The Structure of Hemocyanin I1 from the Horseshoe Crab ...

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

Vol. 261, No. 23, Issue of August 15, pp. 10520-10525.1986 Printed in U.S.A.

The Structure of Hemocyanin I1 from the Horseshoe Crab, Limuluspolyphemus THE AMINO ACID SEQUENCEOFTHESECONDLARGEST

CYANOGEN BROMIDEFRAGMENT* (Received for publication, December 30,

1985)

Patricia Q. Behrens, Hitoshi Nakashimal, Eisuke YokotaS, and AustenF. RiggsQ From the Departmentof Zoology, University of Texas at Austin, Austin, Texas 78712

Fourteen fragments have been isolated from hemocyanin component I1 of Limuluspolyphemus by cleavage withCNBr. The amino acid sequence of the largest fragment, CNBr Ia has been reported (Yokota, E., and Riggs, A. F. (1984) J. Biol. Chem. 259, 4739-4749). The amino acid sequence of the 12 smaller fragments is reported in an accompanying paper (Moore, M. D., Behrens, P. Q., and Riggs, A. F. (1985) J. Biol. Chem. 261, 10511-10519). We have determined the amino acid sequence of the second largest fragment, CNBr Ib. The fragment contains 142 residues and has a molecular weight of 16,095.

Reaction of component I1 of Limulus hemocyaninwith CNBr yields two large fragments, Ia and Ib, togetherwith 12 smaller fragments. The amino acid sequences of Ia and the smaller fragments have been described (2, 3), and a partial sequence of the entire molecule has been compared with the amino acid sequences of other arthropod hemocyanins and the three-dimensional structure of a crustacean hemocyanin (4). We report here the complete determination of the amino acid sequence of CNBr fragment Ib. The complete sequence and a discussion of possible heterogeneity are presented in an accompanying paper ( 5 ) .

domain 111of the hemocyanin (4). This raises the interesting possibility of reconstructing domain111. It seems quite possible that the CNBr fragmentscould self-assemble to form an aggregate similar to domain I11 in the intact molecule. Examination of the architecture of domain I11 (4) suggests that CNBr Ib by itself might form the characteristic structure of domain 111 and that addition of the other CNBr fragments would stabilize this structure.

Acknowledgments-We wish to acknowledge the expert assistance of Marie Ervin, KarenHaschke, and SandraSmith. Some amino acid analyses were performed by the Protein Sequencing Center, University of Texas, Austin, TX 78712 which was established in part by National Institutes of Health Grant GM 21688-0582.

REFERENCES 1. Behrens, P. Q., Moore, M. D., Nakashima, H., Yokota, E., and Riggs, A. F. (1983)Fed. Proc. 42,2157 2. Yokota, E., and Riggs, A. F. (1984)J. Biol. Chem. 259,4739-

The 142 residues of CNBr Ib (Fig. 1) together with CNBr fragmentsIIa, IId, IIIa,IIIc,and IVd (3) compriseall of

4749 3. Moore, M.D., Behrens, P. Q., and Riggs,A. F. (1985)J. Bwl. Chem. 261, 10511-10519 4. Linzen, B., Soeter, N. M., Riggs,A. F., Schneider, H.-J., Schartau, W., Moore, M. D., Yokota, E., Behrens, P. Q., Nakashima, N., Takagi, T., Nemoto, T., Vereijken, J. M., Bak, H. J., Beintema, J. J., Volbeda, A., Gaykema, W. P. J., and Hol, W . G . J . (1985) Science 229, 519-524 5. Nakashima, H., Behrens, P. Q., Moore, M.D., Yokota, E., and Riggs, A. F. (1986)J.Biol. Chem. 261, 10526-10533 6. Tarr, G. (1981)Anal. Biochem. 111,27-32

* This work wassupported by National Institutes of Health Grants GM 21314 and GM 28410 and GrantF-213from the Robert A. Welch Foundation. Preliminary accounts of some of this work have been presented (1-4).The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $Present address: First Dept. of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka 812,Japan. To whom reprint requests should be addressed. Portions of this paper (including “Materials and Methods” and

“Results,” Tables I-V, and Figs. 2-5) are presented in miniprint at the end of this paper. The abbreviations used are: DABITC, 4-N,Ndimethylaminoazobenzene-4’-isothiocyanate; PTH, phenylthiohydantoin; HPLC, high performance liquid chromatography. 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-4242,cite the authors, and include a check or money order for $7.20 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

MATERIALS AND METHODS AND RESULTS’

DISCUSSION

10520

10521

Structure of Limulus Hemocyanin II: The 16-kDa Fragment

I V3 ”””””””””””” 7

”

7

,

I,+?

I ””””“””4”

I

l5+6-

””-

” ” ?

FIG. 1. Summary of sequence analysis of CNBr Ib. Symbols include the following. Automated Edman degradation: +, identification of phenylthiohydantoin-derivative by high performance liquid chromatography. 4-N,N-dimethylaminoazobenzene - 4’ - isothiocyanate manual Edman degradation: -, identified on polyamide sheet. Data printed above the sequence are from steps performed on the intact cyanogen bromide fragment. Peptides are designated T, tryptic; V, S. aureus strain V8 protease; S, BNPS-skatole cleavage peptide.

”””+

v45.-

Miniprinr Suppluncnt IO “Structure of Hemocyanin I1 from the Horseshoe Crab, Linruivr TheAmino Acid Sequence of the SecondLargestCyanogenBromide Fragment” by P.Q. Bchrens, H.Nakashima, E. Yokota, and A.F. Riggs. All materials and procedures have been described (2) except as follows. Enrymoric Digesrion Two trypticdigestsof CNBr Ib wereperformed(Trypsin.TRTPCKgrade, Worthington).

&$emus.

( I ) CNBrIb (3.2 mg) was digested in 1.0 ml 0.5% NH HCO at 25’C with constant stirring for 7.5 hours at an enzyme to substrate ratio of 250 ( d w )

(2) CNBr Ib (30 mg) was digested in 4.0” ml 0.1 MNH HCO at 37°C with constant stirring for 21 hours at an enzyme to substrate ratio of 1:20 (wpw) Two staphylococcal V8 protease digests of CNBr Ib were performed

( I ) CNBr Ib (14.4 mg) was digested in 7.0 ml 0.1 MNH HCO at 37°C with constant stirring for 28 hours at an enzyme to substrate ratio of I : b (“’w) (2) Carboxymethylatedhemocyanin I1 (IO0 mg) was digested in 20.0 ml 50 mM NH HCO at 25°C with constant stirring for 18 hours at an enzyme to substrate ratio of 250 ( d w ) RESULTS CNBr fragment Ib was isolated by HPLC from the Sephmyl S-200 column fraclion Ib(Fig. 2) from wholecarboxymethylatcdCNBrcleavedhemocyanin 11. Other components in this fraction induded CNBr In, several AspPro cleavage fragments and two apparenttrypticfragments ofCNBr Ib ( T I + 2 andT5+ 6). The cyanogen bromide cleavage of whole hemocyanin I1 was carried out in 70% formic acid for 24 hours (2). Thiscould account forthepresenceoftheAsp-Fro cleavage products (thereare three Asp-Prosequences in hemocyanin 11). The trypticfragments may also result from oon-specific cleavage during the long CNBr reaction procedure. from trypticdigestion(Fig. 3). staphylococcal V8 protease CNBrIbpeptides digestion(Fig. 4) andBNPS-skatole cleavage (Fig. 5) were isolatedand purified by reverse phase HPLC. The amino acid sequenceofCNBr I b (Fig, I ) was determined by a mmbination of automated and manual Edman degradations of these peptides. The first 27 residues (with one blank) of the carboxymethylatcd intact CNBr Ib fragment were determined with the sequencer (Table 11). Amino acid composition of trypticpeptidesfromCNBr Ib isshown in Table Ill. Automatedsequencedatafor these peptides is shown in Table 11. The composition and sequence data from tryptic first 20 residues. Amino scid peptides T I + 2, T2 and T3 confirm the identity of the cornpsition of peptides obtained by digestion with Sraphylocorrur a u r m V8 protease is shown in Table IV. Sequence data for these peptides is shown in Table V. ComposiV3 (7-59) extends the sequence from residue 28 tionandsequencedataforpeptide through 54. Sequence and composition data for tryptic peptides T5 (23-45), T5.I (3445), T5+ 6 (23-50), T6 (46-50) and T7 (51.56) confirm the identity of residues 28-54.

1

V7.8.9

77””””

Peptides V3.1 (54.59). V4 (60-63). V4+ 5 (60-68), V5 (64-68), V6 (69-75) and V7 54 through 82. Trypticpeptides TB (57(76-82) extendthesequencefromresidue 6 5 ) . T9 (66-70), T I 0 (72-80) and TI0.I (72-78) provide the overlaps and confirm the identity of residues 57-80. Compositionandsequencedataforpeptides V7+ 8c 9 (76.108). V8 (83-96).V9 (97.108). VI0 (109-lL8). VI1 (119-122). VI2 (123-142) and V12.1 (131.142) extend the sequence from residue 83 to 142, the C-terminal residue of the CNBr Ib fragment. Trypticpeptides T I 1 (81-87). T I 2 (88-92), T I 3 (93120). TI4 (121-136) and TIS (137-140) provide the overlaps and confirm the identity ofresidues 81-140. Compositionandsequencedatafrompeptide SI (from BNPSSkatole cleavage at the only tryptophan present in fragment Ib) confirms the identity of residues 129-142. Problem Areas Peptides T I (1.8). T4 (21-22). VI(1-4) and V2 (5-6) werepresent in their respective digests but were not purified and sequenced because adequate sequence data had been established already for these regions. The compositionoftrypticpeptideT5(total 23 residues)indicates one alanine (23-45). and two leucines.Thisdoesnotagree with thesequenccdataforT5 Although peptide T5 itself was not successfully sequenced completely ( 3 r e p a t r u n s on sequencer--yield dropped dramatically each time after step IS), the composition and sequence data from supporting peptides T5.1 (34-49, T5+ 6 (23-50) and V3 (7-59) all indicate the presence of one leucine and two alanines. This discrepancy may be due to the presence of a mixture of electrophoretically distinct components (2) which wuld contain neutral amino acid changes as well. For purposes of sequencing we treat the mixture as a single component. 6 show an appreciable quanAmino acid compositions of peptides T5.1 and T5+ tity of glycine. However. no traceof glycine was found in eitherpeptide in the sequencing data. Peptide (V6 + V7) is essentially an equimolar mixture of two pptides, each having 7 residues. The mixture was applied to the Sequencer, and gave two clean peaks far each cycle (except cycle 1 which is leucineforbothpeptides).Coincidentallythe two peptides are contiguous in the sequence. Theamino acid compositionofpeptide V9 shows an appreciable quantityof aspartic acid. Sequencer data indicates contamination of V9 with the peptide (V4+ 5) which would explain all the background levels of amino adds in the composition. The (2.2) appears low forthreeresidues.butcorrectingthe molarratiovalueofserine cornposition of V9 for contamination from V4+ 5 , the average molar ratio value for one residue of serine in V9 becomes 0.8. The expected value for three residues would be 2.4. The observed value of 2.2 for three is close. The three acrincs in V9 were all unambiguously identfied by the DAB ITC procedure. The molar ratioforleucine in peptide VI0 is low (2.21) forthreeresidues. Sequence data for VI0 and for the supporting peptide TI3 clearly indicate three Ieucines (residues 110, 111 and 116) in this region. The composition of peptide VI2 is obviously not clean. The sequence data indicaw a low level contaminant comprising residues 28-44 which explains the presence of Glu (0.76), Ala (0.52) and Phe (0.35). These amino acids are not found in Vl2. The composition of peptide V12.1 (131-142) shows background levels of several amino acids, in particular a molar ratio of 1.0 for Glu. Sequence data does not indithe supporting peptides VI2 (123-142) or SI cate the presence of any Glu. Neither of

Structure of Limulus Hemocyanin

10522

11: 16-kDa The

Fragment

(139-142) indicates any Glu. Possibly some incompletely cleaved CNBr Ib + IVd fragment (an extra 7 residues) is present with the CNBr Ib. Digestion with V8 would then produce a peptide with C-terminal a Glu residue (---Lys -Gly-Met-Glu). Sequencer data did not show a terminal Glu, but the final residue of a peptide often cannot be detected. Determination of serinesfromautomatedsequencer data almost always gives very low yields. We consistently observe a large unidentified peak that elutes very late (half a minute after leucine) when using Tarr's isocratic HPLC procedure for separation of PTH amino acids (6). This peak occurs only when Serine is the cleaved amino acid. Similarly threonine is almost always accompanled by a large peak thatelutes in the region ofdehydrothreonine. Cycle yields (Tables 11 and V) do nottakeinto account either of these unidentified peaks. In contrast, serines and threoninesare easily identified by theDAB ITC technique. all DAB ITC Edman degradation results shown i n Fig. I represent clear unamblguous data and provide excellent supportive sequence information. The tryptic digest of CNBr Ib contained one peptide with an unusual cleavagc site: between a Tyr-Ala at positions 33-34 to give peptide T5.1 (34-45). The Staphylococcus nureus strain V8 digest resulted in two peptides with unusual cleavage sites: between Leu Ala at positions 53-54 to give peptide V3.1 (55-59) and between Ser-His at positions 130-131 to give peptide V12.1 (131-142). We have examined the data on CNBr Ib for ewdence of sequence heterogeneity and find that position I S of CNBr Ib may be heterogeneous. Tryptic Peptide TZ has 9 residues(Table 111) and lacks Ser. Another tryptic peptide obtained had exactly the Same compositlon exccpt that it possessed Scr. The composition (asmolar ratios) was: His, 0.91; Arg. 0.68; Asp, 1.87; Scr, 0.89; Pro, 0.73; Gly, 2.05; Ala, 1.08; [le, 0.86. Amounts of other m i n o acids Mre less than 0.15 residues per molecule of peptide. Thispeptide was sequenced by theDABprocedureand gave exaalythe same sequence already found for Peptide T 2 except that step 7 gave DAB spots corresponding to both Scr and Asn with some wry-over of Gly. These observations suggest that some molecules may have Scr at position 425 of hemocyanin 11.

TABLE I. Amino Acid Composition of CNBI Ib. Amino Acid' By

malpia

By rcqucacc

10.9 9.5 6.6 19.6 3.9 10.5

11 10

14.9 7.0

I4 7 9

7 17 6 I1

9.3

8 2 8 6

8.1 UTC

8.3 5.1 15.0 4.9

15

5 4 1 1

4.0 VTC VTC

Total

142

M.W.

16,095

0

r

B

wNg. o

I-

3 0

9

10

20 Ttrnelmin)

ASP-PRO

30

40 Timeimml

11: The 16-kDa Fragment

Structure of Limulus Hemocyanin

10523

TABLE 11. Aulomued Wucnoc Aadyris of CNBr Ib Whole Chain. n d of Tryprie Pwlidea from CNBr Ib.

Tz (100 nmolcr) Residue. Hb

Cycle Number 1

2 3

Glu Oh

4

Glu Leu Glu

5

6 7 9.5 8 9

IO 11

I2 11 I4 I5 16 17

Leu Ly8 Hb Gly lk ~~~

Am FTO

Gly Am Ah

Am

19

su m

20

LyY

21

Ah Art Tyr

18

22 23 24 25 26 27

11.6 4.0 20.6 11.4

GG

(E 19, D 10)

Glu

11.1 10.5

1.9 7.0 2) 4.7 5.9 1.7

L ~ U

L~Y His ~~Gly

(D

Ik (D 2) (N 1J.Y 0.7)

12.1

2.1 1.0 1.6

1.1 1.8 2.5 2.2

Oh Gh Leu

( 0 0.8) (0 I) (H0.9) ( R 0.9)

Am

Pro Gly Am Ah

Aq

17 13.5

(E 13.4)

28.6 12.5 27 n.8 12.8 9.9 21.2 4.1 15.6