Characterization of monoclonal antibodies against human protein C specific for the calcium ion-induced conformation or for the activation peptide region

AbstractThree monoclonal antibodies have been produced that are specific for the activation peptide region m human protein C. These antibodies inhibited the activation of protein C by thrombin and by the thrombin-thrombomodulin complex. A fourth monoclonal antibody specifically recognized the Ca2+-stabilized conformation in protein C. This antibody bound both intact protein C and protein C from which the γ-carboxy-glutamic acid-containing region had been removed by limited proteolysis. These results indicate that this antibody recognizes the conformation in protein C stabilized by Ca2+ bound to the single binding site that is independent of γ-carboxyglutamic acid

Novel γ-carboxyglutamic acid-containing peptides from the venom of Conus textile

Author Posting. © The Author(s), 2006. This is the author's version of the work. It is posted here by permission of Blackwell for personal use, not for redistribution. The definitive version was published in FEBS Journal 273 (2006): 2779-2788, doi:10.1111/j.1742-4658.2006.05355_1.x.The cone snail is the only invertebrate system in which the vitamin K dependent carboxylase (or γ-carboxylase) and its product γ-carboxyglutamic acid (Gla)1 have been identified. It remains the sole source of structural information of invertebrate γ-carboxylase subtrates. Four novel γ- carboxyglutamic acid (Gla)1 containing peptides were purified from the venom of Conus textile and characterized by biochemical methods and mass spectrometry. The peptides Gla(1)-TxVI, Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI each have 6 Cys residues and belong to the O-superfamily of conotoxins. All four conopeptides contain 4-trans-hydroxyproline and the unusual amino acid 6-L-bromotryptophan. Gla(2)-TxVI/A and Gla(2)- TxVI/B are isoforms with an amidated C-terminus that differ at positions +1 and +13. Three isoforms of Gla(3)-TxVI were observed that differ at position +7: Gla(3)-TxVI, Glu7-Gla(3)-TxVI and Asp7-Gla(3)-TxVI. The cDNAs encoding the precursors of the four peptides were cloned. The predicted signal sequences (amino acids –46 to –27) were nearly identical and highly hydrophobic. The predicted propeptide region (–20 to –1) that contains the γ-carboxylation recognition site (γ-CRS) is very similar in Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI, but is more divergent for Gla(1)-TxVI. Kinetic studies utilizing the Conus γ-carboxylase and synthetic peptide substrates localized the γ-CRS of Gla(1)-TxVI to the region –14 to –1 of the polypeptide precursor: the Km was reduced from 1.8 mM for Gla (1)-TxVI lacking a propeptide to 24 μM when a 14-residue propeptide was attached to the substrate. Similarly, addition of an 18-residue propeptide to Gla(2)-TxVI/B reduced the Km 10-fold.This work was supported by grants K2001-03X-04487-27A and K2001- 03GX-04487-27, 08647, 13147 from the Swedish Medical Research Council, the European Union Cono-Euro-Pain (QLK3-CT-2000-00204), the Swedish Foundation for Strategic Research, the Kock Foundation, the Påhlsson Foundation and the Foundation of University Hospital, Malmö

Rezension zu: Katja Kröss, Die politische Rolle der stadtrömischen Plebs

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From gamma-carboxy-glutamate to protein C.

The story I shall recount started in 1969, when I was given the opportunity to join the Department of Clinical Chemistry at the University Hospital in Malmo. I had just finished medical school at the university in the neighboring town of Lund. Parallel to pursuing my medical studies I had spent some time in the Department of Biochemistry. I did not know much about biochemistry, but it was enough for me to realize that I wanted to do laboratory research rather than developing a clinical career. I was happy to accept an offer to start working in the laboratory, particularly as the head of the department, Professor Carl-Bertil Laurell, had an excellent reputation. As it turned out, I came to spend almost all of my professional life in the laboratory

The epidermal growth factor-like domains of factor IX. Effect on blood clotting and endothelial cell binding of a fragment containing the epidermal growth factor-like domains linked to the gamma-carboxyglutamic acid region

The binding of factor IX to cultured bovine endothelial cells was characterized using isolated domains of bovine factor IX. An NH2-terminal fragment that consists of the gamma-carboxyglutamic acid (Gla) region linked to the two epidermal growth factor (EGF)-like domains bound to the endothelial cells with the same affinity as intact factor IX, indicating that the serine protease part of factor IX is not involved in binding. This fragment also inhibited the factor IXa beta'-induced clotting of plasma at a concentration that would suggest a competition for phospholipid binding sites. However, after proteolytic removal of the Gla region from the fragment, the two EGF-like domains inhibited clotting almost as effectively, suggesting a direct interaction between this part of the molecule and the cofactor, factor VIIIa. Using affinity-purified Fab fragments against the Gla region, the EGF-like domains, and the serine protease part, it was observed that the serine protease part of the molecule undergoes a large conformational change upon activation, whereas the Gla region and the EGF-like domains appear to be unaffected. All three classes of Fab fragments were equally efficient as inhibitors of the factor IXa beta'-induced clotting reaction. Part of factor Va and factor VIIIa have significant sequence homology to a lectin. We therefore investigated the effect on in vitro clotting of the recently identified unique disaccharide Xyl alpha 1-3Glc, that is O-linked to a serine residue in the NH2-terminal EGF-like domain of human factor IX (Hase, S., Nishimura, H., Kawabata, S.-I., Iwanaga, S., and Ikenaka, T. (1990) J. Biol. Chem. 265, 1858-1861). However, no effect on blood clotting was observed in the assay system used. Our results are compatible with a model in which the serine protease part provides the specificity of the binding of factor IXa to factor VIIIa-phospholipid, but that the EGF-like domain(s) also contributes to the interaction of the enzyme with its cofactor