Proline-rich cell surface antigens of horseshoe crab hemocytes are substrates for protein cross-linking with a clotting protein coagulin.
Monoclonal antibodies were raised against hemocytes of the horseshoe crab Tachypleus tridentatus. All of the antibodies obtained reacted with the same protein bands on SDS-PAGE of hemocyte lysate. Flow cytometry and biotinylation of surface substances on the hemocytes indicated that the antigens are major peripheral proteins of hemocytes. The antigens were purified from hemocyte lysate and were good substrates for the horseshoe crab hemocyte transglutaminase (HcTGase). Transglutaminases play an important role during the final stage of blood coagulation in mammals and crustaceans. Although HcTGase did not intermolecularly cross-link a clottable protein coagulogen or its proteolytic product coagulin, HcTGase promoted the cross-linking of coagulin with the surface antigens, resulting in the formation of a stable polymer. We determined the nucleotide sequences for two isoproteins of the antigens. The two proteins containing 271 and 284 residues (66% identity) were composed of tandem repeats of proline-rich segments. We named them proxins-1 and -2 after proline-rich proteins for protein cross-linking. Proxins may form a stable physical barrier against invading pathogens in cooperation with hemolymph coagulation at injured sites.
Department of Biology, Faculty of Sciences, Kyushu University, Fukuoka 812-8581, Japan.
Tsukasa
T
Osaki
Nozomu
N
Okino
Fuminori
F
Tokunaga
Sadaaki
S
Iwanaga
Shun-Ichiro
S
Kawabata
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D019921:Biotinylation; D001777:Blood Coagulation; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D015723:Gene Library; D006434:Hemocytes / Q000378:metabolism*; D006458:Hemolymph / Q000378:metabolism; D006737:Horseshoe Crabs; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011392:Proline / Q000737:chemistry*; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013925:Thromboplastin / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D013925:Thromboplastin; D011392:Proline