THEJOURNAL OF BIOLCGICAL CHEMISTRY 0 1994 hy The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269,No. 13, Issue of April 1, pp. 10100-10106, 1994 Printed in U.S.A.

The Shape of High MolecularWeight Kininogen ORGANIZATION INTO STRUCTURAL DOMAINS, CHANGES WITH ACTIVATION, AND INTERACTIONS WITH PREKALLIKREIN, AS DETERMINED BY ELECTRON MICROSCOPY* (Received for publication, November 22, 1993)

John W. WeiselS, Chandrasekaran Nagaswami, John L. Woodhead, Raul A. DeLa CadenaS, Jimmy D. Page§, and Robert W. ColmanP From the Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6058 and the §Sol Sherry Thrombosis Research Center, Departments of Medicine and Pathology, Temple University School of Medicine, Philadelphia, Penns.ylvania 19140

Knowledge of the organization of the kininogen gene and protein structure andfunction correlations has allowed the development of a model of high molecular weight kininogen. Domains 1-3 on the heavy chain are evolutionarily related to cystatin and the lattertwo are inhibitors of cysteine proteases. Proteolytic cleavage in domain 4 to release bradykinin causes a conformational change, exposing a surface-binding region (domain 5 ) on the disulfide-linked light chain. The carboxyl-terminal domain 6 contains azymogen binding sequence for factor XI and prekallikreinwhich, with domain 5, accounts for its cofactor activity. To explore further the domain structure, we have determined the shapes of high molecular weight kininogen and prekallikrein by electron microscopy of rotary shadowed preparations and computer image processing. High molecular weight kininogen appears tobe a lineararray of three linked globular regions about 16 nm long, with the two ends also connected by another thin strand. Both prekallikrein and kallikrein have a compact globular shape, with a subdivision that issometimes visible. Different functional domains of high molecular weight kininogen were identified by monoclonal antibodies against these regions, as well as ligand binding of prekallikrein. These studies indicate that one end globular region is the prekallikrein-binding domain, the other comprises the cysteine protease inhibitor domains and the smaller central nodule is the surface-binding domain. Cleavage of high molecular weight kininogen with plasma kallikrein to yield two-chain high molecular weight kininogen results in a striking change in conformation: the central surface-binding domain swings out so that it isstill adjacent to the prekallikrein-binding domain but no longer in the middle. These structural studies provide insight into the interactionsof these proteins andaspects of the mechanisms of their actions. Human high molecular weight kininogen (HK)’ is a single chain glycoprotein with a molecular mass of 120 kDa (l),first

* This work was supported by Grant HL30954 (to J. W. W.), Clinical Investigator Award HL02681 (to R. A. C.), and Specialized Center of Research in Thrombosis Grant HL45486 (to R. W. C.) from the National Institutes of Health and grants-in-aid (to R.A. C and J. D.P.) and Special Investigatorship(to J. D. P.) from the American Heart Association, Southeastern Pennsylvania Chapter. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed: Dept. of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6058. Tel.: 215-898-3573;Fax: 215-898-9871. The abbreviations used are: HK, high molecular weight kininogen; HKa, cleaved high molecular weight kininogen; D l , domain 1 of HK,

identified as a precursor of the vasoactive nonapeptide bradykinin. HK, however, also has important functions in contact the system of blood coagulation by serving as a cofactor for several enzymatic reactions in the contact phase. Normally, both preblood as kallikrein (PK)(2,3) and FactorXI (4) circulate in the binary complexes with HK. Like many of the reactions in the blood clotting cascade, contact phase reactions are greatlyaccelerated by interaction with a negatively charged surface in vitro. Putative in vivo anionic surfaces are the altered endothelial cell surface or the subendothelial matrixexposed in a damaged vessel. Factor XI1 binds to negatively charged surfaces in the absenceof other proteins, but HK is necessary for efiicient binding of both PK and Factor XI ( 5 ) . Bound Factor XI1 can autocatalytically activate FactorXI1 to XIIa, but this isa slow reaction. On the surface, PK and Factor XI1 are involved in a rapid reciprocal activation reaction (6), such that Factor XIIa activates prekallikrein (bound to HK) to kallikrein, which in turn activates FactorXI1 to Factor XIIa. Factor XIIa then converts Factor XI (bound to HK) to Factor XIa, which in turn activates Factor IX to 1%. HK contains a large amount of 0-linked carbohydrate in its light chain; there is also N-linked carbohydrate in the heavy chain.Many of the oligosaccharide chains are located in a stretch of the light chain beyond the highly positively charged region, closer to the carboxyl-terminal end of the molecule, but these are not requiredfor coagulant activity (7). Although PKand FactorXI are normally boundto HK, intact HK isnot an efficient cofactor for the enzymatic reactions just described. HK is cleaved by kallikrein, producing two-chainHK (HKa), sequentially releasing bradykinin and anotherpeptide (8).The remaining 65-kDa heavy chain is linkedto the 45-kDa light chain by a single disulfide bond. This cleavage of HK appears to involve an increase in the binding of HK to negatively charged surfaces (8). Cleavage of HK by Factor XIa, in contrast, leads to inactivation of the cofactor function of the molecule (9). The carboxyl-terminal portions (D6) of the light chain contain overlapping sequences responsible for the binding of the heavy chain of PK andfactor XIa. The amino-terminal region of the light chain (D5) contains a histidine-glycinerich and histidine-glycine-lysine-rich regions which appear t o be responsible for binding to negatively charged surfaces (10). Kininogen molecules can also act as inhibitors of cysteine proteases, such as papain, calpain, and cathepsins H and L. HK and low molecular weight kininogen are the major cysteine protease inhibitorsof plasma. This protease inhibitory activity resides on the identical heavy chainsof both kininogens. The cloning of cDNAs for human high and low molecular weight D2, domain 2 of HK, D3, domain 3 of HK, D4, domain 4 of H K D5, domain 5 of HK D6, domain 6 of HK, PK, prekallikrein.

10100

HK Shape and Interactions kininogens, and consequent analysis of the gene structure has revealed more about the structures and relationshipsof these proteins. In the identical heavy chains, there are three tandemly repeated groups homologous to cystatin (called D l , D2, and D3) coded for by three exons. Only D2 and D3 contain a critical binding sequence, QWAG, common to other cysteine protease inhibitors. Only D2 inhibits calpain since, in contrast to D3, it contains a specific inhibitory region distinct from QWAG which directly inhibits calpain (11). Recently, it hasbeen discovered that cleaved HK (HKa) isa potentanti-adhesive protein which can displace fibrinogen from neutrophils and platelets(12). It inhibits the spreading of osteosarcoma and melanoma cells on vitronectin and of endothelial cells, platelets, andmononuclear blood cells on vitronectin or fibrinogen (13).The histidine-glycine-lysine-rich domain is critical for this function. It appears that these reactions could be mediated through binding of cleaved HK (HKa) to thrombospondin, since complex formation has been shown recently between the light andheavy chain of HK and thrombospondin ( 14). Many of the experimental results,including the gene structure, suggest that this multifunctional protein is made up of multiple domains or independently folded regions of protein. The kininogens, with such a multiplicity of diverse functions, appear to be a good example of the evolution of complex proteins by duplication and fusion of variousgenes. However, there has been no direct evidence for a simple correspondence between the exons and domains. Electron microscopy of HK, HKa, and PK and two antibodies directed against specific domains was carried out to reveal the shapes of these proteins and allow identification of functional regions. Knowledge of the shapes of these proteins and localization of the structural andfunctional domains could provide insight into mechanismsof their actions.

10101

0

3

3

4

kDa 200

-

92 68

-

43

-

27

-

18

-

.

.

FRONT FIG.1. Sodium dodecyl sulfate polyacrylamide (10%) gel electrophoresis of the purified proteins employed for electron microscopy studies. The 10% gel was stained with Coomassie Brilliant Blue R-250. Lane 1 represents purified plasma prekallikrein (88 kDa) under reducing conditions. Lane 2, under reducing conditions, represents plasma kallikrein-cleaved HK (HKa) with its heavy chain (65 kDa) and light chain (45 kDa); the45-kDa light chain as observed in lane 2 did not stain well with Coomassie Brilliant Blue. Lane 3, under reducing conditions, represents single chainHK (120 kDa); the65-kDa band observed in lane 3 represents partially cleaved HK (