Structural features and functional domains of amassin-1, a cell-binding olfactomedin protein.
Amassin-1 mediates a rapid cell adhesion that tightly adheres sea urchin coelomocytes (body cavity immunocytes) together. Three major structural regions exist in amassin-1: a short beta region, 3 coiled coils, and an olfactomedin domain. Amassin-1 contains 8 disulfide-bonded cysteines that, upon reduction, render it inactive. Truncated forms of recombinant amassin-1 were expressed and purified from Pichia pastoris and their disulfide bonding and biological activities investigated. Expressed alone, the olfactomedin domain contained 2 intramolecular disulfide bonds, existed in a monomeric state, and inhibited amassin-1-mediated clotting of coelomocytes by a calcium-dependent cell-binding activity. The N-terminal beta region, containing 3 cysteines, was not required for clotting activity. The coiled coils may dimerize amassin-1 in a parallel orientation through a homodimerizing disulfide bond. Neither amassin-1 fragments that were disulfide-linked as dimers or that were engineered to exist as dimers induced coelomocytes clotting. Clotting required higher multimeric states of amassin-1, possibly tetramers, which occurred through the N-terminal beta region and (or) the first segment of coiled coils.
Hillier
Brian J
BJ
Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA 92093-0202, USA.
Vacquier
Victor D
VD
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D015815:Cell Adhesion Molecules; D004220:Disulfides; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D011994:Recombinant Proteins; C081451:olfactomedin; D002118:Calcium