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 a chain of human ceruloplasmin studied by McCombs and determined for a fragment of human ceruloplasmin [ferroxidase; Bowman (6) and probably corresponds to the a subunit pro- iron(II)oxygen oxidoreductase, EC 1.16.3.1]. The fragment posed by Simons and Bearn (4) and the chain of (designated Cp F5) contains 159 amino acid residues and has light Freeman a molecular weight of 18,650; it lacks carbohydrate, is rich in and Daniel (5). histidine, and contains one free cysteine that may be part of a The relation of Cp F5 to the structure of the intact cerulo- copper-binding site. This fragment is present in most commer- plasmin chain has to be deduced from indirect observations cial preparations of ceruloplasmin, probably owing to proteo- because of the small amount of single-chain ceruloplasmin lytic degradation, but can also be obtained by limited cleavage available to us and the strong interaction of the fragments. From of single-chain ceruloplasmin with plasmin. Cp F5 probably is the kinetics of the proteolytic cleavage of intact ceruloplasmin an intact domain attached to the COOH-terminal end of sin- gle-chain ceruloplasmin via a labile interdomain peptide bond. and the chemical properties of the fragments, we have sug- A model of the secondary structure predicted by empirical gested that Cp F5 is from the COOH terminus of the single methods suggests that almost one-third of the amino acid resi- chain (8). We have obtained a unique amino acid sequence and dues are distributed in a helices, about a third in ,8-sheet conclude that Cp F5 is probably an intact domain; it appears structure, and the remainder in ft turns and unidentified struc- to be attached to the COOH-terminal end of the ceruloplasmin tures. Computer analysis of the amino acid sequence has not chain by a labile interdomain peptide bond. Prediction of the demonstrated a statistically significant relationship between this ceruloplasmin fragment and any other protein, but there secondary structure by the empirical method of Chou and is some evidence for an internal duplication. 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 Ceruloplasmin [ferroxidase; iron(II):oxygen oxidoreductase, j turns and other structure. EC 1.16.3.1], is a blue, copper-containing a2-glycoprotein that Because the ceruloplasmin preparation studied is derived is normally present in human plasma at a concentration of from a pool of plasma from more than 10,000 donors from the 20-40 mg/100 ml (1). Although the biological role of cerulo- ethnically mixed American population, the finding of a unique plasmin (Cp) is not entirely clear, at least three functions have amino acid sequence indicates that the frequency of genetic been ascribed: ferroxidase activity, copper transport and de- polymorphism is too low to interfere with sequence determi- toxication, and maintenance of copper homeostasis in the nation of this fragment. tissues. These functions are not mutually exclusive, but the most In a previous report (8) we identified a cysteine-containing important is thought to be the action of plasma ceruloplasmin sequence in human Cp F5 that appeared similar to a series of as a ferroxidase in oxidizing ferrous iron to the ferric form cysteine-containing sequences that are apparently homologous which is then incorporated into transferrin (2). The only es- to each other and contain part of the copper-binding sites in tablished biochemical abnormality involving ceruloplasmin is bacterial azurins and plant plastocyanins. Computer analysis its deficiency in Wilson disease (hepatolenticular degeneration). of an intersequence comparison of Cp F5 and azurin from Al- The deficiency is due to a genetic defect in the rate of cerulo- caligenes sp. indeed showed that the most similar segment of plasmin synthesis that leads to abnormal copper metabolism sequence was that which we had identified. However, no sta- and deposition of copper in the tissues (3). tistically significant relationship could be demonstrated be- Despite several reports that it has a subunit structure (1, 4-6), tween the ceruloplasmin fragment and any other protein, in- ceruloplasmin has been shown to be a single polypeptide chain cluding azurin, plastocyanin, and superoxide dismutase, all of with a molecular weight of about 130,000 that is readily cleaved which are copper-containing proteins. Further computer to large fragments by proteolytic enzymes (7, 8). We have iso- analysis indicated a possible internal duplication in the ceru- lated and characterized three fragments of ceruloplasmin that loplasmin. appear to be nonoverlapping and that have approximate mo- lecular weights of 20,000, 53,000, and 67,000 (8). We report MATERIALS AND METHODS here the complete amino acid sequence of the smallest frag- Materials. The purified human ceruloplasmin from normal ment, which we call the histidine-rich fragment and designate pooled sera used for sequence analysis was a preparation made Cp F5. This fragment contains 159 amino acid residues and by the method of Sgouris et al. (11), starting with ethanol lacks carbohydrate. It is present to a varying degree in all the fraction IV-1, and was generously provided by J. J. Hagan commercial preparations we examined and can be prepared from single-chain ceruloplasmin by digestion with plasmin (8). Abbreviation: Cp, ceruloplasmin. Cp F5 is identical in NH2-terminal sequence to the so-called * Present address: National Institute of Agricultural Botany, Hun- tingdon Road, Cambridge, England. The publication costs of this article were defrayed in part by page t Present address: Commonwealth Bureau of Plant Breeding and charge payment. This article must therefore be hereby marked "ad- Genetics, Department of Applied Biology, Pembroke St., Cambridge, vertisement" in accordance with 18 U. S. C. §1734 solely to indicate England. this fact. t To whom reprint requests should be addressed. 1668 Downloaded by guest on September 25, 2021 Biochemistry: Kingston et al. Proc. Natl. Acad. Sci. USA 76 (1979) 1669 (Squibb). This was preparation Cp 1 of our previous paper (8); (which has specificity for peptide bonds having aspartic or several other preparations described there were used for ref- glutamic acid on the carboxyl side) was used to prepare sub- erence purposes, including single-chain ceruloplasmin (Cp 3 peptides of tryptic peptides T3 and T4 and also T6. In addition, and Cp 5) supplied by Yu lee Hao (National Fractionation Cp F5 was citraconylated and digested with trypsin to give a Laboratory of the American National Red Cross). peptide covering the segment from Lys-7 through Arg-58. Purification of Histidine-Rich Fragment Cp F5. Fragment Sequence analysis of these peptides permitted completion of Cp F5 was prepared from Cp 1 after reduction and ami- the missing area. noethylation. Cp 1 (420 mg) was dissolved in a buffer (20 ml) containing 6 M guanidine-HCI, 2 mM EDTA, 0.5 M Tris-HCI, RESULTS AND DISCUSSION at pH 8.0, and reduced under N2 at 50'C with dithiothreitol Amino Acid Sequence. The complete amino acid sequence (360 mg). After cooling to 4°C, three aliquots (80 Al each) of of the human ceruloplasmin fragment Cp F5 derived by the ethylene imine (Pierce) were added at 10-min intervals. The strategy described above is given in Fig. 2. Except for the solution was then dialyzed against deionized water (4 liters, abundance of histidine, there is nothing remarkable about the three times) and lyophilized. The reduced aminoethylated primary structure. The single cysteine residue (Cys-134) must protein was dissolved in 10 ml of 6 M urea/0.2 M formic acid be present in the sulfhydryl form because reduction made no and applied to a column (150 X 4 cm) of Sephadex G-150 or difference in the banding pattern of the original ceruloplasmin G-200 equilibrated with the same solution. The column effluent preparation on electrophoresis in polyacrylamide gel containing was monitored at 280 nm, and good separation of Cp F5 was 0.1% sodium dodecyl sulfate (8). obtained. After volume reduction by ultrafiltration, the frag- The NH2-terminal sequence of Cp F5 is identical to the ment was desalted on Sephadex G-15 in 1 M acetic acid and 21-residue NH2-terminal sequence given by McCombs and Iyophilized. Bowman (6) for a ceruloplasmin polypeptide they designated Methods. The methods for protein characterization, amino a chain. With the exception of this and of three glycopeptides acid analysis, and sequence determination were the same as in from human ceruloplasmin (12), no other sequence data are our previous report (8). published on ceruloplasmin from any species. However, the Strategy for Sequence Determination. In addition to au- amino acid composition of a cysteine-containing peptide iso- tomated sequence analysis of the intact Cp F5 fragment for 18 lated by Egorov et al. (13) from undegraded human cerulo- steps, the strategy for sequence determination involved two plasmin corresponds to the sequence around Cys-134. different routes in order to obtain independent verification of Fragment Cp F5 has previously been referred to by us (8) each position and to secure all necessary overlaps of peptides. as the 20,000 Mr fragment because that was the molecular Because of the presence of seven methionine residues, CNBr weight estimated from polyacrylamide gel electrophoresis. cleavage was chosen for the initial procedure.