Heparin HP Research Articles

A systematic and methodical approach for the efficient purification of recombinant protein from silkworm larval hemolymph

The silkworm, Bombyx mori, is a promising expression system for the production of recombinant proteins, but the purification of these proteins is not easy because of the large amount of host proteins present. To investigate purity, recovery and scale-up ability of the purification of recombinant proteins expressed in silkworm larval hemolymph without any affinity tags, we used mCherry, a red fluorescence protein, as a model. The host cell proteins could be greatly reduced using a three-step chromatography protocol consisting of hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC) and heparin chromatography after heat pretreatment. The thermal treatment had the greatest impact on the removal of host cell extracellular proteins and increasing purity. There were still some minor traces of host cell proteins in the purified sample, which showed that the purification of recombinant proteins from the silkworm hemolymph was still challenging. The proposed protocol and affinity tag purification reduced the overall protein content by 99.84% and 99.95%, respectively, while the amount of DNA was reduced by 98.41% and 99.53%, respectively. Purities of our proposed protocol based on SDS-PAGE and capillary electrophoresis (CE) analyses were 85.45% and 43.60%, respectively, while those of Strep-tag affinity purification were 100% or 63.69%, respectively. Using densitometry, the overall recovery was calculated was 5.78%, which was higher than 4.09% using Strep-tag affinity purification. This proposed protocol, mainly based on thermal treatment, HIC, SEC and HiTrap Heparin HP column chromatography, is applicable to an upscalable purification for the silkworm expression system without employing affinity tag chromatography process.

Determination of native lactoferrin in milk by HPLC on HiTrapTM Heparin HP column

Foods supplemented with lactoferrin are an increasing trend owing to its multiple functions. Therefore, a reliable analytical method for the nutritional assessment and quality control of lactoferrin containing foods must be established. A method for the determination of native lactoferrin in milk using a HiTrapTM Heparin HP column coupled with HPLC was developed and validated. The conditions for lactoferrin enrichment in milk using the HiTrapTM Heparin HP column were optimized. A linear range of 2 to 100 mg L-1 was obtained for lactoferrin standards, with a R2 value of 0.9989. The detection limit (LOD) and quantification limit (LOQ) of the lactoferrin standards were 2.00 and 6.67 mg L-1, respectively. Considering the enrichment factor involved in the pre-treatment procedures, LOD and LOQ for detection of milk sample were 0.57 and 1.90 mg L-1, respectively. In addition, milk samples spiked with lactoferrin at three concentration levels (2, 5, and 10 mg/L) showed overall mean recovery of 88.3%, 90.2% and 95.1%, respectively. The relative standard deviations of intra-day and inter-day precision were 1.3% ~ 4.8%, and 2.1% ~ 5.7%, respectively, indicating that the present method showed good performances. Furthermore, the successful quantification of lactoferrin in raw milk and processed milk demonstrated the usefulness and effectiveness of the proposed method.

Abstract 946: Characterize and identify secreted high molecular weight heparanase from PC3M conditioned medium

Abstract Introduction: Neutrophils, platelets, lymphocytes are typical sources of NAP-2 which is associated with heparanase activity. Heparanase degrades polymeric heparan sulfate molecules into shorter oligosaccharides, participating in extracellular matrix degradation. It is active under acidic conditions during tumor invasion and inflammatory processes. Our lab previously found that some cell lines like androgen independent prostate cancer cells (PC3M) can secrete a protein that reacts with anti-NAP-2 antibody, but has a much higher molecular weight(>50KD) than NAP-2 (MW~6-14 KD), and has heparanase activity. The purpose of this study is to characterize and identify this protein. Procedures: Immunoprecipitation was performed using Pierce DirectIP kit according the manufacturer’s instructions. Heparin binding affinity column used HiTrap™ Heparin HP column from GE Healthcare Bio-Sciences. The binding buffer was 10mM NaAcetate pH 5.0, the eluting buffers were 0.5M-2M NaCl in 10mM NaAcetate pH 5.0. The mass spectrometry was done by KCI Proteomics Core facility. Data: The immunoprecipitation method confirmed that this higher molecular weight protein indeed can be pulled down by the NAP-2 antibody, and after using heparin binding affinity column to purify, we can still get the > 50KD band on the western blot for NAP-2, meaning it is a heparin binding protein. Conclusions: A protein identified with NAP-2 antibody, but with higher molecular weight than native NAP-2 is found in the conditioned medium of PC3M cells that binds heparin, further confirming a potential role in tumor progression for prostate cancer. Citation Format: Donghong Ju, Mary A. Kosir. Characterize and identify secreted high molecular weight heparanase from PC3M conditioned medium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 946. doi:10.1158/1538-7445.AM2017-946

Role of duck plague virus glycoprotein C in viral adsorption: Absence of specific interactions with cell surface heparan sulfate

Many mammalian herpes viruses utilize heparan sulfate (HS) moieties present on cell surface proteoglycans as receptors for cell entry, and this process also requires viral glycoprotein C (gC) homologues. However, our understanding of the role of gC in facilitating attachment of other alpha-herpes viruses such as the duck plague virus (DPV) remains preliminary. To study the role of gC during DPV infection, we used a gC-deleted mutant virus (DPV-ΔgC-EGFP). Examination of the viral copy number by real-time PCR, as well as time course studies of viral adsorption and proliferation revealed that gC was involved in the viral binding to the cell surface. The affinity of viral glycoproteins (gB-DPV, gC-DPV, and gE-DPV) to HS was assessed using a prokaryotic expression system and HiTrap™ Heparin HP column chromatography. In addition, to confirm that gC played a role in the interaction between DPV and HS, viruses were treated with the HS analogue heparin and host cells were treated with its inhibitors heparinase prior to exposure to DPV-ΔgC-EGFP or wild-type strain Chinese virulent duck plague virus (DPV-CHv). The effects of heparin and heparinase on virus infectivity demonstrated that function of gC on viral adsorption is independent of interactions between gC and heparin sulfate on cell surface. All in all, this study demonstrated that the gC of DPV can mediate viral adsorption in an HS-independent manner, which distinguish it from the gC of some other alpha-herpes viruses. Future studies will be required to identify the receptors involved in gC protein binding to cells. This work provides us a foundation for further studies of examining the roles of gC in the adsorption during duck plague virus infection.

A New Platelet-Aggregation-Inhibiting Factor Isolated from Bothrops moojeni Snake Venom.

This work reports the purification and functional characterization of BmooPAi, a platelet-aggregation-inhibiting factor from Bothrops moojeni snake venom. The toxin was purified by a combination of three chromatographic steps (ion-exchange on DEAE-Sephacel, molecular exclusion on Sephadex G-75, and affinity chromatography on HiTrap™ Heparin HP). BmooPAi was found to be a single-chain protein with an apparent molecular mass of 32 kDa on 14% SDS-PAGE, under reducing conditions. Sequencing of BmooPAi by Edman degradation revealed the amino acid sequence LGPDIVPPNELLEVM. The toxin was devoid of proteolytic, haemorrhagic, defibrinating, or coagulant activities and induced no significant oedema or hyperalgesia. BmooPAi showed a rather specific inhibitory effect on ristocetin-induced platelet aggregation in human platelet-rich plasma, whereas it had little or no effect on platelet aggregation induced by collagen and adenosine diphosphate. The results presented in this work suggest that BmooPAi is a toxin comprised of disintegrin-like and cysteine-rich domains, originating from autolysis/proteolysis of PIII SVMPs from B. moojeni snake venom. This toxin may be of medical interest because it is a platelet aggregation inhibitor, which could potentially be developed as a novel therapeutic agent to prevent and/or treat patients with thrombotic disorders.

Purification of foot-and-mouth disease virus by heparin as ligand for certain strains.

The goal of this project was to develop an easily operable and scalable process for the recovery and purification of foot-and-mouth disease virus (FMDV) from cell culture. Heparin resins HipTrap Heparin HP and AF-Heparin HC-650 were utilized to purify FMDV O/HN/CHA/93. Results showed that the purity of AF-Heparin HC-650 was ideal. Then, the O/HN/CHA/93, O/Tibet/CHA/99, Asia I/HN/06, and A/CHA/HB/2009 strains were purified by AF-Heparin HC-650. Their affinity/virus recoveries were approximately 51.2%/45.8%, 71.5%/70.9%, 96.4%/73.5, and 59.5%/42.1%, respectively. During a stepwise elution strategy, the viral particles were mainly eluted at 300mM ionic strength peaks. The heparin affinity chromatography process removed more than 94% of cellular and medium proteins. Anion exchange resin Capto Q captured four FMD virus particles; 40% of binding proteins and 80%-90% of viral particles were eluted at 450mM NaCl. Moreover, ionic strength varied from 30 to 450mM had no effect on the immunity to FMDV. The results revealed that heparin sulfate may be the main receptor for CHA/99 strain attachment-susceptible cells. Heparin affinity chromatography can reach perfect results, especially when used as a ligand of the virus. Anion exchange is useful only as previous step for further purification.

DnaT is a PriC-binding protein

DnaT and PriC are replication restart primosomal proteins required for re-initiating chromosomal DNA replication. DnaT is a component of the PriA-dependent primosome, while PriC belongs to the PriC-dependent primosome. Whether DnaT can interact with PriC is still unknown. In this study, we define a direct interaction between PriC, a key initiator protein in PriC-mediated DNA replication restart, and DnaT, a DnaB/C complex loader protein, from Klebsiella pneumoniae. In fluorescence titrations, PriC bound to single-stranded DNA with a binding-site size of approximately 9 nt. Gold nanoparticle assay showed that the solution of DnaT–PriC changed from red to purple, which indicated the protein–protein interactions due to gold nanoparticle aggregate. In addition, this DnaT–PriC complex could be co-purified by the heparin HP column. Surface plasmon resonance analysis showed that the Kd value of DnaT bound to PriC was 2.9 × 10−8 M. These results constitute a pioneering study of the DnaT–PriC interaction and present a putative link between the two independent replication restart pathways, namely, PriA- and PriC-dependent primosome assemblies. Further research can directly focus on determining how DnaT binds to the PriC–SSB–DNA tricomplex and regulates the PriC-dependent replication restart.

Production of functional human vascular endothelial growth factor165 in transgenic rice cell suspension cultures

Vascular endothelial growth factors (VEGFs) are secreted by tumor cells and other cells exposed to hypoxia, and play a critical role in the development and differentiation of the vascular system. In this study, we investigated the production of functional recombinant human VEGF165 (rhVEGF165) in transgenic rice cell suspension culture. Complementary DNA was synthesized from human leukemia HL60 cells and cloned into expression vectors under the control of the rice α-amylase 3D (RAmy3D) promoter. The rice seed (Oryza sativa L. cv. Dongjin) was transformed with this recombinant vector by the Agrobacterium mediated method and the integration of the target gene into the plant genome was confirmed by genomic PCR. The expression of rhVEGF165 in the rice cells was determined by Northern blot and Western blot analyses. The accumulated rhVEGF165 protein in the culture medium was 19mg/L after 18 days of culturing in a sugar-free medium. The rhVEGF165 was purified using a heparin HP column and its biological activity was tested on human umbilical vein endothelial cells (HUVECs). The purified rhVEGF165 significantly increased the proliferative activity of the HUVECs. Therefore, it was demonstrated that functional rhVEGF165 could be produced using transgenic rice suspension culture vector under the control of the RAmy3D promoter.

DNA Binding Restricts the Intrinsic Conformational Flexibility of Methyl CpG Binding Protein 2 (MeCP2)

Mass spectrometry-based hydrogen/deuterium exchange (H/DX) has been used to define the polypeptide backbone dynamics of full-length methyl CpG binding protein 2 (MeCP2) when free in solution and when bound to unmethylated and methylated DNA. Essentially the entire MeCP2 polypeptide chain underwent H/DX at rates faster than could be measured (i.e. complete exchange in ≤10 s), with the exception of the methyl DNA binding domain (MBD). Even the H/DX of the MBD was rapid compared with that of a typical globular protein. Thus, there is no single tertiary structure of MeCP2. Rather, the full-length protein rapidly samples many different conformations when free in solution. When MeCP2 binds to unmethylated DNA, H/DX is slowed several orders of magnitude throughout the MBD. Binding of MeCP2 to methylated DNA led to additional minor H/DX protection, and only locally within the N-terminal portion of the MBD. H/DX also was used to examine the structural dynamics of the isolated MBD carrying three frequent mutations associated with Rett syndrome. The effects of the mutations ranged from very little (R106W) to a substantial increase in conformational sampling (F155S). Our H/DX results have yielded fine resolution mapping of the structure of full-length MeCP2 in the absence and presence of DNA, provided a biochemical basis for understanding MeCP2 function in normal cells, and predicted potential approaches for the treatment of a subset of RTT cases caused by point mutations that destabilize the MBD.

Human Phenotypically Distinct TGFBI Corneal Dystrophies Are Linked to the Stability of the Fourth FAS1 Domain of TGFBIp

Mutations in the human TGFBI gene encoding TGFBIp have been linked to protein deposits in the cornea leading to visual impairment. The protein consists of an N-terminal Cys-rich EMI domain and four consecutive fasciclin 1 (FAS1) domains. We have compared the stabilities of wild-type (WT) human TGFBIp and six mutants known to produce phenotypically distinct deposits in the cornea. Amino acid substitutions in the first FAS1 (FAS1-1) domain (R124H, R124L, and R124C) did not alter the stability. However, substitutions within the fourth FAS1 (FAS1-4) domain (A546T, R555Q, and R555W) affected the overall stability of intact TGFBIp revealing the following stability ranking R555W>WT>R555Q>A546T. Significantly, the stability ranking of the isolated FAS1-4 domains mirrored the behavior of the intact protein. In addition, it was linked to the aggregation propensity as the least stable mutant (A546T) forms amyloid fibrils while the more stable variants generate non-amyloid amorphous deposits in vivo. Significantly, the data suggested that both an increase and a decrease in the stability of FAS1-4 may unleash a disease mechanism. In contrast, amino acid substitutions in FAS1-1 did not affect the stability of the intact TGFBIp suggesting that molecular the mechanism of disease differs depending on the FAS1 domain carrying the mutation.

Enterovirus 3C Proteases: Structure-based Discovery of Inhibitors

DnaT and PriC are replication restart primosomal proteins required for re-initiating chromosomal DNA replication. DnaT is a component of the PriA-dependent primosome, while PriC belongs to the PriC-dependent primosome. Whether DnaT can interact with PriC is still unknown. In this study, we define a direct interaction between PriC, a key initiator protein in PriC-mediated DNA replication restart, and DnaT, a DnaB/C complex loader protein, from Klebsiella pneumoniae. In fluorescence titrations, PriC bound to single-stranded DNA with a binding-site size of approximately 9 nt. Gold nanoparticle assay showed that the solution of DnaT–PriC changed from red to purple, which indicated the protein–protein interactions due to gold nanoparticle aggregate. In addition, this DnaT–PriC complex could be co-purified by the heparin HP column. Surface plasmon resonance analysis showed that the Kd value of DnaT bound to PriC was 2.9 × 10−8 M. These results constitute a pioneering study of the DnaT–PriC interaction and present a putative link between the two independent replication restart pathways, namely, PriA- and PriC-dependent primosome assemblies. Further research can directly focus on determining how DnaT binds to the PriC–SSB–DNA tricomplex and regulates the PriC-dependent replication restart.

Characterization of DNA polymerase from the hyperthermophilic archaeon Thermococcus marinus and its application to PCR

The family B DNA polymerase gene from the archaeon Thermococcus marinus (Tma) contains a long open reading frame of 3,939 bp that encodes 1,312 amino acid residues. The gene is split by one intervening sequence that forms a continuous open reading frame with the two polymerase exteins. In this study, the Tma DNA polymerase gene both with (precursor form) and without (mature form) its intein was expressed in Escherichia coli, purified by heat treatment and HiTrap Heparin HP column chromatography and characterized. Primary sequence analysis of the mature Tma polymerase showed high sequence identity with DNA polymerases in the genus Thermococcus. The expressed precursor form was easily spliced during purification steps. The molecular mass of the purified Tma DNA polymerases is about 90 kDa, as estimated by SDS-PAGE. Both Tma DNA polymerases showed the same properties. PCR performed with this enzyme was found to be optimal in the presence of 50 mM Tris-HCl (pH 8.4), 40 mM KCl, 12.5 mM (NH(4))(2)SO(4,) 2 mM MgCl(2,) 0.05% Triton X-100 and 0.0075% BSA. Furthermore, long-range PCR and time-saving PCR were performed using various specific ratios of Taq and Tma DNA polymerases (Tma plus DNA polymerase).

Anticancer activity of lactoferrin isolated from caprine colostrum on human cancer cell lines

In the present study, lactoferrin was purified from caprine colostrum and its anticancer effects were investigated on various cancer cell lines, including lung, colon, cervix, stomach and breast cancer cells. The caprine lactoferrin (CLF) was purified by ultrafiltration and sequential chromatography using a CM‐Sephadex C‐50‐120 ion‐exchange column and affinity chromatography on Hi‐TrapTM Heparin HP. The purified CLF exhibited antiproliferative effects on the five tested cancer cell lines in a dose‐dependent manner, with 20–30% survival as compared to the control. Interestingly, the most intense inhibition of cell growth was observed on the ZR‐75‐1 breast cancer cells (IC50 = 27.5 µg/mL). Our findings suggest, for the first time, that purified CLF from caprine colostrum may constitute a novel anticancer agent for the food industry.

Bothrops jararaca antithrombin: Isolation, characterization and comparison with other animal antithrombins

Antithrombin was purified from Bothrops jararaca plasma by affinity chromatography using HiTrap Heparin HP column, and its molecular weight, amino-terminal sequence, carbohydrate content, isoelectric point, inhibition of bovine thrombin, and immunological properties were studied and compared with previously described antithrombins. B. jararaca antithrombin is a single-chain glycoprotein with a total carbohydrate content of 18%. The molecular weight from SDS-PAGE was 61 kDa and the inhibitor exhibited an acidic isoelectric point (4.5). The amino-terminal sequence has been determined as His-Glu-Ser-Ser-Val-Gln-Asp-Ile-Ile-Thr, which is highly homologous to the terminal sequences of other animal antithrombins, indicating high amino acid conservation among several animals. Immunological cross-reactivity was observed among fish, frog, chicken, human, non-venomous snake and B. jararaca antithrombins. B. jararaca antithrombin showed inhibitory activity upon human and B. jararaca coagulation and amidolytic substrate S-2238.

N-Glycans and the N Terminus of Protein C Inhibitor Affect the Cofactor-enhanced Rates of Thrombin Inhibition

Protein C inhibitor (PCI) is a serine protease inhibitor, displaying broad protease specificity, found in blood and other tissues. In blood, it is capable of inhibiting both procoagulant and anticoagulant proteases. Mechanisms that provide specificity to PCI remain largely unrevealed. In this study we have for the first time provided a full explanation for the marked size heterogeneity of blood-derived PCI and identified functional differences between naturally occurring PCI variants. The heterogeneity was caused by differences in N-glycan structures, N-glycosylation occupancy, and the presence of a Delta6-N-cleaved form. Bi-, tri-, and tetra-antennary complex N-glycans were identified. Fucose residues were identified both on the core GlcNAc and as parts of sialyl-Le(a/x) epitopes. Moreover, a glycan with a composition that implied a di-sialyl antenna was observed. PCI was N-glycosylated at all three potential N-glycosylation sites, Asn-230, Asn-243, and Asn-319, but a small fraction of PCI lacked the N-glycan at Asn-243. The overall removal of N-glycans affected the maximal heparin- and thrombomodulin-enhanced rates of thrombin inhibition differently in different solution conditions. In contrast, the Delta6-N-region increased both the heparin- and the thrombomodulin-enhanced rates of thrombin inhibition at all conditions examined. These results thus demonstrate that the N-linked glycans and the N-terminal region of blood-derived PCI in different ways affect the cofactor-enhanced rates of thrombin inhibition and provide information on the mechanisms by which this may be achieved. The findings are medically important, in view of the documented association of PCI with atherosclerotic plaques and the promising effect of PCI on reducing hypercoagulability states.

Structural and Functional Analysis of Slit and Heparin Binding to Immunoglobulin-like Domains 1 and 2 of Drosophila Robo

Recognition of the secreted protein Slit by transmembrane receptors of the Robo family provides important signals in the development of the nervous system and other organs, as well as in tumor metastasis and angiogenesis. Heparan sulfate (HS) proteoglycans serve as essential co-receptors in Slit-Robo signaling. Previous studies have shown that the second leucinerich repeat domain of Slit, D2, binds to the N-terminal immunoglobulin-like domains of Robo, IG1-2. Here we present two crystal structures of Drosophila Robo IG1-2, one of which contains a bound heparin-derived oligosaccharide. Using structure-based mutagenesis of a Robo IG1-5 construct we identified key Slit binding residues (Thr-74, Phe-114, Arg-117) forming a conserved patch on the surface of IG1; mutation of similarly conserved residues in IG2 had no effect on Slit binding. Mutation of conserved basic residues in IG1 (Lys-69, Arg-117, Lys-122, Lys-123), but not in IG2, reduced binding of Robo IG1-5 to heparin, in full agreement with the Robo-heparin co-crystal structure. Our collective results, together with a recent crystal structure of a minimal human Slit-Robo complex ( Morlot, C., Thielens, N. M., Ravelli, R. B., Hemrika, W., Romijn, R. A., Gros, P., Cusack, S., and McCarthy, A. A. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 14923-14928 ), reveal a contiguous HS/heparin binding surface extending across the Slit-Robo interface. Based on the size of this composite binding site, we predict that at least five HS disaccharide units are required to support Slit-Robo signaling.

Inhibition of the Proteolytic Activity of Pregnancy-associated Plasma Protein-A by Targeting Substrate Exosite Binding

The metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) cleaves both insulin-like growth factor (IGF)-binding protein 4 (IGFBP-4) and -5 at a single site in their central domain causing the release of bioactive IGF. Inhibition of IGF signaling is relevant in human disease, and several drugs in development target the IGF receptor. However, inhibition of PAPP-A activity may be a valuable alternative. We have generated monoclonal phage-derived single chain fragment variable (scFv) antibodies which selectively inhibit the cleavage of IGFBP-4 by PAPP-A, relevant under conditions where cleavage of IGFBP-4 represents the final step in the delivery of IGF to the IGF receptor. None of the antibodies inhibited the homologous proteinase PAPP-A2, which allowed mapping of antibody binding by means of chimeras between PAPP-A and PAPP-A2 to the C-terminal Lin12-Notch repeat module, separated from the proteolytic domain by almost 1000 amino acids. Hence, the antibodies define a substrate binding exosite that can be targeted for the selective inhibition of PAPP-A proteolytic activity against IGFBP-4. In addition, we show that the Lin12-Notch repeat module reversibly binds a calcium ion and that bound calcium is required for antibody binding, providing a strategy for the further development of selective inhibitory compounds. To our knowledge these data represent the first example of differential inhibition of cleavage of natural proteinase substrates by exosite targeting. Generally, exosite inhibitors are less likely to affect the activity of related proteolytic enzymes with similar active site environments. In the case of PAPP-A, selective inhibition of IGFBP-4 cleavage by interference with exosite binding is a further advantage, as the activity against other known or unknown PAPP-A substrates, whose cleavage may not depend on binding to the same exosite, is not targeted.

Glycosaminoglycans Facilitate Procathepsin B Activation through Disruption of Propeptide-Mature Enzyme Interactions

Lysosomal cysteine cathepsin B participates in numerous diverse cellular processes. In acquiring its activity, the proregion, which blocks the substrate-binding site in the proenzyme, needs to be cleaved off. Here we demonstrate that polyanionic polysaccharides, glycosaminoglycans (GAGs), can accelerate the autocatalytic removal of the propeptide and subsequent activation of cathepsin B. We show that naturally occurring GAGs such as chondroitin sulfates and heparin, as well as the synthetic analog dextran sulfate, accelerate the processing in a concentration-dependent manner. Heparin oligosaccharides down to the size of tetrasaccharides were efficient in accelerating the procathepsin B processing, whereas disaccharides were without effect. Further, the ability of the GAGs to accelerate procathepsin B processing was sensitive to increasing NaCl concentrations, indicating that electrostatic interaction between the GAGs and procathepsin B are operative in the accelerating effect. Also the processing of the catalytic procathepsin B mutant by wild type cathepsin B was enhanced in the presence of GAGs, suggesting that GAGs induce a conformational change in procathepsin B, converting it into a better substrate. Site-directed mutagenesis showed that His(28), Lys(39), and Arg(40), located within the procathepsin B propeptide, have significant roles in the acceleration of procathepsin B activation induced by short GAGs. Because procathepsin B and GAGs often co-localize in vivo, we propose that GAGs may play a physiological role in the activation of procathepsin B.

A Nuclear Export Sequence Located on a β-Strand in Fibroblast Growth Factor-1

Receptor-bound and endocytosed fibroblast growth factor-1 (FGF-1) is able to cross the vesicle membrane and translocate to cytosol and nucleus. This suggests an intracellular role of FGF-1, which also signals by activating transmembrane FGF receptors. Phosphorylation of internalized FGF-1 by nuclear protein kinase C delta induces rapid export from the nuclei by a leptomycin B-sensitive pathway. In the present work, we have searched for and identified a Leu-rich nuclear export sequence (NES) at the C terminus of FGF-1 required for its nuclear export and able to confer nuclear export activity to a reporter protein in an in vivo system. Mutants where hydrophobic amino acids within the NES were exchanged for alanine exhibited reduced or abolished nuclear export. As demonstrated in co-immunoprecipitation experiments, a complex containing FGF-1, exportin-1, and its co-factor Ran-GTP, was formed in vitro. Formation of this complex in vivo was demonstrated by a peroxisomal targeting assay. Formation of the FGF-1-exportin-1-Ran-GTP complex in vitro as well as nuclear export of FGF-1 in vivo was dependent on phosphorylation of FGF-1, and it was abolished by leptomycin B. The FGF-1 NES was found to be situated along a beta-strand, which has not been reported before, since NESs usually are alpha-helical.

C1 Inhibitor Serpin Domain Structure Reveals the Likely Mechanism of Heparin Potentiation and Conformational Disease

C1 inhibitor, a member of the serpin family, is a major down-regulator of inflammatory processes in blood. Genetic deficiency of C1 inhibitor results in hereditary angioedema, a dominantly inheritable, potentially lethal disease. Here we report the first crystal structure of the serpin domain of human C1 inhibitor, representing a previously unreported latent form, which explains functional consequences of several naturally occurring mutations, two of which are discussed in detail. The presented structure displays a novel conformation with a seven-stranded beta-sheet A. The unique conformation of the C-terminal six residues suggests its potential role as a barrier in the active-latent transition. On the basis of surface charge pattern, heparin affinity measurements, and docking of a heparin disaccharide, a heparin binding site is proposed in the contact area of the serpin-proteinase encounter complex. We show how polyanions change the activity of the C1 inhibitor by a novel "sandwich" mechanism, explaining earlier reaction kinetic and mutagenesis studies. These results may help to improve therapeutic C1 inhibitor preparations used in the treatment of hereditary angioedema, organ transplant rejection, and heart attack.