THE JOURNAL OF BloLoClCAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.

Vol. 261. No. 4. Issue of February 5, pp. 1665-1676,1986 Printed in U. S. A.

The Complete Amino Acid Sequence of the A-chain of Human Plasma a2HS-glycoprotein*

(Received for publication, August 26, 1985)

Yasuyuki YoshiokaS, Fumitake GejyotB, Thomas Martit, Egon E. Rickliy, Willy Burgill, Gwynneth D. OffnerS, Robert F. Troxlert, and Karl Schmidt** From the $Department of Biochemistry, Boston Uniuersity School of Medicine, Boston University Medical Center, Boston, Massachusetts 021 18, the Tlnstitute of Biochemistty, University of Berne, 3012 Berne, Switzerland, and ((Kantonsspital, Aarau, Switzerland

Normal human plasma azHS-glycoprotein has earlier been shown to be comprised of two polypeptide chains. Recently, the amino acid and carbohydrate sequences of the short chain were elucidated (Gejyo, F., Chang, J.-L., Burgi, W., Schmid, K., Offner, G. D., Troxler, R. F., van Halbeck, H., Dorland, L., Gerwig, G. J., and Vliegenthart, J. F. G. (1983) J. Biol. Chem. 258,4966- 4971). In the present study, the amino acid sequence of the long chain of this protein, designated A-chain, was determined and found to consist of 282 amino acid residues. Twenty-four amino acid doublets were found; the most abundant of these are Pro-Pro and Ala-Ala which each occur five times. Of particular interest is the presence of three Gly-X-Pro and one Gly-Pro-X sequences that are characteristic of the repeating se- quences of collagens. Chou-Fasman evaluation of the secondary structure suggested that the A-chain con- tains 29% a-helix, 24% &pleated sheet, and 26% re- verse turns and, thus, approximately 80% of the poly- peptide chain may display ordered structure. Four gly- cosylation sites were identified. The two N-glycosidic oligosaccharides were found in the center region (res- idues 138 and 158), whereas the two 0-glycosidic het- erosaccharides, both linked to threonine (residues 238 and 252), occur within the carboxyl-terminal region. The N-glycans are linked to Asn residues in &turns, while the 0-glycans are located in short random seg- ments. Comparison of the sequence of the amino- and carboxyl-terminal 30 residues with protein sequences in a data bank demonstrated that the A-chain is not significantly related to any known proteins. However, the proline-rich carboxyl-terminal region of the A- chain displays some sequence similarity to collagens and the collagen-like domains of complement subcom- ponent Clq.

* This study was supported by a grant from Hoffmann-LaRoche Inc., Nutley, NJ, Grants GM-10374 and GM-22822 from the National Institutes of Health, Grant HL-13262 from the United States Public Health Service, Grant PCM 83-14386 from the National Science Foundation, and Grant 3.451-0.83 from the Swiss National Science Foundation, Berne, Switzerland. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: Second Depart. of Internal Medicine, Niigata University School of Medicine, Niigata, Japan.

** To whom reprint requests should be addressed: Depart. of Bio- chemistry, Boston University School of Medicine, 80 East Concord St., Boston, MA 02118.

a2HS-glycoprotein (a2HS)’ (discovered by J. F. Heremans and K. Schmid) (1-3), one of the few negative acute-phase reactants (4-8) of human plasma, has been reported to be associated with several important biological functions. This protein promotes endocytosis (9) and possesses opsonic prop- erties (10). Furthermore, because of its high affinity for bar- ium (1) and calcium ions (11, 12), a,HS probably influences the mineral phase of bone, where it was found to be concen- trated up to 300-fold with respect to other plasma proteins (11,12). As a negative acute-phase reactant, it is of particular interest to note that in certain cancer patients the blood level of this protein appears to be an excellent indicator as to whether the immune system of these patients can be stimu- lated by thymosin administration (13).

a2HS has earlier been shown to consist of two polypeptide chains (1, 2). The complete amino acid and monosaccharide sequences of the shorter chain, designated B-chain, have recently been reported (14). The present paper describes the amino acid sequence of the longer chain, the A-chain, of (Y~HS.

EXPERIMENTAL PROCEDURES AND RESULTS~

DISCUSSION

The amino acid sequence of the A-chain of azHS was determined by automated Edman degradation of the polypep- tide chain, fragments derived from acid cleavage of the three Asp-Pro bonds, and tryptic, chymotryptic, Staphylococcus aureus V8 protease and thermolysin peptides, as well as by carboxypeptidase A digestion of the A-chain (Fig. 1). Some “tryptic” peptides resulted from a chymotryptic activity of the trypsin preparation employed (see “Results,” Miniprint). Acid cleavage fragment AF3 (residue 76-256) was particularly use- ful for elucidating the sequence of the middle region of the protein. However, since the only tryptophan is located at

’ The abbreviations used are: cuzHS, azHS-glycoprotein, a human plasma a2-glycoprotein; PE, S-pyridylethyl; TFA, trifluoroacetic acid; PTH, phenylthiohydantoin; HPLC, high performance liquid chro- matography; TPCK, tosylphenylalanyl chloromethyl ketone; DPCC, diphenylcarhamyl chloride; DABTH, dimethylaminoazohenzene- thiohydantoin; PITC, phenylisothiocyanate; DABITC, 4-N,N-di- methylaminoazobenzene-4’-isothiocyanate.

Portions of this paper (including “Experimental Procedures,” “Results,” Tables I-XVII, and Figs. 3-14) are presented in miniprint at the end of this paper. 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. 85M-2876, cite the authors,’and include a check or money order for $14.40 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

1665

1666 Complete Amino Acid Sequence of A-chain of a2HS

Clu-Ile-Glu-Ile-As~-Thr-Leu-Glu-Thr-Thr-Cys-His-V~l-Leu-Asp-Pro-Thr-Pro-Val-Ala-Arg-Cys-Ser-~~l-Ar~-Gln-Leu-Ly~-Glu-His~ T4 ------"-a- +TS 4 C- T6 4 I--

YO

1 so Al~-Clu-Arp-V~l-Arp-Lys-V~l-Cys-Gln-Asp-~ys-Pro-Leu-Leu-Al~-Pro-Leu-~-~p-Thr-Arg-V~l-V~l-HII-A1~-A~~-Lyr-Al~-Al~-L~~-

~~l-~lu-PhO-Thr-V~l-Ser-Cly-Thr-~p-Cys-V~l-Al~-Lys-Glu-Al~-Thr-Glu-Al~-Al~-Lys-Cys-Asn-Leu-Leu-Al~-Glu-Lys-Gln-Tyr-Gly- 210

T14 :: TIS : ! T 1 6 , d

I b- SS ,-I- S6 - I , MSCH2

AFSCH2. ____I

P h ~ - C y s - L ~ s - A l ~ - T h r - ~ ~ ~ - S e r - G l u - L y s - L e u - G l y - G l y - A l ~ - G l u - V ~ l - A l ~ - V ~ l - ~ ~ r - C y s - T h r - V ~ l - P h e - G l n - T h r - G l n - P ~ - ~ ~ ~ - ~ - S e r - G l * - L T18 - TI9

2 4 0

I CT 4

c o 2 70 P r o - G l n - P r o - C l u - G ~ y - A l a - A s ~ - ~ ~ ~ - A l ~ - ~ ~ l - p r o - T ~ r - ~ ~ o - ~ ~ l - V ~ l - ~ s p - P r o - A s ~ - , ~ l a - P r o - ~ r O - ~ e r - ~ r o - ~ r O - ~ ~ U - ~ ~ Y - ~ ~ ~ ~ ~ ~ ~ - ~ ~ Y ~ ~ ~ ~ ~ - T19- - CT4 - TIPS1 4 + TlPSt

I T1 9SZTHZ ,-. 1- T19S2TH5 - )"- T19S2THZAZ -1

Pro-Pro-Al.-Gly-Ser-Pro-Pro-Asp-Ser-His-V.l-Leu "F

TlPS2TH5 _1_____4

FIG. 1. The amino acid sequence of the A-chain of human plasma aaHS. Designations are: -, amino- terminal sequence of A-chain; T, tryptic peptides; CT, tryptic peptides of citraconylated A-chain; CH, chymotryptic peptides; TH, thermolysin peptides; AF, acid cleavage fragments; A, limited acid hydrolysis peptides; and .-, carboxypeptidase digestion of A-chain. Solid lines indicate those regions of peptides that were sequenced.

Complete Amino Acid Sequence of A - c h i n of azHS 1667

2

I

10

FIG. 2. Evaluation of the amino acid sequence of the A-chain of agHS for secondary structures by the procedure of Chou-Fasman. Designations are: a, a-helix; M, @-pleated sheet; 2, &turn; and-, random coil. The distance between two dots represents 1 amino acid residue.

residue 51 and since the A-chain also lacks methionine, no additional selective chemical cleavages were attempted.

The A-chain was sequenced to residue 57 with six blanks (residues 14, 45, 46, 51, 52, and 53) (Fig. 1). The sequence to residue 36 was confirmed by the sequences of tryptic peptides T1 (residue 1-10), T2a (residue 11-29), and T2b (residue 30- 36). The sequence of CT2 (residue 40-81 which was sequenced to residue 75) provided identification of residues 45, 46, 51, 52, and 53 not seen during sequential degradation of the A- chain and provided an 18-residue overlap with the amino- terminal sequence of the A-chain. The sequence of T4a (res- idue 73-81) provided a 3-residue overlap with CT2 and estab- lished the sequence to residue 81. The sequence of the first 40 amino acids of AF3 (residue 76-256) elucidated the se- quence to residue 115 and afforded a 6-residue overlap with T4a. The chymotryptic peptide AF3CH1 (residue 112-133), derived from AF3, yielded a 4-residue overlap with the partial sequence of AF3 and established the sequence of the A-chain

to residue 130. The sequence from residue 126-207 was con- tained in the sequences of T11 (residue 126-141), T12 (residue 142-147), T13 (residue 148-169), T14 (residue 170-193), "15 (residue 194-200), and T16 (residue 201-207). These tryptic peptides were aligned by the sequences of S. aureus V8 pro- tease peptides and a chymotryptic peptide derived from AF3. S3 (residue 140-166) aligned peptides T11, T12, and T13; and S4 (residue 167-182) afforded a 3-residue overlap between "13 and T14. The sequence of AF3CH2 (residue 184-198) provided a 10-residue overlap between T14 and T15. The sequence of S5 (residue 198-206) aligned T15 and T16, and the sequence of S6 (residue 207-218) aligned "16 and T18. "18 (residue 214-219) was aligned with CT4 (residue 220- 250) by the sequence of AF3CH3 (residue 217-223) which established the sequence to residue 250.

The sequence of the proline-rich carboxyl-terminal region was obtained from the sequences of T19, S. aureus V8 protease subfragments of T19, and the thermolysin peptides derived

1668 Complete Amino Acid Sequence of A-chain of a2HS

from the latter subfragments. The sequence of T19S2 (residue 245-282; sequenced to residue 270) provided a 6-residue over- lap with T19S2TH3 (residue 265-280), thereby establishing the sequence of the protein to residue 280. Peptide T19S2TH4 (residue 281-282; not sequenced) derived from T19S2 con- tained 1 residue of valine and 1 residue of leucine. The sequence of this dipeptide was deduced from the carboxyl- terminal sequence of the A-chain, His-Val-Leu-COOH, deter- mined with carboxypeptidase A. This sequence provided a 1- residue overlap with that of T19S2TH3. The sequences of additional peptides, some of which are given in Fig. 1, while others are given under “Results” (Miniprint), were deter- mined, and these further confirmed portions of the sequence of the A-chain. The established sequence of the A-chain revealed a polypeptide chain consisting of 282 amino acid residues and an exact M, of 30,169. Together with the amino acid sequence of the B-chain described earlier (14), the com- plete amino acid sequence of human plasma azHS has now been established.

Several notable features of the sequence of the A-chain deserve further comment. Twenty-four amino acid doublets were observed. The sequences Asn-Asn, Asp-Asp, Gln-Gln, Gly-Gly, Ser-Ser, and Thr-Thr occur once, Glu-Glu twice, Leu-Leu and Val-Val three times, and Ala-Ala and Pro-Pro five times. In no case does any amino acid occur consecutively more than two times. Four of the five Pro-Pro sequences occur within the carboxyl-terminal 22 residues. Significantly, the A-chain possesses one tripeptide Gly-Pro-X (residue 4-6) and three tripeptides Gly-X-Pro (residue 266-268, 269-271, and 274-276) that are characteristic of collagens. It is inter- esting that the three Gly-X-Pro sequences are clustered in the carboxyl-terminal 17 residues. It should be noted that azHS is thus the second plasma protein that has a collagen- like region. Clq, the first such protein (15, 16), possesses a “stem” domain consisting of collagen-like superhelices that bind specifically Clr and Cls. However, the biological signif- icance of the collagen-like region of the A-chain is not yet known. Because of the presence of these three collagen-like tripeptides, the A-chain appears to be related to collagen and Clq. This is borne out by the comparison of the sequences of the amino- and carboxyl-terminal 30 residues with protein sequences in a data bank which demonstrated that the A- chain is not significantly related to any other known protein. Moreover, in the A, B, and C chains of Clq, the sequence Gly-X-Y is sometimes replaced with the sequence Ala-X-Y (15). Similarly, in three places of the A-chain (residues 1,135, and 259) the glycine residue of a collagen-like tripeptide is replaced by an alanine residue which increases the sequence similarity between the A-chain of a,HS and Clq.

The A-chain was found to possess two N-glycosidic (resi- dues 138 and 158) and two 0-glycosidic (residues 238 and 252) heterosaccharides. The identification of the amino acids to which these glycans are linked was accomplished by utilizing the glycopeptides TllaCH2, T13CH2, T19S1, and T19S2TH2 (see “Results”). The strategy used was to determine the amino acid compositions and amino acid sequences of these glyco- peptides and of smaller Pronase glycopeptides derived from the parent peptides. A blank cycle in the sequence of the parent and Pronase glycopeptides could be correlated with an Asn or Thr residue from the difference between the amino acid composition and the phenylthiohydantoins detected in the sequences of glycopeptides (see Tables XVI and XVIk Miniprint).

Evaluation of the secondary structure of the A-chain by the Chou-Fasman method (17, 18) suggested the presence of a high degree of ordered structure consisting of 9 a-helical

segments from 5 to 19 residues in length, 9 segments of p- conformation from 4 to 11 residues in length, and 18 reverse turns (Fig. 2). The a-helix, @-conformation, and reverse turns account for 29,24, and 26% of the polypeptide chain, respec- tively. It is of further interest that the only ordered structure predicted in the 32-residue proline-rich carboxyl-terminal region is 4 reverse turns accounting for 50% of the amino acid residues in this region. It is not known whether these 4 turns form additional structures such as /?-meander, Greek keys, or double Greek keys (16) nor is the extent known to which the three collagen-tripeptides affect the secondary and/or tertiary structure of the carboxyl-terminal region. The two N-glyco- sidic heterosaccharides, located in the center region of the polypeptide chain, are bound to Asn residues located in re- verse turns. This suggests that relatively free movement of these carbohydrate units is possible, assuming that these two p-turns are at the surface of the molecule. The latter assump- tion is consistent with the finding that most p-turns are found at the surface of proteins (19). The two 0-glycans located in the carboxyl-terminal region occur in short sections where ordered structure is not predicted. Thus, the orientation of all four glycans appears to be directed away from the protein. This is in agreement with the fact that sialyl residues are readily cleaved enzymatically from the native protein and that, therefore, all four regions which carry glycans are indeed at the surface of the molecule. On the basis of the above data, one might speculate further that despite the hydrophobic nature of the polypeptide backbone of the A-chain, azHS is very soluble in aqueous solvents, probably due to the presence of the four glycans and as a result of the specific three- dimensional folding of the protein.

Acknowledgments-We are grateful to Professor Frank W. Put- nam, Indiana University, Bloomington, IN, for the computer com- parison of the amino acid sequence of the A-chain with those of other proteins and for evaluating the A-chain for secondary structure according to the procedure of Chou-Fasman. We also wish to thank the Institute of Laboratories, Massachusetts Department of Health, Jamaica Plain, MA, for the generous gift of supernatant solution of Cohn fraction V from which azHS was isolated and to George Crombie for performing amino acid analyses.

REFERENCES

1. Schmid, K., and Burgi, W. (1961) Biochim. Biophys. Acta 47 , 440-453

2. Gejyo, F., and Schmid, K. (1981) Biochim. Biophys. Acta 671, 78-84

3. Schwick, H. G., and Haupt, H. (1984) in The Plasma Proteins

York (Putnam, F. W., ed) Vol. IV, pp. 167-220, Academic Press, New

4. Bradley, W. P., Blasco, A. P., Weiss, J. F., Alexander, J. C., Sivermann, N. A., and Chretien, P. B. (1977) Cancer (Phila.) 40,2264-2272

5. Lebreton, J. P., Joisel, F., Raoult, J. P., Lannuzel, B., Rogez, J. P., and Humbert, G. (1979) J. Clin. Inuest. 64,1118-1129

6. Schelp, F. P., Thanangkul, O., Supawan, V., and Pongpaew, P. (1980) Br. J . Nutr. 43,381-383

7. Ashton, B. A., and Smith, R. (1980) Clin. Sci. (Lond.) 5 8 , 435- 438

8. Koj, A. (1974) in Structure and Function of Plasma Proteins (Allison, A. C., ed) Vol. 1, pp. 71-131, Plenum Press, New York

9. Lewis, J. G., and Andre, C. M. (1980) Immunology 39, 317-322 10. van Oss, C. J., Gillman, C. F., Bronson, P. M., and Border, J. R.

(1974) Immunol. Commun. 3,329-335 11. Triffitt, J. T., Gebauer, U., Ashton, B. A., Owen, M., and Renolds,

J. J. (1976) Nature 262,226-227 12. Ashton, B. A., Hohling, H. J., and Triffitt, J. T. (1976) Calcif.

Tissue Res. 22,27-33 13. Baskies, A. N., Chretien, P. B., Weiss, J. F., Makuch, R. W.,

Beveridge, R. A,, Catalona, W. J., and Spiegel, H. E. (1980) Cancer (Phila.) 45, 3050-3060

Complete Amino Acid Sequence of A-chain of a2HS 1669

14.

15.

16.

17. 18. 19. 20. 21.

Gejyo, F., Chang, J.-L., Biirgi, W., Schmid, K., Offner, G. D., Troxler, R. F., van Halbeek, H., Dorland, L., Gerwig, G. J., and Vliegenthart, J. F. G. (1983) J. Biol. Chern. 258, 4966-4971

Porter, R. R., and Reid, K. B. M. (1979) Ado. Protein Chern. 33,

Ghelis, C., and Yon, J. (1982) Protein Folding, pp. 46-101, Aca- 1-73

Chou, P. Y., and Fasman, G. D. (1974) Biochemistry 13,222-245 demic Press, New York

Fasman, G. D. (1980) Ann. N . Y. Acad. Sci. 348, 147-159 Kuntz, I. D. (1972) J. Am. Chem. SOC. 94,4009-4012 Laemmli, U. K. (1970) Nature 227,680-685 Atassi, M. Z., and Habeeb, A. F. S. A. (1972) Methods Enzymol.

25,546-553

cation of the lvsioe residues with CicIaconic anhvdride was Derforned bv the method of Preoaration and cryptic digestion of cirracanylated PE-A-chain. Chemical modlfi-

ALessi and Habeeb (21) . The PE-A-chain (28 mg) &as dissolved in 1 ml 2 6 H guaoidlne hydrochloride, and *even equal portions of Cifraconic anhydride (total 140 ill, Pierce Chemical Co.) were added LO che reacrion mixture IC 30 mi0 intervals. Throughout Che reaction, the pH was maintained between 8.0 and 8.5 by addition df 5 N NaOH. After the final aliquoc of the reagent had been added. the pU of the solucion we8 maintained at 8.0 for an additional 2 h. The citraconylated PE-A-Chain was then desalted on Sephader G-25 (1.2 x 40 cm,0.05MNH4HC03) aod lyophilized.

Trypain (TPCK-treated, Worthiogton Biochemical Corp.) digestion of 22 mg of citra- conylated PE-A-chain was performed in 2 ml Of 0.2 N N-ethylmarpholine acetate buffer, pH 8.5, at an enzyme to Bubstzate ratio of 11100 ( w h ) at 37OC for 24 h. The reaction was terminated by lyophilization and, in order to remove the cirraconyl gro~ps, the sample was dissolved in 8.8% formic acid and allove.3 t o stand Overnight at room tempera- ture. The resulting peptide mixture was fractionated by gel-filtration on Bia-Gel P-10.

~~

H NII HCO pH 7.8. was digested at 37°C with S. au1eu6 V8 protease (Miles Laboratories, Digestion with S. aweus v8 proreare. PE-*-Chain (SO mg) dissolved in 5 ml of 0.1

Inc.) a r 3 k enzyme to substrate ratio ut l/lOr. -and equal aliquot of enzyme was

by lyophilization. added after Sh, and the reaction was allowed t o proceed for an additional 17 h followed

701 formic acid at a eoncentzatioo of 5 m g / m l d incubated at 40°C for 48 h. The reaction mixture was then diluted IO-fold with deionized water and lyophilized. The obtained preparation w a s dissolved in 0.1 N sodim acetate buffer, pH 5.4, eonrairdng 6 N urea a d fractionated by gel-filtration rhrough Sephader GI00 equilibrated with the 8- buffer.

Acid cleavage with 70% formic acid. PE-A-Chain (60 mg) w a s disaolved in I2 m l of

NOLnclature of peptides. Each peptide vas designated by LL letrer code idicaring the digestion methods wed: tryptic peptides. T; tryptic peptides derived from citra- conyleted protein C T ; s. aureus V8 protease peptides. S; acid cleavage peptides. AI';

A. All peptide. were numbered in the order in which they occur in the sequence of the fhymotrypcie peptidea, CB; the-lysin peptides, m. and limited acid hydrolysia peptides,

p*oceln.

The digastiom of this peptide (1 w o i ) u i t h S . aureus V8 protealie was carried out at 25% Subfrawntation of oeotides. s. -ma V B p r o t o m e digest- of p e p t i d o m 9 -

for 5 h at .II e o r p co subatrate ratio 1/100 ( w h ) and the resulting peptides frastion- ated on Bio-Gel P-30.

ml of 0 1 n NU HCO 45OC io; 3 h a d d' 7'8' fractionated by gel-filtration on Bio-Gel P-30.

enzyme to substrate ratio of l/lOO ( w l w ) . The digest was then

gested with chymotrypsin (Type VII, RCK-treated. Sigma Chemical Co.) in 2 ml of 0.1 Chwtryptio d i g o s t i a of p e p t i d e YIZa rmd AF3 - Peptide Tllr ( 1 mol) vas dl-

I NaztlCOa, pH 7.8. at 37% for 2 h st .D enzyme fo BUbstrefe ratio of lIlO0 (w/w). Afte ly philization. the digest was diesolved in 0.05 N NU HCO pH 7.8. and fraction- ated by gal-filfracioo om Bic-Gel P-30 Peptide AF3 (35 &. d2;aolred in 10 m l of 0.1 I NU E O pH 7.8 was incubated at 37'C for 20 Lin at an enzyme to substrace retio of 1 / 1 d d'rhe reniltiog chmtrypcic peptides purified by KPLC (see below).

formed by the procedure Of hung and Praenkl-Conrat (22). Peptide T19SZlUZ (200 -1) YO. dissolved in 3 m l of 0.03 N HC1 and incubated in vacuo at 10S°C for 17 h. After lyophilization. the hydrolyzate was dissolved ~ e O ~ ; p ~ u ~ t ~ ~ K C O ~ . pH 7.8, and fraction-

disaolved in 0.02 H = KC0 pH 7.8. wL8 applied to a DFAPSephader A-25 COI- (1.0 x ated by gel-filtration through BilrCel P-30. Pr ction A2(Pig. 9.aee below)

27 u) previously equihbr&d with the 8- buffer (data not ah-). The col- was developed with a linear UsCl gradient from 0 toO.SMin 0.02 N NII HCO pK 7.8. The effluent (1.5 ml fracciooe) was mmltored PL 230 om. Pooled fretti& were desalted by gel-filtration Lhmugh a Bio-Gel P-4 colvmn (1.2 x 147 Cm. 0.05 ti NU KO3. on 7.8) to yield purified T19S2TKZAZ.

Th8mZ~tic digsation of p s g t i d s RSS2 - This peptide (5W -1). dissolved in 2 was diaested with themlyaio (Calbioch-Behring Co.) * e

Limited aoid hydmlya ie of p e p i d s PI9S2TU2. - Lhired acid hydrolyeis was per-

22.

23.

24. 25.

26.

27.

Tsung, C. M., and Fraenkel-Conrat, H. (1965) Biochemistry 4,

Allen, G. (1981) Sequencing of Proteins and Peptides, p, 148,

Edman, P., and Begg, G. (1967) Eur. J. Biochem. 1,80-91 Bhown, A. S., Mole, J. E., Weissinger, A., and Bennett, J. C.

(1978) J. Chromatogr. 148, 532-535 Smithies, 0.. Gibson, D., Fanning, E. M., Goodfleish, R. M.,

Gilman, J. G., and Ballantyne, D. L. (1971) Biochemistry 1 0 ,

Chang, J. Y., Brauer, D., and Wittman-Liebold, B. (1978) FEBS

793-801

Elsevier, New York

4912-4921

Lett. 93.205-214

1670 Complete Amino Acid Sequence of A-chain of a2HS

eequenee of cT1 was identical te that of TI. the sequence of CT3 could be deduced un- ambiguously. CT2 of which 36 residues were determined aligned peptide. 1 4 . 14. and Sl. cT4 was identified a~ the carboxyl-terminal CT-peptide becauee it lsckd Lye nnd Arg (Table IX). The sequence of this pcptiue deduced to residue 251. aligned peptides 11952 and T19SZTU2 in the aeauence of the A-chain.

Acid d e ~ v a l e osotidss. M the A-chain contai.3 three Asp-Pro bonds (residue 16- 17. 15-76. a d 256-257). acid cleavage f o m d the expected four peptides which were frsctionrted on Sephdex G100 (Fig. 13) . oaly peptide AF3 (for d u o acid craposition

was required t o Complete the sequence of the A-chain and waa directly subjected to aula- ~ e e Table XIXI) which was ccmpletely separated from the other acid cleavage fr-ts

mated Hdnrn degrdatiao affording 40 step8 (Table XIV). This established the sequence Of the +Chain fIOP residue 76 t o 115 and aligned tryptic peptide* T4s. T5, T6. 77% 78. T9a. T9b. TlO, and CT3 (Pia . 1).

glycopeptide, TilaCU2P1, with the composition (Asx-Thr A r g )and the sequence X-Aepkr- A r g . mese data coofirmed that residue 138 WBB in&eedlaenlto which - glycan i s b o d .

of this peptide included 4 Aor and 1 Ser. Ite complete sequence wpB determined with The second N-glycosylation site occurred i n T13CHZ. The amino acid composition

one blank (residue 158). Three residues of PTU-Am and one residve of Pl'H-Ser were identified in the seqvenee suggesting that Che blank was Aso with a giycso. This conclusion was confi-d by the amino acid composition (-x Ser Gly ) of a short pro- n&se glycopeptide, T13CUP1, derived f x m T13CHZ. Hence. ti?. ahgo&.cchpridt u i z must be linked to Asn (residue 158).

at residue 238. This residue was identified a8 l'hr with an C-glycosidic oligosaccha-

were identifies ae PTU-derIvarives. T19SlP1 contained one Thr and one Ser. Since ride because T19Sl lacks Ann and becauae one of the two l'hc and the single Ser

PTU-Ser we.8 identified in the sequencee of T19S1. the amino acid compo(LitiOn of T19SIP1 confirmed that residue 238 waa a glyeosylated Thr.

analysis of the themlyein pepcidq TI9SZn12. which was sequenced for 12 residues with one blank. Two M x , one S a and one Thr were contained in the d n o acid e-- sition, and two Asp end one Ser were detected as Pl'H-derivatives in the sequence of the rreotide. &nee. Thr (residue 252) is the amino acid to which the glycan is linked.

using the 8- approach. it was found that the first 0-glycosidic glycan occurred

The location of the second &glycosidic hetsrosreeharide unit was determined by

PPACIIOY H m Y l l l

Fig . 4. Ion exchange chtmtography of fraction A (Fig. 3) 00 P DM-Sephadex A-25

with a linear NaCl gradient (0-0.5 M). c o l n (1.0 x 27 aa) rquilibzlted with 0.02 HNki4HC03, pti 7.8. and developed

1 . 5 111. Peptide, I

Complete Amino Acid Sequence of A - c h i n of a2HS 1671

I T19SZTHZ Pept ides

FRACTION NUMBER

Fig. 9. Gel-filtration of the limited seid hydrelyeie peptides derived frm peptide T19SZTHZ on a 810-Gel P-30 col- (1.2 x 149 a) aquilibrrted in 0.05 X NU HCO pH 7.8. Ractions (1.0 ml) yere wnitored far absorbance at 220 m44aod3bled as indicated by the solid bars.

Fig. 11. Gel-filtration of the tryptic digest of citraconylaLed PE-A-chain on a 810- Gel P-10 col- (1.2 x 220 am) equilibrated In 0.1 H m n i m fo-te buffer,

Fig. 12. Gel-filtration of the S. BYI -~YS V8 pratelee digest of PE-A-chain 00 a Bio- Gel P-30 eel- (I 2 x 158 cm) equilibrated in 0.05 M ML HCO pH 7.8 Fractions (1.0 nl)'vere Wnitored for abaorbrnce at 280 t %O w and pooled a8 indicated by the solid bars.

Fig. 13. Gel-filtration of the acid cleavage peptides obtained after incubatloo of PE-A-chain io 70% formic acid OD B Sepheder C l O O c o l m (1.6 x 157 c 3 equilibrated it, 0.1 H sodium acetate buffer, pH 5 . 4 containing 6 H urea. Fractions(l.5 ml) were monitored at 235 M, pooled PO indicated by the solid bars . dialyzed against deionized water and lyophillred.

1672 Complete Amino Acid Sequence of A-chain of a2HS

TABLE I Amino acid composition of A-chain of a2HS

glycoprotein

Amino Acid Amino acid analysisa Sequence

~ ~~

ASP Thr

2 9 . 5 2 9 1 6 . 8 1 7

Ser 1 4 . 3 15 Glu 3 7 . 5 3 7 Pro 2 8 . 5 3 0

G l y 1 5 . 1 1 5 Ala 29 .8 30 Val 2 4 . 0 2 5

1 0 . 5 11 Met 0.0 0 Ile 7 . 3 7 Leu 2 4 . 6 2s

hcysb

Tyr 6 .4 6

LY 5 1 2 . 0 1 4 His 6.0 6

Arg 5 . 8 6 TrpC 0 . 5 1

Phe 8 . 4 8

CHO

Total 2 7 7 . 0 2 8 2

a. This amino acid composition was calculated on the basis of 282 residues per mol o f A-chain.

b. hCys was determined as cysteic acid. c. Trp was determined after hydrolysis with 3 N

mercaptoethanesulfonic acid.

" H H H H " H,C H,G

H H H H

H H H H H H H H H H.G

H.G

H , G

H.G H

8 9 . 2 31.6

3.0

S I . ' 53.3

J6.2 3 3 . 3

32.7 37.0

23.6 33.5 5.0

1 8 . 6

1 5 . 5 1 4 . 4 31.1 26.5 13.7

30.0 27.8

19.6

10.7 3 5 . 7

29.2

11.7 28.6

10.6 9 . 4

9 . 0

H n H H , G

H H H H H H H . G

H

" H H H

" H H H

Complete Amino Acid Sequence of A-chain of azHS 1673

Amino acid sequencer of tryptic peprides from &-chain Table I V

Pgtide TI [Residues 1-101

Sequence: Ala- Pro- His! Gly- Pro- G l y - Leu- I l e - Tyr- Arg

a. The amino acid residue a t this step was determined by [nnol) 2b.8 13.6 . 7.1 9.9 7.9 11.1 12.7 11.7 3.2

1 2 3 4 1 6 1 8 9 1 0

sequencing of intact A-chain (Table l a .

Peptide TZa(Res2dues l l - 2 Y L

Sequence: Gln- Pro- Asn- Cy,+ Asp- Asp- Pro- G1"- Thr- G1u- Glu- Ala. Ala. ~eu. ya1. 1 2 3 4 5 6 7 8 0 1 0 1 1 1 2 1 ~ 1 4 1 s

[nmol) 16.0 6.8 7.7 - 5.6 7.4 2 . 0 J.l 0.5 4.2 3.2 2.3 3.0 1.0 3 . 3

16 I7 I 8 19 Sequence: Ala- Ile- Asp- Tyr

I. The amino acid residue s t this 5rcp was deduced from the amino acid conporit>on of ("mol) 1 . 5 0.8 0 . 4 0.7

this peptlde (Table 111).

Peptide T2b (Rendluer 30-361

ScqUEnCC: I l c - Asn- ~ l n - Asn- p r o - L~~ 1 2 3 4 5 6 7

[n"Ol) 1 9 . 1 41.0 45.1 1 6 . 0 9.1 1.b 0.8

pulltide T4 (Residues 5 0 - 8 1 )

1 2 3 1 5 6 7 8 g Sequence: ~ a l - T ~ P - pro- cln- GI*- p r o - ser- ~ 1 ~ - GIU- \:". Phe. GIU. lie. Glu. lie. (nmoll 6 7 . 7 15.3 29.1 53.7 72.1 17.5 18.4 5 s . o 32.7 29.7 42.8 4b,4 3j.o 28.4 3j,1

I1 12 13 14 15

16 17 1 8 19 10 Sequence: ASP. T h r - Len- Glu- Thi" [ A s p l T h r ~ P r c 2 V a l l A l e l ~ ~ ~ l ~ i ~ , c y ~ l ~ ~ ~ ~

(Una11 18.8 1.1 1 6 . 8 14.J 5.6

PeptideT4a (Residues 73-81)

1 2 3 4 ~ 6 7 8 ~ Sequence: Val- Leu- ASP- Pro- Thr- PTO- Val- Ala- ATg (nmol) 106 104 71.3 30.8 8.5 23.7 24.1 1 3 . 4 3.J

Peptide 7 5 (Residues 8 2 . 8 5 2

1 2 J Q Sequence: Cys- Ser- Val- Arg (nnal) + 12.8 64.6 1 5 . b

PepfIde T6 (Residues 8s-88) '

1 2 3 sequence: Gin- Leu- LY5 a. This peptide vas sequenced by the OABITC/PITC method.

Peptide T7 (Residues 8 9 - 1 0 2 1

Sequence: m u - "is- Ala- Val- Glu- Gly- Asp- C y 5 - Asp- Phe- Gln- Leu- Leu- Lys ( m o l ) 6 6 . 1 13.1 3 6 . 8 1 2 . 5 39.0 14.9 10.6 * 5 . 5 9 . 9 2.4 1.8 1.6 1.2

1 2 J 4 s 6 7 8 ' 3 1 0 1 1 1 2 1 3 1 4

%de T8 [Residues 103-106L"

Sequence: Leu- Asp- Gly- Lys

a . Thls peptide vas Sequenced by the VABITC/PITC method.

1 2 3 4

("mol)

Peptide T9a (Residues 107-Illla

1 1 3 4 5 Sequence: Phe- S e r - Val- Val- Tyr a . Thls pepf~de xes sequenced by DABITCIPITC method.

Peptide T9b (Residues 112-113f

1 2 Sequence: Ala- Lya

B . Thls peptrde was sequenced p the DABITCiPITC method.

Peptide TI0 (Reszdues 114-125)

Sequence: Cysa &g- %;- S e r - Pro- Asp- Serb Ala- Glu- Asp- Val- Arg

a. The 8 m n o acid resrdue at this STOP was determined by sequencing of 1.1 1.1 2.9 . 1.4 1.6 2.0 1.4 0.7

b . The amino aczd residue a t this s t e p ~ i l s deduced from the amino acid

1 2 3 4 5 6 7 a 9 1 0 1 1 12

[nmol) - peptide AF3CHl [Table X V ) .

composition of fhlr peptide [Table 111).

%de Tll(Resxdue lib-1411

1 2 J 4 8 6 7 8 9 IO 11 12 CHO I4 15 Sequence: Lyr- V a l - Cyr- Cln- Asp- Cyr- Pro- Leu- Leu- A l e - Pro- Leu- As": Asp- Thrb (nmai) 30.8 55.1 4 17.0 11.6 I 6.8 17.1 23.1 11.4 J.0 14.9 - 6.5 -

13

Sequence: A:g

a . The amino acid residue a t t h l s step was deduced from the amino acid

b. The arnnlno acid residue a t thlr step w a s determined by sequencing of peprlde TllaCH2

16

inlr'ol)

of rhl, peptide (Table 111).

(Table VI).

PeDtldc T12 (Residues 142-147l

Sequence: V a l - V a l - His- Ala- Ala- L y s (nmall 31.4 35.1 9.4 15.2 13.5 7.1

1 2 3 4 5

Peptide T1J (Residues 148.16%

1 2 3 4 5 6 7 8 9 IO CIIO 1 2 I3 14 1 s Sequence: Ala- Ala- Leu- Ala- Ala- Phe- Asn- Ala- Gln- Asn- Arm? Gly- Ser- Asn- Phe- Inmol) 28.2 28.4 20.4 2 4 . 8 19.9 17.7 7 . 1 14.8 7.2 3.1 - 3.8 1.6 5.0 4.6

._

Sequence: Gln- ~ e u - ~ l u - Glub I l c - Ser- Arg 16 17 1 8 19 20 21 22

(ninall 2.6 4.6 J.2 . J.7 0.8 +

P c p t l d e T11 (Residues 170.193)

Sequence: la. Gln- Leu- Val- Pro- Leu- Pro. pro- s er - Thr- Tyr- Val- Glu- Phe- Thr. (nmol) 1.2 7.U 13.6 10.6 5.4 6 . 2 4.9 1.0 J . 1 1 . 9 3 . 4 9.2 4 . 4 3.5 1.4

1 2 3 4 5 b 7 8 Y 10 11 '12 13 14 IS

Sequence: V a l - Ser- Gly- Thr- Asp- Cyra V a l - Ala- Lys (nmoll 6.8 1.0 2 . 2 I 1.4 . 2.2 1.b 2.4

1 6 11 18 19 20 21 22 2J 24

Peptide T I 5 (Rerlduer 194-2001

1 2 J 4 5 6 7 Sequence: Glu- Ala- Thr- 61"- Ala- Ala- Lys (nmoll 138 3b1 51.1 275 193 9 5 6 121

Peptide Tl8 (Residues 214-119L

1 2 3 4 5 b Sequence: Ala- Thr- Leu- S e r - c l u ~ Lys (nmoll 135 7 8 . 6 93.7 5 . 5 90.1 83 7

Peptide T19 [Residues 233-2821 6

1 2 3 4 5 C H O 7 8 9 1 0 1 1 1 1

(nmol) 21.810.111.81.7 11.4 - 2.8 J.9 2.8 6.7 J.6 + Sequence: Gln- Thr- G l n - Pro- V81- Thr- Set- Gln- Pro- Gln- Pro- Glu-[Arp4ThrlSerl

Amino acld compositions of chymotryptic peptldes Table V

from peptide Tlla a

Amino TllaCHl T l l a C H 2 Total Tlla Acid (127-1331 (134.141) (127-141)

Total b . 1 ( 7 ) 8 . 9 ( 8 1 1 5 1 5

a . Amino acid compoSitions were daterszned as described in footnote a of Table 111.

Amino acid sequences of chymotryptic peptides from peptide Tlla

Peptide TllaCHl [Residues 127-133)

Table VI

Sequence: Leu- Ala- Pro- Leu- Asna Asp- Thr- Arg

a. The amino acid residue at this step was deduced from the amino [nmoll 21.9 4 2 . 4 12.7 24.6 - 8 . 3 2 . 3 0.9

a c i d composition of this peptide (Tfablc V I .

1 2 3 4 C t i O b 7 8

8 . Amino acid compositions were determined as described in footnote a of T a b l e I l l .

Amino acid sequences o f the peptides obtained by subfragmentation of Table V I 1 1

oeotide T I 9

Peptide T19S1 (Residues 2 3 3 - 2 4 4 )

Sequence: Gln- Thr- Gln- Pro- Val- Thrl Ser- Gln- Pro- Gln- Prob- GI$ (llmol) 15.9 3.2 2 0 . 5 7 . 2 1 6 . 7 - 3 . 9 1 4 . 4 6 . 0 7 .4 - -

1 2'. 3 4 5 cio 7 8 9 1 0 11 12

The amino acid residue s t this step was deduced from the amino acid composition of this peptide (Table V I I I .

of peptide T I 9 (Table I v ) . b. The amino acid residues a t these steps were determined by sequencing

Peptide TL9S2 (Residues 2 4 5 - 2 8 2 )

Sequence: Gly- Ala- Asn- Glu- A l a - V a l - Pro? Thrb Pro- Val- Val- Asp- Pro- Asp- Ala- [mol ) 2 2 . 0 50 .4 1 7 . 3 3 2 . 9 3 0 . 3 5 2 . 4 - - 15.5 21.1 1 2 . 6 6 . 4 1.6 5.3 4 . 1

1 2 3 4 5 6 7 CHO 9 10 11 1 2 13 1 4 IS

Sequence: Pro' Pro- Sera Pro* Pro- Leu! Gly- Ala- Pro- Gly? Leu* Pro-(AsplSer2Pro3 1 6 1 7 18 1 9 2 0 21 22 2 3 2 4 25 26 27

(*mol) - 3 . 8 - - 4 . 3 - 1 . 5 2 . 3 3 . 5 - - GlylAla lVal lLeulHis , )

T19SZTH2 T19S2TH3 and T19S2THZA2

of peptide T l O S l T H 2 A l (Table V I I ) .

a . The amino acid residues at these steps were determined by sequencing of peptides

b. The amin: acid residue a t this step was deduced .from the amino acid composition

Peptide T19SZTHZ (Residues 249-264)

a. The amino acid residue at this Step was deduced from the amino acid composition . . of peptide T19S2TH2AI (Table VII).

Peptide T19sTSZJ_!Residues 2 6 5 - 2 8 0 1

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 I S Sequence: Leu- Gly- A l a - Pro- G l y - L e u - Pro- Pro- A l l - G l y - Ser- Pro- Pro- Asp- Ser-

l n m o l ) 2 6 . 5 4 9 . 6 3 3 . 0 18.9 2 9 . 4 1 6 . 1 21.1 2 1 . 1 1 5 . 1 1 5 . 5 4 . 6 1 5 . 8 1 4 . 0 6 . 7 1 . 5

Sequence. His 1 6

["mol) +

Peptide T19SZTHZA2 (Residues 253-2611

Sequence: A l a - Pro- Pro- Ser- Pro- Pro (nololl 1 4 . 1 8.0 8 . 2 1 . 2 2 . 0 2 . 2

1 2 3 4 5 6

Amino acid comporitioosof tryptic peprider TableIX

from citraconylated A-chsina

Acid ( 4 0 - 8 1 ) + C ~ 3 ( 1 0 7 - 1 1 3 ) ( 2 2 0 . 2 8 2 ) Amino CT2 CTl(1-10) CT4

Amino acid sequences of tryptic peprides from citraconylated A-chain Table X

Peptide CT2 (Residues 4 0 - 8 1 )

Sequence: His- Thr- Leu- Asn- Gln- I l e - Alp- Glu- Val- Lys- Val- Trp- PTO- Gtn- C l n - (nrnol) 3.1 0.2 1 9 . 4 5 . 3 8 . 2 1 2 . 7 8 . 5 8 . 4 1 7 . 7 1 0 . 9 18.6 4.4 4 . 1 8 . 2 6.9

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 I S

1 6 1 7 I 8 1 9 20 21 22 2 3 24 25 26 27 28 29 30 Sequence: Pro- Se$ Gly- Glu- Leu- Phe- Glu- I l e - Glu- I Ic - Asp- Thr- Leu- Glu- Thr-

(nrnol) 3 . 4 - 3 . 5 5.5 6 . 5 5 .4 6 . 1 5.1 5 . 3 4 . 0 2 . 8 1 . 4 2.1 3 . 6 1 . 7

Sequence: 1hr.b Cys- HIS- V a l - Leu- Asp- ( T h r 1 A l a l P r o 2 V a l l A r ~

a. The amino acid residue a t this Step was determined by sequencing of peptide T4

b. The amino acid residues at these steps were determined by sequencing of peptide SI

31 32 3 1 34 35 36

("moa] - - - 3.7 1 . 6 1 . 2

(Table I V I ,

(Table XI1 1,

Peptides CT1 (Residues 1-10) and CT3 (Residues 1 0 7 - 1 1 3 )

Sequence 1 2 3 4 5 6 7 8 9 10

(nmol) 2 8 . 1 1 7 . 5 - 1 0 . 7 1 6 . 7 1 4 . 0 ( 1 9 . 7 ) 1 3 . 1 1 1 . 6 * CT1: A l a - Pro- His! Gly- Pro- Gly- Leu- Ile- Tyr- Arg

CT3: Phe- Serb Val- Val- Tyr- A h - Lys ["mol) 3 7 . 5 - 2 9 . 4 2 5 . 3 1 7 . 9 1 4 . 8 ( 1 9 . 7 )

a . The amino acid residue at this s t e p was determined by sequencing of intact A-chain

b. The a m n o acid residue at this step " 8 5 determined by sequencing of peptide 1 9 8 (Table.11).

(Table I v ) .

Peptide CT4 (Residues 2 2 0 - 2 8 2 1

sequence: Leu- ~ l y - ~ l y - A l a - Glu- Val- A l a - Val- Thr- Cysa Thr- V a l - Phe- Gln- Thr- (mol) 1 0 . 9 5 . 3 6 . 3 1 2 . 8 1 1 . 1 1 4 . 1 1 2 . 0 1 S . 7 .. 2.6 - 2.9 1 0 . 5 6 . 3 5 . 2 2 . 5

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5

3.9(41

a . Amino acid compositions were determined as described in footnote a o f T P b l e I 1 b. PE-Cyr meluted with Arg on these analyses. c. Trp was not determined quantitatively.

a. Amino acid compositions were determined as

b. PE-Cy5 coeluted with Arg on this analysis. C. Trp was not determined qumtitatively.

described in footnote a of Table 111.

Complete Amino Acid Sequence of A-chain of azHS 1675

Table X I 1

Amino a c l d sequences of S . aureus V8 protease p e p t l d e s [:-om A - c h a i n

Peptide S 1 (Rerldues 69-83)

Sequence: Thr- Thr- Cysa His- Val- Leu- Asp- Pro- T h r - P r o - Val: la- (SerlArglCysl]

"

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2

(nrnall 5.3 4.2 f 4.0 5.3 4 . 7 3.3 2 . 2 0.4 1.1 3.0 1 . 4

P e p t i d e S 2 (Rerldues 123-1391

1 2 3 4 5 6 7 8 9 1 0 1 1 l Z 1 3 1 4 1 ~ Sequence: A s p - Val- Arga Lys- V a l - Cyra Gln- Asp- Cysa P r o - Leu- Leu- Ala - P r o - L e u - (nmal) 2 0 . 9 1'1.9 - 4.3 9.3 - 2 . 4 3.0 - 2 . 4 2 . 2 4.0 2 . 8 1.8 4.4

CHO 1 7 16

sequence: asnd AS^ (nmo11 - 1.4

a . The a m m o s c l d residues a t t h e s e StCpS were determined by seqrmnclng of p e p r l d c s 110, TI1 (Table 11') and T l l a C H Z ( T a b l e VI).

P e p t i d e S 3 (Reslducs 140-166)

1 2 3 4 5 6 1 8 9 1 O l l 1 2 1 3 1 4 1 5 Sequence: T h r - Arg- V a l - Val- H i s - Ala- Ala- L y s - Ala- Ala- Leu- ala^ A l a - Phe- Asn- (nmoll 5.1 10.8 23.5 35.5 6.2 15.6 16.5 9.3 14.0 ll.b 5.1 7 . 7 6.0 4.8 2 . 7

19

S e q u e n c e : Ala- G l n - Asn- Asn5 Ciy- S e r - AS^^ Phe- ~ l n - L e u - Glu- c lu (nmol l 4.5 2 . 3 1.2 - 0.4 0.2 0.3 0.7 0.4 0.5 0.5 0 . 6

a . 'The amino ackd residue at t h l s s t e p was deduced from the amino acid compos~rlon

16 1 7 18 CHO 20 21 2 2 23 24 25 26 27

o f p e p t i d e T13 (Table 111).

P e p t i d e 54 (Residues 1b7-182)

P e p t i d e S S (RrsLdues 1 8 8 - 2 0 6 1

1 2 3 4 5 6 7 8 9 Sequence: Ala- A l a - L y s - Cys- A s " - Leu- Leu- Ala- Glu ("moll 9.0 7 . 9 4.3 * 1.3 2 . 8 1.3 0.8 0.4

P c p t i d e S6 (Resldues 207-218)

Anino AF3 AFlCIi1 AF3CH2 AF3Cti2a (202-209) A ~ t d (76-7561 (112-1331 (184-2091 AF3CH3 (217-232)

1 . 9 ( 2 )

1.1(11 3.7i4)

1.2i11 4.1(5) 1 . 6 1 2 1

2.3(3)

N-terminal amino ac id sequence a f the large fragment o b t a m e d by a c i d cleavage of

aF-3 ( r e s i d u e s 76-116) t h e A-chain

Table XlV

Sequence: P r o - Thr- Pro- Val- Ala- Arg- Cys- S e r - Val- Arg- Gln- L y s - GI"- H i s - Ala- ("mol) 61.1 16.1 3 6 . 8 27.1 32.2 15.8 32.1 10.1 25.8 12.1 20.1 22.4 16.3 10.3 1 4 . 2

1 2 3 4 5 6 7 8 9 1 0 1 1 I 2 1 3 1 4 1 5

16 1 7 1 8 19 20 21 22 23 24 25 2b 2 7 2 8 29 10 Sequence: Ala- V a l - Glu- Gly- Asp- C y s - Asp- Phe- Gln- Leu- Leu- Lys- Leu- Asp- Gly- (nrnol) 2 0 . 4 16.8 8 . 4 13.4 6 . 2 9.9 4.4 7 . 9 5.3 b.3 6.0 4 . 5 5.8 1 . 2 4.3

Sequence: Lys- Phe- ( X I a Val- V a l - Tyr- Ala- Lys- C y s - Asp 31 3 2 33 34 35 36 37 38 39 40

("mol) 1.8 1.9 - 1.7 + 1.1 * 1 . 3 f 1.1

a . X Indicates an amino acld r e s i d u e whlch Could not be ldentlfied.

Amino a c i d s e q u e n c e s o f c h y m o t r y p t i c p e p t i d e s f r o m p e p t i d e A F 3 T a b l e XV

P-

1 2 3 4 5 6 7 8 9 1 0 S c q u e n c e : A l a - L y s - Cys - Asp- S e r - S e r - P r o - Asp- Sera A l a -

(nmol ) 7 . 9 5 .7 + 2 . 9 0 . 8 0 . 5 1 . 7 1 . 4 - 1.2

S e q u c n c e : V a l - C y s - G l n - A s p - ( P r o l L e u l C y s l )

a . T h e a m i n o a c i d r e s i d u e a t t h i s s t e p w a s d e d u c e d f r o m t h e

1 6 1 7 1 8 1 9

fnmol) 2 . 4 + 1 . 4 0 . 5

o f p e p t i d e T 1 0 ( T a b l e 111) .

P e p t i d e AF3CH2 ( R e s i d u e s 1 8 4 - 2 0 9 )

S e q u e n c e : T h r - V a l - S e r - G l y - T h r - Asp- Cys? V a l - A l a - L y s - (nmol ) 1 . 3 8 . 1 0 . 5 2 .5 0 . 8 1 . 9 - 2 . 8 1 . 6 0 . 6

1 2 3 4 S 6 7 8 9 1 0

(AsplGlu2Ala2LeuZTyrlLys2Cysl)

a. T h e a m i n o a c i d r e s i d u e a t t h i s s t e p was d e d u c e d from t h e o f p e p t i d e T 1 4 ( T a b l e 111).

11 1 2 1 3 1 4 1 5 G l u - Asp- Val- Arg- Lys- 0 . 9 1 . 0 1 . 6 + 0 . 8

a m i n o a c i d c o m p o s i t i o n

11 1 2 1 3 1 4 1 5 Glu - A l a - T h r - G l u - A l a - 0 . 9 1 . 4 0 . 3 1 . 0 1 . 5

a m i n o a c i d c o m p o s i t i o n

P e p t i d e s AF3CH2a ( R e s i d u e s 2 0 2 - 2 0 9 ) a n d AF3CH3 ( R e s i d u e s 2 1 7 - 2 3 2 )

S e q u e n c e s : 1 2 3 4 5 6 7 8 AF3CH3 ,4511- Leu- Leu- A l a - G l u - L y s - G l n - Tyr (nmol) 3 . 2 5 . 0 ( 8 . 3 ) 1 . 4 1 . 3 2 .8 0 . 9 0 . 8

AF3CH4 S e r - Glu- Lys- L e u - G l y - G l y - A la - G lu - (Thr2Ala lVa13Phe lCys1) (nmol ) 1 . S 4 . 7 ( 8 . 3 ) 8 . 3 2 . 8 2 . 9 1 . 8 1 . 5

1676 Complete Amino Acid Sequence of A-chain of a2HS

TllaCHZb TllACHZPl T13CK2 T13CHZP1 T19SlC T19SlPl T19S2THZC T19S2TH2Pl (134-141) (138-141) (154-162) (158-160) (233-244) (238-244) (249-264) (250-264)

Asp 2 . 2 ( 2 ) Thr 1.2(1) Ser Glu Pro 1.1(1)

G l Y Ala 1.1 (1) Val Leu 2.2(2) Phe Arg 1.1(1) CHO +

2.4(2) 4.0(4) 0.9(1)

1 . 0 ( 1 ) 1.1(1)

1.0(1) 1.2(1)

1.0(1) 0.8(1)

+ +

1 . 3 1.7(2J

0.9 1.0(1J 4.8(5) 3.4(3)

1.0

l.O(lJ

+ +

2.0(2) 1.9 0.9 1.0(1) 1.3 0.8 1.1(1J 1.0 2 . 9 2.3 6.8(7) 6.2

1.7(2) 0 . 9 2.5(3) 3.1

+ + +

a. Values in parenthesis indicate amino acid residues identified in the sequence. Of the pronase peptides, onlyTllaCHZP1 was

b. Data from Table V. c. Data from Table VII.

sequenced.

Table XVII

Summary o f amino acid sequences o f the A-chain glycopeptidesa

TllaCH2 (data from Table VI)

Sequence: Leu- Ala- Pro- Leu- X -Asp- Thr- Arg (nmol) 21.9 24.4 12.7 24.6 8 . 3 2.3 0.9

TllaCHZPl

Sequence: X -Asp- Thr- (nmol) 13.2 1.7

T13CH2 __

Arg 4.2

Sequence: Asn- Ala- Gln- Asn- (nmol) 14.4 44.1 15.7 8.0

T19S1 (data from Table VIII) -

X -Gly- 2.8

Ser- Asn- 1.0 3.5

Phe 1.2

Sequence: Gln- Thr- Gln- Pro- Val- X -Ser- Gln- Pro- Gln-[Pro, Glu) (nmol) 15.9 3.2 2 0 . 5 7.2 16.7 3.9 14.4 6.0 7.4

T19S2TH2 (data from Table VIII)

Sequence: Ala- Val- Pro- x -Pro- Val- Val- Asp- Pro- Asp- Ala- Pro-(SerP Pro3) (nmol) 69.4 60.2 3 5 . 8 19.2 43.1 47.4 2 8 . 8 15.9 18.9 7.2 2.5

a) X indicates a cycle where a PTH-derivative was not detected. bJ The PTH-derivative of Ser was positively identified in peptide T19S2THZA2

(see Table VIII).