Role of the low density lipoprotein-related protein receptor in mediation of factor VIII catabolism

In the present study, we found that catabolism of coagulation factor VIII (fVIII) is mediated by the low density lipoprotein receptor-related protein (LPR), a liver multiligand endocytic receptor. In a solid phase assay, fVIII was shown to bind to LRP (K(d) 116 nM). The specificity was confirmed by a complete inhibition of fVIII/LRP binding by 39-kDa receptor-associated protein (RAP), an antagonist of all LRP ligands. The region of fVIII involved in its binding to LRP was localized within the A2 domain residues 484-509, based on the ability of the isolated A2 domain and the synthetic A2 domain peptide 484-509 to prevent fVIII interaction with LRP. Since vWf did not inhibit fVIII binding to LRP, we proposed that LRP receptor may internalize fVIII from its complex with vWf. Consistent with this hypothesis, mouse embryonic fibroblasts that express LRP, but not fibroblasts genetically deficient in LRP, were able to catabolize (125)I-fVIII complexed with vWf, which was not internalized by the cells. These processes could be inhibited by RAP and A2 subunit of fVIII, indicating that cellular internalization and degradation were mediated by interaction of the A2 domain of fVIII with LRP. In vivo studies of (125)I-fVIII.vWf complex clearance in mice demonstrated that RAP completely inhibited the fast phase of the biphasic (125)I-fVIII clearance that is responsible for removal of 60% of fVIII from circulation. Inhibition of the RAP-sensitive phase prolonged the half-life of (125)I-fVIII in circulation by 3.3-fold, indicating that LRP receptor plays an important role in fVIII clearance.     

Identification of a plasmin-interactive site within the A2 domain of the factor VIII heavy chain

Factor VIII is activated and inactivated by plasmin by limited proteolysis. In our one-stage clotting assay, these plasmin-catalyzed reactions were inhibited by the addition of isolated factor VIII A2 subunits and by Glu-Gly-Arg-active-site modified factor IXa. SDS-PAGE analysis showed that an anti-A2 monoclonal antibody, recognizing the factor IXa-interactive site (residues 484-509), blocked the plasmin-catalyzed cleavage at Arg(336) and Arg(372) but not at Arg(740). Surface plasmon resonance-based assays and ELISA demonstrated that the A2 subunit bound to active-site modified anhydro-plasmin with high affinity (K(d): 21 nM). Both an anti-A2 monoclonal antibody and a peptide comprising of A2 residues 479-504 blocked A2 binding by approximately 80% and approximately 55%, respectively. Mutant A2 molecules where the basic residues in A2 were converted to alanine were evaluated for binding of anhydro-plasmin. Among the tested mutants, the R484A A2 mutant possessed approximately 250-fold lower affinity than the wild-type A2. The affinities of K377A, K466A, and R471A mutants were decreased by 10-20-fold. The inhibitory effect of R484A mutant on plasmin-catalyzed inactivation of factor VIIIa was approximately 20% of that of wild-type A2. In addition, the inactivation rate by plasmin of factor VIIIa reconstituted with R484A mutant was approximately 3-fold lower than that with wild-type A2. These findings demonstrate that Arg(484) plays a key role within the A2 plasmin-binding site, responsible for plasmin-catalyzed factor VIII(a) inactivation.     

Cell surface heparan sulfate proteoglycans participate in factor VIII catabolism mediated by low density lipoprotein receptor-related protein

We have demonstrated previously that catabolism of a coagulation factor VIII (fVIII) from its complex with von Willebrand factor (vWf) is mediated by low density lipoprotein receptor-related protein (LRP) (Saenko, E. L., Yakhyaev, A. V., Mikhailenko, I., Strickland, D. K., and Sarafanov, A. G. (1999) J. Biol. Chem. 274, 37685-37692). In the present study, we found that this process is facilitated by cell surface heparan sulfate proteoglycans (HSPGs). This was demonstrated by simultaneous blocking of LRP and HSPGs in model cells, which completely prevented fVIII internalization and degradation from its complex with vWf. In contrast, the selective blocking of either receptor had a lesser effect. In vivo studies of clearance of (125)I-fVIII-vWf complex in mice also demonstrated that the simultaneous blocking of HSPGs and LRP led to a more significant prolongation of fVIII half-life (5.5-fold) than blocking of LRP alone (3.5-fold). The cell culture and in vivo experiments revealed that HSPGs are also involved in another, LRP-independent pathway of fVIII catabolism. In both pathways, HSPGs act as receptors providing the initial binding of fVIII-vWf complex to cells. We demonstrated that this binding occurs via the A2 domain of fVIII, since A2, but not other portions of fVIII or isolated vWf, strongly inhibited cell surface binding of fVIII-vWf complex, and the affinities of A2 and fVIII-vWf complex for the cells were similar. The A2 site involved in binding to heparin was localized to the region 558-565, based on the ability of the corresponding synthetic peptide to inhibit A2 binding to heparin, used as a model for HSPGs.     

Modulation of beta-amyloid precursor protein processing by the low density lipoprotein receptor-related protein (LRP). Evidence that LRP contributes to the pathogenesis of Alzheimer's disease

Beta-amyloid peptide (Abeta), which plays a central role in the pathogenesis of Alzheimer's disease, is derived from the transmembrane beta-amyloid precursor protein (APP) by proteolytic processing. Although mechanisms associated with Abeta generation are not fully understood, it is known that Abeta can be generated within endosomal compartments upon internalization of APP from the cell surface. The low density lipoprotein receptor-related protein (LRP) was previously shown to mediate the endocytosis of APP isoforms containing the Kunitz proteinase inhibitor domain (Kounnas, M. Z., Moir, R. D., Rebeck, G. W., Bush, A. I., Argraves, W. S., Tanzi, R. E., Hyman, B. T., and Strickland, D. K. (1995) Cell 82, 331-340; Knauer, M. F., Orlando, R. A., and Glabe, C. G. (1996) Brain Res. 740, 6-14). The objective of the current study was to test the hypothesis that LRP-mediated internalization of cell surface APP can modulate APP processing and thereby affect Abeta generation. Here, we show that long term culturing of cells in the presence of the LRP-antagonist RAP leads to increased cell surface levels of APP and a significant reduction in Abeta synthesis. Further, restoring LRP function in LRP-deficient cells results in a substantial increase in Abeta production. These findings demonstrate that LRP contributes to Abeta generation and suggest novel pharmacological approaches to reduce Abeta levels based on selective LRP blockade.     

Role of P1 residues Arg336 and Arg562 in the activated-Protein-C-catalysed inactivation of Factor VIIIa

APC (activated Protein C) inactivates human Factor VIIIa following cleavage at residues Arg336 and Arg562 within the A1 and A2 subunits respectively. The role of the P1 arginine in APC-catalysed inactivation of Factor VIIIa was examined by employing recombinant Factor VIIIa molecules where residues 336 and 562 were replaced with alanine and/or glutamine. Stably expressed Factor VIII proteins were activated by thrombin and resultant Factor VIIIa was reacted at high concentration with APC to minimize cofactor inactivation due to A2 subunit dissociation. APC cleaved wild-type Factor VIIIa at the A1 site with a rate approximately 25-fold greater than that for the A2 site. A1 mutants R336A and R336Q were inactivated approximately 9-fold slower than wild-type Factor VIIIa, whereas the A2 mutant R562A was inactivated approximately 2-fold slower. No cleavage at the mutated sites was observed. Taken together, these results suggested that cleavage at the A1 site was the dominant mechanism for Factor VIIIa inactivation catalysed by the proteinase. On the basis of cleavage at Arg336, a K(m) value for wild-type Factor VIIIa of 102 nM was determined, and this value was significantly greater than K(i) values (approximately 9-18 nM) obtained for an R336Q/R562Q Factor VIIIa. Furthermore, evaluation of a series of cluster mutants in the C-terminal region of the A1 subunit revealed a role for acidic residues in segment 341-345 in the APC-catalysed proteolysis of Arg336. Thus, while P1 residues contribute to catalytic efficiency, residues removed from these sites make a primary contribution to the overall binding of APC to Factor VIIIa.     

LRP/amyloid beta-peptide interaction mediates differential brain efflux of Abeta isoforms

LRP (low-density lipoprotein receptor-related protein) is linked to Alzheimer's disease (AD). Here, we report amyloid beta-peptide Abeta40 binds to immobilized LRP clusters II and IV with high affinity (Kd = 0.6-1.2 nM) compared to Abeta42 and mutant Abeta, and LRP-mediated Abeta brain capillary binding, endocytosis, and transcytosis across the mouse blood-brain barrier are substantially reduced by the high beta sheet content in Abeta and deletion of the receptor-associated protein gene. Despite low Abeta production in the brain, transgenic mice expressing low LRP-clearance mutant Abeta develop robust Abeta cerebral accumulations much earlier than Tg-2576 Abeta-overproducing mice. While Abeta does not affect LRP internalization and synthesis, it promotes proteasome-dependent LRP degradation in endothelium at concentrations > 1 microM, consistent with reduced brain capillary LRP levels in Abeta-accumulating transgenic mice, AD, and patients with cerebrovascular beta-amyloidosis. Thus, low-affinity LRP/Abeta interaction and/or Abeta-induced LRP loss at the BBB mediate brain accumulation of neurotoxic Abeta.     

Low density lipoprotein receptor-related protein mediates endocytic clearance of pro-MMP-2.TIMP-2 complex through a thrombospondin-independent mechanism

The low density lipoprotein receptor-related protein (LRP) mediates the endocytic clearance of various proteinases and proteinase.inhibitor complexes, including thrombospondin (TSP)-dependent endocytosis of matrix metalloproteinase (MMP)-2 (or gelatinase A), a key effector of extracellular matrix remodeling and cancer progression. However, the zymogen of MMP-2 (pro-MMP-2) mostly occurs in tissues as a complex with the tissue inhibitor of MMPs (TIMP-2). Here we show that clearance of the pro-MMP-2.TIMP-2 complex is also mediated by LRP, because addition of receptor-associated protein (RAP), a natural LRP ligand antagonist, inhibited endocytosis and lysosomal degradation of (125)I-pro-MMP-2.TIMP-2. Both TIMP-2 and the pro-MMP-2 collagen-binding domain independently competed for endocytosis of (125)I-pro-MMP-2.TIMP-2 complex. Surface plasmon resonance studies indicated that pro-MMP-2, TIMP-2, and pro-MMP-2.TIMP-2 directly interact with LRP in the absence of TSP. LRP-mediated endocytic clearance of (125)I-pro-MMP-2 was inhibited by anti-TSP antibodies and accelerated upon complexing with TSP-1, but these treatments had no effect on (125)I-pro-MMP-2.TIMP-2 uptake. This implies that mechanisms of clearance by LRP of pro-MMP-2 and pro-MMP-2.TIMP-2 complex are different. Interestingly, RAP did not inhibit binding of (125)I-pro-MMP-2.TIMP-2 to the cell surface. We conclude that clearance of pro-MMP-2.TIMP-2 complex is a TSP-independent two-step process, involving (i) initial binding to the cell membrane in a RAP-insensitive manner and (ii) subsequent LRP-dependent (RAP-sensitive) internalization and degradation.     

Kinetic analysis of Dictyostelium discoideum myosin motor domains with glycine-to-alanine mutations in the reactive thiol region.

Three conserved glycine residues in the reactive thiol region of Dictyostelium discoideummyosin II were replaced by alanine residues. The resulting mutants G680A, G684A, and G691A were expressed in the soluble myosin head fragment M761-2R [Anson, M., Geeves, M. A., Kurzawa, S. E., and Manstein, D. J. (1996) EMBO J. 15, 6069-6074] and characterized using transient kinetic methods. Mutant G691A showed no major alterations except for a marked increase in basal Mg2+-ATPase activity. Phosphate release seemed to be facilitated by this mutation, and the addition of actin to G691A stimulated ATP turnover not more than 3-fold. In comparison to M761-2R, mutant constructs G691A and G684A showed a 4-fold reduction in the rate of the ATP cleavage step. Most other changes in the kinetic properties of G684A were small ( approximately 2-fold). In contrast, substitution of G680 by an alanine residue led to large changes in nucleotide binding. Compared to M761-2R, rates of nucleotide binding were 20-30-fold slower and the affinity for mantADP was approximately 10-fold increased due to a 200-fold reduction in the dissociation rate constant of mantADP. The ATP-induced dissociation of actin from the acto.680A complex was normal, but the communication between ADP and actin binding was altered such that the two sites are thermodynamically uncoupled but kinetically actin still accelerates ADP release.     

Low-density Lipoprotein Receptor-related Proteins in a Novel Mechanism of Axon Guidance and Peripheral Nerve Regeneration

The low-density lipoprotein receptor-related protein receptors 1 and 2 (LRP1 and LRP2) are emerging as important cell signaling mediators in modulating neuronal growth and repair. We examined whether LRP1 and LRP2 are able to mediate a specific aspect of neuronal growth: axon guidance. We sought to identify LRP1 and LRP2 ligands that could induce axonal chemoattraction, which might have therapeutic potential. Using embryonic sensory neurons (rat dorsal root ganglia) in a growth cone turning assay, we tested a range of LRP1 and LRP2 ligands for the ability to guide growth cone navigation. Three ligands were chemorepulsive: α-2-macroglobulin, tissue plasminogen activator, and metallothionein III. Conversely, only one LRP ligand, metallothionein II, was found to be chemoattractive. Chemoattraction toward a gradient of metallothionein II was calcium-dependent, required the expression of both LRP1 and LRP2, and likely involves further co-receptors such as the tropomyosin-related kinase A (TrkA) receptor. The potential for LRP-mediated chemoattraction to mediate axonal regeneration was examined in vivo in a model of chemical denervation in adult rats. In these in vivo studies, metallothionein II was shown to enhance epidermal nerve fiber regeneration so that it was complete within 7 days compared with 14 days in saline-treated animals. Our data demonstrate that both LRP1 and LRP2 are necessary for metallothionein II-mediated chemotactic signal transduction and that they may form part of a signaling complex. Furthermore, the data suggest that LRP-mediated chemoattraction represents a novel, non-classical signaling system that has therapeutic potential as a disease-modifying agent for the injured peripheral nervous system.     

Identification of residues Asn89, Ile90, and Val107 of the factor IXa second epidermal growth factor domain that are essential for the assembly of the factor X-activating complex on activated platelets

Activated platelets promote intrinsic factor X-activating complex assembly by presenting high affinity, saturable binding sites for factor IXa mediated by two disulfide-constrained loop structures (loop 1, Cys88-Cys99; loop 2, Cys95-Cys109) within the second epidermal growth factor (EGF2) domain. To identify amino acids essential for factor X activation complex assembly, recombinant factor IXa point mutants in loop 1 (N89A, I90A, K91A, and R94A) and loop 2 (D104A, N105A, and V107A) were prepared. All seven mutants were similar to the native factor IXa by SDS-PAGE, active site titration, and content of gamma-carboxyglutamic acid residues. Kinetic constants obtained by either titrating factor X or factor VIIIa on SFLLRN-activated platelets or phospholipid vesicles revealed near normal values of Km(app) and Kd(app)FVIIIa for all mutants, indicating normal substrate and cofactor binding. In a factor Xa generation assay in the presence of activated platelets and cofactor factor VIIIa, compared with native factor IXa (Kd(app)FIXa approximately 1.1 nm, Vmax approximately 12 nm min(-1)), N89A displayed an increase of approximately 20-fold in Kd(app)FIXa and a decrease of approximately 20-fold in Vmax; I90A had an increase of approximately 5-fold in Kd(app)FIXa and approximately 10-fold decrease in Vmax; and V107A had an increase of approximately 3-fold in Kd(app)FIXa and approximately 4-fold decrease in Vmax. We conclude that residues Asn89, Ile90, and Val107 within loops 1 and 2 (Cys88-Cys109) of the EGF2 domain of factor IXa are essential for normal interactions with the platelet surface and for the assembly of the factor X-activating complex on activated platelets.     

Elimination of substrate inhibition of a β-N-acetyl-d-hexosaminidase by single site mutation

Substrate inhibition hinders chitinolytic β-N-acetyl-d-hexosaminidases in producing N-acetyl-d-glucosamine (GlcNAc), the valuable chemical widely applied in medical and food industries. Here we focused on a promising chitinolytic enzyme, OfHex1 from the insect, Ostrinia furnacalis. By structural analysis of OfHex1, five residues nearby the active pocket including V327, E328, Y471, V484 and W490 were chosen and nine mutants including V327G, E328Q, E328A, Y471V, V484R, W490A, W490H, V327G/V484R/W490A and V327G/Y471V/W490H were constructed and recombinantly expressed in Pichia pastoris. The best-performing mutant, W490A, obtained by a higher yield of 5 mg/L, did not show substrate inhibition even when 5 mM of the substrates, (GlcNAc)_2–4, were applied. The k_cat/K_m values for (GlcNAc)_2–4 are 239.8, 111.3 and 79.8 s^?1 mM^?1, respectively. Besides, the pH stability of the mutant ranges from pH 4 to 11 and the thermal stability is up to 50 °C. This work suggests the W490A mutant might be an ideal biocatalyst for GlcNAc production from chitin.     

Loss-of-function point mutations and two-furin domain derivatives provide insights about R-spondin2 structure and function

R-spondins (Rspos) potentiate Wnt/β-catenin signaling, an important pathway in embryonic development that is constitutively active in many cancers. To analyze Rspo structure and function, we expressed full-length wild-type Rspo2 and Rspo2 point mutants corresponding to Rspo4 variants that have been linked to developmental defects. The Rspo2 mutants had markedly reduced potency relative to the wild-type protein, demonstrating for the first time specific amino acid residues in Rspos that are critical for β-catenin signaling. The diminished activity of Rspo2/C78Y and Rspo2/C113R was attributable to a defect in their secretion, while Rspo2/Q70R exhibited a decrease in its intrinsic activity. Cysteine assignments in a Rspo2 derivative containing only the two furin-like domains (Rspo2-2F) provided the first information about the disulfide-bonding pattern of this motif, which was characterized by multiple short loops and unpaired cysteine residues, and established that the loss-of-function cysteine mutants disrupted disulfide bond formation. Moreover, Rspo2-2F demonstrated potent activity and synergized strongly with Wnt-3a in a β-catenin reporter assay. In contrast, an Rspo2-2F derivative containing the Q70R substitution showed significantly reduced activity, although it still synergized with Wnt-3a in the reporter assay. Rspo2-2F derivatives elicited an unusually sustained phosphorylation (20 h) of the Wnt co-receptor, low density lipoprotein receptor-related protein 6 (LRP6), as well as an increase in cell surface LRP6. Co-immunoprecipitation experiments involving LRP6 and Kremens suggested that these associations contribute to Rspo2 activity, although the lack of major differences between wild-type and Q70R derivatives implied that additional interactions may be important.     

Development and Characterization of Recombinant Ovine Coagulation Factor VIII

Animal models of the bleeding disorder, hemophilia A, have been an integral component of the biopharmaceutical development process and have facilitated the development of recombinant coagulation factor VIII (fVIII) products capable of restoring median survival of persons with hemophilia A to that of the general population. However, there remain several limitations to recombinant fVIII as a biotherapeutic, including invasiveness of intravenous infusion, short half-life, immunogenicity, and lack of availability to the majority of the world''s population. The recently described ovine model of hemophilia A is the largest and most accurate phenocopy. Affected sheep die prematurely due to bleeding-related pathogenesis and display robust adaptive humoral immunity to non-ovine fVIII. Herein, we describe the development and characterization of recombinant ovine fVIII (ofVIII) to support further the utility of the ovine hemophilia A model. Full-length and B-domain deleted (BDD) ofVIII cDNAs were generated and demonstrated to facilitate greater biosynthetic rates than their human fVIII counterparts while both BDD constructs showed greater expression rates than the same-species full-length versions. A top recombinant BDD ofVIII producing baby hamster kidney clone was identified and used to biosynthesize raw material for purification and biochemical characterization. Highly purified recombinant BDD ofVIII preparations possess a specific activity nearly 2-fold higher than recombinant BDD human fVIII and display a differential glycosylation pattern. However, binding to the carrier protein, von Willebrand factor, which is critical for stability of fVIII in circulation, is indistinguishable. Decay of thrombin-activated ofVIIIa is 2-fold slower than human fVIII indicating greater intrinsic stability. Furthermore, intravenous administration of ofVIII effectively reverses the bleeding phenotype in the murine model of hemophilia A. Recombinant ofVIII should facilitate the maintenance of the ovine hemophilia A herd and their utilization as a relevant large animal model for the research and development of novel nucleic acid and protein-based therapies for hemophilia A.     

Coagulant properties of hybrid human/porcine factor VIII molecules.

Human and porcine factor VIII (fVIII) are activated by thrombin to form a heterotrimer composed of subunits designated A1 and A2 derived from the fVIII heavy chain (HC) and a subunit designated A3-C1-C2 derived from the fVIII light chain (LC). Human and porcine fVIII were activated at the same rate to the same peak levels but dissociation of the A2 subunit and concomitant loss of fVIIIa activity at pH 7.4 and 22 degrees C was 3-fold faster with human fVIIIa compared to porcine fVIIIa (0.35 min-1 versus 0.12 min-1, respectively). To determine structural requirements for the increased activity of porcine fVIII, plasma-derived hybrid human/porcine fVIII molecules were isolated. Porcine HC/human LC (pHC/hLC) fVIII had 44-fold higher coagulant activity than reconstituted human fVIII (hHC/hLC), 40-fold higher activity than hHC/pLC, and slightly (1.4-fold) higher activity than reconstituted porcine fVIII (pHC/pLC). Additionally, human and porcine A2 subunits and inactive A1/A3-C1-C2 human and porcine dimers were isolated and reconstitution experiments were done. Addition of the porcine A2 subunit to the human A1/A3-C1-C2 dimer produced coagulant activity similar to that found with porcine fVIIIa and superior to human fVIIIa. These results suggest that human fVIII has weaker coagulant activity than porcine fVIII due to faster dissociation of the A2 subunit and that the A2 subunit itself is responsible for the difference.     

Scanning mutagenesis of interleukin-8 identifies a cluster of residues required for receptor binding

In order to identify residues required for the binding of interleukin-8 (IL-8) to its receptor, mutants were constructed in which clusters of charged amino acids were systematically replaced with alanine along the entire IL-8 sequence. The mutants were tested for their ability to induce a receptor-mediated rise in cytosolic free Ca2+, a property of wild-type IL-8 which can readily be detected by flow cytometry using neutrophils loaded with the calcium probe Indo-1. Eleven of the 12 mutants caused neutrophil calcium mobilization at 5 nM; the exception being a triple alanine mutant at positions K3, E4, and R6, which was inactive at all concentrations tested (150 nM maximum). A second set of mutants was generated in which residues 1-15 were individually mutated to alanine. Mutants E4A, L5A, or R6A were all inactive in the Ca2+ assay at 5 nM and competed poorly with 125I-IL-8 for neutrophil receptor binding; I10A, E4A, L5A, and R6A had approximately 30-, 100-, 100-, and 1000-fold reduced affinity, as compared with control IL-8, respectively. The nuclear magnetic resonance structure of IL-8 indicates that, in solution, the side chains of E4, L5, R6, and I10 point away from the core of the protein and do not participate in any intramolecular hydrogen bonds or salt bridges (Clore, G. M., and Gronenborn, A. M. (1991) J. Mol. Biol. 217, 611-620).     

Mechanisms of interactions of factor X and factor Xa with the acidic region in the factor VIII A1 domain

The 337-372 sequence of the factor VIIIa A1 subunit contains interactive sites for both zymogen factor X and the active enzyme, factor Xa. Solid phase binding studies indicated that factor Xa possessed a >20-fold higher affinity for the isolated A1 subunit of factor VIIIa compared with factor X. Heparin completely inhibited zero-length cross-linking of the 337-372 peptide to factor Xa but not to factor X. In the presence of calcium, factor Xa showed greater affinity for heparin than factor X. Studies using factor Xa mutants in which heparin-binding exosite residues were individually replaced by Ala showed that the R240A mutant was defective in recognition of the Lys36 cleavage site, generating the A137-372 intermediate with approximately 20% the catalytic efficiency of wild type. This defect likely resulted from an approximately 4-fold increase in Km for the A1 substrate because kcat values for the wild type and mutant were equivalent. Cleavage of the A1-A2 domain junction by factor Xa R240A was not blocked by the 337-372 peptide. Studies using mutant factor VIII where clustered acidic residues in the 337-372 segment were replaced by Ala showed that a factor VIIIa D361A/D362A/D363A mutant possessed a approximately 1.6-fold increase in Km for factor X compared with wild type. However, similar Km values were observed for recombinant factor X and R240A substrates. These results indicate that the binding regions of factor X and factor Xa for A1 domain overlap and that both utilize acidic residues 361-363. Furthermore, factor Xa but not factor X interacts with high affinity at this site via residues contained within the heparin-binding exosite of the proteinase.     

The critical role of the 185-189-loop in the factor Xa interaction with Na+ and factor Va in the prothrombinase complex

The S1 site (Asp(189)) of factor Xa (fXa) is located on a loop (residues 185-189) that contains three solvent-exposed charged residues (Asp(185), Lys(186), and Glu(188)) below the active-site pocket of the protease. To investigate the role of these residues in the catalytic function of fXa, we expressed three mutants of the protease in which the charges of these residues were neutralized by their substitutions with Ala (D185A, K186A, and E188A). Kinetic studies revealed that E188A has a normal catalytic activity toward small synthetic and natural substrates and inhibitors of fXa; however, the same activities were slightly ( approximately 2-fold) and dramatically ( approximately 20-50-fold) impaired for the D185A and K186A mutants, respectively. Further studies revealed that the affinity of D185A and K186A for interaction with Na(+) has also been altered, with a modest impairment ( approximately 2-fold) for the former and a dramatic impairment for the latter mutant. Both prothrombinase and direct binding studies indicated that K186A also has an approximately 6-fold impaired affinity for factor Va. Interestingly, a saturating concentration of factor Va restored the catalytic defect of K186A in reactions with prothrombin and the recombinant tick anticoagulant peptide that is known to interact with the Na(+) loop of fXa, but not with other substrates. These results suggest that factor Va interacts with 185-189-loop for fXa, which is energetically linked to the Na(+)-binding site of the protease.     

Aromatic components of two ferric enterobactin binding sites in Escherichia coli FepA

Ferric enterobactin is a catecholate siderophore that binds with high affinity (Kd approximately 10-10 M) to the Escherichia coli outer membrane protein FepA. We studied the involvement of aromatic amino acids in its uptake by determining the binding affinities, kinetics and transport properties of site-directed mutants. We replaced seven aromatic residues (Y260, Y272, Y285, Y289, W297, Y309 and F329) in the central part of FepA primary structure with alanine, individually and in double combinations, and determined the ability of the mutant proteins to interact with ferric enterobactin and the protein toxins colicins B and D. All the constructs showed normal expression and localization. Among single mutants, Y260A and F329A were most detrimental, reducing the affinity between FepA and ferric enterobactin 100- and 10-fold respectively. Double substitutions involving Y260, Y272 and F329 impaired (100- to 2500-fold) adsorption of the iron chelate more strongly. For Y260A and Y272A, the drop in adsorption affinity caused commensurate decreases in transport efficiency, suggesting that the target residues primarily act in ligand binding. F329A, like R316A, showed greater impairment of transport than binding, intimating mechanistic involvement during ligand internalization. Furthermore, immunochemical studies localized F329 in the FepA ligand binding site. The mutagenesis results suggested the existence of dual ligand binding sites in the FepA vestibule, and measurements of the rate of ferric enterobactin adsorption to fluoresceinated FepA mutant proteins confirmed this conclusion. The initial, outermost site contains aromatic residues and probably functions through hydrophobic interactions, whereas the secondary site exists deeper in the vestibule, contains both charged and aromatic residues and probably acts through hydrophobic and electrostatic bonds.     

Pseudomonas exotoxin-mediated selection yields cells with altered expression of low-density lipoprotein receptor-related protein [published erratum appears in J Cell Biol 1995 Aug;130(4):1015]

The alpha 2-macroglobulin (alpha 2M) receptor/low-density lipoprotein receptor-related protein (LRP) is important for the clearance of proteases, protease-inhibitor complexes, and various ligands associated with lipid metabolism. While the regulation of receptor function is poorly understood, the addition of high concentrations of the 39-kD receptor-associated protein (RAP) to cells inhibits the binding and/or uptake of many of these ligands. Previously, we (Kounnas, M.Z., R.E. Morris, M.R. Thompson, D.J. FitzGerald, D.K. Strickland, and C.B. Saelinger. 1992. J. Biol. Chem. 267:12420-12423) [corrected] showed that Pseudomonas exotoxin (PE) could bind immobilized LRP. Also, the addition of RAP blocked toxin-mediated cell killing. These findings suggested that PE might use LRP to gain entry into toxin-sensitive cells. Here we report on a strategy to select PE-resistant lines of Chinese hamster ovary cells that express altered amounts of LRP. An important part of this strategy is to screen PE-resistant clones for those that retain sensitivity to both diphtheria toxin and to a fusion protein composed of lethal factor (from anthrax toxin) fused to the adenosine diphosphate-ribosylating domain of PE. Two lines, with obvious changes in their expression of LRP, were characterized in detail. The 14-2-1 line had significant amounts of LRP, but in contrast to wild-type cells, little or no receptor was displayed on the cell surface. Instead, receptor protein was found primarily within cells, much of it apparently in an unprocessed state. The 14-2-1 line showed no uptake of chymotrypsin-alpha 2M and was 10-fold resistant to PE compared with wild-type cells. A second line, 13-5-1, had no detectable LRP mRNA or protein, did not internalize alpha 2M-chymotrypsin, and exhibited a 100-fold resistance to PE. Resistance to PE appeared to be due to receptor-specific defects, since these mutant lines showed no resistance to a PE chimeric toxin that was internalized via the transferrin receptor. The results of this investigation confirm that LRP mediates the internalization of PE.     

Proteolysis at Arg740 facilitates subsequent bond cleavages during thrombin-catalyzed activation of factor VIII

Thrombin activates factor VIII by proteolysis at three P1 residues: Arg372, Arg740, and Arg1689. Cleavage at Arg372 and Arg1689 are essential for procofactor activation; however cleavage at Arg740 has not been rigorously studied. To evaluate the role for cleavage at Arg740, we prepared and stably expressed two recombinant B-domainless factor VIII mutants, R740H and R740Q to slow and eliminate, respectively, cleavage at this site. Specific activity values for the variants were approximately 50 and 20%, respectively, that of wild-type factor VIII. Activation of factor VIII R740H by thrombin showed an approximately 40-fold reduction in the rate of A2 subunit generation, which reflected an approximately 20-fold reduction in cleavage rate at Arg372. Similarly, a approximately 40-fold rate reduction in cleavage at Arg1689 and consequent generation of the A3-C1-C2 subunit were observed. Rate values for A2 and A3-C1-C2 subunit generation were reduced by >700-fold and approximately 140-fold, respectively, in the R740Q variant. These results suggest that initial cleavage at Arg740 affects cleavage at both Arg372 and Arg1689 sites. Results obtained evaluating proteolysis of the factor VIII mutants by factor Xa revealed more modest rate reductions (<10-fold) in generating A2 and A3-C1-C2 subunits from either variant, suggesting that factor Xa-catalyzed activation of factor VIII was significantly less dependent upon prior cleavage at residue 740 than thrombin. Overall, these results support a model whereby cleavage of factor VIII by thrombin is an ordered pathway with cleavage at Arg740 facilitating cleavages at Arg372 and Arg1689, which result in procofactor activation.