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Mapping the Cysteine Residues and Actin-Binding Regions Of Proc. Nadl. Acad. Sci. USA Vol. 82, pp. 6788-6792, October 1985 Biochemistry Mapping the cysteine residues and actin-binding regions of villin by using antisera to the amino and carboxyl termini of the molecule (cytoskeleton/intestine brush border/peptide mapping/amino acid sequencing/peptide antibody) PAUL MATSUDAIRA*, Ross JAKES, LINDA CAMERON, AND ERIC ATHERTON Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England Communicated by H. E. Huxley, June 21, 1985 ABSTRACT Peptide antisera specific for either the amino- acids, chemical modification sites, ligand binding sites, and or carboxyl-terminal regions of villin were used to locate the chemical or proteolytic fragments. The map of the cysteine position of cysteine residues in immunoblots of villin cleaved residues has enabled us to identify a calcium-regulated with 2-nitro-5-thiocyanobenzoic acid. Maps constructed from actin-binding domain within an amino-terminal villin peptide the cleavage pattern suggest that villin contains six cysteine of Mr 32,000. residues, two located in its amino-terminal peptide of Mr 44,000, and four located in the carboxyl-terminal peptide ofMr MATERIALS AND METHODS 51,000. Gel overlays of the partial cleavage fragments with '2SI-labeled actin identified a calcium-dependent actin-binding Proteins. Villin was purified from chicken intestine epithe- region located within the amino-terminal peptide ofMr 32,000 lia using the procedure described by Bretscher & Weber (1). of villin. The peptide antibody method used, called cleavage The villin proteolytic fragments (villin core, 51T, and 44T) mapping, should be a convenient technique for mapping were purified as described (9). Actin was purified from rabbit residues and ligand binding sites in proteins. skeletal muscle by using the procedure of Taylor and Weeds (10). 2-Nitro-5-thiocyanobenzoic acid (NTCB) was obtained Villin, a Mr 95,000 actin-binding protein of the intestine from Sigma. microvillus cytoskeleton, is unusual because it possesses two Further treatment ofthe 44T fragment of villin with trypsin types of actin-binding activities. In calcium concentrations produces a Mr 14,000 peptide, named 44T-14T. Digestion at >1 ,uM villin severs actin filaments into short lengths while longer times produces a Mr 13,000 peptide, named 44T-13T. in calcium concentrations <1 ,uM villin crosslinks actin Both peptides were purified by CM-52 and Affigel Blue filaments into bundles (1-4). Understanding the mechanism column chromatography (to be reported elsewhere), and the of filament severing and bundling requires identification of first 20 residues of each fragment were sequenced by Edman the villin-, actin-, and calcium-binding sites. degradation using a modified Beckman 890B automated Villin is cleaved by V8 protease into two fragments, villin sequencer (11). core (Mr 87,000) and villin headpiece (Mr 8500). Villin core Preparation of Antigen. The peptide Val-Glu-Leu-Ser-Lys- and headpiece retain the amino and carboxyl termini ofvillin, Lys-Val-Thr-Gly-Lys-Thr-Thr-Tyr-Gly was assembled and respectively. Glenney and co-workers (5, 6) have shown that cleaved as described by Atherton et al. (12). The synthetic a calcium-dependent actin-filament-severing activity is locat- peptide was coupled to keyhole limpet hemocyanin using ed in villin core. The villin headpiece exhibited calcium- bisdiazotized o-tolidine, and excess reagents were removed independent actin-binding but not -bundling or -sever- by dialysis against phosphate-buffered saline (13). ing activity. Villin contains three calcium-binding sites, one Peptide Antisera Production. Antisera were raised in rab- of which is nonexchangeable. One of the exchangeable bits by injecting 1 mg ofthe peptide-hemocyanin conjugate in calcium-binding sites is located in villin core, while the other complete Freund's adjuvant at multiple sites on the back. exchangeable site is located in villin headpiece (7, 8). We This was followed by injections at 2-week intervals in have extended these studies and shown that trypsin produces incomplete Freund's adjuvant. The rabbits were exsangui- a Mr 44,000 amino-terminal fragment of villin (named 44T), nated at week 10. The titers ofthe antisera were quantified by which retains the calcium-dependent actin-severing activity ELISA using either the synthetic peptide or undenatured (9). The calcium dependence of the binding activity suggests villin adsorbed to microtiter plates. Swine anti-rabbit IgG that the exchangeable calcium-binding site in villin core is coupled to peroxidase (Dakopatts) was used as the secondary located within the amino-terminal peptide of Mr 44,000. antibody. The plates were developed with diaminobenzidine, Although our studies locate an actin- and calcium-binding and absorbance was monitored at 490 nm. The specificity of region within the amino-terminal half of villin, much smaller the antisera (R211.3) was determined from immunoblots (14) binding fragments are required to refine further the regulation against villin proteolytic fragments. Antisera (R200.2) against of actin binding by calcium. the villin headpiece peptide were described (9). To map such binding regions easily and accurately, we Quantitation of Cysteine Residues. Villin or its proteolytic have devised a peptide antibody method for localizing certain fragments was reduced and the cysteine residues were amino acids and fragments within the molecule. In our carboxymethylated with iodo[2-14C]acetic acid or iodo[1- approach we have used polyclonal antisera, which recognize 14C]acetamide (Amersham) (15). Incorporation ofradioactiv- either the NH2-terminal or COOH-terminal regions of villin, ity was quantitated by liquid scintillation counting. The to locate the positions of the cysteine residues in villin. This number of cysteine residues was calculated from the specific technique, which we call cleavage mapping, is generally activity of the radiolabeled villin. useful for identifying the positions of certain other amino Abbreviations: NTCB, 2-nitro-5-thiocyanobenzoic acid; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid). The publication costs of this article were defrayed in part by page charge *Present address: Whitehead Institute for Biomedical Research and payment. This article must therefore be hereby marked "advertisement" Department of Biology, Massachusetts Institute of Technology, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Nine Cambridge Center, Cambridge, MA 02142. 6788 Downloaded by guest on September 26, 2021 Biochemistry: Matsudaira et aL Proc. NatL Acad Sci USA 82 (1985) 6789 Cysteine content was also measured by 5,5'-dithiobis(2- ments in addition to the intact protein. Two fragments should nitrobenzoic acid) (DTNB) titration (16) in either NaDodSO4 retain the same amino terminus as the intact protein, while or 6 M guanidine HCl. two other fragments should retain the original carboxyl NTCB Cleavage. Villin was reduced and denatured by terminus. The polypeptides can be separated on NaDodSO4/ dialysis into 6 M guanidine-HCl/10 mM dithiothreitol/0.1 polyacrylamide gels (Fig. lc, lane 1). Immunoblots with mM EDTA at 370C. After 6-12 hr, the sample was rapidly antisera specific to the amino (lane 2) or carboxyl terminus desalted against 6 M guanidine-HCl/0.2 M Tris-HCl (pH (lane 3) should reveal the fragments that retain either termi- 8.0)/0.1 mM dithiothreitol/0.1 mM EDTA. The protein nus of the protein. The molecular weight of each detected concentration was determined by absorbance at 280 nm using fragment then maps the position ofthe cleavage site from the an absorptivity of 1.28 liters/g-cm (7). The reduced and end of the protein identified by the antisera. Mapping from denatured villin was cleaved with NTCB (17, 18). NTCB was both ends should produce the same "cleavage map" of the dissolved in 50 mM sodium phosphate, pH 7.6, mixed with protein. villin at a 5-fold excess over total thiols, and incubated in the Characterization of Amino-Terminal Specific Antiserum. dark at 370C. The reaction was quenched after different times We synthesized the peptide Val-Glu-Leu-Ser-Lys-Lys-Val- by adding acetic acid to 50o (vol/vol) and the cleaved Thr-Gly-Lys-Thr-Thr-Tyr-Gly. This peptide represents the products were dialyzed against 10%6 acetic acid (vol/vol) in amino-terminal 12 residues of villin (9) followed by Tyr-Gly. M, 3500 cutoff dialysis tubing. The cleavage products were The synthetic peptide was crosslinked through its tyrosine lyophilized and stored at -20'C. As control experiments, residue to hemocyanin and the mixture was injected into villin was either incubated in the absence of NTCB under rabbits. The antiserum recognized undenatured villin and the reduced and denaturing conditions or reduced, denatured, synthetic peptide (Fig. 2a). carboxymethylated, and then incubated with NTCB. The The specificity of the antiserum to the amino terminus was cleavage fragments of villin were electrophoresed in tested by immunoblots containing villin proteolytic frag- NaDodSO4 on 10% or 10-22.5% polyacrylamide microslab ments whose locations in the protein have been characterized gels (19). by protein sequencing (Fig. 2b, lane 1). The antiserum (lane Cleavage Mapping. The NTCB cleavage fragments of villin 2) recognized villin, villin core, and 44T. The amino termini were electrophoresed on 1o NaDodSO4/polyacrylamide of these fragments have been confirmed by sequence analy- microslab gels and were immunoblotted with rabbit polyclo- sis, and all contain the villin amino terminus. As expected, nal antisera specific for the amino terminus (R211.3) or the this antiserum did not recognize the 51T fragment, which headpiece region (R200.2) at a dilution of 1:200 or 1:2000, comes from the carboxyl-terminal end of the protein. Our respectively. The filters were incubated at a 1:400 dilution characterized villin headpiece antiserum (9) recognized villin with swine anti-rabbit IgG coupled to peroxidase and then and 51T but not villin core or 44T (lane 3). were developed with 4-chloro-1-naphthol. We isolated two small tryptic fragments of 44T (Mr 14,000 The molecular weights of the peptides were calculated and 13,000) and sequenced their amino termini.
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