Structural/functional properties of the Glu1-HSer57 N-terminal fragment of human plasminogen: conformational characterization and interaction with kringle domains.

The Glu1-Val79 N-terminal peptide (NTP) domain of human plasminogen (Pgn) is followed by a tandem array of five kringle (K) structures of approximately 9 kDa each. K1, K2, K4, and K5 contain each a lysine-binding site (LBS). Pgn was cleaved with CNBr and the Glul-HSer57 N-terminal fragment (CB-NTP) isolated. In addition, the Ile27-Ile56 peptide (L-NTP) that spans the doubly S-S bridged loop segment of NTP was synthesized. Pgn kringles were generated either by proteolytic fragmentation of Pgn (K4, K5) or via recombinant gene expression (rK1, rK2, and rK3). Interactions of CB-NTP with each of the Pgn kringles were monitored by 1H-NMR at 500 MHz and values for the equilibrium association constants (Ka) determined: rK1, Ka approximately 4.6 mM(-1); rK2, Ka approximately 3.3 mM(-1); K4, Ka approximately 6.2 mM-'; K5, K, 2.3 mM(-1). Thus, the lysine-binding kringles interact with CB-NTP more strongly than with Nalpha-acetyl-L-lysine methyl ester (Ka < 0.6 mM(-l), which reveals specificity for the NTP. In contrast, CB-NTP does not measurably interact with rK3. which is devoid of a LBS. CB-NTP and L-NTP 1H-NMR spectra were assigned and interproton distances estimated from 1H-1H Overhauser (NOESY) experiments. Structures of L-NTP and the Glul-Ile27 segment of CB-NTP were computed via restrained dynamic simulated annealing/energy minimization (SA/EM) protocols. Conformational models of CB-NTP were generated by joining the two (sub)structures followed by a round of constrained SA/EM. Helical turns are indicated for segments 6-9, 12-16, 28-30, and 45-48. Within the Cys34-Cys42 loop of L-NTP, the structure of the Glu-Glu-Asp-Glu-Glu39 segment appears to be relatively less defined, as is the case for the stretch containing Lys5O within the Cys42-Cys54 segment, consistent with the latter possibly interacting with kringle domains in intact Glul-Pgn. Overall, the CB-NTP and L-NTP fragments are of low regular secondary structure content-as indicated by UV-CD spectra- and exhibit fast amide 1H-2H exchange in 2H2O, suggestive of high flexibility.     

Phenotypic mixing between different hepadnavirus nucleocapsid proteins reveals C protein dimerization to be cis preferential.

Hepadnaviruses encode a single core (C) protein which assembles into a nucleocapsid containing the polymerase (P) protein and pregenomic RNA during viral replication in hepatocytes. We examined the ability of heterologous hepadnavirus C proteins to cross-oligomerize. Using a two-hybrid assay in HepG2 cells, we observed cross-oligomerization among the core proteins from hepatitis B virus (HBV), woodchuck hepatitis virus, and ground squirrel hepatitis virus. When expressed in Xenopus oocytes, in which hepadnavirus C proteins form capsids, the C polypeptides from woodchuck hepatitis virus and ground squirrel hepatitis virus, but not duck hepatitis B virus, can efficiently coassemble with an epitope-tagged HBV core polypeptide to form mixed capsids. However, when two different core mRNAs are coexpressed in oocytes the core monomers show a strong preference for forming homodimers rather than heterodimers. This holds true even for coexpression of two HBV C proteins differing only by an epitope tag, suggesting that core monomers are not free to diffuse and associate with other monomers. Thus, mixed capsids result from aggregation of different species of homodimers.     

Lysine-50 is a likely site for anchoring the plasminogen N-terminal peptide to lysine-binding kringles.

Interactions between the kringle 4 (K4) domain of human plasminogen (Pgn) and segments of the N-terminal Glu1-Lys77 peptide (NTP) have been investigated via 1H-NMR at 500 MHz. NTP peptide stretches devoid of Lys residues but carrying an internal Arg residue show negligible affinity toward K4 (equilibrium association constant Ka < 0.05 mM(-1)). In contrast, while most fragments containing an internal Lys residue exhibit affinities comparable to that shown by the blocked Lys derivative Nalpha-acetyl-L-lysine-methyl ester (Ka approximately 0.2 mM(-1), peptides encompassing Lys50O consistently show higher Ka values. Among the investigated linear peptides, Nalpha-acetyl-Ala-Phe-Tyr-His-Ser-Ser-Lys5O-Glu-Gln-NH2 (AcAFYHSK5OEQ-NH2) exhibits the strongest interaction with K4 (Ka approximately 1.4 mM(-1)), followed by AcYHSK50EQ-NH2 (Ka approximately 0.9 mM(-1)). Relative to the wild-type sequence, mutated hexapeptides exhibit lesser affinity for K4. When a Lys50 --> Ser mutation was introduced (==> AcYHSS50EQ-NH2), binding was abolished. The Ile27-lle56 construct (L-NTP) contains the Lys50 site within a loop constrained by two cystine bridges. The propensity of recombinant Pgn K1 (rK1) and K2 (rK2) modules, and of Pgn fragments encompassing the intact K4 and K5 domains, for binding L-NTP, was investigated. We find that L-NTP interacts with rK1, rK2, K4, and K5-all lysine-binding kringles-in a fashion that closely mimics what has been observed for the Glul-HSer57 N-terminal fragment of Pgn (CB-NTP). Thus, both the constellation of kringle lysine binding site (LBS) aromatic residues that are perturbed upon complexation of L-NTP and magnitudes of kringle-L-NTP binding affinities (rK1, Ka approximately 4.3 mM(-1); rK2, Ka approximately 3.7 mM(-1; K4, Ka approximately 6.4 mM(1); and K5, Ka approximately 2.1 mM(-1)) are essentially the same as for the corresponding kringle-CB-NTP pairs. Molecular modeling studies suggest that the Glu39-Lys50 stretch in NTP generates an area that complements, both topologically and electrostatically, the solvent-exposed kringle LBS surface.     

Cloning, expression, and purification of histidine-tagged preS domains of hepatitis B virus

The preS domains of the hepatitis B virus are hydrophilic polypeptides that have been implicated, among other functions, in the binding of the virus to hepatocytes and in the induction of virus-neutralizing antibodies. A method of overproducing the preS domains of two different subtypes, adw and ayw, has been developed by adding a 6x His tag at the carboxy-terminal end of the polypeptides. Codons for the 6x His were added in reverse primers used to amplify the corresponding cDNAs. The polymerase chain reaction products were cloned into the expression vectors pET-3d (subtype ayw) and pT7-7 (subtype adw), under the control of the inducible bacteriophage T7 RNA polymerase promoter. Upon induction with isopropyl-beta-d-thiogalactopyranoside, proteins were overexpressed and purified by affinity chromatography on a Ni-nitrilotriacetic acid agarose column. This method yielded 20-40 mg of highly pure and very stable proteins per liter of cell culture. Circular dichroism and fluorescence spectroscopy of isolated preS-his-ayw and preS-his-adw, as well as their ability to bind polymerized human serum albumin, indicate that the 6x His tag does not modify the native-like conformation and, therefore, they may be considered as useful tools to study the function of these viral polypeptide regions.     

Immunolocalisation and expression of proteoglycan 4 (cartilage superficial zone proteoglycan) in tendon.

Cartilage superficial zone protein/proteoglycan (SZP) or proteoglycan 4 (PRG4), has been demonstrated to have the potential for several distinct biological functions including cytoprotection, lubrication and matrix binding. In the present study, we have examined both the immunolocalisation and the mRNA expression pattern of PRG4 in tissue harvested from the compressed and tensional regions of young and mature bovine tendons. Immunohistochemical analyses, utilizing monoclonal antibody 3-A-4 which recognizes a conformational-dependent epitope on native PRG4, demonstrated that PRG4 is present predominantly at the surface of fibrocartilaginous regions of tendon, with the intensity of immunoreactivity in this region increasing with age. RT-PCR analyses revealed that the expression of PRG4 mRNA can be modulated by exposure to cytokines and growth factors. In addition, analyses of human pathological tendon revealed that PRG4 may also be expressed as an alternatively spliced form lacking exons which encode part of the N-terminal matrix-binding and cell-proliferative domain; however, it remains to be determined whether such splice variants are a feature of human tendon, regardless of disease state. Taken together, these data indicate that PRG4 may play an important cytoprotective role by preventing cellular adhesion to the tendon surface as well as providing lubrication during normal tendon function, in a manner complimentary to cartilage PRG4. Structural modifications to SZP, together with a reduction in synthesis during tendon inflammation with injury and disease may account for the formation of tendon adhesions and contribute to the overall dysfunction of the tissue.     

Enzymatic characterization of the full-length and C-terminally truncated hepatitis C virus RNA polymerases: function of the last 21 amino acids of the C terminus in template binding and RNA synthesis

The nonstructural protein NS5B of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp), which plays a central role in viral replication. Most of the reported studies on HCV polymerase in vitro have used a truncated form of the enzyme lacking the C-terminal 21 amino acids (DeltaC(21)-NS5B). In this study, we compared the enzymatic properties of the full-length NS5B (FL-NS5B) and this truncated form. Removal of the C(21) domain enhanced the enzyme stability. Both enzymes are capable of performing de novo and primer-dependent RNA syntheses, but each possesses a unique set of biochemical requirements for optimal RdRp activity. Whereas RNA synthesis by FL-NS5B remained relatively constant at 12-100 mM KCl, synthesis by DeltaC(21)-NS5B rapidly decreased at KCl concentrations greater than 12 mM. The different salt requirement for overall RNA synthesis by these two polymerases can in part be explained by the effect of monovalent ion concentration at the step of template binding, where binding by DeltaC(21)-NS5B but not FL-NS5B decreased proportionally as the KCl concentration increased from 25 to 200 mM. Thus, the C(21) domain appears to contribute to NS5B-RNA template binding, probably through the hydrophobic stacking interaction between its aromatic amino acids and the nucleotide bases of the RNA. This interpretation was supported by the observation that the C(21) polypeptide by itself could also bind to RNA to form binary complexes that were resistant to changes in the KCl concentration. Though both enzymes exhibited similar K(s) values for each of the four NTPs (1-5 microM), DeltaC(21)-NS5B generally required lower NTP concentrations than FL-NS5B for optimal synthesis. Interestingly, DeltaC(21)-NS5B became severely inhibited at elevated NTP concentrations, which most likely is due to competitive binding of the noncomplementary nucleotide to the polymerase catalytic center. Finally, the terminal transferase activity of DeltaC(21)-NS5B was found to be distinct from that of FL-NS5B on several different RNA templates. Together, these findings indicated that the HCV NS5B C(21) domain, in addition to being a membrane anchor, functions in template binding, NTP substrate selection, and modulation of terminal transferase activity.     

Three new members of the RNP protein family in Xenopus.

Many RNP proteins contain one or more copies of the RNA recognition motif (RRM) and are thought to be involved in cellular RNA metabolism. We have previously characterized in Xenopus a nervous system specific gene, nrp1, that is more similar to the hnRNP A/B proteins than to other known proteins (K. Richter, P. J. Good, and I. B. Dawid (1990), New Biol. 2, 556-565). PCR amplification with degenerate primers was used to identify additional cDNAs encoding two RRMs in Xenopus. Three previously uncharacterized genes were identified. Two genes encode hnRNP A/B proteins with two RRMs and a glycine-rich domain. One of these is the Xenopus homolog of the human A2/B1 gene; the other, named hnRNP A3, is similar to both the A1 and A2 hnRNP genes. The Xenopus hnRNP A1, A2 and A3 genes are expressed throughout development and in all adult tissues. Multiple protein isoforms for the hnRNP A2 gene are predicted that differ by the insertion of short peptide sequences in the glycine-rich domain. The third newly isolated gene, named xrp1, encodes a protein that is related by sequence to the nrp1 protein but is expressed ubiquitously. Despite the similarity to nuclear RNP proteins, both the nrp1 and xrp1 proteins are localized to the cytoplasm in the Xenopus oocyte. The xrp1 gene may have a function in all cells that is similar to that executed by nrp1 specifically within the nervous system.     

GroEL recognises sequential and non-sequential linear structural motifs compatible with extended beta-strands and alpha-helices.

The chaperonin GroEL binds a variety of polypeptides that share no obvious sequence similarity. The precise structural, chemical and dynamic features that are recognised remain largely unknown. Structural models of the complex between GroEL and its co-chaperonin GroES, and of the isolated apical domain of GroEL (minichaperone; residues 191-376) with a 17 residue N-terminal tag show that a linear sequential sequence (extended beta-strand) can be bound. We have analysed characteristics of the motifs that bind to GroEL by using affinity panning of immobilised GroEL minichaperones for a library of bacteriophages that display the fungal cellulose-binding domain of the enzyme cellobiohydrolase I. This protein has seven non-sequential residues in its binding site that form a linear binding motif with similar dimensions and characteristics to the peptide tag that was bound to the minichaperone GroEL(191-376). The seven residues thus form a constrained scaffold. We find that GroEL does bind suitable mutants of these seven residues. The side-chains recognised do not have to be totally hydrophobic, but polar and positively charged chains can be accommodated. Further, the spatial distribution of the side-chains is also compatible with those in an alpha-helix. This implies that GroEL can bind a wide range of structures, from extended beta-strands and alpha-helices to folded states, with exposed side-chains. The binding site can accommodate substrates of approximately 18 residues when in a helical or seven when in an extended conformation. The data support two activities of GroEL: the ability to act as a temporary parking spot for sticky intermediates by binding many motifs; and an unfolding activity of GroEL by binding an extended sequential conformation of the substrate.     

Differential expression of the "B" subunit of the vacuolar H(+)-ATPase in bovine tissues.

The B subunit is one of two nucleotide-binding polypeptides found in all members of the vacuolar class of H(+)-translocating ATPases. We have isolated aDNA clone encoding the bovine brain B (58 kDa) subunit and have deduced its amino acid sequence. The bovine brain amino acid sequence is 99% identical to a partial cDNA reported from human brain. Northern blot analysis of RNA isolated from bovine tissues and a bovine kidney cell line reveals that two messages of approximately 3.2 and 2.0 kilobases (kb) are expressed in all tissues examined except brain, where only the 3.2-kb message can be detected. Northern blotting of RNA isolated from human fibroblast and human lung tumor cell lines reveals that three messages of approximately 6.0, 3.2, and 2.0 kb are expressed, whereas only the 3.2-kb message is expressed in a human brain tumor cell line. This is the first demonstration of tissue-specific expression of multiple forms of a vacuolar H(+)-ATPase subunit. We have also isolated a partial cDNA clone from bovine brain which appears to encode an isoform of the B subunit. The deduced amino acid sequence is 82% identical to the major bovine brain B subunit sequence; it does not hybridize with either the 3.2- or 2.0-kb message on Northern blot. Southern blot analysis of bovine genomic DNA with probes derived from both isolated cDNAs indicates that the bovine B subunit is encoded by a multigene family.     

The nuclear 5S RNAs from chicken, rat and man. U5 RNAs are encoded by multiple genes

Preparations of chicken, rat and human nuclear 5S RNA contain two sets of molecules. The set with the lowest electrophoretic mobility (5Sa) contains RNAs identical or closely related to ribosomal 5S RNA from the corresponding animal species. In HeLa cells and rat brain, we only detected an RNA identical to the ribosomal 5S RNA. In hen brain and liver, we found other species differing by a limited number of substitutions. The results suggest that mutated 5S genes may be expressed differently according to the cell type. The set with the highest mobility corresponds to U5 RNA. In both rat brain and HeLa cells, U5 RNA was found to be composed of 4 and 5 different molecules respectively (U5A, U5B1-4) differing by a small number of substitutions or insertions. In hen brain, no U5B was detected but U5A' differing from U5A by the absence of the 3'-terminal adenosine. All the U5 RNAs contain the same set of modified nucleotides. They also have the same secondary structure which consists of two hairpins joined together by a 17 nucleotide long single-stranded region. The 3' half of the molecule has a compact conformation. Together, the results suggest that U5 RNAs are transcribed from a multigene family and that mutated genes may be expressed as far as secondary structure is conserved. The conformation of U5 RNA is likely to be related to its function and it is of interest to mention that several similarities of structure are found between U5 and U1A RNA.     

Virus-like genetic organization and expression strategy for a double-stranded RNA genetic element associated with biological control of chestnut blight.

The complete nucleotide sequence of the largest double-stranded (ds) RNA present in hypovirulent strain EP713 of the chestnut blight pathogen, Cryphonectria parasitica, was determined and the predicted genetic organization was confirmed by translational mapping analysis. The deduced RNA sequence was 12 712 bp in length, excluding the terminal poly(A):poly(U) homopolymer domain. The strand terminating with 3'-poly(A) contained two contiguous large open reading frames (ORF A and ORF B) beginning at nucleotide residues 496-498 and extending to nucleotide positions 11 859-11 861. The junction between ORF A and ORF B consisted of the sequence 5'-UAAUG-3', where UAA served as the termination codon for ORF A and AUG was the 5'-proximal initiation codon within ORF B. ORF A (622 codons in length, excluding the termination codon) was recently shown to encode two polypeptides, p29 and p40, which were generated from a nascent polyprotein by an autocatalytic event mediated by p29 (Choi et al., 1991). A similar autocatalytic event was observed during in vitro translation of ORF B (3165 codons in length) resulting in the release of a 48 kd polypeptide from the amino-terminal portion of the ORF B-encoded polyprotein. These results are discussed in terms of the opportunities they provide for elucidating the molecular basis of transmissible hypovirulence and possible origins of hypovirulence-associated dsRNAs.     

Structure-guided engineering of human thymidine kinase 2 as a positron emission tomography reporter gene for enhanced phosphorylation of non-natural thymidine analog reporter probe

Positron emission tomography (PET) reporter gene imaging can be used to non-invasively monitor cell-based therapies. Therapeutic cells engineered to express a PET reporter gene (PRG) specifically accumulate a PET reporter probe (PRP) and can be detected by PET imaging. Expanding the utility of this technology requires the development of new non-immunogenic PRGs. Here we describe a new PRG-PRP system that employs, as the PRG, a mutated form of human thymidine kinase 2 (TK2) and 2'-deoxy-2'-18F-5-methyl-1-β-L-arabinofuranosyluracil (L-18F-FMAU) as the PRP. We identified L-18F-FMAU as a candidate PRP and determined its biodistribution in mice and humans. Using structure-guided enzyme engineering, we generated a TK2 double mutant (TK2-N93D/L109F) that efficiently phosphorylates L-18F-FMAU. The N93D/L109F TK2 mutant has lower activity for the endogenous nucleosides thymidine and deoxycytidine than wild type TK2, and its ectopic expression in therapeutic cells is not expected to alter nucleotide metabolism. Imaging studies in mice indicate that the sensitivity of the new human TK2-N93D/L109F PRG is comparable with that of a widely used PRG based on the herpes simplex virus 1 thymidine kinase. These findings suggest that the TK2-N93D/L109F/L-18F-FMAU PRG-PRP system warrants further evaluation in preclinical and clinical applications of cell-based therapies.     

Investigation of cis/trans proline isomerism in a multiply occurring peptide fragment from human salivary proline-rich glycoprotein.

The solution-state conformations of eight proline-containing peptide fragments found in human salivary proline-rich glycoprotein (PRG) were investigated in 2 x distilled water (treated with metal ion chelating resin) using 13C-nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. The peptide sequences and acronyms were as follows: PRG9-2 = NH2-G(1)-P(2)-CONH2, PRG9-3 = NH2-G(1)P(2)-P(3)-CONH2, PRG9-4 = NH2-G(1)-P(2)-P(3)-P(4)-CONH2, PRG9-5 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-CONH2, PRG9-6 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-CONH2, PRG9-7 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-CONH2, PRG9-8 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-CONH2 and PRG9-9 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-P(9)-CONH2. Sequence-specific resonance assignments from the 13C-NMR spectra indicated that the trans proline isomer dominated the conformations of the peptides. CD results clearly showed the presence of the poly-L-proline II helix as the major conformation in PRG9-3----PRG9-5, supplemented by beta- and/or gamma-turns in PRG9-6----PRG9-9. These data suggest that in "metal free" water, native PRG could contain several small poly-L-proline II helices along with beta- and/or gamma-turns. Since proline is the major amino acid present in native PRG, these localized conformations may contribute to PRG's global conformation and act as a primary force in determining its biological activities.     

PET imaging of herpes simplex virus type 1 thymidine kinase (HSV1-tk) or mutant HSV1-sr39tk reporter gene expression in mice and humans using [18F]FHBG

The herpes simplex virus type 1 thymidine kinase (HSV1-tk) positron emission tomography (PET) reporter gene (PRG) or its mutant HSV1-sr39tk are used to investigate intracellular molecular events in cultured cells and to image intracellular molecular events and cell trafficking in living subjects. The expression of these PRGs can be imaged using 18F- or 124I-radiolabeled acycloguanosine or pyrimidine analog PET reporter probes (PRPs). This protocol describes the procedures for imaging HSV1-tk or HSV1-sr39tk PRG expression in living subjects with the acycloguanosine analog 9-4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine ([18F]FHBG). [18F]FHBG is a high-affinity substrate for the HSV1-sr39TK enzyme with relatively low affinity for mammalian TK enzymes, resulting in improved detection sensitivity. Furthermore, [18F]FHBG is approved by the US Food and Drug Administration as an investigational new imaging agent and has been shown to detect HSV1-tk transgene expression in the liver tumors of patients. MicroPET imaging of each small animal can be completed in approximately 1.5 h, and each patient imaging session takes approximately 3 h.     

ADAMTSL-3/punctin-2, a novel glycoprotein in extracellular matrix related to the ADAMTS family of metalloproteases.

The complete primary structure of ADAMTSL-3/punctin-2, a novel member of the family designated ADAMTSL (a disintegrin-like and metalloprotease domain with thrombospondin type I motifs-like), was determined by cDNA cloning from a human placenta library. The predicted open reading frame encodes a protein of 1690 amino acids that has considerable similarity to ADAMTSL-1/punctin-1. These multi-domain proteins lack both a protease domain and a disintegrin-like domain but are remarkably similar in their domain organization to the ADAMTS proteases, hence the name ADAMTS-like. Punctin-2 contains thrombospondin type 1 repeats (TSRs), a cysteine-rich domain and a cysteine-free spacer domain in the precise order in which they occur in the ADAMTS proteases. However, the number and organization of the TSRs in punctin-2 is unique with respect to the ADAMTS proteases. Punctin-2 contains 13 TSRs arranged in two arrays separated by a region containing three immunoglobulin-like repeats. Northern blot analysis of RNA from human adult tissues demonstrated that ADAMTSL3 is widely expressed, with highest expression in liver, kidney, heart and skeletal muscle, whereas it is expressed at low levels in mouse embryos. We characterized two punctin-2 polyclonal antisera. Using these and a monoclonal antibody to a C-terminal myc tag, we show that in transfected COS-7 cells, punctin-2 is expressed as a 210-kDa glycoprotein that is located in the extracellular matrix. The domain structure of punctin-2 and its matrix localization suggest that it might play a role in cell-matrix interactions or in assembly of specific extracellular matrices.