Marine invertebrates demonstrate a rich diversity
of form and function of their oxygen transport proteins, reflecting
how the evolution of new proteins resulting from gene duplications
and subsequent mutations is an important process in the adaptive
ability of organisms. The hemocyanin gene family provides a
fertile opportunity to explore new proteins with new functions,
and we are currently studying three related proteins that play
separate and significant roles in the developmental physiology
of the Dungeness crab, Cancer magister. We are interested in
understanding the differential expression and functions of these
proteins during crustacean development and molting as well as
in response to environmental factors including temperature,
salinity and oxygen availability.
Hemocyanin reversibly binds oxygen at its highly conserved
copper-oxygen binding sites and supplies the crab tissues
with oxygen. Our studies on expression of hemocyanin subunits
from megalopa to adult crab have indicated that there are
dramatic changes in subunit composition and oxygen affinity.
These changes are correlated with developmental changes in
salinity tolerance and ion regulation. We are currently investigating
the role of hypoxia in hemocyanin expression in crustaceans
at both the cellular and the organismal level. Cryptocyanin
closely resembles hemocyanin in sequence, but it lacks several
critical copper-binding amino acids and has lost its ability
to bind oxygen. Its presence in high concentrations, particularly
at specific times in the molt cycle, suggests it has been
exploited to carry out new functions. We are examining changes
in expression of cryptocyanin mRNA and protein during the
molt cycle and the role of cryptocyanin in forming a new exoskeleton.
Additionally, we are studying the dynamics of synthesis of
hemocyanin and cryptocyanin at the molecular, tissue and organismal
level, including differential responses to hormonal regulation.
Phenoloxidase, a third member of this protein family, functions
in sclerotization of the new exoskeleton after molting as
well as in immune defense. Its copper-oxygen binding sites
are similar to those of hemocyanin, but it is an intracellular
protein, found in circulating hemocytes. Hemocyanin itself
has phenoloxidase activity, along with potential anti-microbial
activity, which suggests additional functions in response
to environmental stressors. Sequence comparisons at the mRNA
and genomic level are underway as part of our continuing investigations
into the evolutionary and functional relationships among these
proteins.
Selected Publications
Burnett, L., Terwilliger, N., Carroll, A., Jorgensen, D.
and Scholnick, D. (2002) Respiratory and acid-base physiology
of the purple sea urchin, Strongylocentrotus purpuratus, during
air exposure: Presence and function of a facultative lung.
Biol. Bull. 203, 42-50.
Terwilliger, N. B. and Ryan, M. (2001) Ontogeny of Crustacean
Respiratory Proteins. Am. Zool. 41, 1057-1067.
Decker, H., Ryan, M., Janeicke, E. and Terwilliger, N. (2001)
SDS induced phenoloxidase activity of hemocyanins from Limulus
polyphemus, Eurypelma californicum and Cancer magister. J.
Biol. Chem. 276, 17796-17799.
Terwilliger, N.B. and Dumler, K. (2001) Ontogeny of decapod
crustacean hemocyanin: Effects of temperature and nutrition.
J. Exp. Biol. 204, 1013-1020.
Decker, H. and Terwilliger, N.B. (2000) COPs and robbers:
putative evolution of copper oxygen binding proteins. J. exp.
Biol. 203, 1777-1782.
Terwilliger, N.B., Dangott, L., and Ryan, M. (1999) Cryptocyanin,
a crustacean molting protein: Evolutionary link with arthropod
hemocyanins and insect hexamerins. Proc. Natl. Acad. Sci.
USA, 96, 2013-2018.
Terwilliger, N.B. (1999) Hemolymph proteins and molting in
crustaceans and insects. Amer. Zool. 39, 589-599.
Brown, A.C. and Terwilliger, N.B. (1999) Developmental changes
in oxygen uptake in Cancer magister (Dana) in response to
changes in salinity and temperature. J. Exp. Mar. Biol. Ecol.
241, 179-192.
Terwilliger, N.B. (1998) Functional adaptations of oxygen-transport
proteins. J. exp. Biol. 201, 1085-1098.
Brown, A.C. and Terwilliger, N.B. (1998) Ontogeny of hemocyanin
function in the Dungeness crab, Cancer magister II. Hemolymph
modulation of hemocyanin oxygen binding. J. exp. Biol. 201,
819-826.
Durstewitz G. and Terwilliger, N.B. (1997) Developmental changes
in hemocyanin expression in the Dungeness crab, Cancer magister.
J. Biol. Chem. 272, 4347-4350.
Durstewitz G. and Terwilliger, N.B. (1997) cDNA cloning of
a developmentally regulated hemocyanin in the crustacean Cancer
magister and phylogenetic analysis of the hemocyanin gene
family. Mol. Biol. and Evolution 14(3): 266-276.
Terwilliger, N.B. and Durstewitz, G.D. (1996) Molecular studies
of the sequential expression of a respiratory protein during
crustacean development. In "Molecular Zoology: Advances,
Strategies and Protocols", ed. Ferraris J. and Palumbi
S. (Wiley-Liss, New York), pp. 353-368.
Baker, S.M. and Terwilliger, N.B. (1993) Hemoglobin structure
and function in the rat tailed sea cucumber, Paracaudina chilensis.
Biol. Bull. 185, 115-122.
Terwilliger, N.B. and Brown, A.C. (1993) Ontogeny of hemocyanin
function in the Dungeness crab, Cancer magister: The interactive
effects of developmental stage and divalent cations on hemocyanin
oxygenation properties. J. exp. Biol. 183, 1-13.
Brown, A.C. and Terwilliger, N.B. (1992) Developmental changes
in ionic and osmotic regulation in the Dungeness crab, Cancer
magister. Biological Bulletin 182:270-277.
