Proc. Natl. Acad. Sci. USA Vol. 76, No. 4, pp. 1668-1672, April 1979
Biochemistry
Complete amino acid sequence of a histidine-rich proteolytic fragment of human ceruloplasmin (protein structure/ferroxidase/copper binding/genetic polymorphism/evolution)
I. BARRY KINGSTON*, BRIONY L. KINGSTONt, AND FRANK W. PUTNAMt Department of Biology, Indiana University, Bloomington, Indiana 47405
Contributed by Frank W. Putnam, January 22, 1979
ABSTRACT The complete amino acid sequence has been determined for a fragment of human ceruloplasmin [ferroxidase; iron(II)oxygen oxidoreductase, EC 1.16.3.1]. The fragment (designated Cp F5) contains 159 amino acid residues and has a molecular weight of 18,650; it lacks carbohydrate, is rich in histidine, and contains one free cysteine that may be part of a copper-binding site. This fragment is present in most commercial preparations of ceruloplasmin, probably owing to proteolytic degradation, but can also be obtained by limited cleavage of single-chain ceruloplasmin with plasmin. Cp F5 probably is an intact domain attached to the COOH-terminal end of single-chain ceruloplasmin via a labile interdomain peptide bond. A model of the secondary structure predicted by empirical methods suggests that almost one-third of the amino acid residues are distributed in a helices, about a third in ,8-sheet structure, and the remainder in ft turns and unidentified structures. Computer analysis of the amino acid sequence has not demonstrated a statistically significant relationship between this ceruloplasmin fragment and any other protein, but there is some evidence for an internal duplication.
Ceruloplasmin [ferroxidase; iron(II):oxygen oxidoreductase, EC 1.16.3.1], is a blue, copper-containing a2-glycoprotein that is normally present in human plasma at a concentration of 20-40 mg/100 ml (1). Although the biological role of ceruloplasmin (Cp) is not entirely clear, at least three functions have been ascribed: ferroxidase activity, copper transport and detoxication, and maintenance of copper homeostasis in the tissues. These functions are not mutually exclusive, but the most important is thought to be the action of plasma ceruloplasmin as a ferroxidase in oxidizing ferrous iron to the ferric form which is then incorporated into transferrin (2). The only established biochemical abnormality involving ceruloplasmin is its deficiency in Wilson disease (hepatolenticular degeneration). The deficiency is due to a genetic defect in the rate of ceruloplasmin synthesis that leads to abnormal copper metabolism and deposition of copper in the tissues (3). Despite several reports that it has a subunit structure (1, 4-6), ceruloplasmin has been shown to be a single polypeptide chain with a molecular weight of about 130,000 that is readily cleaved to large fragments by proteolytic enzymes (7, 8). We have isolated and characterized three fragments of ceruloplasmin that appear to be nonoverlapping and that have approximate molecular weights of 20,000, 53,000, and 67,000 (8). We report here the complete amino acid sequence of the smallest fragment, which we call the histidine-rich fragment and designate Cp F5. This fragment contains 159 amino acid residues and lacks carbohydrate. It is present to a varying degree in all the commercial preparations we examined and can be prepared from single-chain ceruloplasmin by digestion with plasmin (8). Cp F5 is identical in NH2-terminal sequence to the so-called
a chain of human ceruloplasmin studied by McCombs and Bowman (6) and probably corresponds to the a subunit proposed by Simons and Bearn (4) and the light chain of Freeman and Daniel (5). The relation of Cp F5 to the structure of the intact ceruloplasmin chain has to be deduced from indirect observations because of the small amount of single-chain ceruloplasmin available to us and the strong interaction of the fragments. From the kinetics of the proteolytic cleavage of intact ceruloplasmin and the chemical properties of the fragments, we have suggested that Cp F5 is from the COOH terminus of the single chain (8). We have obtained a unique amino acid sequence and conclude that Cp F5 is probably an intact domain; it appears to be attached to the COOH-terminal end of the ceruloplasmin chain by a labile interdomain peptide bond. Prediction of the secondary structure by the empirical method of Chou and Fasman (9, 10) leads to a model in which approximately 30% of the residues occur in f3 sheets, 25% in a helices, and 45% in j turns and other structure. Because the ceruloplasmin preparation studied is derived from a pool of plasma from more than 10,000 donors from the ethnically mixed American population, the finding of a unique amino acid sequence indicates that the frequency of genetic polymorphism is too low to interfere with sequence determination of this fragment. In a previous report (8) we identified a cysteine-containing sequence in human Cp F5 that appeared similar to a series of cysteine-containing sequences that are apparently homologous to each other and contain part of the copper-binding sites in bacterial azurins and plant plastocyanins. Computer analysis of an intersequence comparison of Cp F5 and azurin from Alcaligenes sp. indeed showed that the most similar segment of sequence was that which we had identified. However, no statistically significant relationship could be demonstrated between the ceruloplasmin fragment and any other protein, including azurin, plastocyanin, and superoxide dismutase, all of which are copper-containing proteins. Further computer analysis indicated a possible internal duplication in the ceru-
loplasmin.
MATERIALS AND METHODS Materials. The purified human ceruloplasmin from normal pooled sera used for sequence analysis was a preparation made by the method of Sgouris et al. (11), starting with ethanol fraction IV-1, and was generously provided by J. J. Hagan Abbreviation: Cp, ceruloplasmin. * Present address: National Institute of Agricultural Botany, Hun-
tingdon Road, Cambridge, England. Genetics, Department of Applied Biology, Pembroke St., Cambridge, England. t To whom reprint requests should be addressed.
t
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
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Present address: Commonwealth Bureau of Plant Breeding and
Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Kingston et al.
(which has specificity for peptide bonds having aspartic or glutamic acid on the carboxyl side) was used to prepare subpeptides of tryptic peptides T3 and T4 and also T6. In addition, Cp F5 was citraconylated and digested with trypsin to give a peptide covering the segment from Lys-7 through Arg-58. Sequence analysis of these peptides permitted completion of the missing area. RESULTS AND DISCUSSION Amino Acid Sequence. The complete amino acid sequence of the human ceruloplasmin fragment Cp F5 derived by the strategy described above is given in Fig. 2. Except for the abundance of histidine, there is nothing remarkable about the primary structure. The single cysteine residue (Cys-134) must be present in the sulfhydryl form because reduction made no difference in the banding pattern of the original ceruloplasmin preparation on electrophoresis in polyacrylamide gel containing 0.1% sodium dodecyl sulfate (8). The NH2-terminal sequence of Cp F5 is identical to the 21-residue NH2-terminal sequence given by McCombs and Bowman (6) for a ceruloplasmin polypeptide they designated a chain. With the exception of this and of three glycopeptides from human ceruloplasmin (12), no other sequence data are published on ceruloplasmin from any species. However, the amino acid composition of a cysteine-containing peptide isolated by Egorov et al. (13) from undegraded human ceruloplasmin corresponds to the sequence around Cys-134. Fragment Cp F5 has previously been referred to by us (8) as the 20,000 Mr fragment because that was the molecular weight estimated from polyacrylamide gel electrophoresis. Calculation of the exact molecular weight from the sequence yields a value of 18,650, which is in good agreement. Carbohydrate Content. The most difficult part of the sequence to determine was the segment from Asp-18 through Lys-30. This acidic stretch contains seven dicarboxylic acid residues, of which five are in the acidic form. In addition to the difficulty of separating subpeptides of this region, nonstoichiometric amounts of glucosamine appeared to be present on amino acid analysis. The sequence determined does not correspond to any of the three glycopeptides reported by Ryden and Eaker (12). However, the tripeptide Asn-Glu-Ser (positions 20-22) contains an example of the triplet acceptor sequence Asn-X-Ser/Thr, to which glucosamine oligosaccharides usually are attached (14). For this reason, and because of the possible presence of glucosamine in the impure peptides from this segment, we gave a sample of Cp F5 to Jacques Baenziger (Washington University School of Medicine, St. Louis, MO) for complete carbohydrate analysis. In personal communication, Dr. Baenziger reported to us that "a preliminary analysis of the
(Squibb). This was preparation Cp 1 of our previous paper (8); several other preparations described there were used for reference purposes, including single-chain ceruloplasmin (Cp 3 and Cp 5) supplied by Yu lee Hao (National Fractionation Laboratory of the American National Red Cross). Purification of Histidine-Rich Fragment Cp F5. Fragment Cp F5 was prepared from Cp 1 after reduction and aminoethylation. Cp 1 (420 mg) was dissolved in a buffer (20 ml) containing 6 M guanidine-HCI, 2 mM EDTA, 0.5 M Tris-HCI, at pH 8.0, and reduced under N2 at 50'C with dithiothreitol (360 mg). After cooling to 4°C, three aliquots (80 Al each) of ethylene imine (Pierce) were added at 10-min intervals. The solution was then dialyzed against deionized water (4 liters, three times) and lyophilized. The reduced aminoethylated protein was dissolved in 10 ml of 6 M urea/0.2 M formic acid and applied to a column (150 X 4 cm) of Sephadex G-150 or G-200 equilibrated with the same solution. The column effluent was monitored at 280 nm, and good separation of Cp F5 was obtained. After volume reduction by ultrafiltration, the fragment was desalted on Sephadex G-15 in 1 M acetic acid and Iyophilized. Methods. The methods for protein characterization, amino acid analysis, and sequence determination were the same as in our previous report (8). Strategy for Sequence Determination. In addition to automated sequence analysis of the intact Cp F5 fragment for 18 steps, the strategy for sequence determination involved two different routes in order to obtain independent verification of each position and to secure all necessary overlaps of peptides. Because of the presence of seven methionine residues, CNBr cleavage was chosen for the initial procedure. Nine CNBr peptides were obtained (Fig. 1); these were purified and submitted directly to automatic sequence analysis, except for the dipeptide CB5. In some instances (CB1, CB2, CB6, and CB9) the CNBr peptides were digested with trypsin, and the tryptic subpeptides were purified and analyzed and their amino acid sequences were determined. In other cases the subpeptides of the CNBr fragment were digested further with another enzyme-e.g., thermolysin for CB6, pepsin for CB8, or Staphyloccocus aureus V8 protease for CB9. The results of sequence analysis by this procedure are diagrammed schematically in Fig. 1. The second route for sequence determination consisted of digestion of the Cp F5 fragment with trypsin, followed by isolation, analysis, and partial or complete sequence determination of the 19 tryptic peptides obtained; these are denoted schematically in Fig. 1. Because of difficulty in purifying the peptides and determining the sequence in the highly acidic region from Asp-18 through Glu-37, staphylococcal protease Residue 0
10
Number I
20
30
I
I
40
50
60
1669
70
80
90
T
l
100
'
120
110
l
'
'
l
l
130
'
140
l
' l
150
'
159
l
CB7 ICB2
CB1
ICB3 I
CB4
151
C9
CB8
CB6
CNB r
Peptides
TlT8 T1| T3 T5 T4 T9 Tryptic Peptides
_1
T2
T4
I-
T6
T10 ITll
l
T13
I
112 1
T1
T15
IT16
I
T17
T T18 'B
T14
FIG. 1. Diagram for sequence determination of Cp F5, The scale at the top gives the amino acid residue number. The nine CNBr peptides are demarcated and are designated CB1-CB9. The extra line indicates CB7, which contains CB6 and CB8 joined together. In a separate procedure 19 tryptic peptides, designated T1-T19, were obtained; some of these are overlapping. Subpeptides of the CNBr or tryptic peptides are not indicated in the diagram. Shaded areas denote positions for which the amino acid sequence was established in each procedure.
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Biochemistry: Kingston et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
10 20 30 H2N-Val -Phe-Asn-Pro-Arg-Arg-Lys -Leu- G1 u-Phe-A1 a- Leu- Leu-Phe- Leu-Val -Phe-Asp-Gl u-Asn-GI u-Ser-Trp-Tyr- Leu-Asp-Asp-Asn- I 1e- Lys-
40 50 60 Thr-Tyr-Ser-Asp-Hi s-Pro-G1 u-Lys-Val -Asn-Lys-Asp-Asp-Gl u-Gl u-Phe- Il e-Gl u-Ser-Asn-Lys-Met-Hi s-Ala-I le-Asn-Gly-Arg-Met-Phe70 80 90 Gly-Asn-Leu-Gl n-Gly-Leu-Thr-Met-Hi s-Val -Gly-Asp-Gl u-Val -Asn-Trp-Tyr-Leu-Met-Gly-Met-Gly-Asn-Gl u- Ile-Asp- Leu-Hi s-Thr-Val 100 110 120 Hi s-Phe-Hi s-Gly-Hi s- Ser-Phe-Gl n- Tyr-Lys -Hi s-Arg-Gly-Val -Tyr- Ser-Se r-Asp-Val -Phe-As p- Ile-Phe-Pro-Gly-Thr-Tyr-Gl n-Thr- Le u130 140 150 Gi u-Met-Phe-Pro-Arg-Thr-Pro-Gly- Ile -Trp-Leu-Leu-Ili s-Cys-Hi s-Val -Thr-Asp-Hi s-Ile-His-Ala-Gly-Met-G1u-Thr-Thr-Tyr-Thr-Val Leu-Gln-Asn-Gl u-Asp-Thr-Lys-Ser-Gly-COOH
FIG. 2. Amino acid sequence of the histidine-rich fragment of human Cp F5.
ceruloplasmin 20,000 Mr fragment would indicate that less than 10% of the peptide could be glycosylated." Expressed as moles of carbohydrate per 20,000 daltons, the fragment contained: 0.05 glucosamine, 0.12 galactose, 0.12 mannose, and 0.00 fucose. It is possible that Asn-20 is partially glycosylated and, although our calculation of the secondary structure of Cp F5 (see later) places Asn-20 at the end of an a helix, residues 20-23 also have a high probability of being in a (3turn. Since it appears likely that carbohydrate is frequently attached to glycoproteins at (3turns (15, 16), Asn-20 may be in a favorable position for glycosylation. However, the bulky tryptophan residue just after the triplet (i.e., Asn-Glu-Ser-Trp) may hinder glycosylation. Genetic Polymorphism. Genetic polymorphism of ceruloplasmin is rare in whites, but in black Americans a variant designated Cp A occurs with an allele frequency of 0.052, compared to a frequency of 0.939 for the common form Cp B and 0.003 and 0.006 for the rare variants Cp C and Cp NH, respectively (1, 3). In white Americans the frequency of Cp B is 0.994, whereas the frequency of Cp A is only 0.006, though Cp A frequency rises to a high of 0.149 in Nigerians (1). Other variants of ceruloplasmin have a very low incidence in all populations studied (1). Because blacks constitute about 11% of the American population, the incidence of Cp A and other known variants in pooled plasma collected by the American Red Cross must be less than 0.6%. Such a value is too low to be detected by the methodology of amino acid sequence analysis. We estimate that because of the cumulative errors in the methodology, genetic variants at an individual frequency of less than 0.05 in pooled plasma would not be detectable by amino acid sequence analysis. It is thus not surprising that we obtained a unique amino acid sequence for the fragment Cp F5. Furthermore, Cp F5 represents only a fragment of the whole ceruloplasmin molecule and may not be the site of any polymorphic substitution. However, one observation suggests to us the possibility that unidentified genetic variants not detectable by the usual method of electrophoretic screening may exist in the American population. In early work with Cp 2 (8), a different preparation of ceruloplasmin than the one used for complete sequence analysis (Cp 1), we obtained a CNBr fragment§ corresponding to the sequence given in Fig. 2 as Phe-Gly-Asn-Leu-Gln-GlyLeu-Thr-Met for positions 60-68; however, the composition of the fragment showed 0.67 residue of phenylalanine and 0.33 of tyrosine, and the dansyl method and sequenator analysis demonstrated both amino acids at the NH2 terminus. Yet, in the complete sequence study neither the CNBr fragment nor the tryptic peptide covering this region gave evidence of ty§ The nonapeptide also had copper.
a
bluish color and appeared to bind
rosine at position 60. We do not know whether the tyrosinecontaining peptide was lost during purification or if it was present only in the first preparation of ceruloplasmin. It is possible that a tyrosine-phenylalanine exchange at position 60 represents a true genetic polymorphism, but it is unlikely that the tyrosyl variant is Cp A because the latter differs in charge from Cp B. Our evidence for proteolytic degradation coupled with the observation of "gull-wing" patterns in immunoelectrophoresis of aged preparations suggests that some genetic variants previously reported may be artifacts. Poulik and Weiss (1) have discussed the questionable genetic significance of one reported genetic variant that has a band with identical electrophoretic mobility to one observed in aged serum that originally had only the Cp B type. Role of Histidine in Histidine-Rich Fragment. Although exceeded in histidine content by the "histidine-rich a2 glycoprotein" of human plasma (17), which has a histidine content of 9.9%, and by the a globin chains of some species that have 10 or more histidines per a chain, the Cp F5 fragment of human ceruloplasmin has a higher histidine content than most other known proteins (8.82%, calculated from the sequence). Furthermore, 7 of the 12 histidines occur in an alternating sequence of His-X-His-i.e., His-Phe-His-Gly-His, His-Cys-His, and His-Ile-His (beginning at positions 91, 133, and 139, respectively). This leads to two short segments that, when other nearby histidines are included, are exceedingly rich in histidine-i.e., there are four histidines in the eight-residue segment from His-88 to His-95 and four more in the nine-residue segment from His-l33 through His-141, which contains the free cysteine. In view of the role of histidine in the binding of copper by serum albumin (18), the proximity of so many histidines close to Cys-134, which is assumed to be a copper-binding site (8), is probably significant. Because of the alternation, all of the histidines could be exposed to the surface in the ( sheet postulated for residues 129-137 and the f turn following it. Prediction of Secondary Structure. The problems of predicting protein structure from amino acid sequence have recently been reviewed by Sternberg and Thornton (19). Nonetheless, they point out that "the method of Chou and Fasman (9, 10) has attracted most attention as it is simple to understand, can be applied without a computer, and has been relatively successful." Hence, we have applied their rules for predicting secondary structure, with the result depicted in Fig. 3. The model depicts a mixed a/,3 secondary structure. Almost one-third of the residues appear to be distributed in five a helices and about a third in ,B sheets. The remainder are in ( turns, coils, or disordered structures, including one segment of 15 residues (residues 100-115) that has no clear a or (3 initiation sites. The charged residues are concentrated at three sites: the
Biochemistry: Kingston et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
6 7
84
108
116 His 139
144 146 Met
120
123
Cys 134
152
159
FIG. 3. Diagram of the secondary structure of Cp F5 as predicted by the method of Chou and Fasman (9, 10). Residues are represented in helical (A.), ,3-sheet (A), and coil (-) conformational states. Chain reversals denote /-turn tetrapeptides (::.
NH2 terminus, the COOH terminus, and the two helices and turn in the segment from Asp-34 through Lys-51. There are several hydrophobic stretches, notably in the first helix from Phe-10 through Phe-17. The alternation of the histidines rea
lieves the hydrophobic character of the sheet beginning at Ile-129. Noncovalent Interaction of Cp F5 with Other Fragments. The several fragments present in commercial preparations interact strongly, and harsh dissociating conditions are required to separate them. Thus, the blue color and the copper are lost during the purification of Cp F5. The association is not due to disulfide bonds because ceruloplasmin preparations that exhibit multiple bands in polyacrylamide gel electrophoresis in 0. 1% sodium dodecyl sulfate give similar patterns before and after reduction although they sediment largely as single components with s2o = 6-7 S in analytical ultracentrifugation (8). The reason for the strong interaction of Cp F5 with the 53,000 Mr and 67,000 Mr fragments is not evident from the primary structure. However, one clue is offered by the model of the secondary structure given in Fig. 3; this illustrates the concentration of charged groups in certain segments of the structure, notably the NH2 terminus, the COOH terminus, and the two a helices and the ,B turn proposed for residues 34-55. Half the residues in the latter segment are either positively or negatively charged. If the largely hydrophobic structures from residues 55-144 are mainly in the interior of the molecule and the ionized residues are at the surface, as would be expected from the general rules for protein conformation (19), then the Cp F5 fragment would be capable of strong electrostatic interaction with the other
1671
fragments of ceruloplasmin. Probably this is its natural role, for it appears to be a separate domain structure of the intact molecule. Intersequence Comparison of Homology. We have proposed (8) that the cysteine-containing COOH-terminal sequence of Cp F5 may be involved in binding one of the two type 1 cupric ions in ceruloplasmin because the sequence from Gly-128 through Val-150 shows similarity to the cysteinecontaining COOH-terminal sequence of blue copper-containing azurins and plastocyanins, which contain a single type 1 copper ion. The cysteine, histidine, and methionine residues, which are invariant in the latter proteins and which have been implicated to be in the copper-binding site (8), could be matched by Cys-134, His-139, and Met-144 in Cp F5. This proposal has since been strengthened by the publication of the results of x-ray crystallographic studies of plastocyanin (20) and azurin (21). In both cases, the copper atom appears to be bound to the cysteine and methionine residues which were matched (8) with Cys-134 and Met-144 in Cp F5 and by two histidine residues one of which was matched with His-139. In our predicted secondary structure shown in Fig. 3, the spacial arrangement of Cys-134, His-139, and Met-144 would give them the potential to act as copper ligands. Furthermore, it is possible to interpret Chou and Fasman's rules for predicting secondary structure in a manner (L.-C. Lin, personal communication) that predicts that Cp F5 will be composed of two a-helical regions at the NH2 terminus and six f-sheet strands separated into two groups of three by an intervening a-helical or random region; these elements could then fold up to produce a structure rather similar to the cylindrical barrel shape formed largely by strands of 3 sheet in poplar plastocyanin (20) and Pseudomonas aeruginosa azurin (21). To search for any additional similarity to other proteins, we requested the National Biomedical Research Foundation to undertake a computer search comparing the sequence of Cp F5 with all sequence data available, not only for the blue copper-containing proteins, but also for all other proteins. With the program RELATE (22), ceruloplasmin was compared to itself and to plastocyanin, azurin, and superoxide dismutase, by using a segment length of 15. In all cases the comparison of real sequences gave a score less than 1 SD from the mean score with 100 randomized sequences. Under the same conditions, azurin against plastocyanin gives a score of 3.9 SD. The ceruloplasmin was rerun against itself by using a segment length of 25. The score was 1.8 SD, which is interesting though not statistically significant (Winona C. Barker, personal communication). However, as we had earlier identified by visual comparison of the segments from 1-53, 54-83, and 108-137, there is an indication that the chain had duplicated. In the computer search, 19 of the top 20 scores came from displacing the sequence by 53 residues. Two 25-residue segments of ceruloplasmin (Met-79 through Gly-i1S, which has six histidioes, and Pro-127 through Leu-151, which contains the cysteine) were searched against the entire data collection of the Atlas of Protein Sequence and Structure (95,365 comparisons). The highest scoring segments retrieved were from a variety of proteins. Nothing stood out significantly from the rest. The cysteine-containing segment of the azurins gave a good score, but the highest scores included a series of ferredoxins and the a and .B globins of many species. The globins and Cp F5 have a high histidine content in common. The ferredoxins are of interest because the highest scoring segment is at the COOH terminus of both Cp F5 and ferredoxin, and the matching residues are Trp-130, Leu-132, and Cys-134, all close to the site proposed for copper binding in Cp F5.
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We thank J. Dwulet, A. Galen, J. Madison, and S. Dorwin for technical assistance, Dr. J. Baenziger for carbohydrate analysis, and Dr. W. C. Barker for computer analysis of the sequence homology. This work was supported by National Institutes of Health Grant AM 19221. 1. Poulik, M. D. & Weiss, M. L. (1975) in The Plasma Proteins, ed. Putnam, F. W. (Academic, New York), 2nd Ed., Vol. 2, pp. 51-108. 2. Frieden, E. & Hsieh, H. S. (1976) Adv. Exp. Med. Biol. 74, 505-529. 3. Gitlin, D. & Gitlin, J. D. (1975) in The Plasma Proteins, ed. Putnam, F. W. (Academic, New York), 2nd Ed., Vol. 2, pp. 321-374. 4. Simons, K. & Bearn, A. G. (1969) Biochim. Blophys. Acta 175, 260-270. 5. Freeman, S. & Daniel, E. (1973) Biochemistry 12,4806-4810. 6. McCombs, M. L. & Bowman, B. H. (1976) Biochim. Biophys. Acta 434,452-461. 7. Ryden, L. (1972) Eur. J. Biochem. 26,380-386. 8. Kingston, I. B., Kingston, B. L. & Putnam, F. W. (1977) Proc. Natl. Acad. Sc$. USA 74,5377-5381. 9. Chou, P. Y. & Fasman, G. D. (1974) Biochemistry 13, 211245.
Froc. Natl. Acad. Sci. USA 76 (1979) 10. Chou, P. Y. & Fasman, G. D. (1978) Annu. Rev. Biochem. 47, 251-276. 11. Sgouris, J. T., Coryell, F. C., Gallick, M., Storey, R. W., McCall, K. B. & Anderson, H. D. (1962) Vox Sang. 7,394-405. 12. Ryden, L. & Eaker, D. (1974) Eur. J. Biochem. 44, 171-180. 13. Egorov, T. A., Svenson, A., Ryden, L. & Carlsson, J. (1975) Proc. NatI. Acad. Sci. USA 72,3029-3033. 14. Clamp, J. R. (1975) in The Plasma Proteins, ed. Putnam, F. W. (Academic, New York), 2nd Ed., Vol. 2, pp. 163-211. 15. Aubert, J. P., Biserte, G. & Loucheux-Lefebvre, M. H. (1976) Arch. Biochem. Biophys. 175,410-418. 16. Huber, R., Deisenhofer, J., Colman, P. M., Matsushima, M. & Palm, W. (1976) Nature (London) 264,415-420. 17. Heimburger, N., Haupt, H., Kranz, T. & Baudner, S. (1972) Hoppe-Seyler's Z. Physiol. Chem. 353, 1133-1140. 18. Peters, T., Jr. (1975) in The Plasma Proteins, ed. Putnam, F. W. (Academic, New York), 2nd Ed., Vol. 1, pp. 133-181. 19. Sternberg, M. J. E. & Thornton, J. M. (1978) Nature (London) 271, 15-20. 20. Colman, P. M., Freeman, H. C., Guss, J. M., Murata, M., Norris, V. A., Ramshaw, J. A. M. & Venkatappa, M. P. (1978) Nature (London) 272, 319-324. 21. Adman, E. T., Stenkamp, R. E., Sieker, L. C. & Jensen, L. H. (1978) J. Mol. Biol. 123,35-47. 22. Barker, W. C., Ketcham, L. K. & Dayhoff, M. 0. (1978) J. Mol. Evol. 10, 265-281.
Biochemistry
Complete amino acid sequence of a histidine-rich proteolytic fragment of human ceruloplasmin (protein structure/ferroxidase/copper binding/genetic polymorphism/evolution)
I. BARRY KINGSTON*, BRIONY L. KINGSTONt, AND FRANK W. PUTNAMt Department of Biology, Indiana University, Bloomington, Indiana 47405
Contributed by Frank W. Putnam, January 22, 1979
ABSTRACT The complete amino acid sequence has been determined for a fragment of human ceruloplasmin [ferroxidase; iron(II)oxygen oxidoreductase, EC 1.16.3.1]. The fragment (designated Cp F5) contains 159 amino acid residues and has a molecular weight of 18,650; it lacks carbohydrate, is rich in histidine, and contains one free cysteine that may be part of a copper-binding site. This fragment is present in most commercial preparations of ceruloplasmin, probably owing to proteolytic degradation, but can also be obtained by limited cleavage of single-chain ceruloplasmin with plasmin. Cp F5 probably is an intact domain attached to the COOH-terminal end of single-chain ceruloplasmin via a labile interdomain peptide bond. A model of the secondary structure predicted by empirical methods suggests that almost one-third of the amino acid residues are distributed in a helices, about a third in ,8-sheet structure, and the remainder in ft turns and unidentified structures. Computer analysis of the amino acid sequence has not demonstrated a statistically significant relationship between this ceruloplasmin fragment and any other protein, but there is some evidence for an internal duplication.
Ceruloplasmin [ferroxidase; iron(II):oxygen oxidoreductase, EC 1.16.3.1], is a blue, copper-containing a2-glycoprotein that is normally present in human plasma at a concentration of 20-40 mg/100 ml (1). Although the biological role of ceruloplasmin (Cp) is not entirely clear, at least three functions have been ascribed: ferroxidase activity, copper transport and detoxication, and maintenance of copper homeostasis in the tissues. These functions are not mutually exclusive, but the most important is thought to be the action of plasma ceruloplasmin as a ferroxidase in oxidizing ferrous iron to the ferric form which is then incorporated into transferrin (2). The only established biochemical abnormality involving ceruloplasmin is its deficiency in Wilson disease (hepatolenticular degeneration). The deficiency is due to a genetic defect in the rate of ceruloplasmin synthesis that leads to abnormal copper metabolism and deposition of copper in the tissues (3). Despite several reports that it has a subunit structure (1, 4-6), ceruloplasmin has been shown to be a single polypeptide chain with a molecular weight of about 130,000 that is readily cleaved to large fragments by proteolytic enzymes (7, 8). We have isolated and characterized three fragments of ceruloplasmin that appear to be nonoverlapping and that have approximate molecular weights of 20,000, 53,000, and 67,000 (8). We report here the complete amino acid sequence of the smallest fragment, which we call the histidine-rich fragment and designate Cp F5. This fragment contains 159 amino acid residues and lacks carbohydrate. It is present to a varying degree in all the commercial preparations we examined and can be prepared from single-chain ceruloplasmin by digestion with plasmin (8). Cp F5 is identical in NH2-terminal sequence to the so-called
a chain of human ceruloplasmin studied by McCombs and Bowman (6) and probably corresponds to the a subunit proposed by Simons and Bearn (4) and the light chain of Freeman and Daniel (5). The relation of Cp F5 to the structure of the intact ceruloplasmin chain has to be deduced from indirect observations because of the small amount of single-chain ceruloplasmin available to us and the strong interaction of the fragments. From the kinetics of the proteolytic cleavage of intact ceruloplasmin and the chemical properties of the fragments, we have suggested that Cp F5 is from the COOH terminus of the single chain (8). We have obtained a unique amino acid sequence and conclude that Cp F5 is probably an intact domain; it appears to be attached to the COOH-terminal end of the ceruloplasmin chain by a labile interdomain peptide bond. Prediction of the secondary structure by the empirical method of Chou and Fasman (9, 10) leads to a model in which approximately 30% of the residues occur in f3 sheets, 25% in a helices, and 45% in j turns and other structure. Because the ceruloplasmin preparation studied is derived from a pool of plasma from more than 10,000 donors from the ethnically mixed American population, the finding of a unique amino acid sequence indicates that the frequency of genetic polymorphism is too low to interfere with sequence determination of this fragment. In a previous report (8) we identified a cysteine-containing sequence in human Cp F5 that appeared similar to a series of cysteine-containing sequences that are apparently homologous to each other and contain part of the copper-binding sites in bacterial azurins and plant plastocyanins. Computer analysis of an intersequence comparison of Cp F5 and azurin from Alcaligenes sp. indeed showed that the most similar segment of sequence was that which we had identified. However, no statistically significant relationship could be demonstrated between the ceruloplasmin fragment and any other protein, including azurin, plastocyanin, and superoxide dismutase, all of which are copper-containing proteins. Further computer analysis indicated a possible internal duplication in the ceru-
loplasmin.
MATERIALS AND METHODS Materials. The purified human ceruloplasmin from normal pooled sera used for sequence analysis was a preparation made by the method of Sgouris et al. (11), starting with ethanol fraction IV-1, and was generously provided by J. J. Hagan Abbreviation: Cp, ceruloplasmin. * Present address: National Institute of Agricultural Botany, Hun-
tingdon Road, Cambridge, England. Genetics, Department of Applied Biology, Pembroke St., Cambridge, England. t To whom reprint requests should be addressed.
t
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
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Present address: Commonwealth Bureau of Plant Breeding and
Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Kingston et al.
(which has specificity for peptide bonds having aspartic or glutamic acid on the carboxyl side) was used to prepare subpeptides of tryptic peptides T3 and T4 and also T6. In addition, Cp F5 was citraconylated and digested with trypsin to give a peptide covering the segment from Lys-7 through Arg-58. Sequence analysis of these peptides permitted completion of the missing area. RESULTS AND DISCUSSION Amino Acid Sequence. The complete amino acid sequence of the human ceruloplasmin fragment Cp F5 derived by the strategy described above is given in Fig. 2. Except for the abundance of histidine, there is nothing remarkable about the primary structure. The single cysteine residue (Cys-134) must be present in the sulfhydryl form because reduction made no difference in the banding pattern of the original ceruloplasmin preparation on electrophoresis in polyacrylamide gel containing 0.1% sodium dodecyl sulfate (8). The NH2-terminal sequence of Cp F5 is identical to the 21-residue NH2-terminal sequence given by McCombs and Bowman (6) for a ceruloplasmin polypeptide they designated a chain. With the exception of this and of three glycopeptides from human ceruloplasmin (12), no other sequence data are published on ceruloplasmin from any species. However, the amino acid composition of a cysteine-containing peptide isolated by Egorov et al. (13) from undegraded human ceruloplasmin corresponds to the sequence around Cys-134. Fragment Cp F5 has previously been referred to by us (8) as the 20,000 Mr fragment because that was the molecular weight estimated from polyacrylamide gel electrophoresis. Calculation of the exact molecular weight from the sequence yields a value of 18,650, which is in good agreement. Carbohydrate Content. The most difficult part of the sequence to determine was the segment from Asp-18 through Lys-30. This acidic stretch contains seven dicarboxylic acid residues, of which five are in the acidic form. In addition to the difficulty of separating subpeptides of this region, nonstoichiometric amounts of glucosamine appeared to be present on amino acid analysis. The sequence determined does not correspond to any of the three glycopeptides reported by Ryden and Eaker (12). However, the tripeptide Asn-Glu-Ser (positions 20-22) contains an example of the triplet acceptor sequence Asn-X-Ser/Thr, to which glucosamine oligosaccharides usually are attached (14). For this reason, and because of the possible presence of glucosamine in the impure peptides from this segment, we gave a sample of Cp F5 to Jacques Baenziger (Washington University School of Medicine, St. Louis, MO) for complete carbohydrate analysis. In personal communication, Dr. Baenziger reported to us that "a preliminary analysis of the
(Squibb). This was preparation Cp 1 of our previous paper (8); several other preparations described there were used for reference purposes, including single-chain ceruloplasmin (Cp 3 and Cp 5) supplied by Yu lee Hao (National Fractionation Laboratory of the American National Red Cross). Purification of Histidine-Rich Fragment Cp F5. Fragment Cp F5 was prepared from Cp 1 after reduction and aminoethylation. Cp 1 (420 mg) was dissolved in a buffer (20 ml) containing 6 M guanidine-HCI, 2 mM EDTA, 0.5 M Tris-HCI, at pH 8.0, and reduced under N2 at 50'C with dithiothreitol (360 mg). After cooling to 4°C, three aliquots (80 Al each) of ethylene imine (Pierce) were added at 10-min intervals. The solution was then dialyzed against deionized water (4 liters, three times) and lyophilized. The reduced aminoethylated protein was dissolved in 10 ml of 6 M urea/0.2 M formic acid and applied to a column (150 X 4 cm) of Sephadex G-150 or G-200 equilibrated with the same solution. The column effluent was monitored at 280 nm, and good separation of Cp F5 was obtained. After volume reduction by ultrafiltration, the fragment was desalted on Sephadex G-15 in 1 M acetic acid and Iyophilized. Methods. The methods for protein characterization, amino acid analysis, and sequence determination were the same as in our previous report (8). Strategy for Sequence Determination. In addition to automated sequence analysis of the intact Cp F5 fragment for 18 steps, the strategy for sequence determination involved two different routes in order to obtain independent verification of each position and to secure all necessary overlaps of peptides. Because of the presence of seven methionine residues, CNBr cleavage was chosen for the initial procedure. Nine CNBr peptides were obtained (Fig. 1); these were purified and submitted directly to automatic sequence analysis, except for the dipeptide CB5. In some instances (CB1, CB2, CB6, and CB9) the CNBr peptides were digested with trypsin, and the tryptic subpeptides were purified and analyzed and their amino acid sequences were determined. In other cases the subpeptides of the CNBr fragment were digested further with another enzyme-e.g., thermolysin for CB6, pepsin for CB8, or Staphyloccocus aureus V8 protease for CB9. The results of sequence analysis by this procedure are diagrammed schematically in Fig. 1. The second route for sequence determination consisted of digestion of the Cp F5 fragment with trypsin, followed by isolation, analysis, and partial or complete sequence determination of the 19 tryptic peptides obtained; these are denoted schematically in Fig. 1. Because of difficulty in purifying the peptides and determining the sequence in the highly acidic region from Asp-18 through Glu-37, staphylococcal protease Residue 0
10
Number I
20
30
I
I
40
50
60
1669
70
80
90
T
l
100
'
120
110
l
'
'
l
l
130
'
140
l
' l
150
'
159
l
CB7 ICB2
CB1
ICB3 I
CB4
151
C9
CB8
CB6
CNB r
Peptides
TlT8 T1| T3 T5 T4 T9 Tryptic Peptides
_1
T2
T4
I-
T6
T10 ITll
l
T13
I
112 1
T1
T15
IT16
I
T17
T T18 'B
T14
FIG. 1. Diagram for sequence determination of Cp F5, The scale at the top gives the amino acid residue number. The nine CNBr peptides are demarcated and are designated CB1-CB9. The extra line indicates CB7, which contains CB6 and CB8 joined together. In a separate procedure 19 tryptic peptides, designated T1-T19, were obtained; some of these are overlapping. Subpeptides of the CNBr or tryptic peptides are not indicated in the diagram. Shaded areas denote positions for which the amino acid sequence was established in each procedure.
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Proc. Natl. Acad. Sci. USA 76 (1979)
10 20 30 H2N-Val -Phe-Asn-Pro-Arg-Arg-Lys -Leu- G1 u-Phe-A1 a- Leu- Leu-Phe- Leu-Val -Phe-Asp-Gl u-Asn-GI u-Ser-Trp-Tyr- Leu-Asp-Asp-Asn- I 1e- Lys-
40 50 60 Thr-Tyr-Ser-Asp-Hi s-Pro-G1 u-Lys-Val -Asn-Lys-Asp-Asp-Gl u-Gl u-Phe- Il e-Gl u-Ser-Asn-Lys-Met-Hi s-Ala-I le-Asn-Gly-Arg-Met-Phe70 80 90 Gly-Asn-Leu-Gl n-Gly-Leu-Thr-Met-Hi s-Val -Gly-Asp-Gl u-Val -Asn-Trp-Tyr-Leu-Met-Gly-Met-Gly-Asn-Gl u- Ile-Asp- Leu-Hi s-Thr-Val 100 110 120 Hi s-Phe-Hi s-Gly-Hi s- Ser-Phe-Gl n- Tyr-Lys -Hi s-Arg-Gly-Val -Tyr- Ser-Se r-Asp-Val -Phe-As p- Ile-Phe-Pro-Gly-Thr-Tyr-Gl n-Thr- Le u130 140 150 Gi u-Met-Phe-Pro-Arg-Thr-Pro-Gly- Ile -Trp-Leu-Leu-Ili s-Cys-Hi s-Val -Thr-Asp-Hi s-Ile-His-Ala-Gly-Met-G1u-Thr-Thr-Tyr-Thr-Val Leu-Gln-Asn-Gl u-Asp-Thr-Lys-Ser-Gly-COOH
FIG. 2. Amino acid sequence of the histidine-rich fragment of human Cp F5.
ceruloplasmin 20,000 Mr fragment would indicate that less than 10% of the peptide could be glycosylated." Expressed as moles of carbohydrate per 20,000 daltons, the fragment contained: 0.05 glucosamine, 0.12 galactose, 0.12 mannose, and 0.00 fucose. It is possible that Asn-20 is partially glycosylated and, although our calculation of the secondary structure of Cp F5 (see later) places Asn-20 at the end of an a helix, residues 20-23 also have a high probability of being in a (3turn. Since it appears likely that carbohydrate is frequently attached to glycoproteins at (3turns (15, 16), Asn-20 may be in a favorable position for glycosylation. However, the bulky tryptophan residue just after the triplet (i.e., Asn-Glu-Ser-Trp) may hinder glycosylation. Genetic Polymorphism. Genetic polymorphism of ceruloplasmin is rare in whites, but in black Americans a variant designated Cp A occurs with an allele frequency of 0.052, compared to a frequency of 0.939 for the common form Cp B and 0.003 and 0.006 for the rare variants Cp C and Cp NH, respectively (1, 3). In white Americans the frequency of Cp B is 0.994, whereas the frequency of Cp A is only 0.006, though Cp A frequency rises to a high of 0.149 in Nigerians (1). Other variants of ceruloplasmin have a very low incidence in all populations studied (1). Because blacks constitute about 11% of the American population, the incidence of Cp A and other known variants in pooled plasma collected by the American Red Cross must be less than 0.6%. Such a value is too low to be detected by the methodology of amino acid sequence analysis. We estimate that because of the cumulative errors in the methodology, genetic variants at an individual frequency of less than 0.05 in pooled plasma would not be detectable by amino acid sequence analysis. It is thus not surprising that we obtained a unique amino acid sequence for the fragment Cp F5. Furthermore, Cp F5 represents only a fragment of the whole ceruloplasmin molecule and may not be the site of any polymorphic substitution. However, one observation suggests to us the possibility that unidentified genetic variants not detectable by the usual method of electrophoretic screening may exist in the American population. In early work with Cp 2 (8), a different preparation of ceruloplasmin than the one used for complete sequence analysis (Cp 1), we obtained a CNBr fragment§ corresponding to the sequence given in Fig. 2 as Phe-Gly-Asn-Leu-Gln-GlyLeu-Thr-Met for positions 60-68; however, the composition of the fragment showed 0.67 residue of phenylalanine and 0.33 of tyrosine, and the dansyl method and sequenator analysis demonstrated both amino acids at the NH2 terminus. Yet, in the complete sequence study neither the CNBr fragment nor the tryptic peptide covering this region gave evidence of ty§ The nonapeptide also had copper.
a
bluish color and appeared to bind
rosine at position 60. We do not know whether the tyrosinecontaining peptide was lost during purification or if it was present only in the first preparation of ceruloplasmin. It is possible that a tyrosine-phenylalanine exchange at position 60 represents a true genetic polymorphism, but it is unlikely that the tyrosyl variant is Cp A because the latter differs in charge from Cp B. Our evidence for proteolytic degradation coupled with the observation of "gull-wing" patterns in immunoelectrophoresis of aged preparations suggests that some genetic variants previously reported may be artifacts. Poulik and Weiss (1) have discussed the questionable genetic significance of one reported genetic variant that has a band with identical electrophoretic mobility to one observed in aged serum that originally had only the Cp B type. Role of Histidine in Histidine-Rich Fragment. Although exceeded in histidine content by the "histidine-rich a2 glycoprotein" of human plasma (17), which has a histidine content of 9.9%, and by the a globin chains of some species that have 10 or more histidines per a chain, the Cp F5 fragment of human ceruloplasmin has a higher histidine content than most other known proteins (8.82%, calculated from the sequence). Furthermore, 7 of the 12 histidines occur in an alternating sequence of His-X-His-i.e., His-Phe-His-Gly-His, His-Cys-His, and His-Ile-His (beginning at positions 91, 133, and 139, respectively). This leads to two short segments that, when other nearby histidines are included, are exceedingly rich in histidine-i.e., there are four histidines in the eight-residue segment from His-88 to His-95 and four more in the nine-residue segment from His-l33 through His-141, which contains the free cysteine. In view of the role of histidine in the binding of copper by serum albumin (18), the proximity of so many histidines close to Cys-134, which is assumed to be a copper-binding site (8), is probably significant. Because of the alternation, all of the histidines could be exposed to the surface in the ( sheet postulated for residues 129-137 and the f turn following it. Prediction of Secondary Structure. The problems of predicting protein structure from amino acid sequence have recently been reviewed by Sternberg and Thornton (19). Nonetheless, they point out that "the method of Chou and Fasman (9, 10) has attracted most attention as it is simple to understand, can be applied without a computer, and has been relatively successful." Hence, we have applied their rules for predicting secondary structure, with the result depicted in Fig. 3. The model depicts a mixed a/,3 secondary structure. Almost one-third of the residues appear to be distributed in five a helices and about a third in ,B sheets. The remainder are in ( turns, coils, or disordered structures, including one segment of 15 residues (residues 100-115) that has no clear a or (3 initiation sites. The charged residues are concentrated at three sites: the
Biochemistry: Kingston et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
6 7
84
108
116 His 139
144 146 Met
120
123
Cys 134
152
159
FIG. 3. Diagram of the secondary structure of Cp F5 as predicted by the method of Chou and Fasman (9, 10). Residues are represented in helical (A.), ,3-sheet (A), and coil (-) conformational states. Chain reversals denote /-turn tetrapeptides (::.
NH2 terminus, the COOH terminus, and the two helices and turn in the segment from Asp-34 through Lys-51. There are several hydrophobic stretches, notably in the first helix from Phe-10 through Phe-17. The alternation of the histidines rea
lieves the hydrophobic character of the sheet beginning at Ile-129. Noncovalent Interaction of Cp F5 with Other Fragments. The several fragments present in commercial preparations interact strongly, and harsh dissociating conditions are required to separate them. Thus, the blue color and the copper are lost during the purification of Cp F5. The association is not due to disulfide bonds because ceruloplasmin preparations that exhibit multiple bands in polyacrylamide gel electrophoresis in 0. 1% sodium dodecyl sulfate give similar patterns before and after reduction although they sediment largely as single components with s2o = 6-7 S in analytical ultracentrifugation (8). The reason for the strong interaction of Cp F5 with the 53,000 Mr and 67,000 Mr fragments is not evident from the primary structure. However, one clue is offered by the model of the secondary structure given in Fig. 3; this illustrates the concentration of charged groups in certain segments of the structure, notably the NH2 terminus, the COOH terminus, and the two a helices and the ,B turn proposed for residues 34-55. Half the residues in the latter segment are either positively or negatively charged. If the largely hydrophobic structures from residues 55-144 are mainly in the interior of the molecule and the ionized residues are at the surface, as would be expected from the general rules for protein conformation (19), then the Cp F5 fragment would be capable of strong electrostatic interaction with the other
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fragments of ceruloplasmin. Probably this is its natural role, for it appears to be a separate domain structure of the intact molecule. Intersequence Comparison of Homology. We have proposed (8) that the cysteine-containing COOH-terminal sequence of Cp F5 may be involved in binding one of the two type 1 cupric ions in ceruloplasmin because the sequence from Gly-128 through Val-150 shows similarity to the cysteinecontaining COOH-terminal sequence of blue copper-containing azurins and plastocyanins, which contain a single type 1 copper ion. The cysteine, histidine, and methionine residues, which are invariant in the latter proteins and which have been implicated to be in the copper-binding site (8), could be matched by Cys-134, His-139, and Met-144 in Cp F5. This proposal has since been strengthened by the publication of the results of x-ray crystallographic studies of plastocyanin (20) and azurin (21). In both cases, the copper atom appears to be bound to the cysteine and methionine residues which were matched (8) with Cys-134 and Met-144 in Cp F5 and by two histidine residues one of which was matched with His-139. In our predicted secondary structure shown in Fig. 3, the spacial arrangement of Cys-134, His-139, and Met-144 would give them the potential to act as copper ligands. Furthermore, it is possible to interpret Chou and Fasman's rules for predicting secondary structure in a manner (L.-C. Lin, personal communication) that predicts that Cp F5 will be composed of two a-helical regions at the NH2 terminus and six f-sheet strands separated into two groups of three by an intervening a-helical or random region; these elements could then fold up to produce a structure rather similar to the cylindrical barrel shape formed largely by strands of 3 sheet in poplar plastocyanin (20) and Pseudomonas aeruginosa azurin (21). To search for any additional similarity to other proteins, we requested the National Biomedical Research Foundation to undertake a computer search comparing the sequence of Cp F5 with all sequence data available, not only for the blue copper-containing proteins, but also for all other proteins. With the program RELATE (22), ceruloplasmin was compared to itself and to plastocyanin, azurin, and superoxide dismutase, by using a segment length of 15. In all cases the comparison of real sequences gave a score less than 1 SD from the mean score with 100 randomized sequences. Under the same conditions, azurin against plastocyanin gives a score of 3.9 SD. The ceruloplasmin was rerun against itself by using a segment length of 25. The score was 1.8 SD, which is interesting though not statistically significant (Winona C. Barker, personal communication). However, as we had earlier identified by visual comparison of the segments from 1-53, 54-83, and 108-137, there is an indication that the chain had duplicated. In the computer search, 19 of the top 20 scores came from displacing the sequence by 53 residues. Two 25-residue segments of ceruloplasmin (Met-79 through Gly-i1S, which has six histidioes, and Pro-127 through Leu-151, which contains the cysteine) were searched against the entire data collection of the Atlas of Protein Sequence and Structure (95,365 comparisons). The highest scoring segments retrieved were from a variety of proteins. Nothing stood out significantly from the rest. The cysteine-containing segment of the azurins gave a good score, but the highest scores included a series of ferredoxins and the a and .B globins of many species. The globins and Cp F5 have a high histidine content in common. The ferredoxins are of interest because the highest scoring segment is at the COOH terminus of both Cp F5 and ferredoxin, and the matching residues are Trp-130, Leu-132, and Cys-134, all close to the site proposed for copper binding in Cp F5.
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We thank J. Dwulet, A. Galen, J. Madison, and S. Dorwin for technical assistance, Dr. J. Baenziger for carbohydrate analysis, and Dr. W. C. Barker for computer analysis of the sequence homology. This work was supported by National Institutes of Health Grant AM 19221. 1. Poulik, M. D. & Weiss, M. L. (1975) in The Plasma Proteins, ed. Putnam, F. W. (Academic, New York), 2nd Ed., Vol. 2, pp. 51-108. 2. Frieden, E. & Hsieh, H. S. (1976) Adv. Exp. Med. Biol. 74, 505-529. 3. Gitlin, D. & Gitlin, J. D. (1975) in The Plasma Proteins, ed. Putnam, F. W. (Academic, New York), 2nd Ed., Vol. 2, pp. 321-374. 4. Simons, K. & Bearn, A. G. (1969) Biochim. Blophys. Acta 175, 260-270. 5. Freeman, S. & Daniel, E. (1973) Biochemistry 12,4806-4810. 6. McCombs, M. L. & Bowman, B. H. (1976) Biochim. Biophys. Acta 434,452-461. 7. Ryden, L. (1972) Eur. J. Biochem. 26,380-386. 8. Kingston, I. B., Kingston, B. L. & Putnam, F. W. (1977) Proc. Natl. Acad. Sc$. USA 74,5377-5381. 9. Chou, P. Y. & Fasman, G. D. (1974) Biochemistry 13, 211245.
Froc. Natl. Acad. Sci. USA 76 (1979) 10. Chou, P. Y. & Fasman, G. D. (1978) Annu. Rev. Biochem. 47, 251-276. 11. Sgouris, J. T., Coryell, F. C., Gallick, M., Storey, R. W., McCall, K. B. & Anderson, H. D. (1962) Vox Sang. 7,394-405. 12. Ryden, L. & Eaker, D. (1974) Eur. J. Biochem. 44, 171-180. 13. Egorov, T. A., Svenson, A., Ryden, L. & Carlsson, J. (1975) Proc. NatI. Acad. Sci. USA 72,3029-3033. 14. Clamp, J. R. (1975) in The Plasma Proteins, ed. Putnam, F. W. (Academic, New York), 2nd Ed., Vol. 2, pp. 163-211. 15. Aubert, J. P., Biserte, G. & Loucheux-Lefebvre, M. H. (1976) Arch. Biochem. Biophys. 175,410-418. 16. Huber, R., Deisenhofer, J., Colman, P. M., Matsushima, M. & Palm, W. (1976) Nature (London) 264,415-420. 17. Heimburger, N., Haupt, H., Kranz, T. & Baudner, S. (1972) Hoppe-Seyler's Z. Physiol. Chem. 353, 1133-1140. 18. Peters, T., Jr. (1975) in The Plasma Proteins, ed. Putnam, F. W. (Academic, New York), 2nd Ed., Vol. 1, pp. 133-181. 19. Sternberg, M. J. E. & Thornton, J. M. (1978) Nature (London) 271, 15-20. 20. Colman, P. M., Freeman, H. C., Guss, J. M., Murata, M., Norris, V. A., Ramshaw, J. A. M. & Venkatappa, M. P. (1978) Nature (London) 272, 319-324. 21. Adman, E. T., Stenkamp, R. E., Sieker, L. C. & Jensen, L. H. (1978) J. Mol. Biol. 123,35-47. 22. Barker, W. C., Ketcham, L. K. & Dayhoff, M. 0. (1978) J. Mol. Evol. 10, 265-281.
