A cell-surface superoxide dismutase is a binding protein for peroxinectin, a cell-adhesive peroxidase in crayfish.
Peroxinectin, a cell-adhesive peroxidase (homologous to human myeloperoxidase), from the crayfish Pacifastacus leniusculus, was shown by immuno-fluorescence to bind to the surface of crayfish blood cells (haemocytes). In order to identify a cell surface receptor for peroxinectin, labelled peroxinectin was incubated with a blot of haemocyte membrane proteins. It was found to specifically bind two bands of 230 and 90 kDa; this binding was decreased in the presence of unlabelled peroxinectin. Purified 230/90 kDa complex also bound peroxinectin in the same assay. In addition, the 230 kDa band binds the crayfish beta-1,3-glucan-binding protein. The 230 kDa band could be reduced to 90 kDa, thus showing that the 230 kDa is a multimer of 90 kDa units. The peroxinectin-binding protein was cloned from a haemocyte cDNA library, using immuno-screening or polymerase chain reaction based on partial amino acid sequence of the purified protein. It has a signal sequence, a domain homologous to CuZn-containing superoxide dismutases, and a basic, proline-rich, C-terminal tail, but no membrane-spanning segment. In accordance, the 90 and 230 kDa bands had superoxide dismutase activity. Immuno-fluorescence of non-permeabilized haemocytes with affinity-purified antibodies confirmed that the crayfish CuZn-superoxide dismutase is localized at the cell surface; it could be released from the membrane with high salt. It was thus concluded that the peroxinectin-binding protein is an extracellular SOD (EC-SOD) and a peripheral membrane protein, presumably kept at the cell surface via ionic interaction with its C-terminal region. This interaction with a peroxidase seems to be a novel function for an SOD. The binding of the cell surface SOD to the cell-adhesive/opsonic peroxinectin may mediate, or regulate, cell adhesion and phagocytosis; it may also be important for efficient localized production of microbicidal substances.
Department of Physiological Mycology, Evolutionary Biology Centre, University of Uppsala, Villavägen 6, SE-75236 Uppsala, Sweden.
M W
MW
Johansson
T
T
Holmblad
P O
PO
Thörnqvist
M
M
Cammarata
N
N
Parrinello
K
K
Söderhäll
D000595:Amino Acid Sequence; D000818:Animals; D003400:Astacoidea; D001483:Base Sequence; D001798:Blood Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D017124:Conserved Sequence; D018076:DNA, Complementary; D006434:Hemocytes / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009195:Peroxidase / Q000378:metabolism; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001798:Blood Proteins; D002352:Carrier Proteins; D015815:Cell Adhesion Molecules; D018076:DNA, Complementary; D011994:Recombinant Proteins; C096867:peroxinectin protein, Pacifastacus leniusculus; D009195:Peroxidase; D013482:Superoxide Dismutase