Fourth Epidermal Growth Factor Research Articles

An unusual mode of concerted evolution of the EGF‐TM7 receptor chimera EMR2

The epidermal growth factor (EGF)-TM7 receptors CD97, EMR1, EMR2, EMR3, and EMR4 form a group of adhesion class heptahelical molecules predominantly expressed by cells of the immune system. These receptors bind cellular ligands through EGF-like domains, localized N-terminal to a large extracellular region. Remarkably, EMR2 possesses a chimeric structure with a seven-span transmembrane (TM7) region most related to EMR3 and an EGF domain region nearly identical to CD97. By comparing EGF-TM7 receptors in primates and dogs, we identified an intriguing pattern of concerted evolution, apparently mediated by gene conversion, among EMR2 and the oppositely orientated and physically adjacent genes CD97 and EMR3. This concerted evolution has continuously maintained the chimeric structure of EMR2 since early mammal radiation. Most highly conserved between EMR2 and CD97 is the fourth EGF domain, which mediates binding to chondroitin sulfate, a ligand specificity shared by both receptors. Another ligand, CD55, is bound effectively only by CD97. We show that different molecular mechanisms (mutations vs. alternative splicing) prevent CD55 binding by EMR2 in hominoids. Our findings illustrate how various and partially opposing evolutionary events have shaped the structure and ligand specificity of a modern mammalian gene family.

Binding of Calcium to Anticoagulant Protein S: Role of the Fourth EGF Module

Protein S is an anticoagulant protein containing a Gla (enclosing gamma-carboxyglutamic acids) module, a TSR (thrombin sensitive region) module, four EGF (epidermal growth factor)-like modules, and a SHBG (sex hormone binding globulin)-like region. Protein S is a cofactor to activated protein C (APC) in the degradation of coagulation factors Va and VIIIa but also has APC-independent activities. The function of the fourth EGF module (EGF4) in protein S has so far not been clear. We have now investigated this module through studies of recombinant wild-type protein S and a naturally occurring mutant (Asn217Ser). The mutant has essentially normal APC anticoagulant activity and a previously reported secretion defect. In the wild-type protein, Asn217 is normally beta-hydroxylated. The binding of calcium to wild-type protein S is characterized by four high-affinity binding sites with K(D) values ranging from 10(-)(7) to 10(-)(9) M. Three of these binding sites are located in EGF modules. Using surface plasmon resonance, competition with a calcium chelator, and antibody-based methods, we found that one high-affinity binding site for calcium was lost in protein S Asn217Ser but that the mutation also affected the calcium-dependent conformation of EGF1. We conclude that binding of calcium to EGF4 of protein S, involving Asn217, is important for the maintenance of the structure of protein S. Also, the abolition of binding of calcium to EGF4, related to Asn217, impairs both the structure and function of EGF1.

Preparation and Characterization of Monoclonal Antibodies to Thrombomodulin

Thrombomodulin (TM) is an endothelial cell surface molecule, capable of specific binding for thrombin. The thrombin/TM complex promotes activation of plasma anticoagulant protein C (PC) and negatively regulates blood coagulation. Along with anticoagulant function, TM has been shown to have additional physiological functions such as regulation of fibrinolysis, cell adhesion, tumor growth, and embryonic development. The extracellular region of TM contains a lectin domain and six epidermal growth factor (EGF)-like domains, which are required for the various functions. To analyze the functions, we established a panel of monoclonal antibodies (MAbs) reactive to each functional domain. We obtained MAbs that react to the lectin domain or the front half of EGF domains from the first to the third using the antigen of a transfected cell line expressing full-length TM. We also obtained MAbs that reacted to the bottom half of the EGF domain from the fourth to the sixth using the antigen of a transfected cell line expressing truncated form of TM lacking the lectin domain and the EGF domains from the first to the third. All obtained MAbs could be used for Western blotting. Endothelial cell function for PC activation can be mimicked by transfected cells positive for TM and the endothelial cell protein C receptor (EPCR). Effects of the established MAbs on thrombin-dependent PC activation on the transfected cells were examined. Strong inhibition was demonstrated by three MAbs, which reacted to the fourth or fifth EGF domain, but not by MAbs to the other domains. The fourth EGF domain is known as the interaction site for PC, and the fifth domain is known to be required for thrombin binding. The sixth EGF domain also has been shown to be required for thrombin binding. An MAb against the domain strongly inhibited thrombin-binding. However, the MAb demonstrated little effect on thrombin dependent PC activation. The contradictory results demonstrated with the MAb to the sixth EGF domain suggest an unknown molecular mechanism for PC activation on the cell surface. A panel of MAbs reactive to each domain could be useful for analyzing the multifunctional molecule thrombomodulin.

Expression of the largest CD97 and EMR2 isoforms on leukocytes facilitates a specific interaction with chondroitin sulfate on B cells

The EGF-TM7 receptors CD97 and EMR2 are heptahelical molecules predominantly expressed on leukocytes. A characteristic of these receptors is their ability to interact with cellular ligands via the N-terminal epidermal growth factor (EGF)-like domains. The first two EGF domains of CD97 (but not EMR2) bind CD55 (decay-accelerating factor), while the fourth EGF domain of both CD97 and EMR2 interacts with the glycosaminoglycan chondroitin sulfate (CS). Using fluorescent beads coated with soluble recombinant CD97 and EMR2 protein, and isoform-specific monoclonal antibodies, we have determined the cellular and molecular characteristics of the interaction with CS. The fourth EGF domain of CD97 and EMR2 is expressed on activated lymphocytes and myeloid cells, whereas the ligand is specifically found on B cells within the peripheral blood. The interaction between CD97/EMR2 and CS may therefore play a role in the interaction of activated T cells, dendritic cells, and macrophages with B cells.

Characterization of recombinant epidermal growth factor (EGF)-like modules from vitamin-K-dependent protein S expressed in Spodoptera cells--the cofactor activity depends on the N-terminal EGF module in human protein S.

Epidermal growth factor (EGF)-like modules in protein S, a physiological anticoagulant protein that functions as a cofactor to activated protein C, have been expressed in Spodoptera cells using baculovirus. EGF modules 1-3, 1-4, 2-3 and 2-4 were produced on a preparative scale. The isolated modules were more than 95% homogeneous, as judged by sequence determination. 45Ca2+-ligand blotting experiments indicated that recombinant proteins that contained the fourth EGF module, i.e. EGF 1-4 and 2-4, bound Ca2+ with high affinity. The 45Ca2+-ligand blotting results, together with results of competitive binding experiments using monoclonal antibodies as structural probes, indicated that the recombinant proteins had been folded to a native conformation. EGF modules 1-3 and 1-4 inhibited the interaction between activated protein C and protein S, whereas modules 2-3 and 2-4 had no effect on this interaction. It is thus apparent that EGF module 1 is crucial for the interaction between protein S and activated protein C. Moreover, EGF modules 1-4 were approximately 10-fold more effective in inhibiting the interaction than modules 1-3, suggesting a very weak interaction between module 4 and activated protein C or that this module is important to keep module 1 in a conformation that is optimal for interaction with activated protein C.

Further localization of binding sites for thrombin and protein C in human thrombomodulin

To elucidate the binding sites for thrombin and protein C in the six epidermal growth factor (EGF) domains of human thrombomodulin, recombinant mutant proteins were expressed in COS-1 cells. Mutant protein EGF456, which contains the fourth, fifth, and sixth EGF domains from the NH2 terminus of thrombomodulin, showed complete cofactor activity in thrombin-catalyzed protein C activation, as did intact thrombomodulin or elastase-digested thrombomodulin. EGF56, containing the fifth and sixth EGF domains, did not have cofactor activity; but EGF45, containing the fourth and fifth EGF domains, had about one-tenth of the cofactor activity of EGF456. Thrombin binding to attached recombinant thrombomodulin (D123) was inhibited by EGF45 as well as by EGF56. A synthetic peptide (ECPEGYILDDGFICTDIDE), corresponding to Glu-408 to Glu-426 in the fifth EGF domain, inhibited thrombin binding to attached thrombomodulin (D123) with an apparent Ki of 95 microM. At Ca2+ concentrations of 0.25-0.3 mM, intact protein C was maximally activated by thrombin in the presence of EGF45, EGF456, or EGF1-6, which contains the first to sixth EGF domains; but such maximum cofactor activity was not observed when gamma-carboxyglutamic acid-domainless protein C was used. These findings suggest that: 1) thrombin binds to the latter half of the fifth EGF domain; and 2) protein C binds to the fourth EGF domain of thrombomodulin through Ca2+ ions.

Novel type of very high affinity calcium-binding sites in beta-hydroxyasparagine-containing epidermal growth factor-like domains in vitamin K-dependent protein S.

Vitamin K-dependent protein S is shown to contain four very high affinity Ca2(+)-binding sites. The number of sites and their affinities were determined from Ca2+ titration in the presence of the chromophoric chelator Quin 2. In 0.15 M NaCl, pH 7.5, the four macroscopic binding constants are K1 greater than or equal to 1 x 10(8) M-1, K2 = 3 +/- 2 x 10(7) M-1, K3 = 4 +/- 2 x 10(6) M-1, and K4 = 9 +/- 4 x 10(5) M-1. At low ionic strength, the corresponding values are K1 greater than or equal to 2 x 10(9) M-1, K2 = 9 +/- 4 x 10(8) M-1, K3 = 2 +/- 1 x 10(8) M-1, and K4 = 9 +/- 4 x 10(7) M-1. To localize the Ca2(+)-binding sites, protein S was subjected to proteolysis using lysyl endopeptidase. This yielded a 20-21-kDa fragment which comprised the third and fourth epidermal growth factor (EGF)-like domains and remained high affinity Ca2(+)-binding site(s). The susceptibility of the EGF-like domains to proteolysis increased when Ca2+ was removed from protein S indicating that the Ca2+ binding is important for the stability and/or conformation of the EGF domains. Three of the four EGF-like domains in protein S contain beta-hydroxyasparagine. In each of these domains there is a cluster of three or four negatively charged amino acid residues which are likely to contribute to the extraordinary high Ca2+ affinity. From sequence homology it is suggested that this novel type of high affinity Ca2(+)-binding site is present in several other proteins, e.g. in the EGF-like domains in the low sensity lipoproteins receptor, thrombomodulin, the Notch protein of Drosophila melanogaster, and transforming growth factor beta 1-binding protein.