©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Involvement of N-Myristoylation in Monoclonal Antibody Recognition Sites on Chimeric G Protein Subunits (*)

(Received for publication, December 6, 1994; and in revised form, January 6, 1995)

John M. Justice (§) M. Michael Bliziotes (¶) Linda A. Stevens Joel Moss Martha Vaughan

From the Pulmonary Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Monoclonal antibody, LAS-2, directed against the alpha subunit of transducin (G), inhibited G-dependent, pertussis toxin-catalyzed ADP ribosylation of G and was specific for G. Immunoblotting studies on proteolytic fragments of G were consistent with an amino-terminal epitope. To define the antibody recognition site, recombinant G was synthesized in Escherichia coli cotransfected with or without yeast N-myristoyltransferase. Amino-terminal fatty acylation of G, verified by use of radiolabeled fatty acid, was required for immunoreactivity. LAS-2 did not react with a chimeric protein consisting of residues 1-9 of G and the remainder G, regardless of its myristoylation. Immunoreactivity was observed when amino acids 1-17 of G were present in a G chimera and the protein was amino-terminally myristoylated; there was no reactivity without myristoylation. It appears that the LAS-2 epitope requires both G-specific sequence in amino acids 10-17 and a fatty acyl group in proximity to these residues. These results are consistent with the hypothesis that the myristoyl group is essential for protein structure; conceivably it ``folds back'' on and stabilizes the amino-terminal structure of G, as opposed to protruding from an amino-terminal alpha-helix and serving as an amino-terminal membrane anchor.


INTRODUCTION

Heterotrimeric guanine nucleotide-binding proteins (G proteins), (^1)consisting of alpha, beta, and subunits, couple cell surface receptors to effectors(1) . GTP/GDP exchange and GTP to GDP hydrolysis, which regulate G protein activity, occur on the alpha subunit (G) and require the presence of receptor and the beta subunit (G)(2, 3, 4, 5) .

The amino-terminal domain of G is important for interaction with G. Removal of 21 amino acids from the amino terminus of the alpha subunit of transducin (G), using Staphylococcal V8 protease, inhibited beta-dependent reactions including GTP hydrolysis, GTP/GDP exchange, and binding of G to rhodopsin(2) . Trypsin, which removes the first 18 amino acids of G, destroyed the ability of beta to stimulate pertussis toxin-catalyzed ADP-ribosylation of G(6) as did amino-terminal deletions in a recombinant G(7) . Amino acids 7-10 of recombinant G were found to be critical for its interaction with G(8) .

Cotranslational acylation of the amino-terminal glycine with myristate occurs in several of the G subunits (9, 10, 11, 12) and facilitates their membrane attachment. Removal of the myristoylated amino terminus of G by tryptic digestion produced a soluble protein from one that was originally membrane bound(13) . G modified with hydrophilic myristate analogues was less likely than native G to associate with membranes (9) . Myristoylation also appears to play a role in protein-protein interaction(14, 15) . Both beta-stimulated ADP-ribosylation and affinity for beta-Sepharose were significantly greater with myristoylated than nonmyristoylated G(8, 16) . N-Fatty acylation of G was found important for its interaction with G(17) . Based on these data, it has been postulated that the myristoyl moiety may be involved in stabilizing the structure of the amino terminus. We describe here an anti-G monoclonal antibody, LAS-2, whose epitope involves the G-binding domain of G and requires amino-terminal fatty acylation as well as specific G amino-terminal sequence.


EXPERIMENTAL PROCEDURES

Materials

[P]NAD (30 Ci/mmol), GA/1 antibody, RM/1 antibody and AS/7 antibody were purchased from DuPont NEN; TPCK-treated trypsin was from Worthington. Glutathione-agarose, NAD, ATP, and soybean trypsin inhibitor were from Sigma, and blue Sepharose CL6B was from Pharmacia Biotech Inc. Isopropyl-1-thio-beta-galactopyranoside was from U. S. Biochemical Corp; pertussis toxin was from List Biological Laboratories. Goat anti-rabbit IgG horseradish peroxidase-coupled antibody was from Pierce, and goat anti-mouse IgG horseradish peroxidase-coupled antibody was from Promega. LB medium was from Digene, ampicillin sodium and kanamycin were from Amresco; and molecular weight standards were from Life Technologies, Inc. SDS-PAGE and Western blotting were done in a NOVEX Minicell apparatus or Bio-Rad Trans-Blot apparatus. Protein concentration was determined using Bio-Rad protein assay with bovine serum albumin as the standard.

Methods

Preparation of Proteins

Transducin was purified from bovine rod outer segments(18) . Blue-Sepharose chromatography was used to separate the G and G subunits.(19) . Rhodopsin was prepared by method of Hong and Hubbell(20) . G and G were isolated from bovine brain (21) . G was a generous gift from Dr. Victor Rebois of NINDS, National Institutes of Health.

Proteolytic Cleavage of Transducin

G was digested with trypsin (22) or Arg-C(23) . Cleavage of G with hydroxylamine was based on the method of Borstein and Balian(24) .

Immunoblots

Intact proteins or proteolytic fragments were resolved by SDS-PAGE(25) ; transfer to nitrocellulose was performed in 25 mM Tris, 192 mM glycine buffer with 20% methanol (26) either in a NOVEX transfer module (30 V for 2 h) or a Bio-Rad Trans-Blot apparatus (100 V for 16 h). Blots were processed as described(27) .

ADP-Ribosylation of G

G (1 µg) and G (1 µg) were incubated in the presence of LAS-1, LAS-2, or control mouse IgG at 4 °C for 1 h before pertussis toxin-catalyzed ADP-ribosylation, which was carried out at 30 °C for 30 min in 50 mM potassium phosphate (pH 7.5), 10 µM [P]NAD (2 µCi/assay), 1 mM ATP with 2 µg of activated pertussis toxin in total volume of 100 µl. Pertussis toxin was activated for 10 min with dithiothreitol immediately before use(6) . Proteins were precipitated with 7.5% trichloroacetic acid and subjected to electrophoresis in 12% SDS-polyacrylamide gels, followed by autoradiography with Kodak XAR film and an intensifying screen at -70 °C for 30 min.

Construction of Coding Regions for Recombinant Proteins

Bovine retinal G cDNA was used as template for PCR amplification of the rG coding region. The sense primer was 5`-ACGACGACGCATATGGGGGCTGGGGCCAGCGCTGAGGAGA-3`, and the antisense primer was 5`-GCGCCACGGTGATCATCAGAAGAGCCCGCAGTCTTT-3`. G cDNA from bovine brain was used as template for PCR preparation of coding regions for rG and the G-G chimeric proteins. The sense primer for the rG PCR was 5`-ACGACGACGCATATGGGATGTACTCTGAGCGCAGAGGAG-3`, and the antisense primer was 5`-GCGCCACGGTGATCATCAGTACAAGCCGCAGCCCCGGAG-3`. For the G9-G chimeric protein, which consists of G sequence in the first 9 amino acids followed by G sequence, the sense primer was 5`-ACGACGACGCATATGGGGGCTGGGGCCAGCGCAGAGGAGCGAGCCGCCCT-3`, and the antisense primer was the same as that used in the rG reactions. To synthesize the G17-G chimera, which contains G sequence in amino acids 1-17 and the remainder G, an initial PCR was carried out using 5`-ACGACGACGCATATGGGGGCTGGGGCCAGCGCTGAGGAGAAGCACTCAAGGGAGCTGGAGAAAAACCTCAAAGAG-3` as the sense primer and the G antisense primer used in other reactions. The product of this PCR was used as template for a second round of PCR amplification using the same 5` primer as in the G amplification and the 3` primer used for G amplifications. PCR products were purified with a PCR purification spin kit (Qiagen), digested with restriction enzymes NdeI and BclI, and gel-purified. The NdeI restriction site at the 5` end and BclI site at the 3` end allowed directional cloning.

Preparation of Recombinant Proteins

The gel-purified, restriction-digested PCR products were inserted into a modified pGEM vector, pT7/Nde, under control of a T7 promoter(28) . Competent DH5alpha Escherichia coli were transfected and screened on LB/ampicillin plates. The presence of insert in the positive clones was verified by PCR. An appropriate colony was selected and used for plasmid isolation and transfection into BL21(DE3) E. coli (Novagen) or competent BL21(DE3) E. coli that had previously been tranfected with the pACYC177/ET3d/yNMT vector(29) , which contains the yeast N-myristoyltransferase coding region under control of a T7 promoter and a kanamycin resistance gene for coselection. Small scale preparations were done by diluting fresh overnight cultures 1:10 and growing at 37 °C in the presence of appropriate antibiotics for 1 h. Isopropyl-1-thio-beta-D-galactopyranoside (final concentration of 0.2 mM) and [^3H]myristic acid (final concentration of 5 µM with a specific activity of 50 Ci/mmol) were added. Growth was continued an additional 3 h before cells were pelleted and suspended in SDS-PAGE sample buffer. Proteins were separated by SDS-PAGE and transferred to nitrocellulose. Presence of incorporated fatty acid was verified by exposure of the gel to Kodak XAR film at -70 °C after treatment with Pro-Mote (Integrated Separation Systems).

Antibodies

Hybridomas LAS-1 and LAS-2 and were grown in serum-free medium (Ultradoma supplemented with glutamine) or RPMI 1640 with glutamine and 5 or 10% fetal bovine serum (BioWhittaker). G(o) polyclonal antibodies (pcGo) were prepared as previously reported(30) .


RESULTS AND DISCUSSION

Murine monoclonal antibodies against purified bovine G were screened for functional effects on G and G interaction. One clone, LAS-2, inhibited G-stimulated ADP-ribosylation of G in a concentration-dependent manner; control immunoglobulin of the same isotype (IgG(2)) and LAS-1 did not inhibit the reaction (Fig. 1).


Figure 1: Inhibition by LAS-2 of G-dependent pertussis toxin-catalyzed ADP-ribosylation of G. Transducin was incubated with LAS-1, LAS-2, or mouse IgG(2) at 4 °C for 1 h before initiation of pertussis toxin-catalyzed ADP-ribosylation, which was carried out at 30 °C for 30 min in 50 mM potassium phosphate (pH 7.5), 10 µM [P]NAD (2 µCi/assay), 1 mM ATP with 2 µg of activated pertussis toxin in total volume of 100 µl. Pertussis toxin was activated for 10 min with dithiothreitol immediately before use(6) . Proteins were precipitated with 7.5% trichloroacetic acid and separated by SDS-PAGE in 12% polyacrylamide gels followed by autoradiography with Kodak XAR film and an intensifying screen at -70 °C for 30 min. Lane1, 3 µg of indicated antibody; lane2, 10 µg of indicated antibody; lane3, 25 µg of indicated antibody. Cont.Ig, control Ig.



To define the LAS-1 and -2 epitopes better, G was cleaved with endoprotease Arg-C(23) , hydroxylamine at alkaline pH (24) , or trypsin(22) ; the fragments were separated by SDS-PAGE, transferred to nitrocellulose, and reacted with antibodies LAS-2 and AS/7. Endoprotease Arg-C digestion generated LAS-2-reactive fragments of 34, 23, and 15 kDa, all of which were refractory to microsequencing, consistent with the presence of a blocked amino terminus (Fig. 2A). Hydroxylamine at alkaline pH cleaves primarily at Asn-Gly bonds(24) ; cleavage of G (Asn-Gly) produced 32-kDa amino-terminal and 7-kDa carboxyl-terminal fragments. Both LAS-1 and -2 reacted with the 32-kDa piece, whereas AS/7 did not, confirming that the LAS-reactive fragment lacked the carboxyl terminus (Fig. 2B, data not shown). Tryptic digestion of G initially removes a 2-kDa amino-terminal fragment(22) ; this modification abolished immunoreactivity with LAS-2. The presence of the remaining 37-kDa fragment was verified by reactivity with the carboxyl-directed polyclonal antibody AS/7 (Fig. 2C). These findings all suggested that the LAS-2 epitope was located within the 18 amino-terminal amino acids removed by trypsin, in agreement with the functional data obtained with G(2, 3, 4, 5) .


Figure 2: Immunoreactivity of LAS antibodies. A, reaction of LAS-2 with submaxillary Arg-C-digested G. G (5 µg) in buffer A (20 mM Tris (pH 7.5), 0.5 mM MgCl(2), 0.05 mM EGTA, 0.5 mM NaN(3), 1 mM dithiothreitol) was incubated with or without 0.625 µg of Arg-C for 4 h at 37 °C; the reaction was stopped by the addition of SDS-sample buffer and boiling for 10 min. Proteins and proteolytic products were separated in 16% polyacrylamide gels and transferred to nitrocellulose, which was then stained with Ponceau S or reacted with LAS-2. Lane1, purified G stained with Ponceau S; lane2, Arg-C-treated G stained with Ponceau S; lane3, Arg-C-treated G reacted with LAS-2. a-c indicate LAS-2-reactive peptides subjected to sequencing by Edman degradation (see text). Positions of protein standards (kDa) are on the left. B, reaction of antibodies with hydroxylamine-treated transducin. 25 µg of G in 50 µl of buffer A plus 58 µl of 3.75 M hydroxylamine, pH 9.25, were incubated at 37 °C for 3 h. 15-µl samples of this mixture were subjected to SDS-PAGE in 4-20% NOVEX gradient gels and then were stained with Coomassie blue or used to transfer separated fragments to nitrocellulose for reaction with antibodies. Positions of standard proteins are on the left; G, G, G, and 32 kDa are indicated in the center. Lane-, 2.5 µg of G; lane+HA, hydroxylamine treatment, both stained with Coomassie blue; laneLAS1, blot of hydroxylamine-treated G reacted with LAS-1; laneAS7, blot of hydroxylamine-treated G reacted with AS7. C, reaction of LAS-2 with G after tryptic digestion. G (4 µg) in 40 µl of buffer A was incubated with or without TPCK-treated trypsin (4 µg/ml) for 45 min at 30 °C, precipitated with 10% trichloroacetic acid, subjected to electrophoresis in 10-20% Tricine gradient gel, and transferred to nitrocellulose. Transferred proteins and fragments were stained with Amido Black (PRO) or reacted with LAS-2 or AS7 antibodies. Presence or absence of trypsin is indicated by a (+) or (-), respectively. Positions of G, and 37-, 21-, 16-, and 5-kDa fragments are indicated at right.



Further mapping was done using recombinant proteins expressed in E. coli. G cDNA inserted into a pGEX-2T expression vector (Pharmacia) yielded high levels of rG in a fusion protein with glutathione S-transferase(31) . Despite reacting with AS/7, this fusion protein failed to react with LAS-2 both before and after cleavage of the thrombin-sensitive link between glutathione S-transferase and G (data not shown). An amino-terminally myristoylated rG was synthesized in E. coli by coexpressing G and yeast N-myristoyltransferase(32) . Myristoylation of rG was verified by detection of ^3H-labeled G after addition of [^3H]myristic acid during protein synthesis. This myristoylated rG reacted with LAS-1 and -2 on immunoblots. rG synthesized without N-myristoyltransferase was not labeled with [^3H]myristate and did not react with LAS-2, although it did react with AS/7 and another transducin monoclonal antibody, 6E4 (27) (Fig. 3). Fidelity of expression and lack of proteolytic modification of the unmyristoylated rG was verified by Edman degradation though five cycles, yielding the expected Gly-Ala-Gly-Ala-Ser sequence (Harvard MicroChem).


Figure 3: Requirement for amino-terminal myristoylation of G for LAS reactivity. Transducin and recombinant proteins, prepared as under ``Methods'' were suspended in sample buffer, subjected to SDS-PAGE in 12% polyacrylamide, transferred to nitrocellulose, and reacted with LAS or 6E4 antibodies. Amounts of G (approx4 µg) in the three lanes were similar based on Coomassie blue staining. Lane1, G; lane2, nonmyristoylated recombinant G; lane3, myristoylated recombinant G.



The LAS-2 antibody was very specific for G. It reacted with only a single band from a bovine retinal homogenate (Fig. 4A) and did not react with G, G, or G on immunoblots (Fig. 4B). A recombinant G, expressed in E. coli, did not react with LAS-2 either in a nonmyristoylated state or when coexpressed with N-myristoyltransferase and myristoylated (Fig. 4C, lanes1 and 2). A chimeric protein, G9-G, consisting of G sequence in amino acids 1-9, and the remainder G sequence was constructed by replacing Cys^3, Thr^4, and Leu^5 of G with Ala^3, Gly^4, Ala^5. This protein was myristoylated when coexpressed with N-myristoyltransferase but did not react with LAS-2 either with or without myristoylation (Fig. 4C, lanes3 and 4). A chimeric protein in which the first 21 amino acids of G were replaced with the first 17 amino acids of G (G17-G) did react with LAS-2, but only when myristoylated; the nonmyristoylated G17-G did not react (Fig. 4, lanes5 and 6).


Figure 4: Immunoreactivity of LAS antibodies. A, specificity of LAS-2 for G among bovine retinal proteins. Bovine retinas were homogenized in 0.25 M sucrose, 10 mM Tris-HCL (pH 7.5), 10 mM EDTA. The homogenate was centrifuged (30 min/14,000 times g). Supernatant proteins (100 µg) were precipitated with 7.5% trichloroacetic acid, suspended in 20 mM phosphate buffer (pH 7.5), subjected to SDS-PAGE (16% polyacrylamide), transferred to nitrocellulose, and stained with Ponceau S (lane1) or reacted with LAS-2 (lane2). B, specificity of LAS-2 monoclonal antibody. G and G were prepared as under ``Methods.'' G and G were isolated from bovine brain(21) . G was a generous gift from Dr. Victor Rebois of NINDS, National Institutes of Health. 1 µg of each of the proteins was subjected to SDS-PAGE (12% polyacrylamide), transferred to nitrocellulose, and reacted with LAS-2. Protein transfer was verified with 6E4 anti-G(27) , pcGo anti-G(30) , and RM/1 anti-G (DuPont NEN) antibodies. Lane1, bovine G; lane2, bovine G; lane3, bovine brain G; lane4, bovine brain G; lane5, G. C, reactivity of LAS-2 with rG and G-G chimeric proteins. rG and chimeric G-G in whole bacterial lysates were subjected to SDS-PAGE, transferred to nitrocellulose, and reacted with LAS-2. There was approximately 1 µg of each recombinant protein in each lane. Lane1, myristoylated G; lane2, rG; lane3, myristoylated G9-G; lane4, G9-G; lane5, myristoylated G17-G; lane6, G17-G; lane7, protein standards (97, 68, 43, 29, and 18 kDa).



The LAS-1 antibody shown in Fig. 1reacted in a manner identical to LAS-2 on all immunoblots including those performed with proteolytic digests of G, native G proteins, and recombinant proteins. It did not, however, interfere functionally with G-G interaction as judged by inhibition of beta-dependent ADP-ribosylation. Why two antibodies with seemingly identical mapping properties differ functionally is unclear, but it may have to do with differing affinities for G.

It appears that monoclonal antibody LAS-2, a G-specific monoclonal antibody, which interferes with G-G interaction, has an epitope that includes specific G amino acid sequence within positions 10-17 and requires a fatty acyl group in proximity to these residues. Just as the first 9 amino acids are not specific for G binding(17) , they do not encompass the LAS-2 epitope, which lies further downstream. The amino terminus of bovine G is modified in a heterogeneous manner by lauroyl (C:12), myristoyl (C:14), (cis-Delta-5)-tetradecaenoyl (C:14:1), or (cis,cis-Delta5,Delta8)-tetradecadienoyl (C:14:2) fatty acids(17, 33) . Kokame et al.(17) reported that the length of the amino-terminal fatty acid was important for the ability of nonapeptides to inhibit the interaction of G and G, although no requirement for specific amino acid sequence was observed.

Upon exchange of GDP for GTP, G disassociates from G, and a conformational difference in crystal structures of these two nucleotide-bound states has been documented (34, 35) . The proteins crystallized, however, lacked the amino-terminal 25 amino acids and thus lacked the G-binding domain and the LAS epitope studied here. It has been postulated that myristate may act to stabilize an amino-terminal amphipathic helix and participate in protein function(38) . Recoverin, a retinal protein that is heterogeneously fatty acylated in a manner similar to G, exposed hydrophobic acyl groups in response to Ca binding, thus behaving as a Ca-myristoyl protein switch(36, 37) . ADP ribosylation factor, a myristoylated 20-kDa GTP-binding protein involved in cellular protein transport may utilize a GTP-myristoyl switch; the myristate may participate in the reversible GTP-dependent association of ADP ribosylation factor with membranes(37, 38) . The fatty acyl group in the N-myristoylated G proteins may play a similar role, depending on whether GTP or GDP is bound. Our findings suggest that within the G amino-terminal domain the fatty acid may ``fold back'' on and stabilize peptide structure, consistent with the hypothesis that the fatty acid does more than merely project from the protein into the lipid bilayer to serve as an amino-terminal membrane anchor.


FOOTNOTES

*
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: NIH, Bldg. 10, Rm. 5N-307, 10 Center Dr., MSC 1434, Bethesda, MD 20892-1434. Tel.: 301-496-1254; Fax: 301-402-1610.

Present address: VA Medical Center, 3710 SW U. S. Veterans Hospital Rd., Portland, OR 97035.

(^1)
The abbreviations used are: G protein, heterotrimeric guanine nucleotide-binding protein; PAGE, polyacrylamide gel electrophoresis; TPCK, L-tosyl-amido-2-phenylethyl chloromethyl ketone; G, transducin; G, alpha subunit of transducin; G, beta subunit of transducin; G, alpha subunit of the abundant G protein obtained from bovine brain; G, alpha subunit of the inhibitory G protein of the adenylyl cyclase system; G, alpha subunit of the stimulatory G protein of the adenylyl cyclase system; PCR, polymerase chain reaction; rG, recombinant G protein.


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