Abstract
Kininogens, the large precursor molecules of the vasoactive kinin peptides, are prototypic multidomain proteins serving numerous functions. To investigate their structure-function relationships, we have raised a panel of monoclonal antibodies against human H-kininogen and L-kininogen and fragments thereof and characterized them with respect to their target epitopes. Of 35 antibodies, 12 were directed to the amino-terminal domains (D1 to D3) of cystatin-like structure, 3 recognized domain D4 bearing the kinin segment, 17 bound to the carboxyl-terminal domains of H-kininogen (D5H and D6H), and 3 bound to the carboxyl-terminal domain D5L of L-kininogen. At least 14 distinct epitopes spread over the kininogen molecules were identified: 9 epitopes located on L-kininogen and 13 epitopes harbored by H-kininogen. Of these, 8 are shared by the two types of kininogens. Fine mapping of the epitopes by proteolytic fragments, recombinant proteins, and anti-idiotypic antibodies demonstrated that most but not all of the antibodies recognize liner epitopes. Synthesis of 28 peptides covering more than one-third of the entire kininogen sequences allowed us to narrow down seven major epitopes to 7-31 residues. Functional analyses identified 14 antibodies interfering with specific biological roles of the kininogens, i.e. cysteine proteinase inhibition, platelet attachment, cofactor binding, contact activation, and kinin delivery. Cross-reactivity studies indicated that three of the epitopes are present throughout the mammalian kininogens and further identified a unique epitope characteristic for the H/L-type of kininogens not present in their T-type. The panel of mapped antibodies provides powerful tools for the characterization of relevant interaction sites exposed by the pleiotropic kininogens and for the development of molecular surrogates mimicking these functional loci.
Highlights
From the SZnstitute for Physiological Chemistry and Pathbiochemistry, Universityof Mainz, Duesbergweg 6, 0-6500 Mainz 1 and the IIMar-von-Pettenkofer-ZnstituteU, niversity of Munich, Pettenkoferstrasse 90a,8000 Munich 2, Germany
To investigate their structure-function relationships, we have raised a panel of monoclonal antibodies against human Hkininogen and L-kininogen and fragments thereof characterized them with respect to their target epitopes
Fine mapping mosaic structure encompasses 11 exons, and 9 of them form of the epitopes by proteolytic fragments, recombinant a group of three triple exons, each of which most probably proteins, and anti-idiotypic antibodies demonstrated arose by gene multiplication from a primordial cystatin gene that most but not all of the antibodies recognize linear (Kitamura et al, 1985)
Summary
ImmuDnioslsoegcitcioanl of Human Kininogens the terminal domains D ~ H / D ~rHath; er, L-kininogen exposes For some of the monoclonal antibodies, purification by protein A-. Modification of Proteins-Monoclonal antibodies were biotinylated using biotin-c-aminocaproyl-N-hydroxysuccinimiidnedimethyl sulfoxide (Subba Rao et al, 1983).Papain was reduced and carboxymethized them with respect to their target domains, their binding ylated followingthe procedure of Anastasi et al (1983).The remaining epitopes, and their functional interferences. Chain generated by chemical cleavage with cyanogen bromide or by Sandrvich ELZSA-Cross-reactivity of the monoclonal antibodies limited proteolysis with plasma kallikrein (see below) was done by with recombinant fusion proteins of H-kininogen and @-galactosidase the Edman degradation method using an Applied Biosystems Model (Vogel et al, 1990) was tested by a sandwich ELISA using immuno-.
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