Jan 15, 2006 - as echothiophate eye drops, and certain nerve gases. Long before a person experiences clinical signs of poisoning, his serum cholinesterase ...
vo1. 262, No. 2, Issue of January 15,pp. 549-557 1987 Printed in d.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY
0 1987 by The American Society of Biological Chemists, Inc
Complete Amino Acid Sequence of Human Serum Cholinesterase* (Received for publication, July 7, 1986)
Oksana Lockridge, Cynthia F. Bartels, Theresa A. Vaughan, CassandraK. Wong, Sheila E. Norton$, and LindaL. Johnson$ From the Pharmacology Department and the $Department of Biological Chemistry, Protein Sequencing Facility, University of Michigan Medical School, Ann Arbor, Michigan 48109-0010
The complete amino acid sequence ofhuman serum cholinesterase(choline esterase I1 (unspecific), EC 3.1.1.8) was determined byEdman degradation of purified peptides. The protein contains 574 amino acids per subunit and nine carbohydrate chains attached to 9 asparagines. Thefour subunits of cholinesterase appear to beidentical. The active site serine is the 198th residue from the amino terminus. The sequence of human serum cholinesterase is 53.8%identical with the from Torpedo califorsequence of acetylcholinesterase nica and 28% identical with thecarboxyl-terminal portion of bovine thyroglobulin.
poisons of the type used in pesticides, insecticides, drugs such as echothiophate eye drops, and certain nerve gases. Long before a person experiences clinical signs of poisoning, his serum cholinesterase activity drops (5-8). The appearance of symptoms is better related to depression of red cellacetylcholinesterase than to depression of serum cholinesterase (8). One of the most thorough studies is by Grob et al. (8) who had a subject group of 75 human volunteers. When 2 mg of diisopropyl fluorophosphate (DFP) was administered either intramuscularly or intrarterially, serum cholinesterase activity dropped to 5% of original activity, whereas red blood cell acetylcholinesterase activity dropped to 65%. The serum cholinesterase activity dropped to near zero within 1 h afterDFP administration, whereas it took 24 h for red blood cellacetylSerum cholinesterase (acylcholine acylhydrolase, EC cholinesterase to be maximally depressed. It is not yet known 3.1.1.8; also known as butyrylcholine esterase, pseudocholin- what structural featuresof serum cholinesterase make it more esterase, and nonspecific cholinesterase) has been recognized reactive than acetylcholinesterase with organophosphate essince 1932 as an enzyme that hydrolyzes choline esters (1). ters. The molecular form of the cholinesterase molecule seDespite its long history, it has not been sequenced until now quenced in this report is the globular tetrameric G4 form. because only small amounts of protein are present in any tissue. Our laboratory introduced an affinity column purifi- Comparison of the amino acid sequence of human serum cation step that made it possible to purify serum cholinester- cholinesterase with the amino acid sequence of acetylcholinase in high yield (2). The new techniques of HPLC‘ purifica- esterase (EC 3.1.1.7) from the electric organ of Torpedo calition of peptides and of HPLC detection of phenylthiohydan- fornicu (9) shows a striking degree of identity. The two protion-derivatives and adaptations of Edman degradation to teins have 574 and 575 amino acids per subunit. 309 amino picomole amounts of peptides have made it possible to com- acids are identical in the primary structure. Only one other sequenced protein hassignificant identity with human cholinplete the amino acid sequence. Cholinesterase was named “pseudocholinesterase” by Men- esterase and that isbovine thyroglobulin which is 28% idendel and Rudney in 1943 (3) because they doubted the possi- tical in its carboxyl-terminal portion. The active site serineis bility that the enzyme could play an essential part in the 198 residues from the amino terminus in humancholinesterhydrolysis of acetylcholine in uiuo. The physiologic role of ase and 200 residues from the amino terminus in Torpedo cholinesterase is still unclear. It is present in nearly every acetylcholinesterase. tissue except erythrocytes. The study of serum cholinesterase EXPERIMENTAL PROCEDURES AND RESULTS~ became important to the anesthesiologist after succinylcholine was introduced as a muscle relaxant. Kalow and Staron DISCUSSION (4) foundthat a rare genetic variant of human serum cholinesterase explained the prolonged apnea observed in certain The complete amino acid sequence of human serum cholinpatients who had been given a normal dose of succinylcholine. esterase (choline esterase I1 (unspecific), EC 3.1.1.8) is shown The human serum cholinesterase variants are a classic ex- in Fig. 1 (Appendix). It consists of 574 amino acids. The ample in pharmacogenetics since they demonstrate that in- overlapping peptides used to establish the sequence are indidividual genetic variation may cause people to respond differ- cated in Fig. 1. ently to thesame drug. Most peptides overlap by 2or more residues. However, Another reason for continued interest in serum cholines- positions 61, 132, 220, and 494 overlap by only 1 residue. terase is its extraordinarysensitivity to organophosphate ester * This work was supported inpart by United States Army Medical
Research and Development Command Contract DAMD17-824-2271 and National Institutes of Health Grant GM 27028. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact. The abbreviations used are: HPLC, high performance liquid chromatography; DFP, diisopropyl fluorophosphate.
Portions of this paper (including “Experimental Procedures,” “Results” Fig. 3, and Tables 1-21) are presented in miniprint at the end of this paper. Figs. 1 and 2 are in the Appendix, pp. 552-553. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 86M-2276, cite the authors, and include a check or money order for $12.80 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.
549
550
Cholinesterase Sequence
There is no overlap between residues 125 and 126. Placement of these peptides is supported by the followingevidence. Position 61 is overlapped by peptides CH3 and CH4 and represents a 1-residue overlap between peptides T3 and T4. Peptides CH3 and CH4 are chymotryptic subfragments derived from the peptic peptide containing residues 29-93. CH3 and CH4 fit only into thedesignated locations within residues 29-93. An additional argument is the great sequence identity between Torpedo acetylcholinesterase and peptide T4 which leavesno doubt that T4 is correctly placed. Position 132 represents a 1-residue overlap between peptides P8, P9, and T6. Sequence identity in this region with Torpedo acetylcholinesterase and bovine thyroglobulin supports thisplacement. Positions 125 and 126 are not overlapped. Placement of PI1 next to P9 is supported by amino acid composition analysis of the tryptic peptide occupying residues 106-131. Position 220 representsa 1-residue overlap. The overwhelming sequence identity with Torpedo acetylcholinesterase in the region of peptide T10 confirms that this peptide is correctly placed. The 1-residue overlap at position 494, which places P33 after P32, is confirmed by the amino acid composition for Staphylococcus aureus peptide 462-497. Carbohydrate chains appear to be attached to asparagine at positions 17,57, 106, 241, 256, 341,455,481, and 486. This conclusion is based on the following observations. 1) During sequencing, glycosylated asparagine showed up as a blank since the carbohydrate chain prevented extraction of the phenylthiohydantoin-derivativeinto thesequencing solvents. 2) The glycosylated asparagine was followed by any amino acid except proline, and then by either threonine or serine to give the sequence Asn-X-Thr/Ser; this tripeptide sequence is common to all N-glycosidically linked carbohydrate chains (10). 3) Amino acid analysis was consistent with the presence of asparagine. 4) Carbohydrate-containing peptides yielded a heavy black residue after acid hydrolysis at 150 "C since these conditions decomposed the sugars. The finding of nine carbohydrate chains is consistent with the report by Haupt et al. (11)that 23.9% of the weight of human serum cholinesterase is due to carbohydrate. These authorsreported a carbohydrate composition of 9.3% galactose plus mannose, 8.4% acetylhexosamine, 6.0% acetylneuraminic, and 0.2% fucose. This can be calculated to represent 50 residues of galactose plus mannose per subunit, 35 acetylhexosamines, 18 neuraminic acids, and 1.2 fucose. The data of Haupt et al. (11) support the conclusion that there are nine carbohydrate chains per cholinesterase subunitand suggest that thesechainsare the complex type terminating in sialic acid. With one exception, all asparagines in the cholinesterase sequence that could theoretically be attached to carbohydrate were found to be glycosylated. The single exception was asparagine 485, which is adjacent to the glycosylated asparagine 486. It is not surprising that asparagine 485 is not glycosylated since N-glycosidic linkages are commonly separated by several amino acids and do not occur on adjacent asparagines. The possibility that additional sugar chains might be present as 0-linkedoligosaccharides was not examined. Comparisons of the amino acid sequences of human serum cholinesterase, T . californica acetylcholinesterase, and bovine thyroglobulin are shown in Fig. 2 (Appendix). These proteins were identified as having significant sequence homology by searching the computerized data base of the Protein Identification Resource, Georgetown University Medical Center, Washington, D. C. The family of proteins known as theserine proteases, which includes trypsin and blood coagulation factors, showed no significant sequence homology. Fig. 2 shows that 309 residues of human serum cholinesterase and Torpedo
acetylcholinesterase are identical. There is identity within every domain. This high degree ofidentity suggests more of a relationship than might have been suspected from the names true and pseudocholinesterases. Bovine thyroglobulin has a total of 2750amino acid residues (12). Comparison of544 amino acids from the carboxylterminal portion of thyroglobulin with the amino acid sequence of human serum cholinesterase shows an identity of 28%. The gene for human serum cholinesterase is on human chromosome 3 (13, 14), whereas the gene for human thyroglobulin is on chromosome 8 (15). Schumacher et al. (9) found 28% identity between thyroglobulin and Torpedo acetylcholinesterase. Seven of the eight half-cystines of human serum cholinesterase are located at the same positions as the halfcystines of Torpedo acetylcholinesterase. Six cysteines are conserved in acetylcholinesterase, cholinesterase, and thyroglobulin, suggesting a similarity in protein folding. The active site serine of human serum cholinesterase is located 198 residues from the amino terminus. Sequence data for the DFP-labeled tryptic peptide are given in Ref. 16. The active site serine of Torpedo acetylcholinesterase is located 200 residues from the amino terminus (9). Locations of other residues important to the activity of cholinesterase are not yet known. The active site histidine (17, 18) is likely to be common in both sequences, and this limits it to being at position 207, 423, 438, or 548. The subunit molecular weight calculated from the amino acid sequence is 65,092. The weight of the carbohydrate chains was reported by Haupt et al. (11)to be 23.9% of the weight of cholinesterase, bringing the subunit molecular weight to a total of 85,534. We have previously shown that theenzyme is a tetramer (2, 19); and thus, the enzyme molecular weight is
TABLE 22 Amino acid Composition of human serum cholinesterase The subunit molecular weight calculated from the sequence is 65,092. This becomes 85,534 when it is adjusted for 23.9% carbohydrate. From sequence
no. residues
Ala Arg Asn ASP CYS Gln Glu Gly His 1.1 Ile 4.8 Leu LYS
34 23 40 24 8 20 37 46 8 27 50 35 11 39 30 37 35 18 20 32 574
mol %
5.9 4.0 7.0 4.2 1.4 3.5 6.4 8.0 1.4 4.7 8.7 6.1 1.9 6.8 5.2 6.4 6.1 3.1 3.5 5.6
From analvsis" mol %
6.0 4.4
Asx 11.3 1.4
Glx 9.7 8.1 9.0 5.3 1.9 6.7 6.3 7.0 6.1 2.1 3.1 5.4
Met Phe Pro Ser Thr Trp TYr Val Total a Amino acid composition analysis was done by AAA Labs, Mercer Island, WA. 2 mg of salt-free cholinesterase (specific activity of 200 units/mg when assayed with 50 W M benzoylcholine in 0.067 M sodium/ potassium phosphate, pH 7.4, at 25 "C) was hydrolyzed for 24, 48, and 96 h in 6 N HCl at 110 "C. Tryptophan was determined from a 48-h alkaline hydrolysis at 135 "C. Half-cystine was determined by performic acid oxidation prior to acid hydrolysis.
Cholinesterase Sequence approximately 342,136. The value of 342,136 is approximate because the carbohydrate weightis not exact. Molecular weights of the monomer and dimer are 90,000 and 180,000, respectively,onsodiumdodecyl sulfate-polyacrylamide gel electrophoresis (19,26). The subunit weightis 85,000 on ultracentrifugation (20), 81,000 and 86,000 on Sephadex gel filtration (21, 22), and 100,000 when titrated with DFP or paraoxon (23). The molecular weight is 348,000 by ultracentrifugation (11).These values agree well with the molecular weight calculated from the amino acid sequence. Table 22 shows the amino acid composition of human serum cholinesterase calculated from the amino acid sequence and compares it to the amino acid composition analysis of the protein. The two methods are in close agreement. This suggests that the sequence analysis did not omit a significant fragment and thatthere is nota second type of subunit with a different sequence. The data lead to the conclusion that the four subunits of human serum cholinesterase are identical. Additional support for this conclusion comes from results of amino-terminal sequencing of the intact cholinesterase molecule. Only one amino-terminal sequence was detected, and this sequence was the same as shown in Fig. 1. Earlier work (19, 20) also supports the conclusion that the four subunits are identical. We have no explanation for the residual dimer band in highly purified cholinesterase, observed when the reduced enzymeis electrophoresedon sodium dodecyl sulfatepolyacrylamide gel (19). This band incorporates radioactive DFP. However, DFP-labeled cholinesterase yields only one radioactivetryptic peptide on HPLC (16);and therefore, there isnoevidence that the residual dimer band represents a second subunit. Recent results have raised the possibility that in some species the acetylcholinesterase molecule may have more than one type of subunit. Rotundo (24) foundtwo subunits of M, 105,000 and 100,000 in chicken brain acetylcholinesterase. Haas and Rosenberry (25) detected 66% glutamic acid and 34% arginine at the amino terminus of human red cell acetylcholinesterase. Our sequencing results indicate only one type of subunit in human serum cholinesterase. Acknowledgments-We thank Professor Palmer Taylor for providing the amino acid sequence of Torpedo acetylcholinesterase prior to publication; Dr. Harry Eckerson for building a computer-controlled step gradient device for HPLC, Steve Adkins for computer analysis of the data base in the Protein Identification Resource, Dr. George Tarr for his manual sequencing method, and Professor Bert N. La Du, in whose laboratory this work wascarried out, for encouragement and support. REFERENCES 1. Stedman, E., Stedman, E., and Easson, L. H. (1932)Biochem. J. 26,2056-2066 2. Lockridge, O., and La Du, B. N. (1978)J. Bwl. Chem. 253,361366
551
3. Mendel, B., and Rudney, H.(1943)Bwchem. J. 37.59-63 4. Kalow, W., and Staron, N. (1957)Can. J. Biochem. Physwl. 35, 1305-1320 5. M~ZUI,A., and Bodansky, 0. (1946)J. BWL Chem 163,261-276 6. Munkner, T., Matzke, J., and Videbaek, A. (1961)Acta Pharm o l . ToxicoL 18,170-174 7. Verberk, M. M. (1977)Toxicol. Appl. Pharmacol. 42, 345-350 8. Grob, D., Lilienthal, J. L., Harvey, A. M., and Jones, B. F. (1947) Buil. Johns Hopkins Hosp. 81,217-244 9. Schumacher, M., Camp, S., Maulet, Y., Newton, M., MacPheeQuigley, K., Taylor, S. S., Friedmann, T., and Taylor, P. (1986) Nature 319,407-409 10. Bause, E. (1983)Biochem. J. 209,331-336 11. Haupt, H., Heide, K., Zwisler, O., and Schwick,H. G. (1966)Blut 14965-75 12. Mercken, L., Simons, M. J., Swillins, S., Massaer, M., and Vassart, G. (1985)Nature 316,647-651 13. Sparkes, R. S., Field, L. L., Sparkes, M. C., Crist, M., Spence, M. A., James, K., and Garry, P. J. (1984)Hum. Hered. 34,96-100 14. Yang, F., Lum, J. B., McGill, J. R., Moore, C. M. Naylor, S. L., Van Bra& P. H., Baldwin, W . D., and Bowman, B. H. (1984) P m . Natl. Acad. Sci. U.S. A. 81,2752-2756 15. Baas, F., Bikker, H., van Kessel, A. G., Melsert, R., Pearson, P. L., de Vijlder, J. J. M., and van Ommen, G. J. B. (1985)Hum. Genet. 69,138-143 16. Lockridge, O., and La Du, B. N. (1986)Biochem. Genet. 24,485498 17. Mounter, L. A., Alexander, H. C., Tuck, K. D., and Dien, L. T. H. (1957)J. Bwl. Chem. 226,867-872 18. Beauregard, G., Lum, J., and Roufogalis, B. D. (1981)Biochem. P h u r m o l . 30,2915-2920 19. Lockridge, O., Eckerson, H. W . , and La Du, B. N. (1979)J. Biol. Chem. 264,8324-8330 20. Muensch, H.,Goedde,H. W . , and Yoshida, A. (1976) Eur. J. Biochem. 70,217-223 21. La Du, B. N., and Dewald, B. (1971)Ado. Enzyme Regul. 9,317332 22. Boutin, D., and Brodeur, J. (1971)Can. J. Physwl. Pharmacoi. 49,777-779 23. Ralston, J. S., Main, A. R., Kilpatrick, B. F., and Chasson, A. L. (1983)Biochem. J. 211,243-250 24. Rotundo, R. L. (1984)J. BwL Chem. 259,13186-13194 25. Haas, R.,and Rosenberry T. L. (1985)A d Biochem. 148,154162 26. Lockridge, O., and La Du, B.N. (1982)J. Biol. Chem. 257, 12012-12018 27. Jansz, H.S.,Brons, D., and Warringa, M. G.P.J. (1959)Biochim. Biophys. Acta 34,573-575 28. Berends, F., Posthumus, C. H., Sluys, I. V. D., and Deierkauf, F. A. (1959)Biochim. Bwphys. Acta 34,576-578 29. Gross, E. (1967)Methods Enzyml. 11,238-255 30. Tarr, G.E. (1986)in Microcharacterization of Polypeptides: A Practical Manual (Shively, J. E., ed) pp. 155-194, Humana Press Inc., Clifton, NJ 31. Tarr, G.E. (1982) in Methods in Protein Sequence Analysis (Elzinga, M., ed) pp. 223-232,Humana Press Inc., Clifton, NJ 32. Black, S. D., and Coon, M. J. (1982)Anal. Biockm. 121, 281285 33. Lipman, D. J., and Pearson, W . R. (1985)Science 227, 14351441 Continued on next page.
552
Cholinesterase Sequence APPENDIX
FIG. 1. Complete amino acid sequence of human serum cholinesterase. T, tryptic peptide; P, peptic peptide; S, S. aureus protease peptide; CB, cyanogen bromide peptide; CH, chymotryptic peptide; CHO, carbohydrate. The active site serine is residue 198. The amino terminal residue is residue 1. io CHO 20 30 Olu-Asp-Asp-I1e-I1e-Ile-Ala-lhr-Lys-Asn~~Gly-Lys-Val-Arg-Gl~-Met-Asn-Leu-Tnr-Vnl-Phe-Gly-Gly-~hr-Val-~hr-Aln-Phe-Leu-Gly-~le-Pro-~yr-Ala-Gln """"""Ti""""""""""-
""""""p2""""""""-""""""""""""""""".~"""".""""+""""p~""""""""40
50
CHO
#
60
70
~ro-Pro-Leu-Gly-Arg-Leu-Arg-Phe-Lys-Lys-P~o-Gln-Ser-Leu-lhr-Lys-lrp-Ser-Asp-lle-lrp-Asn-Ala-lhr-Lys-lyr-Ala-Asn-Ser-Cys-Cys-Gln-Asn-Ile-Asp """"T2""""""""-+"""-"""~~""""""""""-+"""""""-~4"""""""""""
""""p3""""""""""""""""""""""""""""""""""""CBZ"""" """"C",""""""+""""C"2"""""" """"p4""""""""""80
"""-C"4"""".
CH5""
""
""""""""C"3"""""""" 90
100
Gln-Set--Phe-Pro-Gly-Phe-His-Gly-Ser-Glu-Met-~rp-A~n-Pro-Asn-lhr-Asp-Leu-Ser-Glu-Asp-Cys-Leu-Tyr-L~-Asn-Val-Trp-Ile-Pro-Aln-Pro-Lys-Pro-Lys ..--CH5----+--------p5----------------+--------p6---------------------+----p7---------------------*------------p8-------------------------
""""""""""s1-""""""""1i o
CHO
120
140
i30
Asn-Ala-Thr-Val-Leu-Ile-lrp-Ile-Tyr-Gly-Gly~Gly-Phe-Gln-Thr-Gly-Thr-Ser-Ser-Leu-His-Val-lyr-Asp-Gly-Lys-Phe-Leu-Aln-Arg-Val-Glu-Arg-Val-Ile """"""""15""""""""""""""""""76""""----p8--.----------+--------pg-------------------------------------------------+--------pli----------------+----pi2-----------------------""""P10"""""""""" 15 0 160
i7 0
\~al-Val-Ser-Met-Asn-lyr-ArQ-Val-Gly-Ala-Leu-Gly-Phe-Leu-Aln-Leu-Pro-Gly-Asn-P~o-Glu-Ala-Pro-Gly-Asn-Mei-Gly-Leu-Phe-Asp-Gln-Gln-Leu-Ala-Leu """"""T7""""""""""""""""""""""""""""""""""""""""""""""""pi2 """"Pi3"""""""""""""""""" """"""CB3""""""""""""""""""""""""""""""""""""+""""~~4""""""""""""
"-p,4""-
""
180
190
2 io
200
Gln-Trp-Val-Gln-Lys-Asn-Ile-Ala-Ala-Phe-Gly-Gly-Asn-Pro-Lys-Ser-Val-lhr-Leu-Phe-Gly-Glu-Ser-Ala-Gly-Ala-Ala-Ser-Val-Ser-Leu-H1s-Leu-Leu-Ser """"Pi4"""""""""""""""""""""" 220
2 4 0 CHO
230
Fro-G1y-Ser-His-Ser-Leu-Phe-lhr-Arg-Al~-Ile-Leu-Gln-Ser-G1y-Scr-Phe-Asn-Aln-Pro-lrp-Ala-Val-Thr-Ser-Leu-Tyr-Glu-Ala-Arg-Asn-Arg-Thr-Leu-Asn Til""
""
""""""p~~""""""""-""""+""""""""""p~~""""""""""""""""""""+""""p~g"""-""---"--"---250
270
260
CHO
Leu-Ala-Lys-Leu-Thr-Gly-Cys-Ser-Arg-Glu-Asn-Glu-lhr-Glu-Ile-~le-Lys-Cys-Leu-A~~-Asn-Lys-Asp-Pro-Gln-Glu-lle-Leu-Leu-Asn-Glu-Ala-Phe-Val-Val ""T~,""+""""~~~""""""+""""""~~~""""""""+""~~4""+""""""""~~5"""""""""""""""""""" """"""P19""""""""""""""""""""""""""
""""PZO"""" """"P2,""""""
""""53""-
""""~2""""""""""""""""""""""""""290
3 10
300
Pro-Tyr-G1y-Thr-Pro-Leu-Ser-Val-Asn-Phe-Gly-Pro-Thr-V~l-Asp-Gly-Asp-Phe-Leu-Thr-Asp-Met-Pt~o-Asp-~le-Leu-Leu-Glu-Leu-Gly-G1n-Phe-Lys-Lys-Thr """"~3""""""""""""""""""""""""""""""""~4""""""""""""""""""""""""""""""""""""""""""""-
"_
""""""""p~~"""""--C"-""""~p~3"""--""--"--------
320
""""""""""""~g~"""""""""""" 3 4 0 CHO
3 30
350
Gln-Ile-Leu-Va1-Gly-Vnl-Asn-Lys-Asp-Glu-Gly-lhr-Ala-Phe-Leu-Val-lyr-Gly-Ala-Pro-Gly-Phe-Ser-Lys-Asp-Asn-Asn-Ser-Ile-Ile-lhr-Arg-Lys-Glu-Phe --------Ti6-------------------""""""""p23""""""""""""""""""+""""p~4"""""""""""""""""""""""""""""""""""" """"~5""""""-"""--*""--""."~~""""""""""""""""""""""""""~
117""
""
"_
370
360
380
Gln-G1u-G1y-Leu-Lys-Ile-Phe-Phe-Pro-Gly-Val-Ser-Glu-Phe-Gly-Lys-Glu-Ser-Ile-Leu-Phe-Hls-lyr-Thr-Asp-lrp-Val-Asp-Asp-Gln-Arg-PrO-Glu-Asn-lyr """"~~,""""+""""""""T(B""""""""""""+""""""~,g""""""""""""""""""""""""""-""-"
P24----
""
""""57""""""""""""""""-+""""""""sa""""""""""""-
""""P25""""""""
""""""5g""""""""""""""""""""""""-+"sio-4 10
400
390
410
Arg-Glu-Aln-Leu-Gly-A~p-Val-Val-Gly-A~p-Tyr-A~n-Phe-Ile-Cy~-P~o-Ala-Leu-Glu-Phe-lhr-Lys-Lys-Phe-Ser-Glu-lrp-Gly-Asn-Asn-Aln-Phe-Phe-lyr-lYr "-+""""""T20"""""""""""""""""""""""""""""""""" """"S10"""""""""""""""" """"P26"""""""" """"511""""""""""""""""""""""""""""+""""5~~""""""""""""""""""-""""""---
.........................
-P27---
""""513""""""""""""
""""c86""""""""""""""""""""""""""""""
cno 490 460 4 7 0 4 8 0 CHO Tyr-lhr-~y~-Al~-~lu-Glu-~le-Leu-~er-Arg-~er-Ile-V~l-Lys-Ar~-Trp-Ala-Asn-Phe-Al~-Ly~-lyr-Gly-Asn-Pro-Asn-Glu-Thr-Gln-Asn-Asn-Ser-~hr-Ser-~rP ""T23""+""""724"--""""""-+""T~5"""" """"""p~~""""""""+""""""""""~~~""""""""""+""""""""""""p3~""-"""""""--"-------------
~~~""""""~~~~""-"""""""""""~~~"~~~~~~~~~~~~-
""""
""517""""""
""""""cg6"""""" 500
510
520
PrO-Vn1-Pne-Lys-Ser-Thr-G1~-Gln-Lys-lyr-Leu-Tnr-Leu-Asn-Thr-Glu-Ser-lhr-A~g-~le-Met-Thr-Lys-Leu-Arg-Ala-Gln-G1n-Cys-Arg-P~-Trp-Thr-5er-Phe ""127"""" """"""""128""""""""""+""12g~"""""T10"""""" ""p~~""~""""p~~""""""""""""""+""""""p~~""""~"""~""""""""""""~""~~~"~~~~~""""""5~g""""""""""+""""""~~~"""""""""""""""""""""--""-------"---
540
530
550
560
Phe-Pro-Lys-Vnl-Leu-Glu-Met-Thr-Gly-As~-Ile-Asp-Glu-Al~-Glu-lrp-Glu-Trp-Lys-Ala-Gly-P~-H1s-Arg-lrp-Asn-Asn-lyr-Mei-Mei-Asp-Trp-Lys-Asn-Gln "733" ----T30----+--------T31----------------------------------------------------+-----T~2----------""""""P35"""""""""""""" "" 522"" ""
"520"
""""521""""""""""""
""""""CB,"""""""""""""""""""""" 570
""""523"""""""""""""""""""""""""""""-574
Fhe-Asn-Asp-lyr-Thr-Ser-Lys-Lys-Glu-Ser-Cys-Vnl-Gly-Leu """"T33""""""""+""""134""""""""
."""""""-*23""""""""+""""s24"""" """"CB8""""""""""""""""
""""CB8""""
Cholinesterase Sequence
553
FIG. 2. Comparison of amino acid sequences of acetylcholinesterase from T.californica,cholinesterase from human serum, and bovine thyroglobulin. The sequence of acetylcholinesterase is from Ref. 9, and the sequence of thyroglobulin from Ref. 12. Dashes have been placed within sequences to maximize the number of matches. AChE, acetylcholinesterase; ChE, cholinesterase. Torpedo AChE
DDHSELLVNTKSGKVMGTRVPVLSSHISAFLGIPFAEPPVGNMRFRRPEPKKPWSGVWNASTYPNNCOOYVDEOFPGFSGSEMWNPNREMSEDCLYLNIUVP
.. .. .. .. .. ..
.. .. .. .. .. .. .. .. . . . .. .. .. .. . . . .. .. .. . . . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . . . . . . . . . . .
Human ChE
EDDIIIATKNGKVRGMNLTVFGGTVTAFLGIPVAOPPLGRLRFKKPOSLTKWSDIWNATKYANSCCONIDOSFPGFHGSEMUNPNTDLSEDCLYLNVWIP .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..
Thyroglobulin
VPIATffiOLLGRSOAIOVGTSUKPVDOFLGVPYAAPPL~EKRFRAPEHL-NWTGSWEAT~PRARC------UOPGIRTP----TPPGVSEDCLYLNVFVP
Torpedo AChE
SPRPKSTTVMVWIYGGGFYSGSSTLDVYNGKYLAYTEEVVLVSLSYRVGAFGFLALHGSOEAPGNVGLLDORMALOUVHDNIOFFGGDPKTVTIFGESAG
101 100
227 1
.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. APKPKNATVLIWIYGGGFOTGTSSLHVYDGKFLARVERVIVVSMNYRVGALGFLALPGNPEAPGNMGLFDOOLALO~VOKNIAAFGGNPKSVTLFGESAG .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..
202
Human ChE Thyroglobulin
ONMAPNASVLVFFHNAAEGKGSGDRPAVDGSFLAAVGNLIVVTASYRTGIFGFLS-SGSSELSGNWGLLDOVVALTWVOTHIOAFGGDPRRVTLAADRGG
2370
Torpedo AChE
GASVGMHIL--SPGSRDLFRRAILOSGSPNCPWASVSVAEGRRRAVELGRNLNCNLNSDEELIHCLREKKPOELIDVEWNVLPFDSIFRFSFVPVIDGEFFP 302
Human ChE Thyroglobulin
AASVSLHLL--SPGSHSLFTRAILOSGSFNAPWAVTSLYEARNRTLNLAKLTGCSRENETEIIKCLRNKDPOEILLNEAFVVPVGTPLSVNFGPTVDGDFLT 3 0 0 .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ADIASIHLVTTRAANSRLFRRAVLMGGSALSPAAVIRPERAROQAAALAKEVGCPSSSVOEMVSCLROEPARILNDAQTKLLAVSGPFH-YWGPVVDGVYLR 2 4 7 1
Torpedo AChE
TSLESMLNSGNFKKTOILLGVNKDEGSFFLLYGAPGFSKDSESKISREDFMSGVKLSVPH-ANDLG-LDAVTLOVTDWMODNNGIKNRDGLDDIVGDHNVIC 402
Human C h E
DMPDILLELGOFKKTOILVGVNKDEGTAFLVYGAPGFSKDNNSIITRKEFOEGLKIFFPG-VSEFG-KESILFHYTDWVDDORPENYREALGDVVGDYNFIC
400
Thyroglobulin
ETPARVLORAPRVKVDLLIGSSODDGLINRAKAVKOFEESOGRTSSKTAFYOALONSLGGEAADAGVOAAATWYYSLEHDSDDYASFSRALEOATRDYFIIC
2573
Torpedo AChE
PLMHFVNKYTKFGNGTYLYFFNHRASNLVUPEUMGVIHGYEIEFVFGLPL--VKELNYTAEEEALSRRIMHYWATFAKTGNPNEPHSOESKWPLFTTKEOKF
Human ChE Thyroglobulin
.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..
.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . . .. .. .. .. .. .. .. .. .. . .. .. .. .. .. .. .. ..
..
.. .. .. .. .. .. .. .. ..
.. .. ..
.. .. .. .. .. ..
.. .. .. .. ..
.. .. .. .. .. .. .. .. .. .. ..
.. .. .. ..
.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. PALEFTKKFSEWGNNAFFYYFEHRSSKLPWPEWMGVMHGYEIEFVFGLPL--ERRDNYTKAEEILSRSIVKRWANFAKVGNPNETONNSTSWPVFKSTEOKY .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . .
502
PVIDMA---SHWARTVRGNVFMYHAPESYSHSSLELL~-TDVLYAFGLPFYPAYEGOFTLEEKSLSLKIMOYFSNFIRSGNPNYPHEFSRRAPEFAAPUPDF
2670
..
..
..
.. ..
IDLN-TEPMK-------VHORLRVOMCVFUNQFLPKLLNATETIDEAEROWKTEFHRWSSYMMHUKNOFDHY-SRHESCAEL
575
Human ChE
.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. LTLN-TESTR-------IMTKLRAOOCRFWTSFFPKVLEMTGNIDEAEWEWKAGFHRWNNYMMDWKNOFNDYTSKKESCVGL .. .. .. .. ..
Thyroglobulin
VPRDGAESVKELSVLLPNROGLKKADCSFWSKYIOSLKASADETKDGPSADSEEEDOPAGSGLTEDLLGLPELASKTYSK
2750
Torpedo AChE
200
.. .. .. ..
Mlnlprint s.ction
EXPIRIMEYTAL PBOCBDURSS
574
500
Cholinesterase Sequence
554
1.
analpis T1
Glu 2459 L p mp 1172 RO MD
4 5
Con Yter Anal ai. TheProtein Identification Resource, National Bioa&escargh Foundation, Georgetown University Medical Center, Washington, D.C. contain. continuouely updated computerized files of published protein and peptide hormone sequences i n data files NBRF, NEW, PGTRANS (translated from GenBank), KASAT, and JAPAN. In addition, the
Abbreviationo. T-tryptic peptides, S-S.aureus protease peptides, Ppeptic peptide.. CB-cyanogen bromide peptides.-chymotryptic peptides. D F P - d i i ~ o p r ~ p y l f l u ~ ~ ~ p h o n P h ~ tHPLC-high ~, performance liquid chromatography. PTH-phenylthiohyd.ntOin. N-CHO-aarbohydzate attached to asparagine. CHO-carbohydrate.
RESULTS Sequence data and amino acid composition analysis of the active site tryptic peptide have been published 1161. 1 figure shoving HPLC Beparation Of tryptic peptides, and the elution position of the active Bite tryptic peptide is given I n the Same publication (16). Separation of cyanogen bromide peptidea .1 shown in Fig.3 where ~ i g . 3 A ahova the elution po#itions of all the cyanogen bromide fragments and Fig.3- illustrates how peptides were cleaned up p r i o r to .equencing.
6 7 8 9 10 11 12 13 14 15 16 17 18 19 28 21 22 23 24 15 26 27 28 29
T2
raw
-
mt Tzp Am Pro
854
%br
Asp
w Ser
ec
Leu
lyr W
Mn
1.v
38
292 516 459 296 119 278 116 81 65 33 37 65 56 71 32 28
a i G3rmTirmE %br 264 Val 3474 2884 Ile 2614
Deu
rrp 881 981 lyr 2028 Gly 792 Gly 400 Gly 958 Re 322 11-
Dsu 4888 Gly 9328 Ala 44% Ncl 9570 1462 W 8880
Gly 1827 Re 9330 9470 111- 9118 Deu 9330 PTO 9520 GlY 8880 As" 7 5 w PI0 1240 Glu 5939 Ala 7400 Pro 7558 Gly 6710 M Yae0
tSt 4178 Gly 4648 W 4670 P h 5330 h p 3240 Gln 4718 Gln 4230 W 2938 Ala 29#8 Dsu 2498 Gln 2558 hp 318 Val 20100 Gln 1920 L F 1080
Irene
20
48
R O
41 42 43
Na RO
11 14 8 12
Lp
2 3 4 5
6 7 8
11 12 13 14 15
16 17
Wan The peptic peptide P3, which includes residues 29-93, subfragmented with chynotrypsin to get the overlap between residues 60 and 61. Sequence results a r e I n Table 16; amino acid conpellition resYlts are i n Table 11.
886
Glu rsp
7
Tablee 17-21. NO values are shorn for tryptophan even though tryptophan was detected by the "81 destroyed by acid pico-tag method. because most of the tryptophan hydrolysis and tho yield- were not quantitative.
Gl"
M L a n
39
Ammino acid compoeition analysis reeults are given in
n
704 ser 274 3012 Gln 562 *ap 226 Am 5224 I1e 266 6er 4094 Ire 48 Mz 3568 Ka(0 Mz 3812 Ala 168 Gln 2416 Thr 59 Urn 5272 L F 126 n e 4480 rsp 6866 Gln 1276 s e l 1486 Re 1588 R O 1580 GlV 1279 €& 1323 His 388 Gly 1182 Ser 177
38
31 32 33 34 35 36 31
T6 and R.
18 19
4731 4340 3552 3799
Gl" s 5 5 8.r 1812 11. 1651 M 168s Rr 2701 HI. xu94 lyr 1782 lkc Up
500
914
Trp 481
Val9%
up 765 UP%
m n 646 Irg 338 R O 770 Q u 685 M 757 1M 697
R o 2349
Ire 5267 RO
2189
Glu
2454
rrp 1969
mt 1866 Gly 455 Val 1170 mt 1277 HI. 735 Qy 848 ?yI 885
Gl"
740
11.
452
-
Gl" Ren l 99
Re 1%
-
Cholinesterase Sequence
-1y.I.
Table 4. and T28.
Bhn cycle T24
o f -IC
T23, T24. T25. R 6 , T27,
popti&
555
Table 8. and P21.
-ly.Ia
aiur T23
Ala 1847 Glu 1784 Glu 3554 lle 1085 M 643
up 1811
1 2
w3il Tyr 1174 Ru YO Ly. 962
3 4 5 6 7
su
R8
125 T27
T26
Ser 3 4 1
nP 1mV
I l e 2654 -1 1900 Ly. 26W
Ala 1715 M 1W7
Rr
121
Ala 533 LF 671
4%
luq 531
8 9
Lsu 6882
2 3
Gly 6267 Gly 4184
Iru 5964
4
M
N-zm
&m 4293
?hr
Ihr 178 Gln 3-30 M 282
Glu 4912
5 6 7
nca
Ser 2142 1121 Aq m 2
mr
S e x 141 mr 122 Ser 66 rrP 26 R O 42 Val 54 Ra 32
11 12
13 14 15 16 17 18
P15
"a=mr
mr 2149
G ~ U382
YQ15
10
cycle P16
Tur lm
M 798 Gly 473 Mn 516 R o 545
Ly. 19
8 9 10 11 12 13 14 15 16 17
3493 PI0 4935 Ly. 1835
PU
m
H.i
Pll
1w4 12647 W 1768 12657 W 1972 W61 B r 616
23869
16332 ser 1218 2274V Val 2728 Ala 2 1 x 8 Ihr 879 ser 5% w 2211 Val 3417 3639 w 9398
R D
PI9
5
1331
Gly 1334 ser 750 His 721
ssr W
Ra
680 438
559 Ihr 257 lug 299 Ala 523
M 5935 Glu 3636 N a 4388 2 3 4 Ug 1879 33% Nam 1118 ~g 1n3 2289 Ru 751 1426 W 1357 2165 M 611 1270 -109 2s Ala 695 1M9 Ly. 19 867 w 347 5% mr 182 268 GlY (96 54
z
18
phs
Val 781
11410 4158
m mr
5392
Lau 6W Glu 321
4 5 6 7
Ser 2959
M 521
Rr 2492 Ra 2059 R O 1516 ~ y . 658
Ihr 1 8 G l y 125 M n 230
(I
385
11.
n e 537
4 5 6 7
Na
14 15 16 17 19 18 21 21 22 23 24
Ly. 253 M 379 Gly 3% ~ y .115 Val u1
1472 259 997 685 Gly 359
a n a l p i s of @IC
m
Ro ¶)T
519 1192
LKM M Y
"3 z m s
x 281
457 Ly. 1%
Glu 311 up
876
95 155 154 5
Ly. 2s6 R O
146
351
377 269 295 lW
38
a: 1n w54 ryr UU
102 25
Rr 430
a n 252 48 I1
t.u
nuu
sa
am rl9
n
rn
P9
2
v.1
2481
1I
135 82
3 4
nP 536
P11
"
Tiz-zzw Gln 4694
hp 798 11- 935 mr 1434 Tyr 853 Qy 1 W Gly 846 I(u 643 Gly 1524 Ssr 691 Gly l l M m 635 Rr 465 W 332 Gln 331 lbz 327 Ly. LKM Gly 719 N a 234 mr 604 mr 1% ser 566 nl 193 ser 81 mu 1 w U u 5 4 17 11. 1 w 1 Pro 9 4 4 N a 676 RO 792 Ly. 106 R o 6%
n
1 2 3
P12
w
1106
e13
PU,
P14
TirrS mu 3-3s-
Ala 6261 su 347 Ala 4995 lug 1899 Val 5835 Glu 7434 lug Y15 Val 3548 I l e ZBW Yal 3221 Val 159 & I 3 9
mt 629 A m 330
rYr
Uu 1553
M U63
491 rrp
ug 2%
151
Val 1374
MI 162 Gln 1193 GlY 3 8 Ly. 569 Ala 149 M 826 -3s 11. 12a4 Gly 139 Ala 1161 Rr 33 N s 1165
Rr 634 G l y 718 Gly 729 Mn 186 R a 262
pet1W P22, P23, P24. P25, P26, €27.
P11 leu 1734 L a 9369 Glu 483 Val 5141 rmU 481 rYr 4349 Gly 519 GLy 1442 Cln 579 Ala 958 Rm 65 Ro 693 LY. 233 m y 2939 Ly. 221 Rr 967 lbr 411 Ser 160 a n 116 Ly. 21 Ilc 92 up 867 Iru 5 4 K o D Val 260 M 262 Gly 368 €aI lM M1 I29 Ile 622 rn 111 n e 871 L p 117 l W 225 up 91 lug 287 Glu 628 Ly. 191 Gly 3 9 Glu 167 mr 25 phc 65 N. 159 Gln 57 Rr 35 G l U 30 Gly 15
uu1y.i.
Rr 4963 G l y 5498 Ly. 1487 Glu 4266 Ser 1474 11s 1991 w 3367
of P W I C papti-
P29 P31
P30
m,
P31, P31, P32, P33,
P33 P35
TCTHT
@ F i lm sen F 11138 W 14682 Glu 2621 Ile 4139
2186 RL 1271 Val 621 me 41 332 Gly Glu
Val 3111
uo
18
Glu 640 11e 112 w 149
443 Ly. 131 Ala 390 Clu 311
3-3.34 201 218 1272 lDB0 652 171 Ly. 11
-291
1179
lhz
1535
311
Nam
ryr
P34 M 10587
4461 l h Z 9425 Glu 9135 sn 12554 mr 20% rrrl 1453 11. 298 mt 211 M
lug
923 Ser 63995 I l a 1880 Val 2 6 U Ly. 14% Isu 1264 Aq 918 Glu 1519 np 231 A q 1154 Ala 1068 k g 1294 M 5s5 up 735 Rr 516
14 15 16 17
19 20
Table 1. SrZIla d y . 1 . o f pptk p e t i b W . m, PU, P11, P U , and P14.
M 4412 7
aiur cycle P32
I9 11 12 13
Ly. 272 Ly. 4 Y RO 293
su
Ihr 16409 U p 22650 mt 15201 R O 6732 up 21138 n e 7742 Iru 19981
Table 11.
6 7 8
781 112
Lu 241 Ug 199
8869 7651
17136 9647
and P35.
4 5
Ly.= zm-
26
15 16
1711 194s
Lu 238
Gly Ug
P22 P24 LAI 6891
11
-K
W79
1316
Gl" m nu64 Glu 125 1160 11. 14 Ly. 42
12 13 14 15 16 17 18 19 20 21 22 23 24
Ala 1832 Gln 111 Gln 739 b.r 31 Ro 654 Uu 61 561 R o 681
57 Ru 169 l? Val366 YOlD Rr 245 U u - Gly 471 m a 7 Gly 4 3 Val 54 mr 13-3 Rr 15 Val 180 mr 120 Ala 116 Rr 41
ug
s
YZiFmT
M
699 Gly 254 Ly. Val -9
mr
381
C l y 1993 Ly.= 1388 11. 1458
25
5 6 7 8 9 11 11 I2 13 14
5 6 7 8 9 19
N a 110 Glu 1% ?rp 70
3
9 11 11 13 11
4
Glu 110
7Tlrlm- mr
8
C l y 1446 LAI 1341
736 213 189 Ly. 52
sn
Glu 230
19 11 12 13 14 15 16
12R
Val 1727
rn Ihr
Ila 180 up 130
9
2
oc
1 2 3
m
4(96
1-22 1M26
LKM
Illsan cycle P25
Ly. 4367 Gl" 3887 Ser 2831
Fl1
m
5; 357 239
ug
QY
Table 9. and PZ8.
2 3
FlV
ryr 2783
Gln 3245
19 20 21 22 23 24 25 26
-
PLl. P18. P19, -1,
poptic poptib. P15, P16,
Of
Ser 871
m54 R e 321
n l 1v6 Rr 111
Ala Gln Gln
lss
lug
74 71
m Rr
a 5 631
-
P34,
Cholinesterase Sequence
556
1 -=mala 2 3
Ly. 1537
4 5
11. 1616 Rr 5p85
6
ph 59%
i 9
le
11 12 13
78-%n?r Ala 1169 ryr 204 Lar
w 569
x2 716 1495
Us 295 Glu 264 N a 739
w 309
2536
5457 Gly 3495 Val 2524 ser 1313 Q Y 1147
932 1-1 107 625 919 761 14U
RO
14 15 16
G l Y 399 Up 159 n l 2 9
Val 119 Gly 114 Up
ryr
59
39 39
1.p Val Val Qy Up
m
1663 1079 189 le61 454
3Y
673 Rr 423 11. 234 Am
219 547
Qc lm
482
185 13
464 599 499 374
[ru
2 3 4 5 6
urn
w m
GlU
7
821
Pm 148 U a 147
461 142
17 18 19 21 21 22 23 24
GlY
1117 312 112 1317 854 6% 379 751
i
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
13W 1279 1885
1333
"
M 336
1
3145 2254
3
4 5 6 7 8 9
19 I1 12 13 15 14
-RE
333T
2 M
741
M
Ala Rr
489
Ly. 2M 1554
272 331 236 2 6 167 137
Rr
ryr
Tyr Rr
Glu
xTxim tar 3374
Val 6569 Rr 7s82 QY 7325 w 3885 Ro 2287 M 2294 Gl" 3 x 7 Aq 817 Aq 1866 Aw 1412
1994
R O 1152 Up 525 R O SB8
Glu 1255
sl-bo
lyi 814 Ihr 545 Ly. 132
A h 395
16 17 19 18
Q" 353
lrrlw
hpW 1467 M le49 ph 1113 Ala 1205 Ly. 359 ryr 113s QY 1999
Tabla 17.
b i m acld -1tIm
-
;;i; 2.212)
106131 148-180
181-1H
191-219
22C2ll
1.111)
1.012)
1.911)
3.9141
1.912)
3.113)
4.914)
il
