Epidermal Growth Factor-like Region Research Articles

G-protein coupled receptor 15 mediates angiogenesis and cytoprotective function of thrombomodulin

Thrombomodulin (TM) stimulates angiogenesis and protects vascular endothelial cells (ECs) via its fifth epidermal growth factor-like region (TME5); however, the cell surface receptor that mediates the pro-survival signaling activated by TM has remained unknown. We applied pull-down assay followed by MALDI-TOF MS and western blot analysis, and identified G-protein coupled receptor 15 (GPR15) as a binding partner of TME5. TME5 rescued growth inhibition and apoptosis caused by calcineurin inhibitor FK506 in vascular ECs isolated from wild type (WT) C57BL/6 mice. On the other hand, TME5 failed to protect ECs isolated from GPR15 knockout (GPR15 KO) mice from FK506-caused vascular injury. TME5 induced activation of extracellular signal-regulated kinase (ERK) and increased level of anti-apoptotic proteins in a GPR15 dependent manner. In addition, in vivo Matrigel plug angiogenesis assay found that TME5 stimulated angiogenesis in mice. TME5 promoted endothelial migration in vitro. Furthermore, TME5 increased production of NO in association with activated endothelial NO synthase (eNOS) in ECs. All these pro-angiogenesis functions of TME5 were abolished by knockout of GPR15. Our findings suggest that GPR15 plays an important role in mediating cytoprotective function as well as angiogenesis of TM.

Arnica montana Stimulates Extracellular Matrix Gene Expression in a Macrophage Cell Line Differentiated to Wound-Healing Phenotype.

Arnica montana (Arnica m.) is used for its purported anti-inflammatory and tissue healing actions after trauma, bruises, or tissue injuries, but its cellular and molecular mechanisms are largely unknown. This work tested Arnica m. effects on gene expression using an in vitro model of macrophages polarized towards a “wound-healing” phenotype. The monocyte-macrophage human THP-1 cell line was cultured and differentiated with phorbol-myristate acetate and Interleukin-4, then exposed for 24h to Arnica m. centesimal (c) dilutions 2c, 3c, 5c, 9c, 15c or Control. Total RNA was isolated and cDNA libraries were sequenced with a NextSeq500 sequencer. Genes with significantly positive (up-regulated) or negative (down-regulated) fold changes were defined as differentially expressed genes (DEGs). A total of 20 DEGs were identified in Arnica m. 2c treated cells. Of these, 7 genes were up-regulated and 13 were down-regulated. The most significantly up-regulated function concerned 4 genes with a conserved site of epidermal growth factor-like region (p<0.001) and three genes of proteinaceous extracellular matrix, including heparin sulphate proteoglycan 2 (HSPG2), fibrillin 2 (FBN2), and fibronectin (FN1) (p<0.01). Protein assay confirmed a statistically significant increase of fibronectin production (p<0.05). The down-regulated transcripts derived from mitochondrial genes coding for some components of electron transport chain. The same groups of genes were also regulated by increasing dilutions of Arnica m. (3c, 5c, 9c, 15c), although with a lower effect size. We further tested the healing potential of Arnica m. 2c in a scratch model of wound closure based on the motility of bone marrow-derived macrophages and found evidence of an accelerating effect on cell migration in this system. The results of this work, taken together, provide new insights into the action of Arnica m. in tissue healing and repair, and identify extracellular matrix regulation by macrophages as a therapeutic target.

The fifth epidermal growth factor-like region of thrombomodulin exerts cytoprotective function and prevents SOS in a murine model.

The present study found that the fifth epidermal growth factor-like domain of thrombomodulin (TME5) possesses the cytoprotective function in association with an increase in levels of anti-apoptotic myeloid cell leukemia-1 protein in an activated protein C-independent manner in human umbilical vein endothelial cells (HUVECs). Importantly, TME5 counteracted calcineurin inhibitor-induced vascular permeability and successfully prevented monocrotaline-induced sinusoidal obstruction syndrome (SOS) in a murine model. Taken together, TME5 may be useful for preventing or treating lethal complications that develop after hematopoietic stem cell transplantation such as SOS and thrombotic microangiopathy in which endothelial cell damage has a role.

Role of Gamma-Carboxyglutamic Acid (GLA) Domain in Phosphatidylserine (PS)-Regulated Activity of Factor IXa.

Both factors Xa and IXa are vitamin-K-dependent serine proteases that consist of disulfide-linked heavy and light chains. The heavy chain contains the serine protease active site, while the light chain contains the GLA (gamma-carboxyglutamic acid) domain, EGF-I (epidermal growth factor-like region) and EGF-II domains. Effect of PS on FXa: We have extensively studied the interaction of factor Xa with 1, 2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS). C6PS is a soluble analogue of phosphatidylserine, which is present in platelet membranes; it serves as a model for the effect of membrane-bound PS on factor Xa activity and structure. We located three C6PS binding sites to different domains of factor Xa using a combination of activity, circular dichroism, fluorescence, and equilibrium dialysis measurements, showing that :i.the Gla domain binds C6PS only in the absence of calcium (kd ∼ 1 mM);ii.a calcium-dependent, regulatory, PS-binding site exists in the EGFN domain when linked by calcium to the Gla domain; andiii.a second calcium-requiring site exists in the EGFC-catalytic domains and shares residues with the substrate recognition site.We have shown that full functional response to C6PS requires linkage of the Gla, EGFNC, and catalytic domains in the presence of calcium. Recently we have observed that the proteolytic activity of des EGFN factor Xa is not affected in the presence of C6PS, locating more precisely a significant component of the PS-triggered regulatory site to the EGFN domain. Efect of PS on FIXa: We have previously reported that C6PS induces a calcium-dependent conformational change in factor IXa that regulates the amidolytic and proteolytic activities. We have also shown that factor IXa binds 2 molecules of C6PS. Here we have examined the role of the GLA domain in this C6PS regulation of factor IXa using a GLA-domainless variant of factor IXa (GD-IXa). In the absence of the GLA domain, binding to C6PS has no effect on the rate of proteolytic or amidolytic activity of GD-IXa toward both factor X and synthetic substrates. The binding of C6PS to GD-IXa studied using intrinsic tryptophan fluorescence (Kd = ∼50μM) was different from that for native factor IXa (Kd = ∼2μM). The critical micelle concentration (CMC) of C6PS under the conditions of these experiments (∼300μM) was much greater than the C6PS concentrations used in our experiments. We conclude that:1.C6PS does bind to GD-IXa, but at a reduced affinity compared to factor IXa;2.C6PS does not regulate the amidolytic or proteolytic activities of factor IXa in the absence of GLA domain.This could be due to two C6PS sites existing in FIXa, with the regulatory site requiring the Gla domain (as observed for FXa), or to a requirement for the Gla domain for the regulatory activity of a single site. Future studies looking at the stoichiometry of binding of C6PS with GD-IXa will help distinguish between these possibilities.Supported by Supported by NHLBI (HL 072827).

Novel Function of Alternatively Activated Macrophages: Stabilin-1-Mediated Clearance of SPARC

The matricellular protein SPARC (secreted protein acidic and rich in cysteine) has been implicated in development, differentiation, response to injury, and tumor biology by virtue of its regulation of extracellular matrix production/assembly and its antiadhesive and antiproliferative effects on different cell types. Despite numerous biological activities described for SPARC, cell surface receptors for this protein have not been identified. By phage display and in vitro-binding assays, we now show that SPARC interacts with stabilin-1, a scavenger receptor expressed by tissue macrophages and sinusoidal endothelial cells. The interaction is mediated by the extracellular epidermal growth factor-like region of stabilin-1 containing the sequence FHGTAC. Using FACS analysis and confocal microscopy, we demonstrate that stabilin-1 internalizes and targets SPARC to an endosomal pathway in Chinese hamster ovary cells stably transfected with this receptor. In human macrophages, stabilin-1 expression is required for receptor-mediated endocytosis of SPARC. SPARC was efficiently endocytosed by alternatively activated macrophages stimulated by IL-4 and dexamethasone, but not solely by Th1 or Th2 cytokines. A time course of ligand exposure to alternatively activated macrophages revealed that stabilin-1-mediated endocytosis of SPARC was followed by its targeting for degradation, similar to the targeting of acetylated low density lipoprotein, another stabilin-1 ligand. We propose that alternatively activated macrophages coordinate extracellular matrix remodeling, angiogenesis, and tumor progression via stabilin-1-mediated endocytosis of SPARC and thereby regulate its extracellular concentration.

Heregulin ameliorates the dystrophic phenotype in mdx mice.

Duchenne's muscular dystrophy (DMD) is a fatal neuromuscular disease caused by absence of dystrophin. Utrophin is a chromosome 6-encoded dystrophin-related protein (DRP), sharing functional motifs with dystrophin. Utrophin's ability to compensate for dystrophin during development and when transgenically overexpressed has provided an important impetus for identifying activators of utrophin expression. The utrophin promoter A is transcriptionally regulated in part by heregulin-mediated, extracellular signal-related kinase-dependent activation of the GABP(alpha/beta) transcription factor complex. Therefore, this pathway offers a potential mechanism to modulate utrophin expression in muscle. We tested the ability of heregulin to improve the dystrophic phenotype in the mdx mouse model of DMD. Intraperitoneal injections of a small peptide encoding the epidermal growth factor-like region of heregulin ectodomain for 3 months in vivo resulted in up-regulation of utrophin, a marked improvement in the mechanical properties of muscle as evidenced by resistance to eccentric contraction mediated damage, and a reduction of muscle pathology. The amelioration of dystrophic phenotype by heregulin-mediated utrophin up-regulation offers a pharmacological therapeutic modality and obviates many of the toxicity and delivery issues associated with viral vector-based gene therapy for DMD.

TMEFF1 and brain tumors

TMEFF1 is a novel transmembrane protein, containing two follisatin domains and an epidermal growth factor-like region. These structural domains suggest a role for TMEFF1 in growth factor signaling. TMEFF1 fused to enhanced green fluorescent protein revealed that TMEFF1 is expressed on the cell membrane. Northern analysis of normal human tissue showed that TMEFF1 is predominantly expressed in the brain. Study of cancer cell lines from different tissues including the brain, demonstrated moderate to low levels of TMEFF1 in most of these transformed cell lines. Furthermore, quantitative real-time RT-PCR analysis of 54 brain tumors showed that most of these tumors (96%) had lower levels of TMEFF1 expression than normal brain tissue. Interestingly, ectopic expression of TMEFF1 in brain cancer cells resulted in their growth inhibition. These data suggest that TMEFF1 may behave as a tumor suppressor gene in brain cancers.

Immunogenicity and efficacy in aotus monkeys of four recombinant Plasmodium falciparum vaccines in multiple adjuvant formulations based on the 19-kilodalton C terminus of merozoite surface protein 1.

The immunogenicity and protective efficacy of four versions of recombinant C-terminal 19-kDa epidermal growth factor-like region of the major surface protein 1 (rMSP1(19)) of Plasmodium falciparum was studied in Aotus monkeys. Vaccination with each of the four rMSP1(19) constructs elicited high levels of antibodies to MSP1(19) but only one construct, the 19-kDa fragment expressed as a secreted fusion protein from Saccharomyces cerevisiae (yP30P2MSP1(19)), induced a high degree of protective immunity in Aotus nancymai against lethal P. falciparum challenge. Protective formulation required Freund's adjuvant; vaccination with yP30P2MSP1(19) in six other adjuvants that are suitable for human use induced lower levels of antibody response and no protection. These results emphasize the need to continue the search for an adjuvant that is comparable to Freund's adjuvant in potency and is safe for use in humans.

Chemical synthesis of human protein S thrombin-sensitive module and first epidermal growth factor module.

Human plasma protein S is a nonenzymatic cofactor for activated protein C (APC) in the inactivation of coagulation factors Va and VIIIa, and helps to provide an essential negative feedback on blood coagulation. Previous indirect evidence suggested that the thrombin-sensitive region (TSR: residues 47-75, 1 disulfide) and the first epidermal growth factorlike region (EGF1: residues 76-116, 3 disulfides) of protein S may be functionally important for expression of its APC cofactor activity. To study the functional importance of these modules directly, access to the isolated TSR and EGF1 modules would be preferred. Recombinant expression of protein S intact TSR and correctly folded EGF1 has not been possible. Here we describe the synthesis of both TSR and EGF1 modules by stepwise solid phase peptide synthesis using the in situ neutralization/2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluron ium hexafluorophosphate activation procedure for tert-butoxycarbonyl chemistry. For the TSR, correct intramodular disulfide bonding was confirmed. To overcome folding difficulties with the EGF1, a two-step oxidation procedure was used in which the cysteines involved in the middle, crossing, disulfide bond (Cys85-Cys102) remained protected with acetamidomethyl (Acm) groups after hydrogen fluoride treatment of the peptide resin. Selective formation of the first two disulfide bonds (Cys80-Cys93 and Cys104-Cys113) was followed by release of the Acm groups and subsequent formation of the third disulfide bond (Cys85-Cys102). CD studies revealed 54% of beta-sheet/turn in the EGF1 that is characteristic for EGF modules. Deuterium exchange studies suggested a very tightly packed core in EGF1 that is not accessible to the bulk solvent, likely a result from the compact structure caused by its three disulfide bonds. The 30% beta-sheet structure observed in the TSR involved amide protons that could be readily exchanged by deuterons, likely reflecting a more flexible structure of the TSR loop in contrast to the rigid structure of EGF1. The establishment of synthetic access to the TSR and EGF1 of protein S provides a versatile tool to study interactions of these modules with the blood coagulation components of the anticoagulant plasma protein C pathway.

Chemical synthesis of human protein S thrombin‐sensitive module and first epidermal growth factor module

Human plasma protein S is a nonenzymatic cofactor for activated protein C (APC) in the inactivation of coagulation factors Va and VIIIa, and helps to provide an essential negative feedback on blood coagulation. Previous indirect evidence suggested that the thrombin-sensitive region (TSR:residues 47–75, 1 disulfide) and the first epidermal growth factorlike region (EGF1: residues 76–116, 3 disulfides) of protein S may be functionally important for expression of its APC cofactor activity. To study the functional importance of these modules directly, access to the isolated TSR and EGF1 modules would be preferred. Recombinant expression of protein S intact TSR and correctly folded EGF1 has not been possible. Here we describe the synthesis of both TSR and EGF1 modules by stepwise solid phase peptide synthesis using the in situ neutralization/2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate activation procedure for tert-butoxycarbonyl chemistry. For the TSR, correct intramodular disulfide bonding was confirmed. To overcome folding difficulties with the EGF1, a two-step oxidation procedure was used in which the cysteines involved in the middle, crossing, disulfide bond (Cys85-Cys102) remained protected with acetamidomethyl (Acm) groups after hydrogen fluoride treatment of the peptide resin. Selective formation of the first two disulfide bonds (Cys80-Cys93 and Cys104-Cys113) was followed by release of the Acm groups and subsequent formation of the third disulfide bond (Cys85-Cys102). CD studies revealed 54% of β-sheet/turn in the EGF1 that is characteristic for EGF modules. Deuterium exchange studies suggested a very tightly packed core in EGF1 that is not accessible to the bulk solvent, likely a result from the compact structure caused by its three disulfide bonds. The 30% β-sheet structure observed in the TSR involved amide protons that could be readily exchanged by deuterons, likely reflecting a more flexible structure of the TSR loop in contrast to the rigid structure of EGF1. The establishment of synthetic access to the TSR and EGF1 of protein S provides a versatile tool to study interactions of these modules with the blood coagulation components of the anticoagulant plasma protein C pathway. © 1998 John Wiley & Sons, Inc. Biopoly 46: 53–63, 1998

Hypothesis: is soluble P-selectin a new marker of platelet activation?

P-selectin (CD62P, previously also known as GMP140 and PADGEM) is, alongside E-selectin (CD62E) and L-selectin (CD62L), a member of the selectin family of adhesion molecules, sharing common structural features but having different tissue distributions. It has a mass of approximately 140 kDa and is a protein rich in cysteine, containing several complex N-linked oligosaccharides chains. The extracellular domain is arranged as a lectin region (the amino-terminal of 120 amino acid residues), an epidermal growth factor-like region and nine discrete complement regulatory-like regions (each about 60 amino acid residues in length). In addition, there is a cross-membrane segment and an intracytoplasmic tail (Fig. 1). P-selectin is so named because it is a component of the cell membrane of the a granules of platelets, the organelle which stores coagulation-related substances such as b thromboglobulin and platelet factor 4. It is also a component of the membrane of the Weibel-Palade body (the endothelial cell specific organelle which stores von Willebrand factor) and is rapidly mobilised to the cell surface by various stimuli including inflammatory mediators [1–3]. The mucin-like glycoprotein ligand for P-selectin (PSGL-1) bears Oand N-linked carbohydrates, and is found on neutrophils, natural killer cells, monocytes and T-lymphocytes, giving rise to the hypothesis that it is involved in an interaction between these leukocytes and the endothelium, (possibly as part of the rolling/ binding/activation sequence), between platelets and neutrophils, and between platelets and the endothelium [4–8].

Genetic regulation of protective immune response in congenic strains of mice vaccinated with a subunit malaria vaccine.

The C-terminal 19-kDa, epidermal growth factor-like region of the merozoite surface protein 1 (MSP1) has been used as a vaccine to induce protective immunity to Plasmodium yoelii in mice and to Plasmodium falciparum in monkeys. To analyze the mechanisms and genetic regulation of this MSP1 vaccine-induced protection, we studied the immunologic correlates of protection in H-2 recombinant and congenic mouse strains on the B10 background. Multiple H-2-linked loci were found to contribute, each with a different mechanism. One locus mapped to the I-A region based on the strong protection in C57BL/10 mice compared with intermediate protection in B10.A(4R) mice and the lack of a difference between B10.AKM and B10.MBR mice. Differences in efficacy of passively transferred antisera from vaccinated C57BL/10 vs B10.A(4R) mice indicated that the protection regulated by the I-A locus was at least in part Ab dependent. Two loci mapped to the right of I-A (FE, H-2S, or H-2D) based on a correlation with the number of H-2k loci to the right of I-A in mice that were I-Ak. One effect was Ab independent and may correspond to a possible negative effect of the I-Ek locus. T cells from protected and nonprotected strains differed in their production of IFN-gamma and TNF-alpha following immunization with MSP1(19), but it was unclear how the differential patterns of cytokine expression related to the level of protection. Thus, MSP1(19) vaccine-induced protection is regulated by H-2-linked loci corresponding to two different immune mechanisms. These findings may indicate the need for more than one Ag in a vaccine to protect an HLA-diverse population.

Construction and characterization of a chimeric protein consisting of tissue-type plasminogen activator and the epidermal growth factor-like regions of thrombomodulin

We have constructed and characterized a chimeric anticoagulant and fibrinolytic protein by inserting the portion of thrombomodulin (TM) carrying protein C (PC) activating cofactor activity into tissue-type plasminogen activator (t-PA). This was done by replacing the epidermal growth factor (EGF)-like domain of t-PA with the six EGF-like repeats from TM. The 105 kDa chimeric molecule was secreted from transfected COS-7 cells as a single-chain molecule that was recognised by both anti-TM and anti-t-PA antisera. It was shown to have similar PC activating cofactor activity to other soluble derivatives of TM (K m for Protein C of 2.2±0.4 μM), as well as amidolytic and plasminogen activating activity. However, the chimeric protein is not converted to a two chain molecule and is stimulated less by fibrin, due to a lower affinity for lysine. The demonstration that TM activity can be incorporated into a fibrinolytic enzyme is a step towards enhanced fibrinolysis in patients with thrombosis.

Protein S binding to human endothelial cells is required for expression of cofactor activity for activated protein C.

An important feedback mechanism in blood coagulation is supplied by the protein C/protein S anticoagulant pathway. In this study we demonstrate that the binding of human protein S to cultured human umbilical vein endothelial cells (HUVECs) is required for the expression of cofactor activity of protein S toward factor Va inactivation by activated protein C (APC). The initial rate of endothelial cell-mediated factor Va inactivation was 21.7 pM factor Va/50 pM APC min-1, which could be enhanced twice at a protein S concentration of 5 nM. This increase appeared to be specific for protein S because it could be inhibited by C4b-binding protein and polyclonal antibodies against protein S. Furthermore, thrombin-cleaved protein S did not accelerate factor Va inactivation by APC on endothelial cells. The binding of 125I-protein S to endothelial cells was time-dependent, specific, saturable, and required the presence of calcium ions. Scatchard analysis revealed (8.0 +/- 0.3) x 10(5) binding sites per cell with an apparent Kd of 24.4 +/- 2.2 nM. To study the physiological importance of the binding of protein S to human endothelial cells, seven monoclonal antibodies were examined for their ability to influence the protein S cofactor activity and binding capacity. Monoclonal antibodies directed against the gamma-carboxyglutamic acid domain and the thrombin-sensitive region of protein S completely inhibited the protein S cofactor function in factor Va inactivation by APC on HUVECs. These monoclonal antibodies also inhibited 125I-protein S binding to HUVECs. Another monoclonal antibody, directed against an epitope on the third and/or fourth epidermal growth factor-like region, did not influence either protein S cofactor activity or binding of protein S to HUVECs. We conclude that binding of protein S to HUVECs is essential for the expression of its cofactor activity for APC. At least two regions in protein S, the gamma-carboxyglutamic acid domain and the thrombin-sensitive region, are involved in the expression of cofactor activity.

The complete sequence of perlecan, a basement membrane heparan sulfate proteoglycan, reveals extensive similarity with laminin A chain, low density lipoprotein-receptor, and the neural cell adhesion molecule.

A heparan sulfate proteoglycan is a component of all basement membranes. This molecule consists of three heparan sulfate side chains linked to a large core protein of approximately 400 kDa. We have isolated seven overlapping murine cDNA clones that encode the entire mRNA sequence of 12.685 kilobases of this molecule. This sequence has a single open reading frame of 3,707 amino acids that encodes for a protein of 396 kDa. Identical or near identical matchups with nine peptide sequences derived from the core protein of the molecule isolated from the Engelbreth-Holm-Swarm tumor were found with the deduced sequence. Sequence analysis and data base comparison of the deduced sequence show the protein to consist of five different domains, most of which contain internal repeats. Domain I contains a start methionine followed by a typical signal transfer sequence and a unique segment of 172 amino acids that contains the three probable sites of heparan sulfate attachment, SGD. Domain II contains four cysteine- and acidic amino acid-rich repeats that are very similar to those found in the LDL receptor and proteins such as GP330. Domain III consists of cysteine-rich and globular regions, both of which show similarity to those in the short arm of the laminin A chain. Domain IV contains 14 repeats of the immunoglobulin superfamily that are most highly similar to the immunoglobulin-like repeats in the neural cell adhesion molecule. Domain V contains three repeats with similarity to the laminin A chain G domain that are separated by epidermal growth factor-like regions not found in the laminin A chain. As the primary structural data agree with the appearance of the molecule in the electron microscope as a series of globules separated by rods, or "beads on a string," we have adopted the name perlecan for this molecule. The variety of domains in perlecan suggest multiple interactions with other molecules.

Structural requirements for Ca2+ binding to the gamma-carboxyglutamic acid and epidermal growth factor-like regions of factor IX. Studies using intact domains isolated from controlled proteolytic digests of bovine factor IX

Blood coagulation factor IX is composed of discrete domains with an NH2-terminal vitamin K-dependent gamma-carboxyglutamic acid (Gla)-containing region, followed by two domains that are homologous with the epidermal growth factor (EGF) precursor and a COOH-terminal serine protease part. Calcium ions bind to the Gla-containing region and to the NH2-terminal EGF-like domain. To be able to determine the structure and function of the Gla- and EGF-like domains, we have devised a method for cleaving factor IX under controlled conditions and isolating the intact domains in high yield, either separately or linked together. The Ca2+ and Mg2+ binding properties of these fragments were examined by monitoring the metal ion-induced changes in intrinsic protein fluorescence. A fragment, consisting of the Gla region linked to the two EGF-like domains, bound Ca2+ in a manner that was indistinguishable from that of the intact molecule, indicating a native conformation. The Ca2+ affinity of the isolated Gla region was lower, suggesting that the EGF-like domains function as a scaffold for the folding of the Gla region. The Gla-independent high affinity metal ion binding site in the NH2-terminal EGF-like domain was shown to bind Ca2+ but not Mg2+. A comparison with similar studies of factor X (Persson, E., Björk, I., and Stenflo, J. (1991) J. Biol. Chem. 266, 2444-2452) suggests that the Ca2(+)-induced fluorescence quenching is due to an altered environment primarily around the tryptophan residue in position 42.

Partial purification and characterization of bovine liver aspartyl beta-hydroxylase.

In vitro hydroxylation of aspartic acid has recently been demonstrated in a synthetic peptide based on the structure of the first epidermal growth factor domain in human factor IX (Gronke, R. S., VanDusen, W. J., Garsky, V. M., Jacobs, J. W., Sardana, M. K., Stern, A. M., and Friedman, P. A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3609-3613). The putative enzyme responsible for the posttranslational modification, aspartyl beta-hydroxylase, has been shown to be a member of a class of 2-ketoglutarate-dependent dioxygenases, which include prolyl-4- and lysyl-hydroxylases. In the present study, we describe the solubilization with nonionic detergent of the enzyme from bovine liver microsomes and its purification using DEAE-cellulose followed by heparin-Sepharose. No additional detergent was required during purification. The partially purified enzyme preparation was found to contain no prolyl-4- or lysyl-hydroxylase activity. Using a synthetic peptide based on the structure of the epidermal growth factor-like region in human factor X as substrate, the apparent Km values for iron and alpha-ketoglutarate were 3 and 5 microM, respectively. The enzyme hydroxylated the factor X peptide with the same stereospecificity (erythro beta-hydroxyaspartic acid) and occurred only at the aspartate corresponding to the position seen in vivo. Furthermore, the extent to which either peptide (factor IX or X) was hydroxylated reflected the extent of hydroxylation observed for both human plasma factors IX and X.

Chemical and functional characterization of a fragment of C1s containing the epidermal growth factor homology region

C1-s, one of the three subcomponents of C1-, the first component of complement, is a serine protease comprising two disulfide-linked chains, the B chain, containing the catalytic site, and the A chain, involved in Ca2+ binding and Ca2(+)-dependent interaction(s) with the other C1- subcomponents. In an attempt to identify the regions responsible for the latter functions, C1-s was submitted to limited proteolysis with plasmin, a treatment that split the A chain into three major fragments, alpha 1, alpha 2, and gamma. Fragment alpha 2, which comprised the epidermal growth factor-like (EGF-like) region of C1-s, was heterogeneous, starting at serine 97 or phenylalanine 105 and ending at lysine 195. This fragment was reduced and alkylated and then digested with elastase, and three peptides covering positions 131-135, 131-139, and 131-140 were characterized by amino acid analysis, Edman degradation, and mass spectrometry, showing that position 134 of C1-s is occupied partly by an asparagine (47%) and partly by an erythro-beta-hydroxyasparagine, in contrast with the homologous position (150) of C1-r which only contains erythro-beta-hydroxyasparagine. As measured by equilibrium dialysis, native alpha 2, like the other plasmin-cleavage fragments, did not retain the ability of intact C1-s to bind Ca2+. In the same way, plasmin cleavage abolished the ability of C1-s to dimerize or to associate with C1-r in the presence of Ca2+. In contrast, both alpha 2 and the N-terminal alpha 1 fragment, starting at serine 24 of the A chain, were able to compete significantly with intact C1s for the formation of the Ca2(+)-dependent C1-s-C1r-C1-r-C1-s tetramer.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of human blood coagulation factor XII cDNA. Prediction of the primary structure of factor XII and the tertiary structure of beta-factor XIIa.

A human liver cDNA library was screened by colony hybridization with two mixtures of synthetic oligodeoxyribonucleotides as probes. These oligonucleotides encoded regions of beta-factor XIIa as predicted from the amino acid sequence. Four positive clones were isolated that contained DNA coding for most of factor XII mRNA. DNA sequence analysis of these overlapping clones showed that they contained DNA coding for part of an amino-terminal extension, the complete amino acid sequence of plasma factor XII, a TGA stop codon, a 3' untranslated region of 150 nucleotides, and a poly(A)+ tail. The cDNA sequence predicts that plasma factor XII consists of 596 amino acid residues. Within the predicted amino acid sequence of factor XII, we have identified three peptide bonds that are cleaved by kallikrein during the formation of beta-factor XIIa. Comparison of the structure of factor XII with other proteins revealed extensive sequence identity with regions of tissue-type plasminogen activator (the epidermal growth factor-like region and the kringle region) and fibronectin (type I and type II homologies). As the type II region of fibronectin contains a collagen-binding site, the homologous region in factor XII may be responsible for the binding of factor XII to collagen. The carboxyl-terminal region of factor XII shares considerable amino acid sequence homology with other serine proteases including trypsin and many clotting factors. A preliminary structural model of beta-factor XIIa is proposed based on the known high resolution x-ray diffraction structures of trypsin, chymotrypsin, and elastase.