| 1. |
- Hillarp, A, et al.
(författare)
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Novel subunit in C4b-binding protein required for protein S binding
- 1988
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Ingår i: The Journal of biological chemistry. - 0021-9258. ; 263:25, s. 64-12759
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Tidskriftsartikel (refereegranskat)abstract
- C4b-binding protein (C4BP) is a multimeric protein with regulatory functions in the complement system. It also interacts with vitamin K-dependent protein S, which is involved in the regulation of the coagulation system. It has been demonstrated that C4BP consists of seven disulfide-linked, identical 70-kDa subunits, which are arranged to give the molecule a spider-like structure. We now have evidence for the presence of a new subunit in C4BP. On sodium dodecyl sulfate-poly-acrylamide gel electrophoresis it appears as a weakly stainable band with a molecular weight of approximately 45,000. The subunit was isolated by gel filtration in 6 M guanidine hydrochloride of reduced and carboxymethylated C4BP. Its amino-terminal sequence is distinct from previously known protein sequences. The stoichiometry of 45- to 70-kDa subunits was estimated to be 1:9, indicating the presence of one 45-kDa subunit per C4BP molecule. The new subunit was demonstrated to be a disulfide-linked component of the central core of C4BP. It was sensitive to proteolysis by chymotrypsin, and when cleaved the protein S binding ability of C4BP was lost. With protein S bound to C4BP, the 45-kDa subunit was protected from degradation by chymotrypsin, and the protein S binding site remained intact. These data suggest that the new subunit is directly involved in protein S binding.
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| 2. |
- Hillarp, A, et al.
(författare)
-
Protein S binding in relation to the subunit composition of human C4b-binding protein
- 1989
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793. ; 259:1, s. 6-53
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Tidskriftsartikel (refereegranskat)abstract
- The human regulatory complement component C4b-binding protein (C4BP) circulates in plasma either as a free protein or in a bimolecular complex with the vitamin K-dependent protein S. The major form of C4BP is composed of 7 identical, disulfide-linked 70 kDa subunits (alpha-chains), the arrangement of which gives the C4BP molecule a spider-like appearance. Recently, we identified a unique 45 kDa subunit (beta-chain) in C4BP. We have now isolated a subpopulation of C4BP, which does not bind protein S. This C4BP species, which had a molecular weight slightly lower than that of the predominant form, was found to lack the beta-chain. Another lower molecular weight form of C4BP was also purified. It contained the beta-chain and was efficient in binding protein S. Its subunit composition was judged to comprise six alpha-chains and one beta-chain. These results indicate C4BP in plasma to be heterogeneous at a molecular level vis-a-vis subunit composition and/or protein S binding ability and provide support for the concept that the beta-chain of C4BP contains the single protein S binding site.
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| 3. |
- Hillarp, A, et al.
(författare)
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The protein S-binding site localized to the central core of C4b-binding protein
- 1987
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Ingår i: The Journal of biological chemistry. - 0021-9258. ; 262:23, s. 7-11300
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Tidskriftsartikel (refereegranskat)abstract
- Human C4b-binding protein (C4BP) is a regulator of the classical pathway of the complement system. It appears in two forms in plasma, as free protein and in a noncovalent complex with the vitamin K-dependent coagulation protein, protein S. In the electron microscope C4BP has a spider-like structure with a central core and seven extended tentacles, each of which has a binding site for C4b, although the protein S-binding site has not been unequivocally pinpointed. C4BP was subjected to chymotrypsin digestion which yielded two major fragments, one of 160 kDa representing the central core, and one of 48 kDa representing the cleaved-off tentacles. We have now localized the protein S-binding site to the 160-kDa central core fragment. Using immunoblotting with a panel of polyclonal antisera, the isolated central core was shown to be completely devoid of 48-kDa fragments. The protein S-binding site was susceptible to proteolysis by chymotrypsin, but was protected by a molar excess of protein S included during the proteolysis. The 160-kDa central core fragment consisted of identical, disulfide-linked 25-kDa peptides and a proper disulfide bond arrangement was crucial to protein S binding. Using a direct binding assay it was shown that the isolated central core had the same affinity for protein S as intact C4BP.
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