Protein C in bovine plasma after warfarin treatment. Purification, partial characterization, and beta-hydroxyaspartic acid content

Protein C was purified from the plasma of a cow treated with the vitamin K antagonist warfarin. The purified protein appeared not to bind Ca2+ ions in contrast to protein C from an untreated animal. The gamma-carboxyglutamic acid content of the abnormal protein C was reduced to approximately 10% of normal, whereas the beta-hydroxyaspartic acid content was only slightly decreased, suggesting that vitamin K is not involved in the postribosomal hydroxylation of the aspartic acid residue in position 71 of the light chain of protein C. The abnormal and normal proteins were activated at the same rates by thrombin, but normal protein C was more rapidly activated by the thrombin-thrombomodulin complex. Compared to normal protein C, the abnormal one had virtually no anticoagulant activity.     

Characterization of functionally important domains in human vitamin K-dependent protein S using monoclonal antibodies.

Vitamin K-dependent protein S is an anticoagulant plasma protein functioning as a cofactor to activated protein C in the degradation of coagulation factors Va and VIIIa. To determine which regions in protein S are important for its cofactor activity, we have raised and characterized a large panel of monoclonal antibodies against human protein S. Several of the antibodies were directed against Ca2(+)-dependent epitopes, and they were found to be located either in the domain containing gamma-carboxyglutamic acid (Gla), the thrombin-sensitive region, or in the first epidermal growth factor (EGF)-like domain. The first two types of epitopes were exposed at approximately 1 mM Ca2+, whereas the epitope(s) in the EGF-like domains required less than 1 microM Ca2+, suggesting the presence of one or more high affinity Ca2(+)-binding site(s). The antibodies, as well as their Fab' fragments, against all three types of Ca2(+)-dependent epitopes efficiently inhibited the activated protein C cofactor function of protein S, but through different mechanisms. The antibodies against the Gla domain exerted their effects through inhibition of protein S binding to negatively charged phospholipid. Fab'-fragments of antibodies against the thrombin-sensitive region and the first EGF-like domain were the most potent inhibitors of the activated protein C cofactor function but did not inhibit phospholipid binding of protein S. In conclusion, we have identified the domains in protein S that are important for the activated protein C cofactor activity. The Gla domain is instrumental in the binding of protein S to phospholipid, whereas the thrombin-sensitive region and the first EGF-like domain may be directly involved in protein-protein interactions on the phospholipid surface.     

The high affinity calcium-binding site involved in protein C activation is outside the first epidermal growth factor homology domain.

Binding Ca2+ to a high affinity site in protein C and 4-carboxyglutamic acid (Gla)-domainless protein C results in a conformational change that is required for activation by the thrombin-thrombomodulin complex, the natural activator of protein C. It has been hypothesized that this high affinity Ca(2+)-binding site is located in the NH2-terminal epidermal growth factor (EGF) homology region of protein C. We have expressed in human 293 cells a deletion mutant of protein C (E2-PD) which lacks the entire Gla region as well as the NH2-terminal EGF homology region of protein C. Ca2+ inhibits activation of E2-PD or Gla-domainless protein C by thrombin with half-maximal inhibition occurring at Ca2+ concentrations of 103 +/- 11 and 70 +/- 7 microM, respectively, but is required for both E2-PD and Gla-domainless protein C activation by the thrombin-thrombomodulin complex with half-maximal acceleration occurring at Ca2+ concentrations of 87 +/- 8 and 89 +/- 8 microM, respectively. Both E2-PD and Gla-domainless protein C exhibit a reversible, Ca(2+)- but not Mg(2+)-dependent decrease (6 +/- 1%) in fluorescence emission intensity with Kd = 38 +/- 3 microM Ca2+. We conclude that the high affinity Ca(2+)-binding site important for the activation of protein C is located outside of the NH2-terminal EGF homology region and that the metal-binding site in the NH2-terminal EGF homology region may not be a high affinity site in intact protein C.     

Calcium-dependent interaction between the epidermal growth factor precursor-like region of human protein C and a monoclonal antibody

Protein C, like the other vitamin K-dependent plasma proteins that participate in blood coagulation, except prothrombin, has at least one high affinity calcium-binding site that is independent of gamma-carboxyglutamic acid. Calcium binding to this site is required for activation of protein C by the thrombin-thrombomodulin complex. In an attempt to localize this calcium-binding site, we subjected protein C to limited tryptic digestion. A monoclonal antibody that recognizes a calcium-dependent epitope both in intact protein C, in gamma-carboxyglutamic acid-domainless protein C, and in activated protein C, was used to isolate a fragment from the tryptic digest. The fragment was derived from the light chain of protein C and consisted of the two domains that are homologous to the epidermal growth factor precursor. Half-maximal binding of the intact protein and of the isolated fragment by the antibody occurred at 100-200 microM Ca2+. The results suggest the presence of a Ca2+-binding site in the epidermal growth factor homology region of 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.     

Calcium-binding properties of bovine factor X lacking the gamma-carboxyglutamic acid-containing region

In bovine protein C normal activation by the thrombin-thrombomodulin complex requires binding of calcium to one high affinity binding site, contained in a protein fragment that lacks the gamma-carboxyglutamic acid (Gla) region (Esmon, N. L., De Bault, L. E., and Esmon, C. T. (1983) J. Biol. Chem. 258, 5548-5553). In this work, the calcium binding to and the conformational change induced by calcium in the corresponding Gla-domainless fragment of bovine factor X, prepared by limited proteolysis by chymotrypsin, were compared with the calcium-binding properties of Gla-domainless protein C. Equilibrium dialysis experiments demonstrated that the proteolytically modified factor X has one high affinity calcium ion-binding site with Kd = 180 microM, a value almost identical to the Kd for the binding of calcium to proteolytically modified protein C. Measurements of the rate of disulfide bond reduction by thioredoxin showed that the disulfide bonds of both factor X and protein C lacking the Gla domains were more rapidly reduced in the absence than in the presence of calcium. Thus, calcium binding induces a conformational change in both proteolytically modified proteins. Calcium binding to Gla-domainless protein C is accompanied by a quenching of the intrinsic tryptophan fluorescence and by changes in the CD spectrum, indicative of perturbation of the environment of aromatic amino acids by the metal ion. However, no such changes were observed with the proteolytically modified factor X. This difference may be due to the fact that one tryptophan residue (in position 84) is present in the light chain of the proteolytically modified protein C but none in the light chain of the modified factor X. The light chain of factor X has beta-hydroxyaspartic acid in position 64 which is homologous to the beta-hydroxyaspartic acid in position 71 in the light chain of protein C. Our results are compatible with the hypothesis that beta-hydroxyaspartic acid is involved in the Ca2+ ion binding.     

Immunochemical studies of conformational alterations in bone gamma-carboxyglutamic acid containing protein

The Ca2+-dependent transition of the vitamin K dependent bone protein bone Gla-containing protein (BGP) was investigated by use of anti-BGP antibody that reacts with the Ca2+-dependent conformation of BGP. Antibody binding occurred in the presence of Ca2+ or Mg2+ with a Kd(app) of 1.75 mM for Ca2+. Upon removal of Ca2+ with ethylenediaminetetraacetic acid, antibody binding was eliminated. Upon thermal acid decarboxylation of BGP, Ca2+-independent binding of the antibody was restored. Thus, the epitope not expressed by fully carboxylated BGP in the absence of calcium ion was restored either by addition of Ca2+ or by decarboxylation of the protein. Circular dichroic studies of fully carboxylated and fully decarboxylated BGP indicated that addition of Ca2+ to the fully carboxylated protein or decarboxylation to produce the glutamic acid containing equivalent of BGP resulted in increased order structure (apparent alpha-helix) in the protein, and this alteration was coincident with antibody binding. These data suggest that carboxylation of this vitamin K dependent protein may lead to increased disorder in the protein as compared to the glutamic acid containing equivalent. Upon Ca2+ binding a structure more equivalent to the Glu-containing protein is obtained.     

Monoclonal antibodies for human thrombomodulin which recognize binding sites for thrombin and protein C

Monoclonal antibodies for human thrombomodulin, a cofactor for thrombin-catalyzed activation of protein C, were prepared and their epitopes characterized. All six antibodies (MFTM-1-MFTM-6) bound to an elastase-digested active fragment of thrombomodulin, which contains six consecutive EGF domains. Binding of thrombomodulin to these antibodies did not depend on Ca2+ concentration. MFTM-4, MFTM-5, and MFTM-6 strongly inhibited protein C activation by thrombin and thrombomodulin. MFTM-4 and MFTM-5 inhibited thrombin binding to fixed thrombomodulin and bound to a recombinant mutant EGF456 protein, which contained the fourth, fifth, and sixth EGF domains of thrombomodulin. However, MFTM-6 did not inhibit thrombin binding to thrombomodulin and did not bind to EGF456 protein. Binding of thrombomodulin to fixed MFTM-4 or MFTM-5 was competitively inhibited by a recombinant mutant EGF45 protein which contained the fifth and sixth EGF-domains. These results suggest that epitopes of MFTM-4 and MFTM-5 are located in the fifth EGF domain of thrombomodulin. Thus, the binding site for thrombin is located in the fifth EGF domain. These results also suggest that an epitope for MFTM-6 is located at a region near the binding site for gamma-carboxyglutamic acid residues of protein C via Ca2+ on thrombomodulin.     

Immunoaffinity purification of protein C by using conformation-specific monoclonal antibodies to protein C-calcium ion complex

We isolated protein C from a barium citrate-adsorbed fresh plasma and human factor IX concentrate by immunoaffinity chromatography on a column of Sepharose coupled with monoclonal antibodies to protein C. The antibodies used were conformation-specific monoclonal antibodies to the calcium-induced structure of protein C. Protein C was bound to antibodies coupled with Sepharose in the presence of calcium ions and was eluted with EDTA. This immunopurification resulted in a 13,000-fold purification of the fully functional zymogen from plasma. The immunoaffinity-isolated protein C was found to have higher amounts of single-chain protein C than conventionally isolated protein C when analyzed by sodium dodecyl sulfate-polyacrylamide gels under reduced conditions. The factor IX concentrate was applied to this Ca2+-dependent antibody JTC-3-immobilized Sepharose in the presence of 5 mM CaCl2, and protein C with its gamma-carboxyglutamic acid (Gla) domain intact was firstly bound to this column and then eluted by metal chelation with EDTA. When flow-through fractions were applied again in the presence of Ca2+ to this column, modified protein C which had lost its N-terminal 42-residue peptide was weakly bound to this column. It was eluted in the absence of Ca2+. However, only a low percentage of modified protein C was detectable by an enzyme-linked immunosorbent assay using Ca2+-dependent monoclonal antibody JTC-3 and peroxidase-labeled immunopurified polyclonal antibody. These results indicate that factor IX concentrate has both Gla-domain-intact and Gla-domainless protein C. Moreover, it suggests that Ca2+-dependent monoclonal antibody JTC-3 may recognize the coupled conformational change of protein C induced by the combined effect of Ca2+ binding to the Gla domain and to other parts of protein C.     

N Terminus of Sos1 Ras Exchange Factor: Critical Roles for the Dbl and Pleckstrin Homology Domains

We have studied the functional importance of the N terminus of mouse Sos1 (mSos1), a ubiquitously expressed Ras-specific guanine nucleotide exchange factor whose C-terminal sequences bind Grb-2. Consistent with previous reports, addition of a myristoylation signal to mSos1 (MyrSos1) rendered it transforming for NIH 3T3 cells and deletion of the mSos C terminus (MyrSos1-ΔC) did not interfere with this activity. However, an N-terminally deleted myristoylated mSos1 protein (MyrSos1-ΔN) was transformation defective, although the protein was stable and localized to the membrane. Site-directed mutagenesis was used to examine the role of the Dbl and pleckstrin homology (PH) domains located in the N terminus. When mutations in the PH domain were introduced into two conserved amino acids either singly or together in MyrSos1 or MyrSos1-ΔC, the transforming activity was severely impaired. An analogous reduction in biological activity was seen when a cluster of point mutations was engineered into the Dbl domain. The mitogen-activation protein (MAP) kinase activities induced by the various Dbl and PH mutants of MyrSos1 correlated with their biological activities. When NIH 3T3 cells were transfected with a myristoylated Sos N terminus, their growth response to epidermal growth factor (EGF), platelet-derived growth factor, lysophosphatidic acid or serum was greatly impaired. The dominant inhibitory biological activity of the N terminus correlated with its ability to impair EGF-dependent activation of GTP-Ras and of MAP kinase, as well with the ability of endogenous Sos to form a stable complex with activated EGF receptors. The N terminus with mutations in the Dbl and PH domains was much less inhibitory in these biological and biochemical assays. In contrast to wild-type Sos1, nonmyristoylated versions of Sos1-ΔN and Sos1-ΔC did not form a stable complex with activated EGF receptors. We conclude that the Dbl and PH domains are critical for Sos function and that stable association of Sos with activated EGF receptors requires both the Sos N and C termini.     

Proteolytic formation and properties of a fragment of protein C containing the gamma-carboxyglutamic acid rich domain and the EGF-like region

The function of the epidermal growth factor (EGF) like domains in the vitamin K dependent plasma proteins is largely unknown. In order to elucidate the function of these domains in protein C, we have devised a method to isolate the EGF-like region from the light chain connected to the NH2-terminal region, containing the gamma-carboxyglutamic acid (Gla) residues. This was accomplished by tryptic cleavage of protein C that had been reversibly modified with citraconic anhydride to prevent cleavage at the lysine residue (in position 43) that is located between the two regions. The isolated fragment consists of residues 1-143 from the light chain of protein C connected by a disulfide bond to residues 108-131 from the heavy chain. Upon Ca2+ binding to the isolated Gla-EGF fragment from bovine protein C, the tryptophan fluorescence emission was quenched in a manner indicating binding to at least two classes of binding sites. These were presumably the Gla-independent Ca2(+)-binding site located in the EGF-like region and the lower affinity sites in the Gla region. A comparison with the tryptophan fluorescence quenching that occurred upon Ca2+ binding to the separately isolated EGF-like and Gla regions suggested that the EGF-like region influenced the structure and Ca2+ binding of the Gla region. The isolated Gla-EGF fragment functioned as an inhibitor of the anticoagulant effect of activated protein C in a clotting assay, whereas no inhibition was observed with either the Gla region or the EGF-like region.     

The interaction of thrombomodulin with Ca2+.

Thrombomodulin (TM) is a cofactor for protein C activation by thrombin and each residue of a consensus Ca2+ site in the sixth epidermal growth factor domain (EGF6) is essential for this cofactor activity [Nagashima, M., Lundh, E., Leonard, J.C., Morser, J. & Parkinson, J.F. (1993) J. Biol. Chem. 268, 2888-2892]. Three soluble analogs of the extracellular domain of TM, solulin (Glu4-Pro490), TME1-6 (Cys227-Cys462) and TMEi4-6 (Val345-Cys462) were prepared for equilibrium dialysis experiments by exhaustive dialysis against Ca2+-depleted buffer. However, all three analogs still contained one tightly bound Ca2+ (Kd approximately 2 microm), which could only be removed by EDTA. Epitope mapping with Ca2+-dependent monoclonal antibodies to EGF6 provided further localization of this tight Ca2+ site. Equilibrium dialysis of the soluble TM analogs in [45Ca2+] between 10 and 200 microm revealed a second Ca2+ site (Kd = 30 +/- 10 microm) in both solulin and TME1-6, but not in TMEi4-6. Ca2+ binding to this second site was unaffected by bound thrombin and we attribute it to the consensus Ca2+ site in EGF3. A 75-fold decrease in the binding affinity of thrombin to TM was observed with immobilized solulin treated with EDTA to remove the high affinity Ca2+ by measuring kassoc and kdiss rates in a BIAcoretrade mark instrument. Ca2+-dependent conformational transitions detected by CD spectroscopy in the far UV indicate a more ordered structure upon Ca2+ binding. Bound Ca2+ stabilized soluble TM against protease digestion at a trypsin-like protease-sensitive site between Arg456 and His457 in EGF6 compared with protease treatment in EDTA. Finally, TM containing EGF domains 4-6, but lacking the interdomain loop between EGF3 and 4 (TME4-6), has an identical Ca2+ dependence for the activation of protein C as found for TMEi4-6, indicating this interdomain loop is not involved in Ca2+ binding.     

The interaction of a Ca2+-dependent monoclonal antibody with the protein C activation peptide region. Evidence for obligatory Ca2+ binding to both antigen and antibody

Protein C undergoes Ca2+-induced conformational changes required for activation by the thrombin-thrombomodulin complex. A Ca2+-dependent monoclonal antibody (HPC4) that blocks protein C activation was used to study conformational changes near the activation site in protein C. The half-maximal Ca2+ dependence was similar for protein C and gamma-carboxy-glutamic acid-domainless protein C for binding to HPC4 (205 +/- 23 and 110 +/- 29 microM Ca2+, respectively), activation rates (214 +/- 22 and 210 +/- 37 microM), and intrinsic fluorescence of gamma-carboxyglutamic acid-domainless protein C (176 +/- 34 microM). Protein C heavy chain binding to HPC4 was half-maximal at 36 microM Ca2+, although neither the heavy chain nor HPC4 separately bound Ca2+ with high affinity. The epitope was lost when the activation peptide was released. A synthetic peptide, P (6-17), which spans the activation site, exhibited Ca2+-dependent binding to HPC4 (half-maximal binding = 6 microM Ca2+). Thus, each decrease in antigen structure resulted in a reduced Ca2+ requirement for binding to HPC4. Tb3+ and Ca2+ binding studies demonstrated a Ca2+-binding site in HPC4 required for high affinity antigen binding. These studies provide the first direct evidence for a Ca2+-induced conformational change in the activation region of a vitamin K-dependent zymogen. Furthermore, Ca2+ binding to HPC4 is required for antigen binding. The multiple roles of Ca2+ described may be useful in interpretation of other metal-dependent antibody/antigen interactions.     

Epidermal growth factor activates calcium channels by phospholipase A2/5-lipoxygenase-mediated leukotriene C4 production.

Epidermal growth factor (EGF) induces a Ca2+ influx in many cell types, but the underlying mechanisms are so far unresolved. We report that: EGF-induced Ca2+ channel activity is eliminated by lipoxygenase inhibition and is mimicked by artificial induction of lipoxygenase activity; addition of leukotriene C4 can fully mimic EGF in its ability to activate Ca2+ channels; and EGF induces a rapid accumulation of intracellular leukotriene C4. In addition, we show that EGF-induced, Ca(2+)-dependent membrane hyperpolarization and junB proto-oncogene expression are dependent on lipoxygenase activity, whereas EGF-induced cytoplasmic alkalinization is not. We conclude that PLA2/5-lipoxygenase-mediated leukotriene C4 production constitutes a novel and specific signal transduction pathway in growth factor action.     

The calcium-binding loops of the tandem C2 domains of synaptotagmin VII cooperatively mediate calcium-dependent oligomerization

Synaptotagmin VII (Syt VII), a proposed regulator for Ca2+-dependent exocytosis, showed a robust Ca2+-dependent oligomerization property via its two C2 domains (Fukuda, M., and Mikoshiba, K. (2001) J. Biol. Chem. 276, 27670-27676), but little is known about its structure or the critical residues directly involved in the oligomerization interface. In this study, site-directed mutagenesis and chimeric analysis between Syt I and Syt VII showed that three Asp residues in Ca2+-binding loop 1 or 3 (Asp-172, Asp-303, and Asp-357) are crucial to robust Ca(2+)-dependent oligomerization. Unlike Syt I, however, the polybasic sequence in the beta4 strands of the C2 structures (so-called "C2 effector domain") is not involved in the Ca2+-dependent oligomerization of Syt VII. The results also showed that the Ca2+-binding loops of the two C2 domains cooperatively mediate Syt VII oligomerization (i.e. the presence of redundant Ca2+-binding site(s)) as well as the importance of Ca2+-dependent oligomerization of Syt VII in Ca2+-regulated secretion. Expression of wild-type tandem C2 domains of Syt VII in PC12 cells inhibited Ca2+-dependent neuropeptide Y release, whereas mutant fragments lacking Ca2+-dependent oligomerization activity had no effect. Finally, rotary-shadowing electron microscopy showed that the Ca2+-dependent oligomer of Syt VII is "a large linear structure," not an irregular aggregate. By contrast, in the absence of Ca2+ Syt VII molecules were observed to form a globular structure. Based on these results, we suggest that the linear Ca2+-dependent oligomer may be aligned at the fusion site between vesicles and plasma membrane and modulate Ca2+-regulated exocytosis by opening or dilating fusion pores.     

Aspartic acid 349 in the fourth epidermal growth factor-like structure of human thrombomodulin plays a role in its Ca(2+)-mediated binding to protein C.

The last three consecutive epidermal growth factor (EGF)-like structures of human thrombomodulin constitute the functional domain for protein C-activating cofactor activity and anticoagulant activity. Using site-directed deletion mutagenesis, we found that amino acid Asp349 of TME456, a recombinantly produced protein consisting of EGF-like structures 4, 5, and 6, is essential for retaining full protein C-activating cofactor activity. To investigate the role of Asp349 in the protein C-activating cofactor activity of human thrombomodulin, we have constructed two mutants of TMD123, a recombinantly produced protein consisting of domains D1, D2, and D3 of thrombomodulin, using site-directed point mutagenesis of the thrombomodulin coding sequence. In mutant TMD123A, the Asp349 codon was replaced with an Ala codon and in mutant TMD123E, the Asp349 codon was replaced with a Glu codon. The partially purified mutant proteins were assayed for their protein C-activating cofactor activity at various Ca2+ concentrations. TMD123 and TMD123E protein showed similar high levels of cofactor activity and similar patterns of Ca2+ dependence, while TMD123A had lower cofactor activity and did not show any Ca2+ dependence. We concluded that Asp349 in the fourth EGF-like structure of human thrombomodulin plays a role in its Ca(2+)-mediated binding to protein C.     

The C2A domain of double C2 protein gamma contains a functional nuclear localization signal

The C2 domain was originally defined as a homologous domain to the C2 regulatory region of Ca2+ -dependent protein kinase C and has been identified in more than 50 different signaling molecules. The original C2 domain of protein kinase Calpha functions as a Ca2+ binding module, and the Ca2+ binding to the C2 domain allows translocation of proteins to phospholipid membranes. By contrast, however, some C2 domains do not exhibit Ca2+ binding activity because of amino acid substitutions at Ca2+ -binding sites, and their physiological meanings remain largely unknown. In this study, we discovered an unexpected function of the Ca2+ -independent C2A domain of double C2 protein gamma (Doc2gamma) in nuclear localization. Deletion and mutation analyses revealed that the putative Ca2+ binding loop 3 of Doc2gamma contains six Arg residues ((177)RLRRRRR(183)) and that this basic cluster is both necessary and sufficient for nuclear localization of Doc2gamma. Because of the presence of the basic cluster, the C2A domain of Doc2gamma did not show Ca2+ -dependent phospholipid binding activity. Our findings indicate that by changing the nature of the putative Ca2+ binding loops the C2 domain has more diversified function in cellular signaling than a simple Ca2+ binding motif.     

Neutrophil recognition requires a Ca(2+)-induced conformational change in the lectin domain of GMP-140.

GMP-140, a receptor for myeloid cells that is expressed on surfaces of thrombin-activated platelets and endothelial cells, is a member of the selectin family of adhesion molecules that regulate leukocyte interactions with the blood vessel wall. Each selectin contains an N-terminal domain homologous to Ca(2+)-dependent lectins and mediates cell-cell contact by binding to oligosaccharide ligands in a Ca(2+)-dependent manner. The mechanisms by which Ca2+ promotes selectin-dependent cellular interactions have not been defined. We demonstrate that purified GMP-140 contains two high affinity binding sites for Ca2+ as measured by equilibrium dialysis (Kd = 22 +/- 2 microM). Occupancy of these sites by Ca2+ alters the conformation of the protein as detected by a reduction in intrinsic fluorescence emission intensity (Kd = 4.8 +/- 0.2 microM). This Ca(2+)-dependent conformational change exposes an epitope spanning residues 19-34 of the lectin domain that is recognized by a monoclonal antibody capable of blocking neutrophil adhesion to GMP-140 (half-maximal antibody binding at approximately 20 microM Ca2+). Furthermore, a synthetic peptide encoding this epitope, CQNRYTDLVAIQNKNE, inhibits neutrophil binding to GMP-140. Mg2+ also alters the conformation of the protein, but not in a manner that will support leukocyte recognition in the absence of Ca2+. There is a strong correlation between the Ca2+ levels required for neutrophil adhesion to GMP-140, for occupancy of the two Ca(2+)-binding sites, for the fluorescence-detected conformational change, and for exposure of the antibody epitope in the lectin domain. We conclude that binding of Ca2+ to high affinity sites on GMP-140 modulates the conformation of the lectin domain in a manner that is essential for leukocyte recognition.     

The structure of cell adhesion molecule uvomorulin. Insights into the molecular mechanism of Ca2+-dependent cell adhesion.

We have determined the amino acid sequence of the Ca2+-dependent cell adhesion molecule uvomorulin as it appears on the cell surface. The extracellular part of the molecule exhibits three internally repeated domains of 112 residues which are most likely generated by gene duplication. Each of the repeated domains contains two highly conserved units which could represent putative Ca2+-binding sites. Secondary structure predictions suggest that the putative Ca2+-binding units are located in external loops at the surface of the protein. The protein sequence exhibits a single membrane-spanning region and a cytoplasmic domain. Sequence comparison reveals extensive homology to the chicken L-CAM. Both uvomorulin and L-CAM are identical in 65% of their entire amino acid sequence suggesting a common origin for both CAMs.     

The effect of phospholipids on the activation of protein C by the human thrombin-thrombomodulin complex.

Human thrombomodulin, an endothelial-cell-membrane glycoprotein, has been purified from placenta by Triton X-100 extraction and by affinity chromatography on concanavalin A-Sepharose and thrombin-Sepharose. It has been characterized by its ability to promote the activation of human protein C by human alpha-thrombin in the presence of Ca2+ and fulfilled the requirements of a cofactor. Reconstitution of thrombomodulin into phospholipid vesicles containing anionic phospholipids resulted in an increased rate of activation of protein C. Cardiolipin and vesicles containing phosphatidylcholine/phosphatidylserine (1:1, w/w) were the most effective. The apparent Km of the thrombin-thrombomodulin complex for protein C was 2 microM. It was not changed in the presence of phospholipid, whereas the Vmax. could be apparently increased up to 3.2-fold depending on the phospholipid and on its concentration, the catalytic-centre activity reaching 15.7 mol of activated protein C formed/min per mol of thrombin. Above their optimal concentrations, phospholipids inhibited the amidolytic activity of activated protein C. Phospholipids had no effect on the activation of 4-carboxyglutamic acid-domainless protein C, a proteolytic derivative of protein C lacking the 4-carboxyglutamic acid residues. These results show that the positive effect of anionic phospholipids in the activation of protein C by the thrombin-thrombomodulin complex involves a Ca2+-dependent interaction between protein C and phospholipids. They suggest that the enhancement of thrombomodulin activity by such phospholipids may be of functional significance.