抄録
Thrombin is a multifunctional serine protease in haemostasis. It is a two-chain glycoprotein containing an Asn53-binding carbohydrate chain, consisting of lactosamin and N-acetyl neuraminic acid (NeuAc). The glycosylated site is located in the insertion loop Lue45-Asn57 near the catalytic site cleft. However, the function of the carbohydrate chain remains unknown. In this report, the enzymatic property of the carbohydrate chain moiety-modified thrombin was compared to that of intact thrombin in order to clarify the function of the carbohydrate chain. The carbohydrate chain, especially NeuAc at its non-reductive terminal was chemically modified by PDBA-hydrazide. The intact thrombin ([M+H]+=37,494, measured by MALDI/TOF mass spectrometer) released fibrinopeptide A and B during fibrin clot formation. The PDBA-modified thrombin ([M+H]+=38,012) formed fibrin clot, however, fibrinopeptide A was primarily released while little fibrinopeptide B was released. The result led us to propose that the access of the fibrinogen Bβ chain to the catalytic site cleft was restricted by the modification of the carbohydrate chain. This chain played an important role in the protease activity, especially the interaction between thrombin and macromolecular substrate such as fibrinogen. The chemical modification of NeuAc of the carbohydrate chain may cause the structural alteration of the catalytic site cleft of thrombin. [Jpn J Physiol 54 Suppl:S114 (2004)]
