The human kininogen gene (KNG) mapped to chromosome 3q26-qter by analysis of somatic cell hybrids using the polymerase chain reaction

Summary Kinins, peptide products of kininogens, may be involved in hypertensive and diabetic diseases, and inflammatory disorders. The human kininogen gene (KNG) has been mapped to chromosome 3, using a panel of human-hamster somatic cell hybrids by polymerase chain reaction of hybrid DNA with gene-specific primers. KNG was further assigned to 3q26-3qter, using DNA from a second panel of chromosome 3 deletion mapping cell hybrids.     

Structure and expression of two kininogen genes in mice

Kininogens serve dual functions by forming a scaffold for the assembly of the protein complex initiating the surface-activated blood coagulation cascade and as precursors for the kinin hormones. While rats have three kininogen genes, for mice, cattle, and humans only one gene has been described. Here, we present sequence and expression data of a second mouse kininogen gene. The two genes, kininogen-I and kininogen-II, are located in close proximity on chromosome 16 in a head-to-head arrangement. In liver and kidney, both genes are expressed and for each gene three alternative splice variants are synthesized. Two of them are the expected high and low molecular weight isoforms known from all mammalian kininogens. However, for both genes also a third, hitherto unknown splice variant was detected which lacks part of the high molecular weight mRNA due to splicing from the low molecular weight donor site to alternative splice acceptor sites in exon 10. The physiological functions of the six kininogen isoforms predicted by these findings need to be determined.     

Canine Plasma Kininogen: Evidence for the Presence of Two Kininogens and Purification of High Molecular Weight Kininogen and Characterization as Multi-Functional Molecule

The ratio of kininogen that is substrate of plasma kallikrein to kininogen, which is not substrate of plasma kallikrein in canine plasma, was about 1:3.6 by differential assay of kininogens. When the plasma was gel-filtered through a column of Sephacryl S-300 superfine, two fractions, which released kinin by trypsin, were obtained. These results indicate that two kininogens with different molecular weights are present in the plasma and they show different susceptibility to plasma kallikrein. One kininogen was purified by ion-exchange and zinc-chelating affinity chromatographies. Purified kininogen showed a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing condition and its molecular weight was 125 kDa. Released kinin from the kininogen by trypsin was bradykinin. The kininogen inhibited papain and ficin but did not inhibit bromelain at the concentration used. The kininogen bound to carboxymethylated-papain and this binding was dissociated by 3M NaSCN. Canine plasma shortened the abnormal clotting time of human high molecular weight kininogen-deficint plasma. The kininogen also shortened the abnormal clotting time of the plasma. From these results, the purified kininogen was high molecular weight kininogen and it was multi-functional protein.     

Mapping of functional domains of human high molecular weight and low molecular weight kininogens using murine monoclonal antibodies

Thirty-four monoclonal antibodies directed against human high molecular weight (HMW) and low molecular weight (LMW) kininogens and their derivatives were obtained, and the specificities of the antibodies were assayed by enzyme-linked immunosorbent assay (ELISA). By use of HMW kininogen, kinin-free HMW kininogen, kinin-free and fragment 1.2 (fr 1.2) free HMW kininogen, fr 1.2-light chain of HMW kininogen, LMW kininogen, kinin-free LMW kininogen, heavy chain of LMW kininogen, and light chain of LMW kininogen, the monoclonal antibodies were characterized and classified into four groups: (A) 20 monoclonal antibodies reacting with only the heavy chain, a common region of HMW and LMW kininogens; each of these monoclonal antibodies possessed the specificity to domain 1 (2 monoclonal antibodies), domain 2 (2 monoclonal antibodies), domain 3 (7 monoclonal antibodies), and both domains 2 and 3 (7 monoclonal antibodies) of the heavy chain; (B) 7 monoclonal antibodies reacting with fr 1.2, a unique histidine-rich region; (C) 5 monoclonal antibodies reacting with the light chain of HMW kininogen; (D) 2 monoclonal antibodies reacting with the light chain of LMW kininogen. Two monoclonal antibodies in the first group (group A), designated HKG H7 and H12, effectively suppressed the thiol proteinase inhibitor activity of HMW kininogen to papain and calpains and of LMW kininogen to papain, but the others did not affect it. Further, all the monoclonal antibodies which recognized the fr 1.2 or light chain of HMW kininogen (groups B and C) suppressed the clotting activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Porcine high molecular weight kininogen: its purification and properties as a thiol proteinase inhibitor as compared to human high molecular weight kininogen

1. High mol. wt kininogen (HMW kininogen) was purified to a homogeneous state from porcine plasma. 2. The protein exhibited a strong inhibitory activity for thiol proteinases such as ficin, papain and calpain I, whereas it did not inhibit serine proteinases, trypsin and chymotrypsin. 3. The mol. wt, isoelectric point, amino acid and carbohydrate compositions, stabilities to temperature and pH, kinetic constants, and immunological properties of the porcine HMW kininogen were determined and compared with those of human HMW kininogen.     

激肽原基因的选择性剪接与功能研究进展

激肽原基因在不同物种中有一定的差异, 其编码的蛋白质产物也有所不同。在人类、牛和小鼠中存在的激肽原基因K是一种选择性剪接基因, 其可以编码两种激肽原产物, 而在大鼠中, 除了激肽原基因K之外还有一类激肽原基因T, 其只能编码一种激肽原。激肽原属于半胱氨酸蛋白酶抑制剂超家族中家族3的成员, 是一类具有多个结构域的多功能蛋白, 在人体内多种功能协同作用, 维持着机体的正常生理状态。激肽原的凝血和抗凝血两种相互拮抗的功能既能使破损的血管得到修复, 防止流血不止, 又能抑制血管内血栓的形成。本文将对目前研究较多的人、牛、小鼠和大鼠的激肽原基因的选择性剪接以及激肽原的凝血、抗凝血等多方面的功能做一综述, 为更进一步地阐明激肽原基因的进化机制, 加深对基因选择性剪接机制的了解, 揭示激肽原功能的认识等方面有所启示, 并为调节血管通透性、调节血压和抗肿瘤等新药的开发提供有益的参考。     

Hydrolysis of human high-molecular-mass kininogen by human plasma kallikrein. Proposal of a new model concept for the course of reaction in presence and absence of C1(-)-inhibitor

Hydrolysis of high-molecular-mass kininogen was studied by following the changes in the amounts of substrate, intermediates and products as a function of time using quantitative polyacrylamide-gel electrophoresis (silver staining). The experimental data was analysed on the basis of the concept that the overall reaction is composed of three hydrolysis reactions, two positional-change processes of intermediates at the active site, and two product-substrate exchange processes. It is proposed C1(-)-inhibitor to form two types of complexes with kallikrein, one with non-covalent and one with covalent bonds. With an adequately chosen set of reaction-partner concentrations and four different kinds of experimental conditions with respect to kininogen and inhibitor addition to kallikrein, the following results were obtained: 1) Non-covalently bound inhibitor has no effect on the first and the second hydrolysis reaction, but efficiently interferes with the third hydrolysis reaction; 2) Nicked kininogen (first intermediate; one of the two bradykinin bonds split) for the second bond to be hydrolysed undergoes a positional change during which it remains strongly bound to the enzyme, never exchanges with kininogen, and is not displaced by non-covalently bound inhibitor; 3) Intermediate kinin-free kininogen (second intermediate; both bradykinin bonds split and bradykinin released) prior to turning over into stable kinin-free kininogen (final product; histidine-rich fragment split off and released) undergoes a positional change involving dissociation and reassociation so that non-covalently bound inhibitor finds access to the active site; 4) Intermediate kinin-free kininogen to sustain multiple turnovers exchanges with kininogen via a stable complex of such structure that during this process non-covalently bound inhibitor cannot or can only slightly interfere; 5) Stable kinin-free kininogen to sustain multiple turnovers exchanges with intermediate kinin-free kininogen via free enzyme with the effect that non-covalently bound inhibitor efficiently interferes; 6) As hydrolysis proceeds more and more inhibitor becomes covalently bound, gradually leading to complete inactivation of the enzyme.     

Hypothesis. The molecular 'double-pivot' mechanism for water oxidation

High-molecular-weight (HMW) kininogen was purified from guinea-pig plasma by measuring its ability to correct the prolonged clotting time in human HMW kininogen deficient plasma (Fitzgerald trait). The purified HMW kininogen demonstrated a homogeneous band in disc gel electrophoresis in the presence of sodium dodecyl sulfate under reducing or non-reducing conditions with an apparent molecular weight of 100,000. Kinin released from HMW kininogen by treatment with guinea-pig plasma kallikrein was identified as bradykinin by reverse-phase HPLC and amino-acid analysis. The capacity of HMW kininogen as a thiol-proteinase inhibitor was realized by its dose-dependent inhibitory activity to papain. The Ki value for papain was estimated to be 42 pM. The kinin-free HMW kininogen maintained the inhibitor and clotting-factor activities with similar capacities to those of the HMW kininogen molecule. Heavy chain (H-chain) and light chain (L-chain) of HMW kininogen were prepared from reduced and alkylated kinin-free HMW kininogen by HPLC. The S-alkylated H-chain, but not L-chain, demonstrated the inhibitor activity with the Ki value 6.9 nM for papain, whereas the S-alkylated L-chain, but not H-chain, maintained the clotting activity one-third of the capacity of HMW kininogen. Specific antibodies recognized HMW kininogen, but also a probable low-molecular-weight kininogen(s) with an apparent molecular weight of 60,000 in the guinea-pig plasma. All of these properties are consistent with the reports on human, bovine and rat HMW kininogen.     

Interstitial-tissue localization of high-molecular-weight kininogen in guinea-pig skin

A rabbit antibody against the light-chain of guinea-pig high-molecular-weight (HMW) kininogen, which was specific to HWM kininogen and did not recognize low-molecular-weight kininogen, was prepared. This antibody demonstrated the presence of HMW kininogen antigen at the interstitial-tissue space in the guinea-pig skin by means of immunohistochemistry. The interstitial-tissue HMW kininogen antigen was extracted from the skin. This antigen molecule in the skin extract behaved identically as HWM kininogen of plasma in slab-polyacrylamide gel electrophoresis under the presence of sodium dodecyl sulfate followed by immunoblotting. Therefore, it was concluded that HMW kininogen was present in the interstitial-tissue fluid in the skin. The amount of HMW kininogen in the skin extract was quantified by a sandwich enzyme-linked immunosorbent assay with the anti-light-chain antibody and a goat anti-guinea-pig HMW kininogen antibody. On the assumption that the interstitial-tissue volume is 50 ml/100 g wet skin tissue, the average concentration of HMW kininogen in the interstitial-tissue fluid of the skin was calculated to be 23% of the plasma concentration. On the other hand, the proportion of intravascular HMW kininogen (derived from blood remaining in the vessels of the harvested skin) in relation to the total HMW kininogen in the skin extract was quantified by measuring the radio-labelled HMW kininogen which had been injected intravenously as a tracer of the intravascular HMW kininogen. About 5% of the total HMW kininogen in the skin extract was calculated to be derived from the intravascular blood volume of the skin, indicating that the majority of the HMW kininogen in the skin extract was derived from the extravascular-tissue space.     

High molecular weight kininogen utilizes heparan sulfate proteoglycans for accumulation on endothelial cells

Kininogens, the high molecular weight precursor of vasoactive kinins, bind to a wide variety of cells in a specific, reversible, and saturable manner. The cell docking sites have been mapped to domains D3 and D5(H) of kininogens; however, the corresponding cellular acceptor sites are not fully established. To characterize the major cell binding sites for kininogens exposed by the endothelial cell line EA.hy926, we digested intact cells with trypsin and other proteases and found a time- and concentration-dependent loss of (125)I-labeled high molecular weight kininogen (H-kininogen) binding capacity (up to 82%), indicating that proteins are crucially involved in kininogen cell attachment. Cell surface digestion with heparinases similarly reduced kininogen binding capacity (up to 78%), and the combined action of heparinases and trypsin almost eliminated kininogen binding (up to 85%), suggesting that proteoglycans of the heparan sulfate type are intimately involved. Consistently, inhibitors such as p-nitrophenyl-beta-d-xylopyranoside and chlorate interfering with heparan sulfate proteoglycan biosynthesis reduced the total number of kininogen binding sites in a time- and concentration-dependent manner (up to 67%). In vitro binding studies demonstrated that biotinylated H-kininogen binds to heparan sulfate glycosaminoglycans via domains D3 and D5(H) and that the presence of Zn(2+) promotes this association. Cloning and over-expression of the major endothelial heparan sulfate-type proteoglycans syndecan-1, syndecan-2, syndecan-4, and glypican in HEK293t cells significantly increased total heparan sulfate at the cell surface and thus the number of kininogen binding sites (up to 3. 3-fold). This gain in kininogen binding capacity was completely abolished by treating transfected cells with heparinases. We conclude that heparan sulfate proteoglycans on the surface of endothelial cells provide a platform for the local accumulation of kininogens on the vascular lining. This accumulation may allow the circumscribed release of short-lived kinins from their precursor molecules in close proximity to their sites of action.     

Influence of plasma proteinase inhibitors and aprotinin on trypsin-induced bradykinin release in vitro in man

The consumption of kininogen (measured as kinin-releasable material) was studied in an experimental model in vitro. Analyses were made following the addition of increasing amounts of human cationic trypsin to human serum and plasma. The consumption of kininogen was correlated with the degree of saturation of the plasma proteinase inhibitors alpha 2-macroglobulin (alpha 2-M) and alpha 1-proteinase inhibitor (alpha 1-PI) with trypsin in the presence and absence of aprotinin (Trasylol). The level of kininogen fell dramatically when alpha 2-M was saturated to 70% in spite of 90% free alpha 1-PI. Trypsin-alpha 2-M complexes had no effect on kininogen levels. 60 mumol/l of aprotinin, i.e. approximately 3 X 10(6) KIU/l, blocked only 60% of the trypsin-induced kininogen consumption in serum, while 15 mumol/l of aprotinin blocked 100% of this consumption in plasma. With increasing concentration of aprotinin in serum, a decreasing consumption of alpha 2-M and especially of alpha 1-PI was observed on the addition of trypsin. The high aprotinin concentration needed to block trypsin-induced kininogen cleavage in human serum or plasma may explain the poor clinical effect of aprotinin to date in human acute pancreatitis.     

Purification and heterogeneity of human kininogen. Use of DEAE-chromatography, molecular sieving and antibody specific immunosorbents.

Various methods of preparing human kininogen were investigated with an aim to limit the immunoreactive contaminant proteins to permit purification by immunosorption. A five-step procedure is described giving 7.5% yield of highly purified kininogen (pharmacological purity 14--20) from pooled human plasma, and containing approximately 30% alpha-2HS-glycoprotein and 2.8% albumin. Alpha-2HS could not be removed by polyacrylamide gel electrophoresis or isoelectric focusing in column. Analysis of heterogeneity of kininogen after chromatography on DEAE-Sephadex using various linear gradients and gel filtration on Sephadex G-100 suggested that a minor component may be an aggregate, not included in the yield. It remains uncertain whether this component derives from an occasionally observed high molecular form of active kininogen in the primary purification steps in the 7-12 S sieve fractions from Sephadex G-200, and excluded from further purification by pooling. Purification with immunosorbents was investigated using batch operations with antibody specific polymers prepared with antisera insolubilized with ethylchloroformate. It was found that the adsorption-desorption procedure was favourable for immunization purposes in producing highly specific immunologically pure kininogen. The kininogen obtained by this method or by the removal of contaminant alpha-2HS and albumin with the corresponding antibody specific polymers gave similar heterogenous patterns by polyacrylamide gel electrophoresis, indicating a main band of kininogen and several faintly stained bands which responded only to anti-kininogen. With 200 mug of the kininogen protein purified by immunosorption using monospecific antiserum the kininogen precipitation titre was 1:8 after 6--8 weeks in rabbits. With a polymer prepared with 4 ml anti-kininogen serum (1:8) and incubated with 800 mug highly purified kininogen approximately half the protein was desorbed with 2 M and 3 M sodium iodide in the first adsorption-desorption procedure.     

Localization of DNA sequences governing alternative mRNA production of rat kininogen genes

The two types of the rat kininogen genes show different modes of mRNA production. The K gene encodes two distinct mRNAs for high molecular weight (HMW) and low molecular weight (LMW) kininogens. These two mRNAs are generated by differential usage of the 3'-terminal exon (LMW exon) and the one next to this exon (HMW exon) through alternative polyadenylation and splicing. In contrast, the two T genes selectively generate the LMW form of the mRNA, although the T genes are extremely homologous to the K gene, including the sequence (psi HMW region) corresponding to the HMW exon of the K gene. In this study, we constructed a series of chimeric kininogen genes by exchanging equivalent restriction fragments of the K and T genes and examined the sequences and the mechanisms governing the different expression patterns of the kininogen genes by introducing the chimeric genes into heterologous COS cells. The results indicate that the formation of the two forms of the mRNA is controlled by two separate 3' sequences of the kininogen genes. One is located within the internal sequence of the HMW/psi HMW region, whereas the other is within the LMW exon and its preceding region. Our data also suggest that the different expression patterns of the kininogen genes are primarily governed by differing splicing efficiency.     

Zinc ions promote the binding of factor XII/factor XIIA to acidic phospholipids but have no effect on the binding of high-Mr kininogen

Binding of high-Mr kininogen and factor XII/factor XIIa to phospholipids coated on to polystyrene microtiter plates was investigated by ELISA. Both high-Mr kininogen and factor XII/factor XIIa bound specifically to the phospholipid surface. Binding was observed to negatively charged phospholipids only. The binding of high-Mr kininogen was not affected by the presence of zinc ions. At a surface concentration of 20% phosphatidylinositol phosphate in phosphatidylcholine a dissociation constant (kD) of 10 nM for the binding of high-Mr kininogen was calculated. The amount of bound purified alpha-factor XIIa could be increased 4-5-fold in the presence of zinc ions. The lowest zinc ion concentration giving maximal binding was 0.1 mM. The binding of alpha-factor XIIa was inhibited by high-Mr kininogen. Independent of the presence of zinc ions or high-Mr kininogen, a kD of 7.9 nM was calculated for alpha-factor XIIa binding. The binding of prekallikrein was dependent upon the presence and the concentration of high-Mr kininogen. In plasma containing aprotinin, the binding of high-Mr kininogen was apparently inhibited in the presence of zinc ions, which was a prerequisite for the binding of factor XII. This apparently inhibitory effect of zinc ions on the binding of high-Mr kininogen was probably due to the increased binding of factor XII, which displaced high-Mr kininogen.     

Purification and immunochemical properties of human low molecular weight kininogen.

A low molecular weight (LMW) kininogen was isolated from pooled human serum by chromatography on DEAE-Sephadex A-50, CM-Sephadex C-50, Sephadex G-150, and Sephadex G-100. It was shown to be homogeneous by ultracentrifugation, polyacrylamide gel electrophoresis, and immunoelectrophoresis. The sedimentation coefficient, S020,W, of purified LMW kininogen was 3.85 s, and its molecular weight was determined to be 78,000 by Sephadex G-100 gel-filtration. The LMW kininogen contained 79.3% protein, 8.0% hexose, 3.9% hexosamine, and 4.9% sialic acid. In order to determine the immunochemical properties of LMW kininogen, specific antiserum was prepared in rabbits. The antigenic determinant of LMW kininogen was not related to the sialic acid and kinin moieties in the kininogen molecule, but could not be distinguished from that of high molecular weight (HMW) kininogen. In the quantitative single radial immunodiffusion test, a sialic acid-free LMW kininogen reacted to a greater extent with the antiserum than the native LMW kininogen. The kininogen level in human serum was estimated by single radial immunodiffusion. The antiserum cross-reacted with monkey serum, but not with sera from dogs, rats, and mice, horses, pigs, guinea pigs, oxen, and rabbits.     

Rat plasma high-molecular-weight kininogen. A simple method for purification and its characterization

High-molecular-weight kininogen has been isolated from rat plasma in three steps in a relatively high yield. The purified preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence and presence of 2-mercaptoethanol, and the apparent Mr was estimated as 100,000. On incubation with rat plasma kallikrein, rat high Mr kininogen yielded a kinin-free protein consisting of a heavy chain (Mr = 64,000) and a light chain (Mr = 46,000), liberating bradykinin. The kinin-free protein was S-alkylated, and its heavy and light chains were separated by a zinc-chelating Sepharose 6B column. The amino acid compositions of rat high Mr kininogen and its heavy and light chains were very similar to those of bovine high Mr kininogen and its heavy and fragment 1.2-light chains, respectively. A high histidine content in the light chain of rat high Mr kininogen indicated the presence of a histidine-rich region in this protein as in bovine high Mr kininogen, although this region was not cleaved by rat plasma kallikrein. Rat high Mr kininogen corrected to normal values the prolonged activated partial thromboplastin time of Brown-Norway Katholiek rat plasma known to be deficient in high Mr kininogen and of Fitzgerald trait plasma. The kinin-free protein had the same correcting activity as intact high Mr kininogen. Rat high Mr kininogen also accelerated approximately 10-fold the surface-dependent activation of rat factor XII and prekallikrein, which was mediated with kaolin, amylose sulfate, and sulfatide. These results indicate that rat high Mr kininogen is quite similar to human and bovine high Mr kininogens in terms of biochemical and functional properties.     

Studies on human high molecular weight (HMW) kininogen. II. Structural change of HMW kininogen by the action of human plasma kallikrein

We have investigated in detail the cleavage of human high molecular weight (HMW) kininogen by human plasma kallikrein and revealed the formation of a nicked kininogen and a novel kinin-free protein (KFP) as intermediate cleavage products. The cleavage of a single chain HMW kininogen (Mr=120,000) by plasma kallikrein was a three-step reaction. The first cleavage yielded a nicked kininogen composed of two disulfide-linked 62,000 and 56,000 daltons chains. The second cleavage yielded kinin and an intermediate kinin-free protein, KFP-I, which was apparently of equal size to the nicked kininogen. The third cleavage yielded a stable kinin-free protein, KFP-II, composed of two disulfide-linked 62,000 and 45,000 daltons chains. The liberation of an 8,000 daltons fragment was identified when the 56,000 daltons chain isolated by SP-Sephadex C-50 chromatography of reduced and alkylated KFP-I was cleaved by plasma kallikrein into the 45,000 daltons chain. Although the antiserum against HMW kininogen cross-reacted with low molecular weight (LMW) kininogen, the antiserum against the 45,000 daltons chain was specific for HMW kininogen. These results suggest that the antigenic determinant groups common to HMW and LMW kininogens are located in the 62,000 daltons heavy chain, while those specific for HMW kininogen are located in the 45,000 daltons light chain, which is known to retain blood coagulation activity.