Proc. Natl. Acad. Sci. USA Vol. 96, pp. 2013–2018, March 1999 Biochemistry Cryptocyanin, a crustacean molting protein: Evolutionary link with arthropod hemocyanins and insect hexamerins (phenoloxidaseyEcdysozoa) NORA B. TERWILLIGER*†,LAWRENCE DANGOTT‡, AND MARGARET RYAN* *Oregon Institute of Marine Biology, University of Oregon, Charleston, OR 97420 and Department of Biology, University of Oregon, Eugene, OR 97403; and ‡Department of Chemistry, Texas A&M University, College Station, TX 77801 Edited by K. E. van Holde, Oregon State University, Corvallis, OR, and approved December 10, 1998 (received for review September 14, 1998) ABSTRACT Cryptocyanin, a copper-free hexameric pro- known as larval serum proteins or storage proteins because of tein in crab (Cancer magister) hemolymph, has been charac- their high concentrations in larval stages and incorporation terized and the amino acid sequence has been deduced from into new body structures, including cuticle, during metamor- its cDNA. It is markedly similar in sequence, size, and phosis or nonfeeding periods of adult development (6, 8, 9). structure to hemocyanin, the copper-containing oxygen- Although these insect hemolymph proteins lack a binuclear transport protein found in many arthropods. Cryptocyanin copper-binding site, a common ancestral molecule for the does not bind oxygen, however, and lacks three of the six highly copper-containing hemocyanins and the copperless hexam- conserved copper-binding histidine residues of hemocyanin. erins has been postulated based on sequence similarities, Cryptocyanin has no phenoloxidase activity, although a phe- subunit size, hexameric shape, and a conserved exon–intron noloxidase is present in the hemolymph. The concentration of boundary (7, 10–14). cryptocyanin in the hemolymph is closely coordinated with the We have found a protein in crustacean blood that reaches molt cycle and reaches levels higher than hemocyanin during hemolymph concentrations even greater than hemocyanin premolt. Cryptocyanin resembles insect hexamerins in the during each molt cycle of C. magister and disappears from the lack of copper, molt cycle patterns of biosynthesis, and hemolymph during intermolt. It is so structurally similar to potential contributions to the new exoskeleton. Phylogenetic hemocyanin that early studies often overlooked it. Because this analysis of sequence similarities between cryptocyanin and protein masquerades as hemocyanin in structure but neither other members of the hemocyanin gene family shows that combines reversibly with oxygen nor has the 340 nm absor- cryptocyanin is closely associated with crustacean hemocya- bance maximum characteristic of oxyhemocyanin, we have nins and suggests that cryptocyanin arose as a result of a named it cryptocyanin. A ‘‘nonrespiratory protein’’ had been hemocyanin gene duplication. The presence of both hemocy- noted previously in hemolymph of several crustaceans and anin and cryptocyanin in one animal provides an example of chelicerates (15–21) but has not been fully characterized. A how insect hexamerins might have evolved from hemocyanin. large protein like cryptocyanin present in such high concen- Our results suggest that multiple members of the hemocyanin trations in the circulation must have profound effects on many gene family—hemocyanin, cryptocyanin, phenoloxidase, and aspects of arthropod physiology including cardiovascular func- hexamerins—may participate in two vital functions of molting tion and ion regulation. It may also participate in forming the animals, oxygen binding and molting. Cryptocyanin may new exoskeleton during the molt, similar to some insect provide important molecular data to further investigate evo- hexamerins. Could this crustacean protein resolve the evolu- lutionary relationships among all molting animals. tionary link between the oxyhemocyanins and the insect hexamerins? Another arthropod protein, phenoloxidase, has recently Hemocyanin, the blue copper-containing oxygen-transport been shown to have a close structural relationship to hemo- molecule, usually is described as the predominant protein in cyanin (22–25). Arthropod prophenoloxidases contain the the hemolymph of many crustaceans. It has been reported to . copper A (CuA) and copper B (CuB) binding sites of arthro- contribute 90% of the total hemolymph protein. The prop- pod hemocyanins, and they share sequence similarities with erties of this large extracellular oligomer, found in three classes arthropod hemocyanins and hexamerins. Proteins with phe- of Arthropoda (Crustacea, Chelicerata, and Myriapoda), have noloxidase (tyrosinase) activity are widely distributed among been extensively studied for many years, as have those of the animals, plants, fungi, and prokaryotes (26). They incorporate structurally distinct but functionally similar molluscan hemo- oxygen into other molecules, converting monophenols to cyanin (for review, see refs. 1–3). Cancer magister, the Dunge- o-diphenols and diphenols to the corresponding o-quinones. In ness crab, contains a typical arthropod hemocyanin. Its six ' ' arthropods, phenoloxidases function in defense responses and heterogeneous subunits, ranging from 82 to 67 kDa, as- in exoskeleton sclerotization at ecdysis. Molluscan hemocya- semble into two distinct populations of molecules, 16S hex- nins have phenoloxidase activity (27, 28), as do arthropod amers and 25S two-hexamers (4, 5), that are not in association– hemocyanins, although the latter must be activated by partial dissociation equilibria with one another. Each subunit con- unfolding or proteolysis (refs. 29 and 30; N.B.T., H. Decker, tains one binuclear copper site and combines reversibly with and M.R., unpublished data). Some crustaceans and insects one molecule of oxygen. have a separate prophenoloxidase in hemolymph andyor blood Insects lack a functional hemocyanin. This class of Ar- cells, which must be activated to phenoloxidase via a series of thropoda does have other hemolymph proteins, the hexam- erins, whose quaternary structures resemble hexameric hemo- This paper was submitted directly (Track II) to the Proceedings office. cyanin (for review, see refs. 6 and 7). Some hexamerins are also Abbreviations: CuA and CuB, copper binding sites in arthropod hemocyanins. The publication costs of this article were defrayed in part by page charge Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AF091261). payment. This article must therefore be hereby marked ‘‘advertisement’’ in †To whom reprint requests should be addressed at: Oregon Institute accordance with 18 U.S.C. §1734 solely to indicate this fact. of Marine Biology, Boat Basin Drive, Charleston, OR 97420. e-mail: PNAS is available online at www.pnas.org. [email protected]. 2013 Downloaded by guest on October 1, 2021 2014 Biochemistry: Terwilliger et al. Proc. Natl. Acad. Sci. USA 96 (1999) proteolytic reactions (31, 32). Does cryptocyanin exhibit any razoliumy5-bromo-4-chloro-3-indolyl phosphate (NBTyBCIP; phenoloxidase activity? Zymed). In this study, we have characterized purified cryptocyanin of Amino Acid Sequence Analysis. Cryptocyanin was further C. magister, determined its cDNA sequence, and compared its purified after BioGel A-1.5m chromatography by C4 reverse derived amino acid sequence with those of arthropod hemo- phase HPLC. HPLC-purified cryptocyanin and two cyanogen cyanins, hexamerins, and prophenoloxidases to explore their bromide fragments were submitted to automated Edman structural, functional, and evolutionary relationships. We have degradation on an Applied Biosystems Model 477A modified also begun to investigate the expression of cryptocyanin gas-phase sequenator equipped with an on-line Applied Bio- throughout the molt cycle of juvenile and adult crabs. We find systems 120A PTH amino acid analyzer (Worcester Founda- a close synchrony between stage of molt cycle and hemolymph tion Protein Sequencing Center). SDSyPAGE-purified cryp- levels of cryptocyanin that suggests that cryptocyanin is a tocyanin band 1 was excised from Immobilon membranes after hormonally regulated molting protein. Thus, cryptocyanin and electroblotting and sequenced on an Applied Biosystems related molecules may provide important molecular data to Model 470A liquid-phase protein sequenator and an Applied further investigate the monophyly of the newly proposed clade Biosystems Model 120 PTH Analyzer (University of Oregon of molting animals, the Ecdysozoa (33), in addition to enhanc- Biotechnology Laboratory). ing our understanding of the molecular phylogeny of the PCR Amplification of Cryptocyanin cDNA. Hypodermis, hemocyanins. muscle, and hepatopancreas tissues from C. magister were dissected and immediately frozen in liquid nitrogen. RNA was MATERIALS AND METHODS isolated by using the SNAP total RNA isolation kit (Invitro- gen). Reverse transcription–PCR was carried out as in ref. 39. Animals. Adult and juvenile C. magister (Dana) were col- Degenerate PCR primers were designed based on the cryp- lected from Coos Bay, Oregon, and maintained in running tocyanin N-terminal and CNBr fragment amino acid se- seawater at ambient temperature and salinity at the Oregon quences. The sense primer was 59-CGG-ATC-CGA-TyCGA- Institute of Marine Biology. GyACC-AyGyTyCGA-TyCGG-AyGyTyCG-39; the antisense Protein Purification. Fresh hemolymph was purified by primer was 59-GGA-ATT-CCG-AGyAyCA-GyAAyGG- BioGel A-1.5m column chromatography as in ref. 19. Fractions GGA-TGA-ACyAyGA-C-39. These primers allowed