comigrates with the largest reactive band in skeletal muscle lysate. Lanes 1– 5 contain a-actinin, purified from human placenta, in increasing amounts, 0.5, 1, 2, 4, and 8 mg protein per lane, respectively. Lane 6 contains human skeletal muscle lysate, 54 mg protein. Lane 7 contains prestained protein molecular weight standards, 105, 70, 43, and 28 kDa in vertical descending order. Anti-P189 was 1 mg/ml. (B) Competition of the a-actinin immunoreactivity with anti-P189 on immunoblots using purified human a-actinin. Lanes 1 and 2 contain A2058 human melanoma cell lysate, 80 mg protein per lane, reacted with anti-P189, 2 mg/ml. In lane 2 anti-P189’s reactivity was competed with human placental aactinin, 189 mg/ml. Lane 3 contains prestained protein molecular weight standards, 105, 71, 44, and 28 kDa, in vertical descending order. Samples were reduced, electrophoresed in 10% polyacrylamide gels with SDS, and transferred to nylon membranes.

for proteins. Reactivity competed by the sequence variants identified the epitope, ESESES, as being responsible for anti-P189’s reactivity in three tissue proteins (Table 1 and Fig. 5). However, inclusion of the adjacent methionine (MESESES) may have slightly enhanced competition for P189’s reactivity for a-actinin. The entire peptide is an epitope in recombinant AAMP. The RRLRRM portion of the peptide is the stronger part of

the epitope in recombinant AAMP but appears to be unavailable to the antibody in the tissue protein. Anti-P189’s immunohistochemical specificity for fast skeletal muscle fibers. Immunoreactivity of anti-P189 on tissue sections shows specificity for fast-twitch skeletal muscle fibers (Fig. 6). The specificity was deter-

FIG. 3. Expression of AAMP and a-actinin in a time course of T lymphocyte activation demonstrated with anti-P189 reactivity on immunoblots. Lanes 1–6 contain reduced lysates of cultured T cells, 31.5 mg protein/lane, harvested at time points of 0, 1, 24, 48, 72, and 96 h, respectively, following stimulation with phytohemagglutinin, 1 mg/ml. Lane 7 contains prestained protein standards with the molecular weights, 105, 70, 43, and 28 kDa in vertical descending order. The upper and lower blots were reacted with anti-P189 and antirAAMP, respectively, 1 mg/ml each. Although a-actinin’s expression remains the same, AAMP’s expression increases at 24 h and is maximal at 72 h. Anti-rAAMP showed a similar pattern of reactivity with AAMP but did not crossreact with a-actinin. Loading standardized for an equal number of cells rather than protein yielded similar results. Samples were reduced, electrophoresed in a 10% polyacrylamide gel with SDS, and transferred to a nylon membrane.

FIG. 4. Immunoblots that show the effects of thermolysin digestion on anti-P189’s reactivity with a-actinin compared to recombinant AAMP. Anti-a-actinin (1:1000), anti-P189 (2 mg/ml), and antirAAMP (0.1 mg/ml) were reacted with lanes 1– 3, 4 –8, and 9 –10, respectively. Lanes 1 and 4 contain undigested human placental aactinin, 4.5 mg/lane. Lanes 2 and 5 contain thermolysin-digested aactinin, 4.5 mg/lane. Lanes 6 and 9 contain undigested recombinant AAMP (purified from a bacterial fusion protein), 1.25 mg/lane. Lanes 7 and 10 contain thermolysin-digested recombinant AAMP, 3.96 mg/ lane. A lesser amount of loading for undigested recombinant AAMP is shown for better visualization of antibody reactivities. Lanes 3 and 8 contain prestained protein molecular weight standards, 206, 105, 71, 44, and 28 kDa, in descending vertical order. Samples were reduced, electrophoresed in 10% polyacrylamide gels with SDS, and transferred to nylon membranes.

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FIG. 5. Representative immunoblots showing anti-P189 reactivity competed with peptide variants of P189 for epitope localization. Lanes 1–4, 6– 8, and 13 contain human brain tissue lysate (265 mg protein/lane except for lane 13, which contains 40 mg). Lanes 10 and 12 contain human skeletal muscle lysate, 81 mg protein each. Lanes 5, 9, and 11 contain four prestained protein molecular weight standards, 105, 70, 43, and 28 kDa, in vertical descending order. Anti-P189 reactivity in lanes 1, 6, 12, and 13 was not competed with peptides as indicated by dashes (-) above the lanes. Peptides used for competition (501 greater concentration than antibody by weight) in lanes 2 and 10, 3, 4, 7, and 8 were P359, P358, P357, P369, and P360, respectively, as indicated above the lanes. Sequences for each peptide are listed. Peptides containing ESESES competed anti-P189’s reactivity in tissue lysates. Inclusion of the methionine (MESESES) in the P357 competing peptide provided more consistent blockage of anti-P189 reactivity for a-actinin than P359 with ESESES on repeated assays. Anti-P189 was 1 mg/ml. Samples were reduced, electrophoresed in 10% polyacrylamide gels with SDS, and transferred to nylon membranes.

mined by comparing anti-P189 stained sections with matching tissue sections stained with anti-skeletal muscle myosin heavy chain specific for type II (fasttwitch) fibers. The corresponding fibers stained with both antibodies. The staining was enhanced at periodic bands in the fast fibers that were identified as Z disks. Slight staining of these structures is also sometimes seen in the slow fibers. Simultaneous staining reactions of tissue sections from the same paraffin block with anti-a-actinin (sarcomeric) showed reactivity with the same structures in both fast and slow fibers (not shown). Double labeling also showed that both staining reactions are at the Z disks. Staining with anti-a-ac-

tinin (sarcomeric) requires predigestion with protease (trypsin). When this treatment is sufficient to give good reactions for anti-a-actinin (sarcomeric), it decreases (but does not completely abolish) anti-P189’s reactivity. DISCUSSION

The development of a polyclonal, anti-peptide antibody (anti-P189) for a newly identified protein, AAMP, has led to the discovery of an epitope shared by three proteins potentially involved with cell movement. Two proteins are known, AAMP and a-actinin. The third is a newly identified 23-kDa protein restricted to skeletal

TABLE 1 Peptide Competition of Anti-P189 Competition of anti-P189 reactivity on immunoblots Competing peptides

Tissue proteins

No.

Sequence

Recombinant AAMP

a-Actinin

AAMP

Skeletal muscle protein

189 350 357 358 359 360 369

RRLRRMESESES MSRERESRERSL QQLQQMESESES RRLRRMQSQSQS RRGRRGESESES RRLRRMEAEAEA RLRRMESESE

4/ 0 0 2/ 0 3/ 3/

4/ 0 4/ 1/ 3-4/ 1/ 0

4/ 0 4/ 2/ 4/ 2/ 0

4/ 0 4/ 1/ 4/ 1/ 0

Note. The degree of peptide competition of antibody reactivity is indicated by grades: 4/ Å complete or ú95%, 80% õ 3/ õ95%, 30% õ 2/ õ80%, 1/ Å detectable and õ30%, and 0 indicates no competition. Skeletal muscle protein, 23 kDa.

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FIG. 6. Anti-P189’s specificity for skeletal muscle fast-twitch fibers demonstrated with immunohistochemistry. Anti-P189 stains skeletal muscle in a checkerboard type pattern in A and C. The staining reaction in C matches the same pattern seen on the same tissue reacted with monoclonal anti-fast skeletal myosin (1:400) in D. Rabbit immunoglobulin (the same concentration as anti-P189 in A) was used as a negative control antibody in B. Anti-P189 stained periodic bands in the fast fibers represent Z disks that were identified by staining with sarcomeric anti-a-actinin (not shown). Early batches of anti-P189 obtained during the first few months of rabbit injections were used fresh in A, 0.4 mg/ml. A later purification of anti-P189 stored at 0707C was used in C, 3 mg/ml. Mayer’s hematoxylin and methyl green were used as counterstains for the top and bottom rows, respectively. Magnifications were 1501 for A and 751 for B, C, and D.

muscle. Database searches have not detected any 23kDa proteins that contain this epitope, ESESES, in the primary sequence. Although AAMP is widely distributed, it is in the lysates of brain tissue and activated T lymphocytes where AAMP’s ESESES epitope from the carboxyl end of peptide 189 reacts most strongly with anti-P189 (Figs. 1A and 3). The remainder of peptide 189’s sequence, RRLRRM, is unavailable as an epitope in human tissues. It is immunoreactive in recombinant AAMP produced in bacteria (Table 1 and Fig. 4). There is very strong affinity for binding to glycosaminoglycans (heparin binding Kd Å 14 pmol) in the RRLRRM region, which may mask it in tissues [1]. AAMP, 52 kDa in tissue, is slightly larger than its predicted molecular weight of 49 kDa, which may indicate the presence of a small amount of carbohydrate that does not readily separate from AAMP polypeptide in this region. Determination of AAMP’s DNA sequence obtained with 5* RACE–PCR using cDNA transcribed from human brain mRNA has confirmed that the entire coding sequence for peptide 189 is in AAMP [1]. Another explanation could be that

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since thrombin digestion easily occurs in this region, the RRLRRM epitope is destroyed in lysate preparation but this seems unlikely. Protease inhibitors used in preparing cell and tissue lysates should prevent this from happening. In addition, the ESESES epitope would be unaffected and equally available in all tissues with AAMP, rather than being preferentially reactive in brain and activated T lymphocytes. a-Actinin’s secondary structure has been found previously to contain other shared epitopes. Another crossreacting antibody for a-actinin has been found that was generated to a synthetic peptide (SEDYGKDL). It was derived from a-spectrin in a region which shows only a low level of homology (four contiguous identities) to a-actinin [24, 25]. A shared epitope with dystrophin was also found with the use of a peptide. A 207-aminoacid residue peptide derived from human dystrophin has been used to generate antibodies from rabbits (affinity-purified) that cross-react with a-actinin. A comparison of nondenatured and denatured peptides used as antigens showed that the cross-reacting antibodies were generated only to nondenatured peptide, which

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suggests that the shared antigenic determinants between a-actinin and dystrophin are structurally dependent. The reactive regions in these two proteins show very little sequence homology but are hypothesized to be structurally homologous [23]. In our study, the sensitivity of the ESESES epitope in a-actinin to limited protein cleavage via thermolysin digestion (Fig. 4) supports the observation that the epitope is not present in the primary sequence of a-actinin and therefore it is attributed to a-actinin’s secondary structure that persists in the presence of sodium dodecyl sulfate. Assembled or discontinuous antigenic determinants formed by residues brought together by folded polypeptide chain(s) are numerous on the surfaces of globular regions in proteins [30]. Antigenic determinants of proteins found by using synthetic peptides as immunogens have been shown to represent regions exposed to solvent in crystallographic studies of protein structure [31]. These can be regions that interact with other proteins. The anti-P189 reactive protein that is restricted to skeletal muscle appears to be a fast-twitch skeletal muscle fiber protein (Fig. 6). Anti-rAAMP does not react significantly with skeletal muscle fiber, although it does show faint positive staining with smooth muscle fibers following microwave treatment (not shown). The staining pattern with or without microwave treatment in skeletal muscle for anti-P189 is specific for fasttwitch fibers. It may correspond to anti-P189’s reactivity with the 23-kDa protein seen only on immunoblots of skeletal muscle (Fig. 1A). Two candidate skeletal muscle proteins of similar molecular weights, myosin regulatory light chain (DTNB chain) (19 kDa) and myosin essential light chains (alkali chains 1 and 2) (29.3 and 24.1 and 24.1 kDa), in their purified forms have not shown reactivity with anti-P189 (not shown). A predicted fast-twitch isoform of a-actinin exists [23, 32] and also remains a possibility for the immunoreactive pattern seen with anti-P189. However, many isoforms of a-actinin found in multiple tissue types react on immunoblots and yet the fast fiber immunohistochemical reaction is limited to the only tissue (skeletal muscle) that contains the 23-kDa protein. Since the same sixresidue epitope is present in both proteins, preabsorption studies with one protein to increase specificity for the other are not helpful. Preabsorption studies with peptides have shown almost identical reactivities for both a-actinin and the 23-kDa protein (Table 1 and Fig. 5). Reactivity for a-actinin and AAMP was also seen with antibody obtained from a previously injected rabbit that has been sacrificed due to development of a tumor (skeletal muscle was not checked for the 23-kDa protein reactivity). Another immunohistochemical staining reaction with anti-P189, which is seen after microwaving, is with the lumenal surfaces of uterine

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TABLE 2

a-Actinin Ligands Ligands F-actin Vinculin Zyxin Integrin b1 subunit Integrin b2 subunit L-selectin ICAM-1 Thrombospondin Nebulin Clathrin Phosphatidylinositol 3-kinase Phosphatidylinositol 4,5-bisphosphate

Motility-related functions Crosslinked by a-actinin to form a gel Membrane–cytoskeletal attachments Membrane–cytoskeletal attachments Cell adhesion Cell adhesion Leukocyte–endothelial adhesion Cell adhesion and motility Cell motility Sarcomere filament Flexible coat for vesicles Regulates cytoskeletal reorganization Stimulates a-actinininduced fibrin gelation

Ligand reference [48, 49] [50] [51] [35, 36] [37] [34] [33] [52] [53] [54] [39] [38]

endometrial glands. A corresponding distribution can also be seen with anti-rAAMP and is therefore attributed to AAMP [1]. Anti-P189’s reactivity with immunoblots of T lymphocyte lysates from an activation assay show an increase in AAMP’s protein expression that correlates with an increase in mRNA expression seen on Northern blots of multiple T cell activation studies (not shown). Anti-P189 also reacts with a-actinin which is expressed constitutively in that it does not increase its expression with activation and therefore serves as an internal control protein. AAMP’s expression is increased at 24–96 h after stimulation with phytohemagglutination (Fig. 3). Not only is the entire protein’s expression increased over this time span but the anti-P189 epitope remains available. AAMP differs among tissue types in regard to the reactivity of this epitope in that it is only detectable in brain and activated T lymphocytes. Immunofluorescence studies with polyclonal antirAAMP have shown that AAMP is distributed diffusely in the cytoplasm of permeabilized, motile cells such as cultured human metastatic melanoma cells and endothelial cells (not shown). The ESESES epitope of AAMP is of interest in that both of the two other proteins that share it are involved with cell motility or movement. The anti-P189 reactive skeletal muscle fiber protein is distributed diffusely and in periodic bands (Z disks identified with staining for the sarcomeric type of aactinin) in fast-twitch fibers, thus indicating that it may have something to do with muscle contraction. The ability of calcium-sensitive a-actinin to crosslink F-actin is thought to help with regulating the gel –sol transition of cytoplasm in cell locomotion [21]. Besides F-

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AAMP SHARES AN a-ACTININ AND SKELETAL FIBER PROTEIN EPITOPE

actin, a-actinin is known to bind to various other proteins (Table 2). Many of these are known participants in the regulation of cell adhesion and movement. aactinin’s epitope that is shared with AAMP could indicate a functional role for this newly identified protein in one or several of a-actinin’s binding interactions. Similar regions of proteins can share ligands and/or function similarly. The binding regions in some of aactinin’s target proteins are small (RKIKK in ICAM-1 [33], KKSKRSMNDPY in L-selectin [34], FAKFEKEKMN in the b1 integrin subunit [35–37], and a similar sequence in the b2 integrin subunit [37]). The reactive sites in a-actinin may also be small and formed by its secondary structure. Modulation of a-actinin’s Factin gelating activity by phosphatidylinositol 4,5-bisphosphate suggests that a conformational change occurs when it is bound [38, 39]. By transfecting a-actinin’s cDNA into simian virus 40-transformed 3T3 cells, modulation of its levels have been found to affect cell motility and tumorigenicity. These cells had been found to be low level expressors of a-actinin (six fold lower levels than nontransformed 3T3 cells). Clones expressing a-actinin at levels found in nontumorigenic 3T3 cells displayed a flatter phenotype, a decreased ability to grow in suspension culture in soft agar, and a marked reduction in their ability to form tumors in syngeneic BALB/c mice and in athymic nude mice. Clones with the highest level of a-actinin expression (twofold higher than 3T3 cells) were completely suppressed in their ability to form tumors in syngeneic mice [40]. Cells overexpressing a-actinin by 40–60% displayed a significant reduction in cell motility. Transfection of 3T3 cells with an antisense a-actinin cDNA to suppress its levels led to increased cell motility and these cells formed tumors following injection into nude mice [41]. Calcium-sensitive a-actinin as a component of stress fibers seen in motile cells may help these fibers to function in a muscle-like fashion for maintenence of cell shape and adhesion in the presence of calcium. An intracellular calcium flux could trigger the rapid breakdown and reorganization of actin-filament networks and filament/membrane junctions of nonmuscle cells [42]. a-Actinin is associated with eukaryotic cell structures involved in a variety of cell movements, such as cleavage furrows in embryos and the contractile vacuoles and phagosomes found in yeast. Their formation requires rearrangement of actin filaments from a network into oriented configurations [43, 44]. In addition, modulations in the cytoskeletal a-actinin/ actin ratio are seen in polymorphonucleated cells stimulated for motility [45]. a-Actinin’s role as a link between actin and other proteins at the cell membrane may vary as a direct or indirect connector with integrins. There is speculation that different types of linkages may correlate with various modes of adhesion such as the rapid on and

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off attachments necessary for motility or the more stable attachments needed by stationary cells [46]. The discovery of an unexpected epitope in a-actinin not predicted by its primary structure may provide new insight in regard to its conformation, identifies a potential functional region shared by proteins that influence cell movement, and provides the generation of a new antibody that reacts strongly on immunoblots with aactinin isoforms in all tissues. Regions of high antigenicity in proteins have been found to correlate with functional specialization [47]. The epitope in AAMP which is consistently exposed in brain and activated T lymphocytes may share functional similarity with aactinin in these tissue types. This epitope in AAMP is near its amino terminus and proximal to the WD40 domains that are predicted to be structural regions involved in stable interactions. In this location the ESESES site could be readily available for participation in dynamic protein –protein interactions. We thank the NIH Visual Arts Department for their help in preparing the figures for this paper.

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Received October 3, 1995 Revised version received March 15, 1996

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