WO2000009550A9 - Vanadate resistance glycosylation 4 gene - Google Patents
Vanadate resistance glycosylation 4 geneInfo
- Publication number
- WO2000009550A9 WO2000009550A9 PCT/US1999/018402 US9918402W WO0009550A9 WO 2000009550 A9 WO2000009550 A9 WO 2000009550A9 US 9918402 W US9918402 W US 9918402W WO 0009550 A9 WO0009550 A9 WO 0009550A9
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- WIPO (PCT)
- Prior art keywords
- vrg4
- yeast
- nucleotide
- sugar
- seq
- Prior art date
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to a yeast Vanadate Resistance Glycosylation 4
- VRG 4 gene and homologs thereof and protein encoded therein useful in methods of identifying inhibitors of a GDP-mannose transporter for use as anti-fungal compounds.
- the Golgi complex is the site at which the terminal glycosylation of both proteins and lipids occurs. Unlike mammalian cells, in the yeast S. cerevisiae, glycoproteins and sphingolipids are exclusively modified by the addition of ⁇ c mannose residues in the Golgi. Glycoproteins can undergo two types of modifications in which oligosaccharides are linked to either asparagine residues (N- linked) or serine/threonine residues (O-linked) (for review see (1,2). Both of these glycosylation pathways initiate in the endoplasmic reticulum (ER) and terminate in the Golgi. After transport of the protein to the Golgi, most N-linked
- oligosaccharides are elongated by a series of different mannosyltransferases to form glycoproteins that contain outer chains of 50 or more mannose residues.
- the c ,6- linked outer chain is highly branched with ⁇ l,2- and ⁇ l,3-linked mannoses. As in higher eukaryotes, it appears that the various mannosyltransferases that catalyse
- IPCs inositol-phosphorylceramides
- MIPC and M(IP) 2 C contain a single mannose attached to the inositol (6). though little is known about the mannosyltransferase(s) that catalyses this reaction. 5
- the mannosyl donor for all of these Golgi-localized reactions is the nucleotide sugar GDP-mannose, whose site of synthesis is the cytoplasm. Before it can be utilized by the different lumenal mannosyltransferases, GDP-mannose must be transported into the Golgi by a specific nucleotide sugar transporter (7). Once the sugar is donated to lumenal mannosyltransferase acceptors, the nucleoside diphosphate GDP is converted to a monophosphate by a nucleoside diphosphatase
- GDAI yeast GDPase-encoding gene
- nucleotide sugar transport activities have been reported, which differ from one another in their substrate specificity and subcelluar localization (9). Since the cytoplasm is the sole site at which nucleotide sugars are synthesized, they must be transported into the various organelles in which glycosylation occurs. Mammalian cells require the transport of many different nucleotide sugars due to the diversity of carbohydrate processing in the ER and Golgi. Carbohydrate chains may contain galactose, sialic acid, fucose, xylose, N-acetylglucosamine, and N- acetylgalactosamine. In contrast, in S. cerevisiae, glycosylation in the Golgi is largely restricted to mannosylation which in principle requires only a single transporter.
- the VRG4 gene is an essential gene that is required for a number of different Golgi-specific functions, including N-linked glycosylation (10-12), secretion, protein sorting and the maintenance of a normal endomembrane system (12).
- the present invention discloses that the transport of GDP-mannose into the Golgi is the principal function of the VRG4 gene product in yeast.
- the present invention discloses that pathogenic yeast also contain a VRG 4 gene homolog.
- the VRG 4 gene is essential for viability.
- a simple system to assay GDP-mannose transport is disclosed, using permeabilized yeast spheroplasts. Methods for identifying putative inhibitors of VRG 4 gene and gene product are disclosed. Summary of the Invention
- the invention provides a method of measuring an activity of a nucleotide- sugar transporter derived from yeast comprising providing a nucleotide sugar to a source, said source comprising a nucleotide-sugar transporter associated with a phospholipid membrane, and determining the amount of nucleotide-sugar bound to or transported through said membrane.
- the invention further provides a method of measuring an activity of a nucleotide-sugar transporter derived from yeast comprising providing a nucleotide- sugar to a source, said source comprising a nucleotide-sugar transporter associated with a phospholipid vesicle, and determining the amount of nucleotide-sugar transported into or accumulated within the vesicle.
- One object of the invention is to provide a method of measuring an activity of a Golgi nucleotide-sugar transporter from yeast comprising providing a nucleotide-sugar to a source derived from permeabilized yeast spheroplasts, the source comprising a nucleotide-sugar transporter and yeast golgi, and determining the amount of golgi-associated nucleotide-sugar as an indicator of nucleotide-sugar transporter activity.
- Another object of the invention is to provide a method of identifying inhibitors of golgi nucleotide-sugar transporter activity in yeasts comprising providing a putative inhibitor to a source derived from permeabilized yeast spheroplasts, the source comprising a nucleotide-sugar transporter and yeast golgi, with a nucleotide-sugar, and determining the amount of golgi-associated nucleotide- sugar in the presence of the inhibitor compared to the amount of golgi-associated nucleotide sugar in the absence of candidate inhibitor.
- One aspect of the invention is permeabilized yeast cells useful in methods of assessing nucleotide-sugar transport.
- the present invention encompasses permeabilized yeast cells useful in methods of assessing GDP-mannose transport and useful in methods of identifying inhibitors of GDP-mannose transport.
- the present invention provides permeabilized yeast cells containing a dpm 1 mutation useful in methods of measuring transporter activity.
- An aspect of the invention is a kit comprising permeabilized yeast cells and optionally a nucleotide sugar useful in methods of measuring golgi nucleotide-sugar transporter activity.
- One aspect of the invention are anti-fungal compounds that inhibit GDP- mannose transporter activity in yeast.
- Another aspect of the invention is an isolated VRG4 gene and portions thereof encoding a golgi GDP-mannose transporter in a pathogenic yeast.
- Another aspect of the invention is antibody specifically reactive to a VRG4 protein or immunogenic portion thereof.
- a further aspect of the invention is a pharmaceutical composition
- a pharmaceutical composition comprising an antibody specifically reactive to a VRG4 protein or immunogenic portion thereof useful in inhibiting nucleotide-sugar transporter function.
- nucleic acid probes for the detection of a wild-type VRG4 gene or alterations or mutations in the VRG4 gene.
- nucleic acid probes are complementary to the wild-type VRG4 gene coding sequences and can form mismatches with mutant or altered NRG4 genes, thus allowing their detection by enzymatic cleavage, chemical cleavage, or by shifts in electrophoretic mobilities.
- the method comprises isolating cells and/or infected tissue from a human and detecting the normal or wild-type VRG4 genes, mR ⁇ A or their expression products from the cells and/or infected tissue, wherein the detection of the gene, mR ⁇ A or expression products in the cell and/or infected tissue is indicative of a yeast infection.
- Another aspect of the invention is to provide a method of determining efficacy of treatment of a yeast infection in humans comprising isolating cells and/or infected tissue from a human and detecting alterations, reduction or absence ° of the normal or wild-type VRG4 gene, mRNA or expression product is indicative of efficacy of treatment by an antifungal compound.
- Yet another object of the invention is to provide a method of supplying normal or wild-type VRG4 gene function to a cell which has lost such normal gene 10 function by virtue of a mutation or alteration in the endogenous wild-type VRG4 gene, which comprises introducing a exogenous wild-type VRG4 gene or functional portion thereof into a cell which has lost said gene function, or which contains an aberrant gene, such that the wild-type gene is expressed in the cell.
- c Another object of the invention is to provide a method of supplying VRG4 gene function to a cell which lacks such a gene, which comprises introducing a portion or part of a wild-type VRG4 gene into a cell which lacks said gene function, such that the gene portion or part is expressed in the cell.
- FIG. 1 Immunoblot analysis of chitinase in vrg4 mutant and wild type extracts. Proteins were extracted from the culture supernatants of RSY255 (VRG4, lane 1), NDY5 (vrg4-2, lane 2), or HTY10 (mnnlO-2, lane 3) by acetone 5 precipitation, fractionated by 8% SDS-PAGE and subjected to immunoblot analysis using anti-chitinase antisera as described in Materials and Methods. The arrow denotes the mobility of wild type chitinase.
- FIG. 1 Analysis of sphingolipids in vrg4-2 and wild type cells.
- Sphingolipids in RSY255 (VRG4) or NDY5 (vrg4-2) were pulse labeled with [ 3 H] 0 myoinositol and chased for 20 or 40 minutes with unlabeled inositol, extracted and separated by thin layer chromatography as described in Materials and Methods.
- the assignment of PI, IPCs, MIPC and M(IP) C, denoted by arrows, is based upon a comparison of their mobility on TLC with those reported in the literature and upon 5 their relative abundance (41).
- Figure 3A and 3B GDP-mannose transport in permeabilized yeast cells as a function of time and protein concentration.
- FIG. 3 A shows the transport of GDP-[ 3 H]mannose as a function of time, in which reactions were carried out in a final protein concentration of 0.5 ⁇ g/ ⁇ l.
- Figure 3B shows the protein dependence of the reactions, carried out for six min at 30°C under conditions described above. 20 pmoles of GDP mannose transport corresponds to a lumenal uptake of 27% of the GDP-mannose in the
- Figure 5A-5C Western immunoblot and cytological analysis of the 0 Vrg4 protein.
- Figure 5A Whole cell protein extracts were prepared from yeast cells (SEY6210) harboring plasmids expressing Vrg4-HA3p on a CEN plasmid (pRHL-HA3), a 2 ⁇ plasmid (pYRHL-HA3) or the vector alone (pYEp352) or untransformed and fractionated by 10 % SDS-PAGE and subjected to western blot 5 analysis using anti-HA antibodies, as described in Materials and Methods.
- Figure 5A Whole cell protein extracts were prepared from yeast cells (SEY6210) harboring plasmids expressing Vrg4-HA3p on a CEN plasmid (pRHL-HA3), a 2 ⁇ plasmid (pYRHL-HA3) or the vector alone (pYEp352) or untransformed and fractionated by 10 % SDS-PAGE and subjected to western blot 5 analysis
- FIG. 6 The Nucleotide (SEQ. ID No: 1) and Predicted Amino Acid Sequence (SEQ. ID No: 2) of VRG4 gene from Saccharomyces cerevisiae.
- the five potential glycosylation sites, at amino acid positions 81, 119, 242, 246, and 249. are denoted by asterisks.
- Four potential membrane-spanning domains, 5 comprised of at least 20 uncharged residues and flanked by charged residues are 0 underlined. This sequence was found to be the same as that of the VAN 2 gene
- Figure 7A-7B The Full Length Nucleotide (SEQ. ID No: 3) and
- FIG. 8 An Alignment of the S. cerevisiae and C. albicans VRG4 proteins. Shown here is a portion of the C. albicans VRG4 homolog (SEQ. ID No:
- FIG. 9 The physical map of the pSK " Ca RG plasmid containing a , c portion of the VRG4 gene from Candida albicans.
- FIGS. 10A and 10B Co-immunoprecipitation of stable Vrg4p multimers in detergent extracts.
- Epitope tagged Vrg4p-containing complexes were extracted from yeast cells by treatment with 1 % digitonin (PANEL 10A) or 1% Triton X-100 (PANEL 10B) and immunoprecipitated with anti-HA antibodies.
- PANEL 10A 1 % digitonin
- PANEL 10B Triton X-100
- Extracts were prepared from a yeast strain (SEY6210) expressing VRG4-myc alone (YEplac81-Vrg4myc 3 ) (lane 1), co-expressing both 5 VRG4-myc (YEplacl81-Vrg4myc 3 ) and VRG4-HA (YEp352-Vrg4-HA 3 ) (lane 2), or expressing either VRG4-myc (YEplacl81-Vrg4myc 3 ) or VRG4-HA (YEp352-Vrg4- HA 3 ) and mixed after extraction but prior to immunoprecipitation (lane 3).
- Extracts were prepared from SEY6210 co-expresing VRG4-myc (YEplacl81-Vrg4myc 3 ) and GDAI -HA (pY023-GDAl-HA 3 ) (PANEL 10A, lane 4) or from strains expressing 0 either of these genes and extracts were mixed prior to immunoprecitation (PANEL 10B, lane 4).
- FIG 11 A and 11B Expression of the cloned vrg4-2 (A286D) mutant allele.
- the vrg4-2 mutant allele was cloned and epitope-tagged as described in 5 Materials and Methods.
- PANEL 1 1 A Isogenic wild type (RSY255) or vrg4-2 mutatnt cells (NDY5) transformed with plasmids containing either VRG4-HA (YEp- RHL-HA 3 ) or vrg4-A286D-HA (YEpVrg4-A286D-HA 3 ) were streaked onto YPAD media plus or minus 50 ⁇ g/ml hygromycin B.
- PANEL 1 IB Isogenic wild type (RSY255) or vrg4-2 mutatnt cells (NDY5) transformed with plasmids containing either VRG4-HA (YEp- RHL-HA 3 ) or vrg4-A
- Vrg4-A286D mutant protein stably interacts with itself and with the wild type Vrg4 protein.
- Extracts were prepared from a yeast strain (SEY6210) expressing VRG4-myc alone (YEplacl81-Vrg4-myc 3 ) (lane 1), co-expressing both VRG4-HA (YEpRHLHA3) and vrg4-A286D-myc (YEplacl81-Vrg4-A286D-myc 3 ) (lane 2), both vrg4A286D-HA and vrg4-A286D-myc (YEp352-Vrg4-A2826-HA 3 and YEplacl81Vrg4-A286D-myc 3 ) (lane 3), or both VRG4-HA and VRG4-myc
- Vrg4p-containing complexes were extracted from yeast cells by treatment with 1 % Digitonin and immunoprecipitated with anti-HA antibodies. After loading equivalent amounts of protein in each lane and fractionating by SDS-PAGE, precipitates were - immunoblotted with anti-myc antibodies and detected by chemiluminescence.
- FIG. 13A-13D The Vrg4-A286D mutant protein localizes to the Golgi.
- Ochl-HAp (a Golgi marker) (13B) and Ost4-HAp (an ER marker) (13A).
- Fixed cells were treated with anti-HA antibodies, followed by FITC-conjugated anti- mouse IgG.
- the vrg4-2 allele contains a single mutation (A286D) in a region of the protein that is highly conserved among other NSTs.
- PANEL 14A A hydropathy profile of the Vrg4 protein, with the location of the A286D mutation indicated with a circle.
- PANEL 14B An alignment of the region surrounding the A286D mutation (depicted with an asterisk) in other nucleotide sugar transporters.
- Group I proteins are closely related to the GDP- mannose transporters defined by the Leishmania donovani Lpg2 protein and the S cerevisiae Vrg4 protein.
- Group II proteins are most highly related to those that transport UDP-sugars. The accession numbers for related but uncharacterized ORFs are indicated. The identification of these proteins was obtained using BLAST version 2.0s [Altschul, 1997 #242].
- the present invention discloses that the Vanadate Resistance Glycosylation 4 (VRG4) gene isolated from Saccharomyces cerevisiae encodes a protein that transports GDP-mannose from the cytoplasm into the lumen of the golgi complex. GDP-mannose is the mannosyl donor for all of the glycosylation events that occur in the yeast golgi. As such, the VRG4 protein may be said to be the master regulator of glycosylation in yeast.
- the VRG4 protein of the present invention as essential for viability of the yeast Golgi. Mammalian cells do not have a GDP- mannose transporter. Since the VRG4 protein is a yeast-specific gene product, it represents a specific anti-fungal drug target. Thus anti-fungal compounds specifically targeted against the VRG4 gene or gene product have little or no side effects on mammalian cells.
- the present invention encompassed the VRG4 gene isolated from yeast.
- VRG4 genes isolated from pathogenic yeast.
- the pathogenic yeast including but not limited to Candida albicans, Candida tropicalis, Torulopsis glabrata, Cryptococcus neoformans, Aspergillus. Microsporium. Trichophyton, Epidermophyton, Pityrosporum, Histoplasma, Blastomyces and the like.
- the VRG4 gene is derived from Candida albicans. Homologs and functional portions of the VRG4 gene are included in the ambit of the invention. Functional portion as used herein, is any portion which has nucleotide-sugar transporter activity, preferably GDP-mannose transporter activity.
- nucleotide sequence of the VRG4 gene is depicted in Figures 6 and 7.
- nucleotide sequence of the VRG4 gene of the present invention is in no way limited to the sequences depicted in Figures 6 and 7, but may include the complementary sequence as well as variations in the nucleotide sequence as are known in the art as a result of code degeneracy that results in a functionally equivalent sequence.
- naturally occurring allelic variations in a given species are also encompassed by the present invention.
- the present invention also encompasses a mutant VRG 4 gene.
- mutant VRG4 genes are mutations which renders the gene totally nonfunctional or renders selected portions of the gene nonfunctional.
- the mutant VRG4 gene has one or more mutations in a GDP-mannose binding domain.
- the mutation includes one or more substitutions or deletions, and the like, which render the GDP-mannose binding domain incapable of binding GDP-mannose.
- the mutant VRG4 gene comprises a single base pair change in the region coding for the GDP-mannose binding domain.
- the mutant VRG4 gene comprises a single C to A base pair change at nucleotide position 857 of the wild-type VRG4 gene.
- a VRG4 gene from a yeast may be cloned by methods known in the art such as those used to clone the VRG4 gene of Saccharomyces cerevisiae (12). Of particular interest is the cloning of the full length VRG4 gene from pathogenic yeast such as from Candida albicans.
- the full length VRG4 gene of Candida albicans may be isolated from a genomic Candida albicans library using standard methods known in the art.
- E. coli is used to isolate the full length gene.
- the full length clone is obtained by screening E. coli colonies using the partial Candida gene as a probe by the conventional method of colony hybridization.
- the isolated VRG4 gene may be incorporated into vectors including but not limited to yeast vectors, yeast shuttle vectors, plasmid vectors for expression in mammalian cells and the like.
- the vectors contain the appropriate promoters and selectable markers as are known in the art for expression in the host cell.
- the vector is the plasmid SK Ca VRG4 containing a partial VRG4 gene of C. albicans deposited August 1 1, 1998 with the American Type Culture Collection, 10801 University Boulevard, Manassas, VA under Accession No. ATCC 203137 under the terms of the Budapest Treaty.
- the present invention includes host cells transformed or transfected with the VRG4 gene or portion thereof.
- Host cells include both eukaryotic and prokaryotic host cells provided they contain the correct elements for host-specific expression.
- host cells include but are not limited to bacterial cells, yeast cells, mutant yeast, mammalian cells such as BHK-21, COS-7, CV-1, Hela and the like.
- Saccharomyces cerevisiae is a host cell in which the endogenous
- VRG4 gene is replaced by the homolog VRG4 gene isolated from Candida albicans.
- the present invention encompasses the VRG4 gene product from yeast.
- the VRG4 gene product is a protein of about 36.9 kDa and peptides thereof.
- the VRG4 gene product of the present invention is associated with Golgi GDP-mannose transport in yeast.
- the amino acid sequence of the VRG4 gene product from Saccharomyces cerevisiae is depicted in Figure 6.
- Functional portions of the VRG4 protein are within the ambit of the present invention. Such isolated portions are those which facilitate transport of GDP-mannose across the Golgi.
- a full length nucleic acid sequence of the VRG4 gene and predicted amino acid sequence of the VRG4 protein from Candida albicans is depicted in Figure 7 A and 7B.
- the alignment of the amino acid sequence from C. albicans with S. cerevisiae is provided in Figure 8.
- the two proteins display 65% identity and 78% similarity along their entire length.
- a functional portion of the VRG4 protein encompass the GDP-mannose binding domain.
- the GDP-mannose binding domain comprises the consensus sequence: W Xaa Xaa Xaa Xaa T Xaa Xaa T T Y S
- Xaa at position 20 is Leu or He
- Xaa at position 22 is Leu or He
- Xaa at positions 2-5, 7-8, 14, 22-25, 27-29 and 31 is one of any naturally occurring amino acid (SEQ. ID No: 23).
- the GDP-mannose binding domain comprises SEQ. ID No: 7, SEQ. ID No: 9, or SEQ. ID No: 1 1.
- mutant GDP-mannose binding domain Another aspect of the invention is a mutant GDP-mannose binding domain.
- the mutant may be the result of one or more substitutions, deletions and the like which affects the function of the domain.
- the mutant GDP- mannose binding domain comprises SEQ. ID No. 8, which contains a single amino acid substitution in the consensus sequence.
- the full length VRG4 protein from Candida albicans may be easily obtained by recombinant techniques or the protein may be isolated from yeast cells by chromatographic techniques known in the art such as immunoaffinity chromatography and the like.
- a recombinant VRG4 protein or portion thereof is made by a method comprising incorporating an isolated VRG4 gene or functional nucleic acid sequence thereof into a vector, transforming or transfecting a host cell with the vector and culturing the host cell under conditions that allows expression of the recombinant VRG4 protein or portion thereof.
- the VRG4 protein or functional portion thereof may be used in assays for measuring VRG4 transporter activity and in assays for identifying inhibitors of GDP-mannose transport activity. Such inhibitors may be used as anti-fungal compounds in the treatment of yeast infections.
- the VRG4 protein or immunogenic portions thereof are also useful in eliciting anti-VRG4 antibody. Such antibody is useful in diagnostic assay and as a therapeutic to inhibit transporter activity.
- the present invention encompasses a method of measuring a nucleotide- sugar transporter activity.
- the nucleotide-sugar transporter under natural conditions, is unique to yeast cells and is not present under natural conditions in mammalian cells or bacterial cells. Of particular interest is a nucleotide-sugar transporter from pathogenic yeasts such as Candida albicans, Cryptococcus, Aspergillus and the like.
- the transport of an amount of exogenously added nucleotide-sugar is measured which is associated with the Golgi.
- the nucleotide-sugar transporter of the present invention is capable of causing the transport of a nucleotide-sugar into the Golgi.
- the amount of nucleotide-sugar accumulating within the Golgi, in particular the lumen of the Golgi is indicative of activity by the transporter.
- the method of the present invention may be used to determine the activity of any nucleotide-sugar transporter which is associated with golgi.
- the activity of a GDP-mannose transporter is determined.
- the method of assaying GDP-mannose transporter activity comprises adding an amount of GDP-mannose to a source of GDP-mannose transporter and a membrane source.
- the GDP-mannose transporter is associated with the membrane source in a manner such that the binding and transport of the GDP-mannose may be determined from the membrane source.
- the GDP-mannose is detectably labeled in such a manner to allow it to be easily detected and quantitated when associated with the GDP-transporter.
- labels include but are not limited to enzymes, radioisotopes, chemiluminescent compounds, bioluminescent compounds, and the like provided the labels do not interfere with transport.
- a second reagent may be added to detect the GDP-mannose.
- the second reagent such as an antibody, may be labeled.
- the source of the GDP-mannose transporter is from natural sources or recombinant sources.
- the GDP-mannose transporter may be expressed from an endogenous GDP-mannose transporter gene or an exogenous GDP-mannose transport gene or a functional portion thereof.
- the GDP- mannose transporter is associated in a membrane such as the Golgi membrane, or ° synthetic membrane such as a liposome membrane.
- Liposome membranes useful in the present invention may be made by methods known in the art such as those described in U.S. Patent Nos. 4,663,161 and 5,766,626.
- the membrane source for use in the method of measuring transport activity and inhibitors of transport activity include but are not limited to permeabilized yeast 5 cells, mammalian cells, liposomes having associated therewith a GDP-mannose transporter, and the like.
- the membrane source is a mammalian cell transformed or transfected with a yeast nucleotide-sugar transporter.
- permeabilized yeast cells are used in the method of determining nucleotide-sugar transporter activity.
- the yeast cells for use in the method of measuring transport activity are yeast spheroplasts, devoid of cell walls.
- the yeast spheroplasts are permeabilized spheroplasts which comprise a leaky plasma membrane within which is contained an intact Golgi, an endomembrane
- the permeabilization of the yeast spheroplasts provides a leaky plasma membrane to allow access of the nucleotide-sugar into the cell.
- the yeast cells may be permeabilized by means which slightly disrupts the integrity of the plasma
- yeast spheroplasts are permeabilized using liquid nitrogen.
- the permeabilized yeast spheroplasts for use in a method of measuring 25 Golgi nucleotide-sugar transporter activity are devoid of any other system which may utilize GDP-mannose or compete with the GDP-mannose transport.
- the permeabilized spheroplasts lack/or have been genetically altered to inactivate a dolichol phosphate-mannose synthase.
- the permeabilized spheroplasts have a dolichol phosphate- mannose synthase mutation which renders the synthase inactive.
- the permeabilized spheroplasts for use in the method of measuring Golgi nucleotide-sugar transporter activity preferably Golgi GDP-mannose transporter activity, contain a functionally active endogenous Golgi GDP-mannose transporter ⁇ ' *> or an exogenous source of a functionally active Golgi GDP-mannose transporter.
- the permeabilized spheroplasts utilizes an exogenous source of active Golgi GDP-mannose transporter provided by an exogenous gene encoding a Golgi GDP-mannose transporter
- an endogenous gene encoding a Golgi GDP-mannose transporter if present, is mutated so as to prevent expression of the endogenous Golgi GDP-mannose transporter.
- the permeabilized spheroplasts have a functional VRG4 gene derived from a pathogenic yeast, preferably derived from Candida albicans.
- the permeabilized spheroplasts are from a Saccharomyces strain which has a dpml gene mutation (dpml " ) and a mutation in the endogenous VRG4 gene (VRG4 ⁇ ), each mutation rendering the respective gene inactive.
- the yeast strain is JPY263D of Saccharomyces cerevisiae having the genotype dpm ⁇ ⁇ /VRG4 ⁇ deposited August 1 1, 1998 with The American Type Culture Collection under Accession No. ATCC 74461 under the terms of the Budapest Treaty.
- VRG4 gene may be incorporated into JPY263D yeast strain by techniques known in the art and transporter activity determined using the methods of the present invention.
- the method of the present invention is an improvement over methods described in the past because of its simplicity and efficiency.
- Past methods relied on the use of enriched, partially purified Golgi membranes that are of a low specific activity.
- the prior art method involved growing up liters (10) of cell cultures, resulting in the recovery of only small amounts of active membranes.
- the prior art method starting with 1 liter of cells only provided enough material for about 4-5 reactions.
- the present method allows one to start with much smaller cell culture volumes, yet provides enough material to perform many assays of the present invention as the Golgi membranes enclosed in permeabilized spheroplasts retain higher levels of activity.
- 1 liter provided enough material for about 100-200 assays.
- a quick, reliable and quantitative assay is essential as it allows large number of candidate inhibitors to be screened at a time.
- Vectors suitable for use in the invention comprise at least one expression control element operationally linked to the nucleic acid sequence or part thereof.
- Expression control elements are inserted in the vector to control and regulate the expression of the nucleic acid sequence.
- Non-limiting examples of expression control elements include, but are not limited to. the lac system, operator and promoter regions of phage lambda, yeast promoters, and promoters derived from vaccinia virus, adenovirus, retroviruses, or SV40.
- Other operational elements include, but are not limited to, appropriate leader sequences, termination codons, polyadenylation signals, and other sequences required for the appropriate transcription and subsequent translation of the nucleic acid sequence in a given host system.
- the correct combinations of expression control elements will depend on the host system used.
- the expression vector contains any additional elements necessary for the transfer and subsequent replication of the nucleic acid-containing expression vector in the host system.
- additional elements include, but are not limited to, origins of replication and selectable markers.
- Such expression vectors are commercially available or are readily constructed using methods known to those in the art (e.g., F. Ausubel et al., 1987, in "Current Protocols in Molecular Biology", John Wiley and Sons, New York, New York).
- the recombinant expression vector containing all or part of the VRG4 nucleic acid sequence are transformed, transfected or otherwise inserted into a host organism or cell.
- the host cells transformed with the VRG4 nucleic acid sequence of the invention include eukaryotic and mammalian cells, such as animal, plant, insect and yeast cells, and prokaryotic cells, such as E. coli, or algal cells as known in the art.
- the means by which the vector carrying the gene may be introduced into a cell includes, but is not limited to, micro injection, electroporation, transduction, or transfection using DEAE-dextran, lipofection, calcium phosphate, or other procedures known to those skilled in the art (Sambrook et al. (1989) in "Molecular Cloning.
- eukaryotic expression vectors that function in eukaryotic cells are used.
- mammalian expression vectors that function in a mammalian host cells are used.
- yeast expression vectors that function in yeast host cells are used.
- Non-limiting examples of vectors include vaccinia virus vectors, adenovirus vectors, herpes virus vectors, and baculovirus transfer vectors.
- Preferred eukaryotic cell lines include, but are not limited to, COS cells, CHO cells, HeLa cells, and NIH/3T3 cells. Particularly preferred host cells are yeast cells.
- the expressed recombinant VRG4 protein or portions thereof may be detected by methods known in the art, some of which include Coomassie blue staining, silver staining, and Western blot analysis using antibodies specific for the
- the recombinant protein expressed by the transformed host cells can be obtained as a crude lysate or can be purified by standard protein purification procedures known in the art, including differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis, affinity and immunoaffinity chromatography and the like. (Ausubel et. al., 1987, In “Current Protocols in Molecular Biology” John Wiley and Sons, New York, New York).
- the recombinant protein may be purified by passage through a column containing a resin which has bound thereto antibodies specific for the VRG4 protein (Ausubel et. al., 1987, In “Current Protocols in Molecular Biology” John Wiley and Sons, New York, New York).
- an active VRG4 protein was purified by using an HA- epitope tagged gene which was constructed and tested for activity by complementation of the VRG4 mutant phenotype. This HA-tagged protein was then over-expressed and purified by affinity-chromoatography using 12CA5 bound resin.
- the wild type VRG4 gene or alterations of the wild type VRG4 gene is detected.
- Alteration of a wild-type gene according to the present invention encompasses all forms of mutations, including deletions. The alteration may also be due to rearrangements, such as insertions, inversions, and deletions, or to point mutations. Deletions may be of the entire gene or only a portion of the gene.
- the method of the present invention may be used to determine efficacy of antifungal treatment in a mammal in which an alteration, reduction or elimination of the wild type VRG4 gene, mRNA or gene product is indicative of efficacy in reducing or eliminating a yeast infection.
- Mutations induced by antifungal therapy leads to non-functional or absent gene products which in turn also lead to inhibition and loss of viability of yeast.
- the detection of point mutations may be accomplished by molecular cloning of the allele (or alleles) present in the yeast and sequencing that allele(s) using techniques well known in the art.
- polymerase chain reaction PCR
- PCR polymerase chain reaction
- the DNA sequence of the amplified sequences can then be determined by conventional methods.
- the polymerase chain reaction itself is well known in the art (see, e.g., Saiki et al., 1988, Science, 239:487; U.S. Patent No. 4,683,203; and
- combinations of oligonucleotide pairs based on the VRG4 nucleotide sequence may be used as PCR primers to detect VRG4 mRNA in a biological sample using the reverse transcriptase polymerase chain reaction (RT-PCR) process for amplifying selected RNA nucleic acid sequences as detailed in Ausubel et al., 1987, In “Current Protocols in Molecular Biology” Chapter 15, John Wiley and Sons, New York, New York.
- the oligonucleotides can be synthesized by automated instruments sold by a variety of manufacturers or can be commercially prepared based upon the nucleic acid sequence of the invention.
- Biological samples for testing may include cells, tissues, organs, blood, serum, stool, sputum, amniotic fluid, mucous secretions and urine.
- insertions and deletions of VRG4 gene are detected by using a non-complementation assay of a mutant as is known in the art
- the invention also relates to antibody specifically immunoreactive with the
- GDP-mannose transporter preferably immunoreactive with VRG-4 protein or epitope thereof.
- This invention comprises an antibody preparation or antibodies which are immunoreactive with the VRG4 protein having the amino acid sequence depicted in Figure 6, 7 or 8, or a unique portion or peptide thereof.
- the antibodies are either monoclonal or polyclonal in origin.
- the antibodies may be raised against native VRG4 protein or peptides,
- VRG4 fusion proteins or peptides or mutant VRG4 proteins or peptides.
- VRG4 proteins or peptides used to generate the antibodies may be from natural or recombinant sources or produced by chemical synthesis using synthesis techniques known in the art. Natural VRG4 proteins can be isolated from yeast cultures, isolated golgi, from mammalian biological samples containing or suspected to contain yeast and the like. Recombinant VRG4 proteins or peptides may be produced and purified by conventional methods. Synthetic VRG4 peptides may be custom ordered or commercially made based upon the predicted amino acid sequence of the present invention ( Figure 6, 7 or 8) or synthesized by methods known to one skilled in the art (Merrifield, R.B., 1963, J. Amer. Soc. 85:2149).
- the peptide may be conjugated, complexed, or otherwise covalently linked to a carrier molecule to enhance the antigenicity of the peptide.
- carrier molecules include, but are not limited to, albumins (e.g., human, bovine, fish, ovine), and keyhole limpet hemocyanin ("Basic and Clinical Immunology", 1991, Eds. D.P. Stites, and A.I. Terr, Appleton and Lange, Norwalk Connecticut, San Mateo, California).
- the antibodies should be specific and immunoreactive with VRG4 epitopes, preferably epitopes not present on other yeast protein or human protein, to avoid crossreactivity. However, antibodies can be generated against particular epitopes which are found to be common to other proteins, if desired or necessary to detect related structures or molecules.
- the antibodies will immunoprecipitate VRG4 proteins from solution as well as react with VRG4 proteins on Western or immunoblots of polyacrylamide gels on membrane supports or substrates.
- the antibodies will detect VRG4 proteins in paraffin or frozen tissue sections, or in cells which 0 have been fixed or unfixed and prepared on slides, coverslips, or the like, for use in immunocytochemical, immunohistochemical, and immunofluorescence techniques.
- Exemplary antibody molecules for use in the detection methods or as a therapeutic of the present invention are intact immunoglobulin molecules, substantially intact immunoglobulin molecules, or those portions of immunoglobulin molecules that contain the antigen binding site, known in the art as F(ab), F(ab) 1 2 , and F(v) immunoglobulin fragments.
- Polyclonal or monoclonal antibodies may be produced by methods conventionally known in the art (e.g., Kohler and Milstein, 1975, Nature, 256:495-497; Campbell "Monoclonal Antibody 0 Technology, the Production and Characterization of Rodent and Human
- Monoclonal antibodies may be human monoclonal antibodies, chimeric monoclonal 5 antibodies, or humanized monoclonal antibodies made by techniques that are well known in the art. (Takeda 1985 Nature 314:452; U.S. Patent No. 5,585,089, U.S. Patent No. 5,530,101.)
- the antibodies or antigen binding fragments may also be produced by genetic engineering.
- the technology for expression of both heavy and light chain genes in E. coli is the subject of PCT patent applications, publication
- Antibody molecules of the present invention may be intact immunoglobulin molecules, or portions thereof that contain the antigen binding site.
- Single chain antibody may be constructed by methods known in the art 25 (U.S. Patent No. 4,946,778; Davis, G.T. et al. 1991 Biotechnology 9:165-169;
- the antibody molecules may be of any class including IgG, IgM and IgA.
- the antibody of the present invention may be used as a diagnostic reagent to ,. confuse detect and quantitate the Golgi GDP-mannose transporter, and to determine Golgi
- Standard immunoassay may be used with the anti-GDP-mannose transporter antibody for detection and quantitation of the Golgi GDP-mannose transporter in biological samples.
- VRG4 protein-specific antibodies are used in 5 immunoassays to detect the novel VRG4 protein in biological samples.
- the antibodies of the present invention are contacted with a biological sample and the formation of a complex between the VRG4 protein and the antibody is detected.
- suitable immunoassays include radioimmunoassay, Western blot assay, immunofluorescent assay, enzyme linked immunoassay (ELISA), chemiluminescent assay, immunohistochemical assay, immunocytochemical assay, and the like (see, e.g., "Principles and Practice of Immunoassay", 1991, Eds. Christopher P. Price and David J.
- Such assays may be direct, indirect, competitive, or noncompetitive as described in the art (see, e.g., "Principles and Practice of Immunoassay", 1991, Eds. Christopher P. Price and David J.
- Proteins may be isolated from test specimens and biological samples by conventional methods, as described in "Current Protocols in Molecular Biology", 1987, Eds. Ausubel et al., John Wiley and Sons, New York, New York.
- the antibody of the present invention may be provided in the form of a kit and may also include GDP-mannose and/or other assay reagents.
- the antibody of the present invention may be used as a therapeutic to specifically inhibit the function of the Golgi GDP-mannose transporter. Binding of the antibody to the transporter prevents glycosylation in yeast and results in the loss of viability of the yeast cells.
- the antibody may be provided intraperitoneally, intravenously, intramuscularly, subcutaneously, mucosally or topically administered.
- the antibody is administered to a patient with a yeast infection for a period of time sufficient to reduce or eliminate the infection.
- the antibody may be modified so as to enhance the transport of the antibody across the cell wall of the yeast so as to aid accessibility of the antibody to the transporter in the golgi.
- topical administration, or as an oral mouthwash the antibody may be administered in combination with a detergent to allow entry of the antibody through the cell wall.
- primer pairs of the invention are useful for the determination of the nucleotide sequence of the VRG4 gene using the polymerase chain reaction.
- the pairs of single stranded DNA primers can be annealed to sequences within or surrounding the VRG4 gene, or a discrete segment of the gene in order to prime amplifying DNA synthesis of the VRG 4 gene itself.
- PCR primers may be on the order of about 15-40 bp, more preferably, on the order of about 18-30 bp to PCR an approximately 100-600 bp, more preferably, a 100-200 bp stretch of DNA.
- a complete set of primers allows synthesis of all of the nucleotides of the VRG4 gene coding sequences.
- Allele specific primers can also be used. Such primers anneal only to particular VRG4 mutant alleles, and thus will only amplify a product in the presence of the mutant allele as a template.
- Non- limiting examples of VRG4 sequence primers for use in the invention may be the nucleic acid sequence or portion thereof as shown in Figures 6, 7A and 7B.
- primers may have enzyme restriction site sequences appended to their 5' ends.
- all nucleotides of the primers are derived from VRG4 sequences or sequences adjacent to VRG4. except for the few nucleotides necessary to form a restriction enzyme site.
- enzymes and enzyme restriction sites are well known in the art.
- the primers themselves can be synthesized using techniques which are well known in the art. Generally, the primers can be made using synthesizing machines which are commercially available.
- Nucleic acid probes provided by the present invention are useful for a number of purposes. They can be used in Southern hybridization analyses to probe genomic DNA, and in the RNase protection method for detecting point mutations. The probes can be used to detect PCR amplification products, and can also be used to detect mismatches with the VRG4 gene or mRNA using other techniques. Examples of nucleic acid sequences that can be used as probes include, but are not limited to, native DNA, recombinant DNA, and synthetic oligonucleotides.
- DNA sequences can be labeled with 32 P using Klenow enzyme, polynucleotide kinase or polymerases, such as TAQ used in the PCR reactions.
- Klenow enzyme polynucleotide kinase or polymerases, such as TAQ used in the PCR reactions.
- Non-radioactive labeling techniques for signal amplification including methods for attaching chemical moieties to pyrimidine and purine rings (Dale, R.N.K. et al., 1973, Proc. Natl. Acad. Sci. USA, 70:2238-2242; and Heck, R.F., 1986, S. Am. Chem. Soc.
- kits for detecting wild type VRG4 genes and alternations thereof.
- the kit allows for hybridization to the entire VRG4 gene, or to particular regions thereof.
- the probes may overlap with each other or may be contiguous.
- Kits may contain other reagents useful for carrying out the assay, such as buffers, enzymes, control samples, and the like.
- Nucleic acid probes may also be complementary to the VRG4 gene or to mutant alleles of the VRG4 gene. Such probes are useful to detect similar homologs or mutations on the basis of hybridization. As also described, the VRG4 probes can be used in Southern hybridizations to genomic DNA to detect gross chromosomal changes, such as deletions and insertions. In addition, the probes can be used to detect VRG4 mRNA from yeasts in tissues to determine if expression is diminished as a result of alteration of wild type VRG4 genes as a result of antifungal therapy.
- Antisense oligonucleotides may be derived from the VRG4 gene as depicted in Figures 6, 7A and 7B or derived from the nucleic acid sequence encoding the GDP-mannose binding domain depicted in Figures 14A and 14B.
- Antisense oligonucleotides are useful in specifically inhibiting the formation of the VRG4 gene product. Antisense oligonucleotides may be made by methods known in the art.
- a method is also provided to supply wild type VRG4 function to a cell which is devoid of a VRG4 gene or to a cell that carries a mutant VRG4 alleles.
- the wild type VRG 4 gene or a part of the gene may be introduced into the cell in a vector.
- Vectors for the introduction of genes both for recombination and for extrachromosomal maintenance are known in the art, and any suitable vector or vector construct may be used in the invention.
- Methods for introducing DNA into cells such as electroporation, calcium phosphate co-precipitation and viral transduction as mentioned hereinabove are known in the art; therefore, the choice of method may lie with the competence and preference of the skilled practitioner.
- Cells transformed with the wild type VRG4 gene are used as model systems to study nucleotide-sugar transport to screen for antifungal drugs for treatments against yeast infections.
- the protein or peptides of VRG4 may function as competitive inhibitors of the native VRG4 protein and as such may be used as an antifungal compound to treat infections caused by yeast.
- Protein and peptides can be produced by expression of the isolated DNA sequence, in particular genomic DNA, in yeast, for example, using known expression vectors.
- VRG4 can be extracted from FRG ⁇ -producing cells, such as yeast cells.
- the protein may be cleaved using enzymes or reducing agents may be used to form peptides.
- Inhibitor peptides may be selected using the methods described herein .
- the techniques of synthetic chemistry can be employed to synthesize VRG4 protein or peptides.
- Active inhibitory VRG4 molecules can be introduced into cells by microinjection or by the use of liposomes, for example. Alternatively, some of the active molecules may be taken up by cells, actively or passively by diffusion.
- Extracellular application of VRG4 gene product may be sufficient to affect yeast growth.
- Agents may be added to facilitate the entry of the VRG4 protein or peptide across the cell wall of the yeast and to facilitate the association of the exogenously supplied VRG4 protein or peptide with the golgi.
- agents include but are not limited to liposomes antibody and the like.
- the isolated VRG4 protein or portion thereof may be used in the form of a pharmaceutical composition, along with standard excipients as are known in the art, for inhibition of the endogenous VRG4 gene product of a yeast.
- the inhibitors of the present invention are useful in preventing and treating yeast and fungal infections in mammals, including humans.
- Yeast and fungal diseases that may be treated using the inhibitors of the present invention include but are not limited to candidiasis, aspergillosis, phycomycoses, nocardiosis, cryptococcosis, histoplasmosis, blastomycosis, coccidioidomycosis, paracoccidioidomycosis, onychomycosis dermatophyte infections and the like.
- the inhibitors of the present invention may be administered in the form of a pharmaceutical composition, alone, or as a mixture, and may be administered in combination with one or more other fungicidal and fungistatic compounds.
- the fungicidal and fungistatis compounds include but are not limited , _- to ketoconazole, flucytosine, fluconazole, itraconazole, amphotericin B and the like.
- Means of administering the VRG4 protein or parts thereof include, but are not limited to, oral, sublingual, intravenous, intraperitoneal, percutaneous, intranasal, intrathecal, subcutaneous, intracutaneous, mucosal or enteral.
- Local administration to the afflicted site may be accomplished through means known in 0 the art, including, but not limited to, topical application, injection, and implantation.
- the inhibitor is provided in a dose sufficient to inhibit nucleotide-sugar transport into the Golgi of the yeast or fungi, preferably a dose effective in inhibiting a Golgi GDP-mannose 5 transporter present in the yeast or fungi. Such inhibition in transport results in inhibition of growth and loss of viability of the yeast or fungi causing the infection.
- inhibitors of a Golgi nucleotide-sugar transporter include but are not limited to a nucleotide-sugar analog, stilbeine or derivatives thereof provided the inhibitor specifically inhibits a Golgi nucleotide-sugar transporter in fungi or 0 yeast and is nontoxic or has minimal effect on mammalian cells.
- the inhibitors of the present invention may be competitive or noncompetitive inhibitors.
- the inhibitors of the present invention are useful in inhibiting VRG4 transport activity in fungi and yeast. Such inhibition of VRG4 transport activity in 5 the Golgi result in inhibition of growth and ultimate lack of inability of fungi and yeast.
- the fungi and yeast amenable to inhibition by the inhibitors of VRG4 transport activity include but are not limited to Candida, Torulopsis, Cryptococcus,
- Plasmid Constructions All DNA manipulations were carried out according to standard protocols (45). The 2.1-kb EcoRI/Hindlll fragment containing the entire VRG4 gene and regulatory sequences was subcloned into the vector pRS316 (46) to generate the CEN based plasmid, pRHL, containing the selectable marker, URA3. This plasmid was labeled with [ j2 P]dCTP (Amersham
- the disruption plasmid pG5::LEU was constructed by inserting a
- the integrative plasmid pG5i was constructed by cloning the
- the plasmid was linearized at a unique Hpal site in the VRG4 gene and transformed into strain NDY5.
- DNA sequence analysis of the 2.1-kb fragment revealed the presence of two open reading frames. Further analyses mapped the complementing activity to the larger open reading frame, within a 1.6-kb Hindlll/EcoRV fragment. The nucleotide and predicted amino acid sequence of this region is shown in Figure 6.
- the VRG4 DNA sequence encodes a predicted protein of about 36.9 kDa. There are five potential recognition sites for N-linked glycosylation (indicated by asterisks in Figure 6). Hydrophobicity analysis (33) suggests that the protein is hydrophobic, containing multiple membrane-spanning domains.
- a plasmid designated SK ' Ca VRG4 comprising a partial VRG4 gene of Candida albicans was deposited with the American Type Culture Collection, P.O. Box 3605, Manassas, Virginia 20108 U.S.A. (ATCC), on August 11, 1998 under Accession No. ATCC 203137 under the terms of the Budapest treaty.
- the physical map of the Sk ' Ca VRG4 plasmid is shown in Figure 9.
- Yeast strain JPY263D of Saccharomyces cerevisiae which lacks dpml and has a mutant endogenous VRG 4 gene was used as a host cell for incorporation of the VRG4 gene of Candida albicans.
- the yeast strain was deposited with ATCC on August 11 , 1998 under Accession No. ATCC 74461 under the terms of the Budapest Treaty.
- the partial VRG4 gene was used to isolate a full length genomic VRG4 gene from a genomic Candida albicans library and a clone comprising a full length genomic VRG4 gene was isolated.
- Yeast strains used in this study are listed in Table 1. Media preparation and standard yeast genetic methods used for sporulation, tetrad dissection and strain constructions have been described (13). YPAD liquid medium was supplemented with 0.5 M KC1 for the growth of the vrg4 mutant strains (10,12). Hygromycin B (Boehringer Mannheim) was added to YPAD agar after autoclaving to a final concentration of 30 ⁇ g/ml. Yeast strains were transformed using the lithium acetate procedure (14).
- An epitope -tagged allele of VRG4 was created in several steps. First, PCR was used to amplify the VRG4 gene while replacing the stop codon with an Nsil site. A 1.5 kb Hin ⁇ llllNsil fragment containing the VRG4 gene was then ligated into the
- pYRHL-HA3 contains the HA3 -tagged VRG4, under its own promoter in a 2 ⁇ , URA3 plasmid. It was constructed by ligating the H dIII/Nb ⁇ l fragment from pR ⁇ L- ⁇ A3 into the HindllVXbal sites of YEp352.
- a DNA fragment containing the entire HVG1 gene and flanking sequences was cloned by PCR amplification of genomic yeast DNA. This 1.3 kb fragment, flanked by BamRl/Hindl ⁇ l sites was cloned into pRS316 to generate the plasmid, pHVGl . Similarly, PCR amplification of yeast genomic DNA was used to generate a 1,026 bp fragment containing only the HVG1 ORF. This fragment, flanked by a BamHl/Hindlll site was cloned into YIp56X to place it under the control of the TPI promoter (16). A deletion of the HVG1 gene was carried out by replacing the entire HVG1 open reading frame with the S.
- pombe HIS5 gene which is functionally analogous to the S. cerevisiae HIS3 gene (17).
- PCR amplification of a fragment containing the S. pombe HIS5 gene (kindly provided by Sean Munro, MRC, LMB), with HVG1 primer ends, was used to generate a linear fragment containing the HIS5 gene flanked by 50 bp of sequence homologous to HVGl. This linear fragment was used to transform strain SEY6210. His + transformants were isolated and the deletion was confirmed by PCR (data not shown).
- Western Immunoblotting Western Immunoblotting
- Spheroplasts were treated with 0.1% Triton X-100 for 5 min at room temperature. After attaching to glass slides, cells were plunged into - 20°C methanol for 6 minutes, followed by -20°C acetone for 30 sec Slides were incubated overnight in primary antibody (12CA5 culture supernatant, diluted 1 :10), washed 12 times with PBS and then incubated in anti-mouse IgG:FITC or anti- rabbit: IgG:FITC (Jackson ImmunoResearch, PA), diluted 1 :200, for 1-2 hours. After washing, cells were overlayed with mounting media containing 25 ng/ml DAPI.
- Radiolabeling of cells and lipid analysis Cultures were grown to an OD 600 of 1 in Wickerham's minimal medium (18), containing 2% glucose and lacking myo-inositol (WH-I). Labeling was initiated by the addition of 5 ⁇ Ci/ml of [ H]-myoinositol (American Radiolabeled Chemicals, St Louis, MO). Cells were metabolically labeled for 10 min at 30 °C and chased by the addition of 4 volumes of WH containing 40 ⁇ g/ml unlabeled myoinositol. Reactions were stopped by the addition of ice cold NaN 3 . Cells were washed once in NaN 3 , suspended in 100 ⁇ l NaN 3 and broken by vortexing with glass beads.
- the lysate was removed from the glass beads and lipids were extracted by adding 600 ⁇ l chloroform/methanol (1 :1) to 90 ⁇ l of the cell extract to achieve a final concentration of (10: 10:3) chloroform/methanol/aqueous solution.
- Permeabilized yeast cells suitable for use in determining Golgi nucleotide- sugar transport, were prepared as described (20) with modifications. 100-200 ml of cells were grown in YPAD medium containing 0.5 M KC1 at 30°C to an OD 6 oo of 1- 2. After harvesting, cells were suspended at 50 OD unit/ml in (100 mM Tris-HCl, pH 9.4; 10 mM DTT) and kept at room temperature for 5 min.
- the cells were centrifuged and resuspended at 50 OD unit/ml in (0.75 X YPA, 0.5% glucose, 0.7 M sorbitol, 10 mM Tris-HCl, pH 7.5) and 10 U lyticase/OD unit of cells was added to form spheroplasts. After 20 minutes incubation at 30°C, over 80% of the yeast cells were converted to spheroplasts. Spheroplasts were centrifuged at 1,500 X g for 3 min and resuspended in 0.75 X YPA containing 0.7 M sorbitol and 1% glucose.
- GDP-mannose transport was measured in permeabilized spheroplasts. Reactions contained 20 mM HEPES (pH6.8); 150 mM potassium acetate, 250 mM sorbitol, 5 M magnesium acetate; 3 ⁇ M GDP mannose and 50 nCi GDP- [ 3 H] -mannose (15
- Protein concentrations were determined using the BCA reagent (Pierce Chemical Co, Rockford, IL) After incubating at 30°C for 6 min, the reaction was stopped by adding 0.5 ml of ice-cold buffer H and samples were placed on ice. Membranes were pelleted by centrifugation at either 14,000 X g or 100,000 X g in an ultracentrifuge (Beckman Optima TL). Free radioactive solutes were removed by washing the membrane pellet three times with 1.0 ml of ice-cold buffer H.
- GDPase was assayed as described (7) in solubilized PI 00 fractions prepared from PYCs from strains JPY25 6c (VRG4) or JPY26 3d (vrg4-2).
- Inorganic phosphate was determined by the method of Ames (21). One unit of GDPase is defined as the activity that releases 1 nmole of inorganic phosphate per minute. Background values, determined by assaying reactions that lacked substrate or protein were subtracted to give the values described.
- the vrg4 mutant is defective in both N- and O-linked sugar modifications VRG4 is required for N-linked glycosylation (10-12).
- VRG4 is required for N-linked glycosylation (10-12).
- Chitinase is a secreted protein that contains carbohydrates that are exclusively O- linked. Therefore, any effect on O-linked glycosylation can be detected by an electrophoretic mobility shift (22).
- Whole cell extracts were prepared from isogenic wild type and vrg4 cells and assayed by immunoblotting, using anti-chitinase antiserum.
- chitinase mobility was also examined in cells containing an mnnlO-2 mutation, which are defective only in N-linked glycosylation (23).
- a mobility shift was detected in chitinase from vrg4-2 when compared to wild type cells, but not in mnnlO-2 cells ( Figure 1). This result demonstrates that the vrg4 mutation affects O-linked glycosylation and therefore is required for the glycosylation of both classes of proteins.
- Example 5 The vrg4 mutant is defective in sphingolipid mannosylation vrg4 cells display an aberrant morphology of intracellular membranes when viewed by electron microscopy (12). In vrg4 mutants, membranes accumulate but stain poorly with potassium permanganate. This observation suggested that the VRG4 gene product may be required for maintaining the normal protein/lipid ratio of these Golgi membranes whose staining properties are altered by the vrg4 mutation. The synthesis of sphingolipids in yeast requires vesicular transport to the Golgi, and suggests that their synthesis occurs in this compartment (24). Therefore, it was of interest to determine whether the vrg4 mutation affected sphingolipid biosynthesis. In S.
- sphingolipids there are three major classes of sphingolipids. These include the inositolphosphorylceremides (IPCs) and the mannosylinositolphosphorylceramides (MIPC, and M(IP) 2 C) (see reference 5 for review).
- IPCs inositolphosphorylceremides
- MIPC mannosylinositolphosphorylceramides
- M(IP) 2 C mannosylinositolphosphorylceramides
- GDP-mannose transport was characterized in permeabilized yeast cells containing a dpml mutation that results in a 90%-95% decrease of dolichol- phosphate -mannose synthase (Dpml) activity in vitro (25).
- Dpml dolichol- phosphate -mannose synthase
- This mutant background was required to eliminate a competing reaction catalyzed by Dpmlp, in which GDP-mannose donates mannose to form dolichol-phosphate-mannose (Dol- P-Man) that in turn acts as the mannose donor for glycosylation in the ER.
- This ER reaction which is quite efficient in vitro, would otherwise obscure the Golgi transport of GDP-mannose (7).
- the physical properties of lumenal radioactive material was examined by analyzing the transport reaction products after phase partitioning. This 5 separates lipid-linked oligosaccharides, which partition into the organic phase from protein-linked oligosaccharide, which are insoluble in chloro form/methanol. After allowing the transport reaction to occur for six minutes, PYCs were extracted to separate lipid, protein and water soluble products as described by Waechter et al (27). By this assay, most of the radioactive products (87%) that associated with the membranes were water soluble (GDP-mannose) or chloroform/methanol insoluble (protein). We conclude that GDP-mannose transport in PYCs appears to have all of the hallmarks previously described for this activity in crude Golgi membranes (7).
- the VRG4 gene product is required for lumenal Golgi GDP-mannose translocation.
- Vrg4p is therefore specifically required for GDP- mannose uptake.
- Membranes were prepared from the isogenic strains JPY25 6c (VRG4) or JPY26 3d 0 (vrg4-2) and assayed for hydrolysis of GDP, as described (7, 21).
- VRG4 is a resident Golgi protein
- the VRG4 gene product is required for a number of different Golgi functions (12). If these effects are due to its role in nucleotide sugar uptake, Vrg4p would be predicted to reside in the Golgi complex.
- the VRG4 gene was tagged at the carboxy terminus with three tandem copies of the HA epitope (see Materials and Methods). Even when tagged with three copies of the HA epitope, when expressed as a single copy,Vrg4-HA3p in whole cell extracts was barely detectable by immunoblot analyses (Figure 5A).
- Vrg4-HA3p The intracellular location of Vrg4-HA3p was examined by indirect immunofluorescence, using antibody directed at the HA epitope. A punctate pattern of fluorescence, characteristic of the Golgi complex, was observed in cells expressing Vrg4-HA3p ( Figure 5B). This staining pattern was similar to another
- Vrg4-HA3p-expressing cells observed, the average number of HA-staining spots per cell observed by shifting the plane of focus was 20-25. This was confirmed by performing a Z-series in which the analysis of optical sections of l ⁇ thickness through individual cells indicated an average number of 25 spots per cell (data not shown). Cells expressing Ochl-HA3p contained between 7-10 spots/cell and no qualitative differences were observed in cells overexpressing Ochlp. From these results, we conclude that Vrg4p resides in the Golgi complex.
- VRG4 encodes a highly conserved protein. Thirteen different members have been identified including the Leishmania LPG2 and the Kluyveromyces lactis MNN2 gene products (12, 29, 30). In the case of Lpg2p and Mnn2p, both proteins have been implicated as nucleotide sugar transporters (29-31). A search of the S. cerevisiae genome data base identified several other yeast ORFs with sequence similarity to Vrg4, suggesting that these putative proteins may function in nucleotide sugar transport. These putative yeast proteins are listed by ORF name in Table III.
- HVG1 Homologous to VRG4
- HVG1 Homologous to VRG4
- Vrg4 protein sequence was used to search the S. cerevisiae genome data base using the BLAST algorithm (42) and the identified proteins were aligned using DNASTAR MegAlign program with the Clustal algorithm.
- Vrg4p Because of the high degree of identity to Vrg4p, it was of interest to examine the Hvgl protein and the phenotype of the null mutant.
- the predicted ORF and flanking sequences were cloned by PCR amplification of yeast genomic DNA (see Materials and Methods).
- the HVG1 gene was introduced into the vrg4-2 mutant strain (NDY5) in either a single and high copy expression plasmid. Though the encoded gene products are remarkably similar, the HVG1 gene does not complement the glycosylation and slow growth phenotype of the vrg4-2 mutant or the inviability of the null vrg4 allele.
- Hvglp performs a function that is redundant to another, as yet unidentified protein(s) or that its function is completely dispensable for vegetative growth of yeast.
- Example 10 We have undertaken a functional analysis of Vrg4p as a model for understanding nucleotide sugar transport in the Golgi.
- Vrg4p was analyzed epitope tagged alleles of VRG4 which were fused with either the myc or HA epitope.
- Results from co-immunoprecipitation experiments demonstrate that the Vrg4 protein multimerizes with specificity and high affinity, both in vivo and in vitro.
- the molecular weight of the Vrg4p-containing complex calculated by gel filtration is twice that of the monomer, suggesting that the active enzyme is a dimer of identical subunits.
- mutant protein is catalytically inactive for nucleotide sugar - transport, it maintains the ability to multimerize, is localized normally to the Golgi, and is as stable as its wild type counterpart.
- Sequence analysis of the vrg4-2 allele reveals a single base pair alteration that changes an alanine to an aspartate residue. This alanine is embedded in a region that is highly conserved in other GDP-mannose transporters but has diverged in transporters of other nucleotide sugars.
- Yeast Strains and Media Standard yeast media and genetic techniques were used (48). Hygromycin B sensitivity was tested on yeast extract/peptone/ adenine sulfate/dextrose plates (YPAD) supplemented with 50 ⁇ g/ml hygromycin B (Boehringer Mannheim) as described (49).
- the wild type strain used was SEY6210 (MAT ⁇ ura3-52 leu2-3, ⁇ 12 his3-A200 trpl- ⁇ 901 lys2-80l suc2A9) NDY5 (MAT ⁇ ura3-52 leu2-2 ⁇ 1 vrg4-2)
- a 1.35-kB fragment containing the vrg4-2 open reading frame and 237 base pairs of 5' and 72 base pairs of 3' flanking sequences was amplified by PCR using LA Taq thermophilic DNA polymerase (TaKaRa Shuzo, Japan) from genomic DNA isolated from the vrgA-2 strain, NDY5 (12), using the following primers: 5'CGTAATGAATCGCAATATACG3' (SEQ. ID No: 25) and 5 TGCATTAGATGCCTCTATAA3' (SEQ. ID No: 26).
- LA Taq polymerase has the same high fidelity as Pfu, but like Taq polymerase, lacks the 3' to 5' proofreading exonuclease activity.
- pRS316Vrg4-A286D-HA 3 encodes the Vrg4-A286D mutant protein tagged with three copies of the HA epitope at the C-terminus in pRS316. It was constructed by replacing a Hp ⁇ llMfel fragment of pRHL-HA 3 (51), with the Hp ⁇ llMfel fragment from pCRIIvrg4-2.
- YEp352-Vrg4-A286D-HA 3 which contains the HA-tagged vrg4-2 allele on a 2 ⁇ /URA3 plasmid
- a Hindlll/Xb ⁇ l fragment from pRS316 Vrg4-A286D-HA 3 containing the entire Vrg4-A286D-HA 3 , was subcloned into YEp352 (52).
- pRHL-myc 3 To construct pRHL-myc 3 , the VRG4 gene was cloned in-frame to three copies of the myc epitope. A fragment containing the VRG4 ORF, lacking the stop codon and flanked by a 5' Hindlll and a 3' Nsil site was isolated by PCR. This fragment was cloned into Hindlll-Pstl digested pSK " P/X myc 3 , a Bluescript SK " derivative (Stratagene). pSK " P/X myc3 carries a 172 bp fragment containing sequences that encode three tandem copies of the myc epitope (EQKLISEEDL) (SEQ.
- the SKNRG4-myc 3 construct contains an in-frame fusion of the three copies of the myc epitope to the carboxy terminus of Vrg4p.
- SK VRG4-myc 3 was used to generate pRHL-myc 3 which contains VRG4-myc ⁇ , on an EcoRllHindlll fragment in pRS316.
- a myc-tagged vrg4-A286D allele was constructed in several steps.
- the resulting plasmid, pRS316-Vrg4-A286D-myc3 contains the myc-tagged vrg4-2 allele in pRS316.
- HA-tagged GDA 1 plasmid was created by introducing a S ⁇ /1 lEco RI site 5' and 3' to the GDA ORF by PCR. After digestion with Sail and EcoRI, this fragment was ligated into the SaW EcoRI site of pSK ' P/X HA 3 plasmid (15) to produce PSKODAI-HA 3 . This results in the in-frame fusion of GDA with sequences encoding 3 copies of the HA epitope at the 3' terminus, followed by a stop codon. The sequence of PSKODAI-HA 3 was confirmed by DNA sequencing as described above.
- Exponentially growing yeast cells (A600: 1-3) were harvested and converted to spheroplasts with lyticase (SIGMA), as described (59).
- Spheroplasts were resuspended in 400 ⁇ l of ice cold lysis buffer (150 mM NaCl, 10 mM HEPES-KOH (pH7.5), 5 mM MgCl 2 , 1 mM PMSF) containing either 1% digitonin or 1% Triton X-100 to solubilize membrane proteins, and centrifuged for 5 min at 4°C at 14 kG to remove debris.
- ice cold lysis buffer 150 mM NaCl, 10 mM HEPES-KOH (pH7.5), 5 mM MgCl 2 , 1 mM PMSF
- 1% digitonin or 1% Triton X-100 to solubilize membrane proteins
- Lysis was carried out by dounce homogenization (25 strokes) on ice and unbroken cells were removed from the lysate by centrifugation for 5 min in a micro fuge. Membranes were isolated by centrifugation at 100 kG for 30 min in a Beckman optima TL ultracentrifuge. The membrane pellet was resuspended in 150 ⁇ l lysis buffer and used for protease protection assays (see below). Co-immunoprecipitation, western immunoblotting and immunofluorescence
- the HA-tagged proteins were immunoprecipitated by incubating 400ul of the detergent extract (described above) with 200 ⁇ l of a hybridoma cell cultpre supernatants containing the 12CA5 monoclonal anti-HA antibody and 25 ⁇ l of protein A-Sepharose (Pharmacia) at room temperature for 2 hours.
- the protein A- Sepharose beads and associated proteins were centrifuged and washed three times with the same lysis buffer ( 1% digitonin or 1% Triton X-100; 150 mM NaCl, 50 mM HEPES-KOH ( ⁇ H7.5), 5 mM MgCl 2 , 1 mM PMSF).
- immunoprecipitates were fractionated by 10% SDS-PAGE, transferred to Immobilon-PVDF membranes (Millipore) and immunoblotted with anti-myc A- 14 polyclonal antibodies (Santa Cruz Biotechnology). Secondary anti-rabbit antibodies conjugated to horseradish peroxidase (Amersham) were used at a 1:3000 dilution and detected by chemiluminescence (ECL, Amersham) followed by autoradiography.
- Vrg4 protein multimerizes in vivo and in vitro
- Vrg4 protein functions as a monomer or in a higher order structure
- a co-immunoprecipitation assay was first used to determine whether the Vrg4 protein can interact with itself.
- a yeast strain was constructed that co- expressed both an HA- and myc-tagged allele of VRG4 on high copy plasmids. Both of these tagged alleles can complement the hygromycin B sensitivity of a vrg4 mutant, although not as well as the untagged alleles, indicating that these epitopes do not significantly alter the normal function of Vrg4p.
- Membrane proteins from this strain were solubilized with 1 % digitonin and immunoprecipitated with the 12CA5 anti-HA monoclonal antibody.
- Vrg4-mycp To measure the relative amount of Vrg4-myc protein that associated with the HA-tagged Vrg4p, the precipitates were fractionated by SDS-PAGE and immunoblotted with a rabbit antiserum against the myc epitope (Figure 10A). Vrg4-mycp efficiently co-precipitated with Vrg4-HA, since it could only be detected in the presence of extracts containing Vrg4-HA ( Figure 10A, compare lanes 1 and 2). Similar results were obtained if the anti-myc antibody was used for the immunoprecipitation and the anti-HA antibody was used for western blotting (not shown), indicating that co-precipitation is not dependent on the antibody.
- Vrg4p is very hydrophobic, containing six to eight predicted membrane spanning domains. As a control for non-specific aggregation due to its hydrophobicity, we examined whether we could detect an interaction of Vrg4p with other membrane proteins. Neither Gdal-HAp ( Figure 10B, lane 4), Yndl-HAp (not shown) which are Golgi localized GDPases with single transmembrane domains, nor Pmalp (not shown), a plasma membrane protein that contains 10 predicted transmembrane domains, co-precipitated with Vrg4-mycp, suggesting that Vrg4p oligomerization is not due to nonspecific hydrophobic interactions.
- Vrg4p-containing complex had assembled in vivo.
- digitonin extracts were prepared from strains that expressed either Vrg4-mycp or Vrg4-HAp. These extracts were combined together prior to immunoprecipitation with anti-HA antibody. Following fractionation by SDS-PAGE, no Vrg4-mycp could be detected in the precipitate ( Figure 10A, lane 3), demonstrating that the complex had stably formed in vivo and did not disassemble in the presence of digitonin in vitro.
- Vrg4p-containing complex behaved differently in Triton X-100 than in digitonin.
- stable Vrg4p oligomers could be extracted from yeast solubilized with 1% Triton X-1OO since Vrg4-mycp efficiently co-precipitated with Vrg4HA and could only be detected in the presence of extracts containing Vrg4HAp ( Figure 10B, lanes 1 and 2).
- Vrg4mycp that co-precipitated with Vrg4-HAp after mixing was three fold reduced from that which co-precipitated from extracts that were prepared from cells co-expressing these two proteins ( Figure 10B, lanes 2 and 3), suggesting that the Vrg4p-containing complex is less stable in Triton X-1OO than in digitonin.
- Vrg4 multimerization in Triton is specific since its association with other membrane proteins, such as Gdal- mycp ( Figure 10B, lane 4) or Yndl-mycp (not shown) was not observed.
- the mutant protein encoded by the vrg4-2 allele is stable and retains the ability to form protein interactions.
- vrg4-2 strains display a level of nucleotide sugar transport activity in vitro that is about twenty five fold reduced from those of wild type strains.
- the vrg4-2 allele was cloned by PCR amplification of genomic DNA from a vrg4-2 mutant strain and tagged with either the HA-or myc epitope appended to the C-terminus (see Materials and Methods). The isolation of the mutant allele was confirmed by sequence analysis (see below), by testing its inability to complement the hygromycin B growth sensitivity of the vrg4-2 mutant strain ( Figure 11A), and by western blot analysis ( Figure 1 IB).
- Vrg4-A286Dp the steady state level of the HA-tagged mutant protein was compared to the normal Vrg4-HA protein by western blot analysis with the anti-HA antibody ( Figure 1 IB).
- Multimerization of the mutant Vrg4 A286D-HA was examined using a co- immunoprecipitation assay.
- Yeast strains were constructed that co-express in either the mutant and wild type Vrg4 proteins that were HA- and myc-tagged, respectively, or that co-express the HA- and myc- tagged mutant Vrg4-A286D protein.
- the relative affinity of the mutant protein for itself and for the wild type Vrg4 protein was compared by quantitating the amount of Vrg4 A286Dmycp that precipitated with anti-HA antibody in detergent extracts.
- Vrg4A286D mutant protein could multimerize as well as the wild type Vrg4 protein since equal amounts of Vrg4 A286D-mycp precipitated with both Vrg4-HAp and Vrg4-A286D-HAp
- the mutant protein displayed the same punctate pattern characteristic of the yeast Golgi that is observed for the wild type Vrg4 protein that is distinct from the perinuclear, ER staining ( Figure 13A-13D) suggesting that the mutant protein is correctly localized to the Golgi. Therefore, the inactivity of the mutant Vrg4 A286D protein is not due to its mislocalization. Sequence analysis of the mutant vrg4-2 allele
- the vrg4-2 allele contains a mutation that affects nucleotide sugar transport, but that does not affect homo- oligomerization, Golgi localization or protein stability.
- sequence of the vrg4-2 mutant allele was determined. The mutant gene was cloned using a PCR approach (see Materials and
- Example 11 Figure 15A and 15 B show complementation of the S cerevisiae vrg-4 mutant by the Candida VRG4 gene.
- This figure demonstrates that the isolated gene is not just a structural homologue but also a functional homologue, i.e., that the isolated C. albicans VRG4 gene is a bonaflde GDP-mannose transporter.
- the second important point of this datum is that the Candida gene functions in S cerevisiae, which means that method aimed at inhibition of the Candida protein can be performed in this nonpathogenic strain, rather than in Candida.
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EP99942152A EP1105418A1 (en) | 1998-08-14 | 1999-08-12 | Vanadate resistance glycosylation 4 gene |
JP2000564999A JP2002522061A (en) | 1998-08-14 | 1999-08-12 | Vanadate-resistant glycosylation 4 gene |
AU55593/99A AU5559399A (en) | 1998-08-14 | 1999-08-12 | Vanadate resistance glycosylation 4 gene |
CA002339338A CA2339338A1 (en) | 1998-08-14 | 1999-08-12 | Vanadate resistance glycosylation 4 gene |
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