Biomedical subjects

W Garten

Publications and source records attributed to W Garten.

At least 19 recordsLinked to original sources

PubMed1998

Processing of the Borna disease virus glycoprotein gp94 by the subtilisin-like endoprotease furin.

Open reading frame IV (ORF-IV) of Borna disease virus (BDV) encodes a protein with a calculated molecular mass of ca. 57 kDa (p57), which increases after N glycosylation to 94 kDa (gp94). The unglycosylated and glycosylated proteins are proteolytically cleaved by the subtilisin-like protease furin. Furin most likely recognizes one of three potential cleavage sites, namely, an arginine at position 249 of the ORF-IV gene product. The furin inhibitor decRVKRcmk decreases the production of infectious BDV significantly, indicating that proteolytic cleavage of the gp94 precursor molecule is necessary for the full biological activity of the BDV glycoprotein.

Amino Acid Chloromethyl Ketones↗

PubMed1997

Biosynthesis, intracellular transport and enzymatic activity of an avian influenza A virus neuraminidase: role of unpaired cysteines and individual oligosaccharides.

Intracellular transport, glycosylation, tetramerization and enzymatic activity of the neuraminidase (NA) of fowl plague virus (FPV) were analysed in vertebrate cells after expression from a vaccinia virus vector. Tetramerization occurred with a half-time of 15 min, whereas passage through the medial Golgi apparatus and transport to the plasma membrane occurred with half-times of 2 and 3 h, respectively, suggesting a step in NA maturation beyond tetramerization that limits the rate of transport to the medial Golgi. NA transport rates were about fourfold slower than those of haemagglutinin (HA). Slow transport and processing of FPV NA was not altered by coexpression of FPV HA, nor was the transport rate of HA influenced by NA. The slow transport kinetics of NA were also observed in FPV-infected CV-1 cells. As deduced from the coding sequence, FPV NA has the shortest stalk of all naturally occurring NAs described to date and contains only three potential N-glycosylation sites, which are all located in the globular head domain. Elimination of each of the three N-glycosylation sites revealed that the two oligosaccharides at positions 124 and 66 are of the complex type, whereas the one at Asn-213 remains in mannose-rich form. The glycosylation mutants showed also that oligosaccharides at positions 124 and 213 of FPV NA modulate enzymatic activity. Transport of NA is not influenced by single elimination of any of the three oligosaccharide attachment sites. Mutational analysis of the three Cys residues not involved in intrachain disulfide pairing revealed that Cys-49 in the stalk of the NA molecule is responsible for the formation of disulfide-linked dimers. Analysis of cysteine mutants of FPV NA also demonstrated that disulfide-linked dimers are not absolutely necessary for the formation of enzymatically active tetramers but may stabilize the quaternary structure of NA.

Animals↗

PubMed1997

The role of eukaryotic subtilisin-like endoproteases for the activation of human immunodeficiency virus glycoproteins in natural host cells.

Proteolytic activation of the precursor envelope glycoproteins gp160 of human immunodeficiency virus type 1 (HIV-1) and gp140 of HIV-2, a prerequisite for viral infection, results in the formation of gp120/gp41 and gp125/gp36, respectively. Cleavage is mediated by cellular proteases. Furin, a member of the eukaryotic subtilisin family, has been shown to be an activating protease for HIV. Here, we compared the presence of furin and other mammalian subtilisins in lymphatic cells and tissues. Northern blot analyses revealed that furin and the recently discovered protease LPC/PC7 were the only subtilisin-like enzymes transcribed in such cells. Furin was identified as an enzymatically active endoprotease present in different lymphocytic, as well as monocytic, cell lines. When expressed from vaccinia virus vectors, the proprotein convertases were correctly processed, transported, and secreted into the media and enzymatically active. Coexpression of different subtilisins with the HIV envelope precursors revealed that furin and LPC/PC7 are able to cleave HIV-1 gp160. Moreover, both enzymes proteolytically processed the envelope precursor of HIV-2. gp140 was also cleaved to some extent by PC1, which is not, however, present in lymphatic cells. Furin- and LPC/PC7-catalyzed cleavage of HIV-1 gp160 resulted in biologically active envelope protein. In conclusion, among the known members of the subtilisin family, only furin and LPC/PC7 fulfill the requirements of a protease responsible for in vivo activation of HIV envelope glycoproteins.

Animals↗

PubMed1997

Oligosaccharides in the stem region maintain the influenza virus hemagglutinin in the metastable form required for fusion activity.

The influenza A virus hemagglutinin (HA) has three conserved oligosaccharides located in the stem region at asparagine residues 12, 28, and 478. The biological role of these oligosaccharides has been investigated by mutational analysis of HA of fowl plague virus that was expressed from a simian virus 40 vector in the presence of ammonium chloride for protection from acid denaturation in the trans-Golgi network. Resistance to endoglycosidase H and cleavage of HA into the subunits HA1 and HA2 have been analyzed as markers for intracellular transport. Cell surface exposure has been determined by hemadsorption following neuraminidase treatment, by immunofluorescence staining, and by fluorescence-activated cell sorter analysis. When all three stem oligosaccharides were removed, transport was almost completely blocked. When two of the three stem oligosaccharides, particularly those at asparagine residues 12 and 28, were missing, HA was transported to the surface but showed extremely low fusion activity. With mutants lacking one stem oligosaccharide, fusion was reduced to a lesser extent. Removal of stem oligosaccharides resulted also in an increase in the pH optimum required for fusion. On the other hand, no reduction in fusion activity was observed when oligosaccharides in the head region of the HA spike were removed. These results indicate that the conserved oligosaccharides in the stem stabilize HA in the form susceptible to the conformational change necessary for fusion.

Glycosylation↗

PubMed1996

Structural domains involved in human cytomegalovirus glycoprotein B-mediated cell-cell fusion.

A novel fusion assay was established to determine fusion activity with cocultivated human foreskin fibroblasts of stable transfectants derived from human astrocytoma cells (U373) expressing authentic or mutagenized human cytomegalovirus glycoprotein B (HCMV gB; gpUL55). Compared to transfectants expressing authentic HCMV gB, those expressing gB forms with a deletion of hydrophobic domain 1 (hd1; aa 714-747) or with deletions of specific segments in the cytoplasmic tail (aa 811-825 and 871-906) exhibited significantly reduced heterologous fusogenicity. HCMV gB-specific monoclonal antibodies (MAbs) as well as MAb against cellular annexin II prevented fusion of the transfectant expressing authentic gB. Comparable surface exposure of HCMV gB or its derivatives was demonstrated in all transfectants by FACS analysis. Our observations are compatible with the notion that indigenous fusion activity of HCMV gB depends on the extracellular hd1 domain and on the conformation of the cytoplasmic tail.

Binding Sites↗

PubMed1995

Two independent targeting signals in the cytoplasmic domain determine trans-Golgi network localization and endosomal trafficking of the proprotein convertase furin.

Furin, a subtilisin-like eukaryotic endoprotease, is responsible for proteolytic cleavage of cellular and viral proteins transported via the constitutive secretory pathway. Cleavage occurs at the C-terminus of basic amino acid sequences, such as R-X-K/R-R and R-X-X-R. Furin was found predominantly in the trans-Golgi network (TGN), but also in clathrin-coated vesicles dispatched from the TGN, on the plasma membrane as an integral membrane protein and in the medium as an anchorless enzyme. When furin was vectorially expressed in normal rat kidney (NRK) cells it accumulated in the TGN similarly to the endogenous glycoprotein TGN38, often used as a TGN marker protein. The signals determining TGN targeting of furin were investigated by mutational analysis of the cytoplasmic tail of furin and by using the hemagglutinin (HA) of fowl plague virus, a protein with cell surface destination, as a reporter molecule, in which membrane anchor and cytoplasmic tail were replaced by the respective domains of furin. The membrane-spanning domain of furin grafted to HA does not localize the chimeric molecule to the TGN, whereas the cytoplasmic domain does. Results obtained on furin mutants with substitutions and deletions of amino acids in the cytoplasmic tail indicate that wild-type furin is concentrated in the TGN by a mechanism involving two independent targeting signals, which consist of the acidic peptide CPSDSEEDEG783 and the tetrapeptide YKGL765. The acidic signal in the cytoplasmic domain of a HA-furin chimera is necessary and sufficient to localize the reporter molecule to the TGN, whereas YKGL is a determinant for targeting to the endosomes. The data support the concept that the acidic signal, which is the dominant one, retains furin in the TGN, whereas the YKGL motif acts as a retrieval signal for furin that has escaped to the cell surface.

Amino Acid Sequence↗

PubMed1995

Endoproteolytic cleavage of its propeptide is a prerequisite for efficient transport of furin out of the endoplasmic reticulum.

The trans-Golgi network (TGN) proprotein convertase furin is synthesized in a zymogenic form and is activated by intramolecular, autoproteolytic cleavage of the propeptide from its precursor. To obtain insight in possible functions of the furin propeptide, we have studied biosynthesis, propeptide cleavage, biological activity, and intracellular localization of human and bovine furin. Analysis of autocatalytic cleavage site mutants of furin revealed that efficient propeptide cleavage requires the presence of the complete furin cleavage consensus sequence Arg-X-Lys-Arg. In studies of a mutant in which the P1 + P4 + P5 residues of the autoproteolytic cleavage site were substituted, no substrate processing activity could be demonstrated, indicating a complete block of maturation. In immunofluorescence analysis, this mutant was found in the endoplasmic reticulum (ER), suggesting ER retention of profurin. This ER retention, however, appeared saturable. Furin proteins encoded by oxyanion hole mutant N188A and negative side chain mutant D248L, which possess autoprocessing activity but lack substrate processing activity, were found in the Golgi and the ER, respectively. Finally, analysis of a furin mutant, in which all three potential sites for N-linked glycosylation were altered, revealed autocatalytic cleavage, substrate processing, and transport to the Golgi. Our results indicate that cleavage of the propeptide occurs in the endoplasmic reticulum and is necessary but not sufficient for transport of furin out of this compartment.

Amino Acid Sequence↗

PubMed1995

Proteolytic processing of human cytomegalovirus glycoprotein B (gpUL55) is mediated by the human endoprotease furin.

Inhibition of endoproteolytic cleavage of glycoprotein B (gB; gpUL55) of human cytomegalovirus was achieved by treatment of infected fibroblasts with decanoyl peptidyl chloromethyl ketone (decRVKR-CMK), which inhibits the action of cellular subtilisin-like endoproteases with the amino acid recognition motif R x K/R R. Uncleaved gB precursor molecules of 160 kDa that were accumulated were endoglycosidase H resistant, suggesting that correct cellular transport occurred in the presence of the drug. The inhibitor also prevented endoproteolytic gB processing in CV-1 cells infected with a recombinant vaccinia virus-gB construct (VVgB). Evidence for direct involvement of the ubiquitous subtilisin-like endoprotease furin in gB cleavage was obtained from the observation that coinfection of CV-1 cells with WgB and a recombinant vaccinia-human furin construct reestablished endoproteolytic activity which was normally absent late after infection with WgB alone.

Cells, Cultured↗

PubMed1995

Constitutive expression of human cytomegalovirus glycoprotein B (gpUL55) with mutagenized carboxy-terminal hydrophobic domains.

Stable transfectants were selected from human astrocytoma cells (U373) after transfection with recombinant expression vectors carrying the human cytomegalovirus (HCMV) glycoprotein B (gB; gpUL55) gene with alternative deletions of hydrophobic domain segment 1 (hd1) or segment 2 (hd2) of the carboxy-terminal potential bipartite membrane anchor domain. Comparative analysis of HCMV gB forms from cell lines gB(Mhd1) and gB(Mhd2), expressing mutagenized gB, and those from cells expressing authentic gB showed that deletion of hd1, but not that of hd2, interfered with efficient proteolytic cleavage of the gB precursor. Both mutagenized gB forms exhibited correct transport to the cell surface. Deletion of hd2, but not that of hd1, caused loss of membrane anchoring of the gB molecule, resulting in secretion of the respective gB form into the culture medium. The carboxy-terminal cleavage product of the soluble gB molecule, which migrated more slowly than its authentic counterpart, was modified by complex carbohydrate side chains and formed disulphide-linked complexes. Our observations indicate that hd2 is essential as well as sufficient for membrane anchoring of the HCMV gB molecule. For hd1, a potential fusogenic role is suggested by the conserved positional pattern of glycine residues, which is comparable to that of known fusion peptides of other viruses.

Amino Acid Sequence↗

PubMed1995

N1 neuraminidase of influenza virus A/FPV/Rostock/34 has haemadsorbing activity.

The neuraminidase (NA) gene of influenza virus A/FPV/Rostock/34 virus (H7N1) was cloned and expressed in Sf9 cells using a baculovirus vector. The expression product had the expected molecular mass and showed neuraminidase activity. NA expressed in Sf9 cells also showed haemagglutinating activity as indicated by its ability to induce haemadsorption of chicken red blood cells. Haemadsorption depended on the presence of neuraminic acid on the erythrocytes, but was not blocked by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, a specific inhibitor of the enzymatic activity. These observations suggest that the N1 neuraminidase has two distinct reactive sites: a catalytic site and a receptor binding site. This concept was confirmed by site-directed mutagenesis which showed that the haemadsorbing activity of FPV NA was abolished when amino acids located on two surface loops at the putative binding site were exchanged. Substitutions on either of the two loops were sufficient for this effect. The mutated NAs retained full neuraminidase activity. In contrast, a mutated NA lacking neuraminidase activity still had haemadsorbing activity.

Animals↗

PubMed1994

Activation of an influenza virus A/turkey/Oregon/71 HA insertion variant by the subtilisin-like endoprotease furin.

The hemagglutinin (HA) gene of the influenza A turkey/Oregon/71 variant Tc1 adapted to primary chicken embryo cells contains an insertion of 54 nucleotides that encodes a peptide adjacent to the HA cleavage site, which is responsible for increased cleavability by ubiquitous cellular proteases. After coexpression with human furin from cDNA by vaccinia virus vectors and by an endogenous protease, the HA of Tc1, which possesses the amino acid sequence R-T-A-R at the cleavage site, is proteolytically processed. Site-directed mutagenesis of the cleavage site indicated that the arginine in position -4 is critical for HA activation by furin. Deletion of the insert revealed that the amino acid sequence -1 to -4 predisposes the protein for furin recognition.

Base Sequence↗

PubMed1994

Processing of viral glycoproteins by the subtilisin-like endoprotease furin and its inhibition by specific peptidylchloroalkylketones.

The spike glycoproteins of many enveloped viruses are proteolytically cleaved at the carboxytermini of sequences containing the basic motif R-X-K/R-R. Cleavage is often necessary for the fusion capacity of the glycoproteins and, thus, for virus infectivity. Among these viruses are pathogenic avian influenza viruses, human parainfluenza virus, human cytomegalovirus, and human immunodeficiency virus; it has been demonstrated that these viruses can be activated by furin. Indigenous furin has been identified in T-lymphocytes, which are host cells for HIV. Furin has been localized in the TGN and on the surface of cells after vectorial expression. Peptidylchloroalkylketones have been designed that inhibit with high specificity cleavage and fusion activity of viral glycoproteins, as well as virus replication.

Amino Acid Chloromethyl Ketones↗

PubMed1994

Host cell proteases controlling virus pathogenicity.

The majority of viral glycoproteins that undergo post-translational proteolysis are cleaved by ubiquitous intracellular proteases; however, a minority are cleaved by secreted proteases available only in a few host systems. The interplay of viral glycoproteins and cellular proteases may have a pivotal role in the spread of infection, host range and pathogenicity.

Amino Acid Sequence↗

PubMed1994

Maturation of the trans-Golgi network protease furin: compartmentalization of propeptide removal, substrate cleavage, and COOH-terminal truncation.

We have cloned a bovine cDNA encoding the trans-Golgi network (TGN) protease furin and expressed it via recombinant vaccinia viruses to investigate intracellular maturation. Pulse-chase labeling reveals that the 104-kD pro-furin bearing high mannose N-glycans is rapidly processed into the 98-kD protease whose N-glycans remain sensitive to endoglycosidase H for a certain period of time. Furthermore, in the presence of brefeldin A, pro-furin cleavage occurs. From these data we conclude that the ER is the compartment of propeptide removal. Studies employing the ionophore A23187 and DTT show that autocatalysis is Ca2+ dependent and that it does not occur under reducing conditions. Pro-furin produced under these conditions never gains endo H resistance indicating that it is retained in the ER. Coexpression of furin with the fowl plague virus hemagglutinin in the presence of brefeldin A and monensin reveals that furin has to enter the Golgi region to gain substrate cleaving activity. N-glycans of furin are sialylated proving its transit through the trans-Golgi network. A truncated form of furin is found in supernatants of cells. Truncation is inhibited in the absence of Ca2+ ions and in the presence of acidotropic agents indicating that it takes place in an acidic compartment of cells. Comparative analysis with furin expressed from cDNA reveals that the truncated form prevails in preparations of biologically active, endogenous furin obtained from MDBK cells. This observation supports the concept that secretion of truncated furin is a physiological event that may have important implications for the processing of extracellular substrates.

Amino Acid Sequence↗

PubMed1994

Rescue of vector-expressed fowl plague virus hemagglutinin in biologically active form by acidotropic agents and coexpressed M2 protein.

The hemagglutinin of the Rostock strain of fowl plague virus was expressed in CV-1 cells by a simian virus 40 vector, and its stability in the exocytotic transport process was examined by a fusion assay. A 50-fold increase in the fusion activity of the hemagglutinin was observed when expression occurred in the presence of ammonium chloride, Tris-HCl, or high doses of amantadine. When chloroquine, another acidotropic agent, was used, the hemagglutinin exposed at the cell surface had to be activated by trypsin, because intracellular cleavage was inhibited by this compound. Hemagglutinin mutants resistant to intracellular cleavage did not require acidotropic agents for full expression of fusion activity, when treated with trypsin after arrival at the cell surface. These results indicate that fowl plague virus hemagglutinin expressed by a simian virus 40 vector is denatured in the acidic milieu of the exocytotic pathway and that cleavage is a major factor responsible for the pH instability. Coexpression with the M2 protein also markedly enhanced the fusion activity of the hemagglutinin, and this effect was inhibited by low doses of amantadine. These results support the concept that M2, known to have ion channel function, protects the hemagglutinin from denaturation by raising the pH in the exocytotic transport system. The data also stress the importance of acidotropic agents or coexpressed M2 for the structural and functional integrity of vector-expressed hemagglutinin.

Ammonium Chloride↗

PubMed1994

Proteolytic cleavage of wild type and mutants of the F protein of human parainfluenza virus type 3 by two subtilisin-like endoproteases, furin and Kex2.

The fusion (F) protein of human parainfluenza virus type 3 contains the tribasic cleavage site R-T-K-R, which was altered by site-directed mutagenesis. Wild-type F protein and various mutants were expressed by recombinant vaccinia viruses. The endogenous endoprotease present in CV-1 cells cleaves F variants containing the furin recognition motif R-X-K/R-R but not variants containing the dibasic site K-R or a single R at the cleavage site. A similar cleavage pattern was obtained when the subtilisin-like endoproteases Kex2 and furin were coexpressed with the wild type and mutants of the F protein. Peptidylchloromethylketone inhibitors mimicking basic cleavage sites prevent cleavage of the precursor Fo by the endogenous protease only when the furin-specific motif is present in the peptidyl portion. The data support the concept that furin is a cellular protease responsible for the activation of the F protein of human parainfluenza virus type 3.

Amino Acid Sequence↗

PubMed1993

A H1 hemagglutinin of a human influenza A virus with a carbohydrate-modulated receptor binding site and an unusual cleavage site.

Two receptor binding variants of the influenza virus A/Tübingen/12/85 (H1N1) were separated by their different plaque formation in MDCK cells. Hemagglutination of variant I was restricted to red blood cells of guinea pigs, whereas variant II also hemagglutinated chicken cells. The variants differed also in their ability to bind to alpha 2,6-linked sialic acid. Evidence is presented that this difference is determined by a complex carbohydrate side chain at asparagine131 near the receptor binding site which is absent in variant II. With both variants, the arginine found at the cleavage site of all other human isolates analyzed so far was replaced by lysine.

Amino Acid Sequence↗

PubMed1993

Role of conserved glycosylation sites in maturation and transport of influenza A virus hemagglutinin.

The role of three N-linked glycans which are conserved among various hemagglutinin (HA) subtypes of influenza A viruses was investigated by eliminating the conserved glycosylation (cg) sites at asparagine residues 12 (cg1), 28 (cg2), and 478 (cg3) by site-directed mutagenesis. An additional mutant was constructed by eliminating the cg3 site and introducing a novel site 4 amino acids away, at position 482. Expression of the altered HA proteins in eukaryotic cells by a panel of recombinant vaccinia viruses revealed that rates and efficiency of intracellular transport of HA are dependent upon both the number of conserved N-linked oligosaccharides and their respective positions on the polypeptide backbone. Glycosylation at two of the three sites was sufficient for maintenance of transport of the HA protein. Conserved glycosylation at either the cg1 or cg2 site alone also promoted efficient transport of HA. However, the rates of transport of these mutants were significantly reduced compared with the wild-type protein or single-site mutants of HA. The transport of HA proteins lacking all three conserved sites or both amino-terminally located sites was temperature sensitive, implying that a polypeptide folding step had been affected. Analysis of trimer assembly by these mutants indicated that the presence of a single oligosaccharide in the stem domain of the HA molecule plays an important role in preventing aggregation of molecules in the endoplasmic reticulum, possibly by maintaining the hydrophilic properties of this domain. The conformational change observed after loss of all three conserved oligosaccharides also resulted in exposure of a normally mannose-rich oligosaccharide at the tip of the large stem helix that allowed its conversion to a complex type of structure. Evidence was also obtained suggesting that carbohydrate-carbohydrate interactions between neighboring oligosaccharides at positions 12 and 28 influence the accessibility of the cg2 oligosaccharide for processing enzymes. We also showed that terminal glycosylation of the cg3 oligosaccharide is site specific, since shifting of this site 4 amino acids away, to position 482, yielded an oligosaccharide that was arrested in the mannose-rich form. In conclusion, carbohydrates at conserved positions not only act synergistically by promoting and stabilizing a conformation compatible with transport, they also enhance trimerization and/or folding rates of the HA protein.

Biological Transport↗