Classifications

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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
  • G16H10/00—ICT specially adapted for the handling or processing of patient-related medical or healthcare data
  • G16H10/60—ICT specially adapted for the handling or processing of patient-related medical or healthcare data for patient-specific data, e.g. for electronic patient records
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/23—Immunoglobulins specific features characterized by taxonomic origin from birds
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/54—F(ab')2
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
  • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
  • C12Y207/11009—Goodpasture-antigen-binding protein kinase (2.7.11.9)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/91—Transferases (2.)
  • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
  • Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Definitions

• NCl non-collagenous domain of the ⁇ 3 chain of the basement membrane collagen IV [ ⁇ 3 (IV)NCl] depends in part on phosphorylation.
• Goodpasture Antigen Binding Protein GPBP
• GPBP Goodpasture Antigen Binding Protein
• WO 00/50607; WO 02/061430 is a novel non-conventional protein kinase that catalyzes the conformational isomerization of the ⁇ 3 (IV)NCl domain during its supramolecular assembly, resulting in the production and stabilization of multiple ⁇ 3 (IV)NCl conformers in basement membranes.
• Elevated levels of GPBP have been associated with the production of non- tolerized ⁇ 3 (IV)NCl conformers, which conduct the autoimmune response mediating Goodpasture ("GP") disease.
• IV non- tolerized ⁇ 3
• GP antigen autoantibodies against the non-collagenous C-terminal domain (NCl) of the type IV collagen ⁇ 3 chain
• Goodpasture antigen or "GP antigen”
• the identification of GPBP provided methods for identification of compounds for the treatment of autoimmune disorders, cancer, protein misfolding-mediated disorders and aberrant apoptosis, and also provided potential therapeutics for these disorders.
• the identification of novel GPBP isoforms would be advantageous in at least these fields.
• the present invention provides isolated polypeptides of 90% or greater purity consisting of the amino acid sequence of SEQ ID NO: 2 (91 kD GPBP).
• the present invention provides substantially purified recombinant polypeptides comprising the general formula X-SEQ ID NO:2, wherein X is a detectable polypeptide.
• the detectable polypeptide is selected from the group consisting of fluorescent polypeptides and polypeptide members of a binding pair.
• the present invention provides substantially purified nucleic acids encoding the polypeptides of this second aspect of the invention.
• the present invention provides substantially purified nucleic acids encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO:2 (91 kD GPBP).
• the substantially purified nucleic acids consist of the nucleic acid of SEQ ID NO: 1 , or a mRNA product thereof.
• the present invention provides recombinant expression vectors comprising the substantially purified nucleic acid of any aspect of the invention.
• the present invention provides host cells transfected with a recombinant expression vector of the invention.
• the present invention provides a substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO:2 (91 kD GPBP) or SEQ ID NO:4 (77 kD GPBP) , wherein the polypeptide of SEQ ID NO:2 or SEQ ID NO:4 comprises one or more post-translational modifications (PTMs) directly and/or indirectly involving amino acids residues 305-344 GGPDYEEGPNSLINEEEFFDAVEAALDRQDKIEEQSQSEK (SEQ ID NO: 10) (numbering based on position within 77 kD GPBP).
• the one or more PTMs comprise covalent PTMs.
• the one or more PTMs comprise covalent PTMs within amino acids 305-344 (SEQ ID NO: 10). In one preferred embodiment the one or more PTMs directly or indirectly involve residues 320-327 (EEFFDAVE, SEQ ID NO:5). In another preferred embodiment, the one or more PTMs comprise one or more covalent PTMs within residues 320-327 (EEFFDAVE, SEQ ID NO:5). In various preferred embodiments of this aspect, the substantially purified polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:2 (91 kD GPBP) or SEQ ID NO:4 (77 kD GPBP).
• the present invention provides substantially purified polypeptides comprising the amino acid sequence of SEQ ID NO:2 (91 kD GPBP) or SEQ ID NO:4 (77 kD GPBP), wherein the polypeptide of SEQ ID NO:2 or SEQ ID NO:4 comprises one or more PTMs directly and/or indirectly involving residues 371-396 ,
• PYSRSSSMSSIDLVSASDDVHRFSSQ (SEQ ID NO:9) (numbering based on positions within 77 kD GPBP).
• the one or more PTMs comprise covalent PTMs.
• the one or more PTMs comprise covalent PTMs within amino acids 371-396 (SEQ ID NO:9).
• the one or more PTMs directly or indirectly involve residues 388-392 (DDVHR, SEQ ID NO:6).
• the one or more PTMs comprise one or more covalent PTMs within residues 388-392 (SEQ ID NO: 6)
• the polypeptide further comprises one or more PTMs directly and/or indirectly involving amino acids residues 305-344
• GGPDYEEGPNSLINEEEFFDAVEAALDRQDKIEEQSQSEK (SEQ ID NO: 10) (numbering based on position within 77 kD GPBP); preferably the one or more PTMs comprise covalent PTMs, and even more preferably the one or more PTMs comprise covalent PTMs within amino acids 305-344 (SEQ ID NO: 10).
• the one or more PTMs directly or indirectly involve residues 320-327
• the one or more PTMs comprise one or more covalent PTMs within residues 320-327 (EEFFDAVE, SEQ ID NO:5).
• the substantially purified polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:2 (91 kD GPBP) or SEQ ID NO:4 (77 kD GPBP).
• the present invention provides substantially purified monoclonal antibodies that selectively bind to a polypeptide of the sixth or seventh aspect of the invention.
• the present invention provides substantially purified monoclonal antibodies that specifically binds to the polypeptide of SEQ ID NO:2 and not to the polypeptide of SEQ ID NO:4.
• the monoclonal antibody binds to an epitope within the amino acid sequence DGWKGRLPSPLVLLPRSARC (SEQ ID NO:7)
• the present invention provides methods for detecting circulating Goodpasture antigen binding protein (GPBP), comprising
• the present invention provides methods for detecting urinary Goodpasture antigen binding protein (GPBP), comprising
• the present invention provides methods for isolating native 77 kD GPBP, comprising:
• the present invention provides methods for isolating native 91 kD GPBP, comprising:
• the present invention provides methods for isolating native GPBP isoforms, comprising:
• these methods can be used, for example, to substantially purify native 77 kD GPBP and native 91 kD GPBP from plasma and urine, respectively, as disclosed in more detail in the examples that follow.
• the GPBP-binding molecules comprise GPBP antibodies.
• the antibodies comprise the novel monoclonal antibodies of the present invention.
• the eluting step comprises use of a denaturing eluting buffer.
• COL4A3BP encodes for polypeptides of 77-, 91- and 120-kDa.
• FLAG-tagged GPBP or GPBP ⁇ 26/CERT (10-20 ng) were analyzed by Western blot with the indicated antibodies.
• B cell extracts (50 ⁇ g) were analyzed as in A.
• C extracts (10 ⁇ g) from control cells (-) or cells expressing pc-n4'were analyzed as in A.
• D extracts (50 ⁇ g) from untransfected cells (-) or from cells transfected with the indicated siRNA-expressing plasmid were analyzed as in A.
• Glyceraldehyde-3- phosphate dehydrogenase (GAPDH) was used as a loading control and siRNA specificity.
• cell extracts (10 ⁇ g) (ex vivo) or individual transcription/translation mixtures (in vitro) expressing the indicated plasmid construct were analyzed by Western blot using mAb e26 (ex vivo) or by fluorography (in vitro). Lysates from untransfected cells (ex vivo) or mixtures without plasmid (in vitro) were used as Control.
• indicated are the sequence of the N terminal open reading frame (ORF) of GPBP in one-letter code (SEQ ID NO: 15) and the corresponding mRNA nucleotide sequence (SEQ ID NO: 14) in capital letters. The gray and black letters indicate the 5'-UTR and ATR, respectively.
• the 91- and 120-kDa GPBP isoforms are insoluble membrane-bound polypeptides.
• A intact cells were incubated with ⁇ GPBP-Alexa Fluor 647 antibodies ( ⁇ GPBP-AF647) in the presence of GPBPpepl or equimolecular amount of a nonrelevant peptide (Contpep) and Rhodamine 123 for mitochondrial staining of living cells, and analyzed by confocal microscopy. Scale bar, 21 ⁇ M.
• B cells were detached and incubated with blocking solution in the absence (control) or presence of biotinylated ⁇ GPBP antibodies ( ⁇ GPBP). The cell surface-bound antibody was detected with streptavidin-FITC and flow cytometry.
• mAb e26 and mAb 14 were used for GPBP and GPBP ⁇ 26/CERT detection. Since we did not detect expression of 77-kDa GPBP in the cytosol, mAb 14 reactivity in this compartment can be attributed to GPBP ⁇ 26/CERT.
• Figure 4. The 77-kDa GPBP isoform interacts with type IV collagen in cultured cells. In A, HEK 293 or HEK 293-FLAG- ⁇ 3(IV) cells were cross-linked, lysed and ⁇ FLAG extracted.
• FIG. 5 Export of 77-kDa GPBP to the extracellular compartment.
• A HeLa cells were transfected with the indicated plasmid constructs, and the indicated proteins visualized by standard indirect immunofluorescence. DNA was stained with 4'-6'- diamino-2-phenylindole (DAPI) for nuclear visualization. Original magnification x 400.
• B and C extracts (10 ⁇ g) from cells expressing the indicated plasmid constructs (lysates) or FLAG-immunoprecipitates from the corresponding culture media (media IP) were analyzed by Western blot using the indicated antibodies.
• FIG. 6 The 91-kDa GPBP regulates 77-kDa GPBP secretion in cultured cells.
• A extracts (10 ⁇ g) from two independent clones expressing (c8) or not expressing (c 19) recombinant 91 -kDa GPBP were analyzed by Western blot with mAb 14 antibodies, which react poorly with native 91-kDa counterpart (Fig. IB).
• B the same clones were transfected with pc-FLAG-GPBP, and cell extracts (lysates) or FLAG- immunoprecipitates from the corresponding culture media (media IP) were analyzed by Western blot using the indicated antibodies. Similar conclusions were obtained when assaying cl4, an independent HEK 293 clone expressing levels of recombinant 91-kDa GPBP similar to c8 (not shown).
• FIG. 7 GPBP ⁇ 26/CERT but not GPBP is sensitive to sphingomyelinase cell treatment.
• HeLa cells were transfected with the indicated plasmid contracts and treated (+) or not (-) with spingomyelinase, lysed, FLAG-immunoprecipitated, and analyzed by Western blot with ⁇ FLAG antibodies (bSMase). Immunoprecipitates from untreated cells were incubated (+) or not (-) with phosphatase and similarly analyzed ( ⁇ PPase).
• FIG. 8 The levels of circulating 77-kDa GPBP are up-regulated in Goodpasture patients and in animal models of immune complex-mediated glomerulonephritis.
• A material isolated by immunoaffinity chromatography from a Goodpasture patient plasmapheresis was analyzed by Western blot in the presence ( ⁇ GPBP) or absence (Control) of GPBP-specific antibodies, hi B, is the plot representing the standard curve obtained from an ELISA performed as in Material and Methods indicated using recombinant GPBP.
• C and D are scatter plots of intensity of fluorescence (I.F.) in arbitrary units (A.U.) measured by similar ELISA.
• A indicated in one-letter code is the primary structure of the FFAT motif and flanking region in GPBP (residues 316-333) (SEQ ID NO:8) and the homologous region in GPBP ⁇ FFAT (SEQ ID NO:29) where dashes indicate the deleted residues within FFAT motif (boxed).
• B cell extracts (10 ⁇ g) expressing the indicated proteins were analyzed by Western blot using the indicated antibodies.
• Fig. 10 Recombinant GPBP expression induces accumulation of GPBP polypeptides in the cytosol.
• Cells were transfected with the indicated plasmid constructs, collected one day after transfection, subjected to fractionation as indicated in Material and Methods in the Example 1 and analyzed by Western blot as in Fig. 3 C using the indicated antibodies. Arrows and numbers indicate the position and M r in kDa of the different GPBP polypeptides. The 120-kDa polypeptide was mainly found in lysosomal fraction and in a more limited amounts in microsomal fraction, further suggesting that it represents a covalently modified-derived version of the 91 -kDa generated in the secretory pathway.
• Figure 12 Western blot analysis of GPBP isolated from plasma samples using chemical techniques.
• the GPBP partially purified from approximately 1.25 mL of human plasma (see Example 2) was analyzed by Western blot under reducing conditions using HRP-labeled mAb N 27. Arrows and numbers indicated the position and the estimated M r for reactive polypeptides.
• FIG 13 GPBP isolated from urine of a control donor using immunoaffinity chromatography. Two hundred and fifty milliliters of urine from a control donor (previously cleared by centrifugation and neutralized with Tris), were loaded onto a 1 mL column of Sepharose 4B-conjugated with 200 ⁇ g of rabbit polyclonal anti-GPBP antibodies. The column was washed with 30 mL of TBS and the bound material was eluted with Gentle ImmunopureTM Elution Buffer (Pierce). The material eluted was dialyzed against TBS and further analyzed by Western blot using GPBP-specific chicken polyclonal antibodies ( ⁇ GPBPch) and HRP-labelled anti-chicken IgY (secondary antibody).
• ⁇ GPBPch GPBP-specific chicken polyclonal antibodies
• HRP-labelled anti-chicken IgY secondary antibody
• A is represented a scatter plot on a log-log scale of the indicated concentrations of GPBP versus fluorescence intensity (F.I.) expressed in arbitrary units (A.U.).
• B is represented the linear regression line calculated with the indicated concentrations and their respective F.I. values plotted on linear scale, that was used to determine GPBP sample concentration in D.
• C is represented raw data obtained analyzing donor samples with: secondary antibody (Cont), nonspecific chicken IgY and secondary antibody (IgY), or with ⁇ GPBPch and secondary antibody ( ⁇ GPBPch).
• D the table shows corresponding transformed data using the curve obtained in B.
• CM resin was eluted with IM NaCl, 50 mM Tris pH 7.5 (CM, IM NaCl), and DEAE resin was subsequently eluted with 0.35M NaCl, 50 mM Tris pH 7.5 (DEAE, 0.35M NaCl) and IM NaCl, 50 mM Tris pH 7.5 (DEAE, IM NaCl).
• Equivalent amounts of each sample including the supernatant of the DEAE extraction (Spt CM/DEAE) were analyzed by Western blot with GPBP-specific chicken polyclonal antibodies and HRP-labelled anti-chicken IgY ( ⁇ GPBPch).
• Nonspecific reactive polypeptides were identified by staining an in-parallel analysis using only HRP-labelled anti-chicken IgY (Cont). Bars and numbers or arrows and numbers indicate the position and size (kDa) of MW standards (left) or polypeptides specifically reacting with anti-GPBP antibodies and that were detected only in SptCM/DEAE (right), respectively.
• FIG. 16 Western blot analysis of intracellular and extracellular FLAG-GPBP produced in HEK 293 cells using individual N1-N28 monoclonal antibodies. At the upper composite, 10 ⁇ g of total protein extract from HEK 293 cells expressing recombinant FLAG-GPBP were subjected to Western blot analysis using Nl -N28 antibodies (1-28). A major polypeptide of- 77-kDa representing the full length recombinant GPBP polypeptide and variable presence of derived polypeptides of lower IVL (45-77 kDa) were observed.
• 1-28 is N1-N28, and anti-mouse-HRP and chemiluminescence were used for developing purposes.
• Figure 17. Western blot analysis of HEK 293 cell extracts using N1-N27 monoclonal antibodies. Fifty ⁇ g of HEK 293 cell extract were analysed by Western blot using the indicated antibodies. The antibodies recognized four distinct GPBP- related polypeptides: the 77-kDa canonical polypeptide, a 45-kDa fragment, an 88-kDa band, and a 91 -kDa polypeptide also targeted by mAb e26.
• the polypeptide pinpointed by the arrow was recognized by the secondary antibody (anti-mouse IgG HRP-labelled) and therefore does not represents a GPBP product.
• the origin of 88-kDa polypeptide is unknown although its M r suggest that it represents a phosphorylated version of the 77- kDa canonical polypeptide.
• FIG. 18 Cloning of GPBP deletion mutants.
• A on the primary structure of GPBP (SEQ ID NO:4) we indicate the C terminus (bent arrows) of the thirteen 3' terminal FLAG-GPBP cDNA deletion mutants (1-13), obtained by standard PCR and recombinant DNA procedures.
• B is shown the sequence of GPBP encompassing the C-terminal regions of deletion mutants 7 (upper box) and 8 (lower box). In each lane, the number of the last residue is indicated.
• ⁇ l is a FLAG-GPBP deletion mutant lacking residues 285-304 and similarly ⁇ 2- ⁇ 4 mutants lack residues 305-324, 325-344 and 345- 364, respectively (SEQ ID NOS: 30-33).
• a peptide representing the bold sequence (SEQ ID NO: 8) efficiently competed mAb 14 binding to GPBP and a GPBP mutant containing the sequence Ala Ala VaI instead of the underlined residues failed to react with mAb 14.
• native protein means the protein naturally produced by the cell, including any post-translational modifications (PTMs), and includes non-denatured protein, or denatured protein (as, for example, naturally produced protein substantially purified and subjected to one or more denaturing agents to, for example, run on a SDS-PAGE gel).
• PTMs post-translational modifications
• substantially purified polypeptide means that the polypeptide has been separated from its in vivo cellular environments. It is further preferred that the isolated polypeptides are also substantially free of gel agents, such as polyacrylamide, agarose, and chromatography reagents.
• the present invention provides isolated polypeptides of 90% or greater purity consisting of the amino acid sequence of SEQ ID NO: 2 (91 kD GPBP).
• the inventors have determined that the hypothesized sequence of 91 kD GPBP previously proposed in WO 2004/070025 is incorrect, and have now isolated native 91 kD protein and determined its correct amino acid sequence, which is shown in SEQ ID NO:2.
• Figure 19 shows the sequence of 91 kD GPBP, and in bold cursive underlined form, and from N to the C terminus, the amino acid residues comprising the epitopes of Ab 24, mAb 14 and mAb e26 respectively.
• the first residue (Met) of canonical 77-kDa GPBP (SEQ ID NO:4) is highlighted in bold and boxed in the figure.
• 91-kDa and 77-kDa GPBP are identical in amino acid sequence from the highlighted "Met" residue through the end of the protein.
• the inventors have obtained compelling evidence that the mRNA of GPBP undergoes canonical (AUG) and noncanonical (ACG) translation initiation to generate two primary polypeptides of 77- and 91-kDa, respectively.
• the results from this study also support that both products enter the secretory pathway.
• the 77-kDa reaches the extracellular compartment and exists in a soluble immunoprecipitable form
• the 91-kDa remains mainly insoluble, associated with cellular membranes and likely reaches the external side of plasma membrane.
• the present invention provides additional evidence for the 91-kDa GPBP to exist in a soluble form in the plasma and urine revealing that the 91-kDa GPBP can be released from the cellular membranes.
• polypeptides of this aspect of the invention can be used, for example, to produce antibodies against 91-kDa GPBP, and as targets for identification of compounds that interfere with GPBP activity, making them useful therapeutics for various disorders, including Goodpasture Syndrome.
• mRNA alternative translation initiation is a strategy to direct GPBP to multiple locations including secretory pathway, plasma membrane and extracellular compartment.
• the polypeptides can be used, for example, to generate specific antibodies for detection of different isoforms of native GPBP present in serum or in urine, which can thus be used as, for example, diagnostic agents for autoimmune and other disorders.
• the polypeptides of the invention can also be used, for example, as tools to identify candidate compounds for inhibiting various specific types of native GPBP isoforms and also to identify candidate compounds for treating, for example, autoimmunity and protein misfolding-mediated disorders, as discussed in more detail below.
• fraction or greater purity means that contaminating proteins make up no more than 10% of the isolated polypeptide; in various preferred embodiments, no more than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% of the isolated polypeptide (e.g., isolated polypeptides of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or greater purity consisting of the amino acid sequence of SEQ ID NO: 2). It is further preferred that the isolated polypeptides are also substantially free of gel agents, such as polyacrylamide and agarose.
• the isolated polypeptides are present in solution, frozen, or as a dried powder.
• the isolated polypeptides of this first aspect are optionally labeled with a detectable, non-polypeptide label, including but not limited to fluorescent labels or radioactive labels.
• the present invention provides substantially purified recombinant polypeptides comprising or consisting of the general formula X-SEQ ID NO:2, wherein X is a detectable polypeptide, hi this aspect, the correct amino acid sequence for 91 kD GPBP (SEQ ID NO: 2) is expressed as a fusion protein with a detectable polypeptide.
• the polypeptides of this aspect of the invention can be used, for example, to track 91 kD GPBP in cells, and as a detectable target for identification of compounds that interfere with GPBP activity, making them useful therapeutics for various disorders, including Goodpasture Syndrome.
• a "recombinant polypeptide” means that the detectable polypeptide is not derived from GPBP or expressed from a GPBP mRNA, and thus fuses a heterologous detectable peptide with the correct 91 kD GPBP polypeptide.
• a “detectable polypeptide” is any heterologous peptide that can be detected, thus permitting detection of the recombinant polypeptide.
• the detectable polypeptide comprises a fluorescent protein. Any fluorescent protein known in the art can be used in the invention. For example, green fluorescent proteins of cnidarians, which act as their energy-transfer acceptors in bioluminescence, are suitable fluorescent proteins for use in the fluorescent indicators.
• a green fluorescent protein (“GFP”) is a protein that emits green light
• a blue fluorescent protein (“BFP”) is a protein that emits blue light.
• GFPs have been isolated from the Pacific Northwest jellyfish, Aequorea victoria, the sea pansy, Renilla reniformis, and Phialidium gregarium. See, Ward, W. W., et al., Photochem. Photobiol., 35:803 808 (1982); and Levine, L. D., et al., Comp. Biochem. Physiol., 72B:77 85 (1982).
• a variety of Aequorea-related GFPs having useful excitation and emission spectra have been engineered by modifying the amino acid sequence of a naturally occurring GFP from Aequorea victoria. See, Prasher, D.
• GFP mutants with shifted wavelengths of excitation or emission. See, Heim, R. & Tsien, R. Y. Current Biol. 6:178 182 (1996).
• Suitable pairs for example a blue-shifted GFP mutant P4-3 (Y66H/Y145F) and an improved green mutant S65T can respectively serve as a donor and an acceptor for fluorescence resonance energy transfer (FRET).
• FRET fluorescence resonance energy transfer
• the detectable polypeptide comprises a non-GPBP epitope for which antibodies are commercially available, including but not limited to the FLAG (Sigma Chemical, St. Louis, MO), myc (9E10) (Invitrogen, Carlsbad, CA), 6-His (Invitrogen; Novagen, Madison, WI), glutathione S-transferase (GST) (Santa Cruz Biotechnology, Santa Cruz, California), and HA (hemaglutunin) (Boehringer Manheim Biochemicals).
• the isolated polypeptide may preferably further comprise a linker sequence between the detectable polypeptide and the polypeptide of SEQ ID NO:2.
• the linker is not a portion of GPBP or encoded by a GPBP mRNA.
• a linker can be of any desirable length, and preferably is between 1 and 20 amino acids, if present; more preferably between 1 and 15, 1-10, 1-5, 1-4, 1-3, or 1-2 amino acids, if present.
• the linker can be used, for example, to optimally position the detectable polypeptide and the 91 kD GPBP sequence and to include specific sequence for protease recognition site to allow removal of detectable polypeptide.
• the isolated polypeptide may further comprise any additional residues necessary for expression, such as an N-terminal methionine residue or peptide sequences to deliver the polypeptide to different cellular and extracellular compartments.
• the substantially purified polypeptides of the invention can be made by any method known to those of skill in the art, but are preferably made by recombinant means based on the teachings provided herein.
• a coding region of interest as disclosed herein can be cloned into a recombinant expression vector, which can then be used to transfect a host cell for recombinant protein production by the host cells.
• the present invention provides substantially purified nucleic acids encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO:2 (91 kD GPBP).
• the substantially purified nucleic acid sequence may comprise RNA or DNA.
• substantially purified nucleic acids are those that have been removed from their normal surrounding nucleic acid sequences in the genome or in cDNA sequences. Such substantially purified nucleic acid sequences may comprise additional sequences useful for promoting expression and/or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals.
• the substantially purified nucleic acid coding region consists of the nucleic acid of SEQ ID NO: 1 , or a mRNA product thereof, hi another preferred embodiment, the present invention provides substantially purified nucleic acids encoding the polypeptide of any embodiment of the substantially purified recombinant polypeptides comprising or consisting of the general formula X-SEQ ID NO:2, as discussed in the second aspect of the invention. In a fourth aspect, the present invention provides recombinant expression vectors comprising the substantially purified nucleic acid of any aspect of the invention operatively linked to a promoter.
• Recombinant expression vector includes vectors that operatively link a nucleic acid coding region or gene to any promoter capable of effecting expression of the gene product.
• the promoter sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive).
• the construction of expression vectors for use in transfecting prokaryotic cells is also well known in the art, and thus can be accomplished via standard techniques.
• the expression vector must be replicable in the host organisms either as an episome or by integration into host chromosomal DNA.
• the expression vector comprises a plasmid.
• the invention is intended to include other expression vectors that serve equivalent functions, such as viral vectors.
• the present invention provides host cells that have been transfected with the recombinant expression vectors disclosed herein, wherein the host cells can be either prokaryotic or eukaryotic.
• the cells can be transiently or stably transfected.
• transfection of expression vectors into prokaryotic and eukaryotic cells can be accomplished via any technique known in the art, including but not limited to standard bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection.
• the present invention provides a substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO:2 (91 kD GPBP) or SEQ ID NO:4 (77 kD GPBP) , wherein the polypeptide of SEQ ID NO:2 or SEQ ID NO:4 comprises one or more post-translational modifications (PTMs) directly and/or indirectly involving amino acids residues 305-344 GGPDYEEGPNSLINEEEFFDAVEAALDRQDKIEEQSQSEK (SEQ ID NO: 10) (numbering based on position within 77 kD GPBP).
• PTMs post-translational modifications
• the inventors provide the first purification of native 77 and 91 kD GPBP and have determined that existing monoclonal antibodies that bind to recombinant versions of 77 kD- and 91 kD-GPBP do not bind to purified native versions, verifying that structural differences exist between recombinant and native forms of the 77 kD GPBP and between recombinant and native forms of the 91 kD GPBP.
• the polypeptides of this aspect of the invention can be used, for example, to produce antibodies against native GPBP forms, and as targets for identification of compounds that interfere with native GPBP activity, making them useful therapeutics for various disorders, including
• the one or more PTMs comprise covalent PTMs.
• the one or more PTMs comprise covalent PTMs within amino acids 305-344 (SEQ ID NO: 10).
• the one or more PTMs directly or indirectly involve residues 320-327 (EEFFDAVE, SEQ ID NO:5).
• the one or more PTMs comprise covalent PTMs within residues 320-327 (EEFFDAVE, SEQ ID NO:5) (numbering based on position within 77 kD GPBP).
• the one or more PTMs comprise one more PTMs present in residue 320, 321, and/or 327; most preferably, the one or more PTMs present at these residues comprise covalent PTMs.
• the substantially purified polypeptide possesses an amino acid sequence consisting of SEQ ID NO:2 (91 kD GPBP) or SEQ ID NO:4 (77 kD GPBP).
• post-translational modification means a modification in the structure of a protein after its translation.
• the PTM comprises addition of a functional group, including but not limited to carboxylation, methylation, citrullination, phosphorylation, glycosylation, and formation of atypical isoaspartyl.
• the PTM comprises an isomerization, leading to a conformational change.
• Any suitable method for making the covalently modified polypeptide of SEQ ID NO: 2 or SEQ ID NO:4 based on the teachings of the present disclosure can be used, including isolating from natural sources of GPBP as disclosed herein, and recombinant production of GPBP followed by suitable covalent modification within the relevant region of amino acid residues, using standard methods known to those of skill in the art.
• the present invention provides substantially purified polypeptides comprising the amino acid sequence of SEQ ID NO:2 (91 kD GPBP) or SEQ ID NO:4 (77 kD GPBP), wherein the polypeptide of SEQ ID NO:2 or SEQ ID NO:4 comprises one or more PTMs directly and/or indirectly involving residues 371-396 PYSRSSSMSSIDLVSASDDVHRFSSQ (SEQ ID NO:9) (numbering based on positions within 77 kD GPBP).
• the inventors provide the first purification of native 77 kD and 91 kD GPBP and have determined that existing monoclonal antibodies that bind to recombinant version of 77 kD and 91 kD GPBP do not bind to the purified native 77 kD and 91 kD GPBP versions, verifying that structural differences exist between recombinant and native forms of the 77 and 91 kD GPBP.
• the polypeptides of this aspect of the invention can be used, for example, to produce antibodies against native GPBP, and as targets for identification of compounds that interfere with native GPBP activity, making them useful therapeutics for various disorders, including Goodpasture Syndrome.
• the one or more PTMs comprise covalent PTMs.
• the one or more PTMs comprise covalent PTMs within amino acids 371-396 (SEQ ID NO:9).
• the one or more PTMs directly or indirectly involve residues 388- 392 (DDVHR, SEQ ID NO:6).
• the one or more PTMs comprise one or more covalent PTMs within residues 388-392 (SEQ ID NO: 6)
• the polypeptide further comprises one or more PTMs directly or indirectly involving residues 320-327 (EEFFDAVE, SEQ ID NO:5)
• the one or more PTMs within residues 320-327 are covalent PTMs.
• the substantially purified polypeptide possesses an amino acid sequence consisting of SEQ ID NO:2 (91 kD GPBP) or SEQ ID NO:4 (77 kD GPBP). Any suitable method for making the covalently modified polypeptide of SEQ ID NO:2 or SEQ ID NO:4 can be used, including isolating from natural sources of GPBP as disclosed herein, and recombinant production of GPBP followed by suitable covalent modification within the relevant region of amino acid residues, using standard methods known to those of skill in the art.
• the present invention provides substantially purified monoclonal antibodies that selectively bind to the substantially purified polypeptides of the sixth or seventh aspect of the invention.
• the inventors have for the first time isolated native 77- and 91 kD GPBP species that when substantially purified do not bind to existing GPBP-specific monoclonal antibodies.
• existing monoclonal antibodies do not detect GPBP in plasma or urine samples in ELISAs, nor are they capable of use for purification of plasma or urine GPBP.
• the monoclonal antibodies of the invention are useful, for example, in ELISA-based assays for GPBP detection in urine or plasma, and for purification of GPBP from plasma or serum.
• the inventors further demonstrate herein that these native 77 kD GPBP and native 91 kD GPBP species are pos-translationally modified, and that at least some of these PTMs render substantially purified, native GPBP non-reactive to existing monoclonal GPBP antibodies.
• Exemplary monoclonal antibodies according to this aspect of the invention are provided in the examples that follow.
• the "monoclonal antibodies” of the invention can be any type of monoclonal antibody, including but not limited to standard monoclonal antibodies, humanized monoclonals, chimeric monoclonals, and fragments thereof.
• substantially purified means that the recited monoclonal antibodies make up at least 80% of the antibodies in a substantially purified sample; more preferable at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.
• selective bind means preferential binding of the GPBP monoclonal antibody to native GPBP epitope, as opposed to one or more other biological molecules, structures, cells, tissues, etc., as is well understood by those of skill in the art.
• Monoclonal antibodies can be produced by obtaining spleen cells from the animal [See Kohler and Milstein, Nature 256, 495-497 (1975)].
• monoclonal antibodies (mAb) of interest are prepared by immunizing inbred mice with native 77 kD GPBP, native 91 kD GBPB, or an antigenic fragment thereof, including, but not limited to, one or more epitopes comprising or consisting of the PTM-containing peptides EEFFDAVE (SEQ ID NO.5), DDVHR (SEQ ID NO:6), LINEEEFFDAVEAALDRQ (SEQ ID NO:8), PYSRSSSMSSIDLVSASDDVHRFSSQ (SEQ ID NO:9), and GGPDYEEGPNSLINEEEFFDAVEAALDRQDKIEEQSQSEK (SEQ ID NO: 10).
• the monoclonal antibodies bind one or more epitopes comprising one or more PTMs, selected from the group consisting of PTM-containing EEFFDAVE (SEQ ID NO:5), DDVHR (SEQ ID NO:6), LINEEEFFDAVEAALDRQ (SEQ ID NO: 8), PYSRSSSMSSIDLVSASDDVHRFSSQ (SEQ ID NO:9), and GGPDYEEGPNSLINEEEFFDAVEAALDRQDKIEEQSQSEK (SEQ ID NO: 10).
• the one or more PTMs are covalent PTMs.
• the monoclonal antibodies bind to an epitope that comprises one or more PTMs (preferably covalent PTMs) present in residue 320, 321 , and/or 327 (numbering based on 77 kD GPBP).
• PTMs preferably covalent PTMs
• the mice are immunized by the IP or SC route in an amount and at intervals sufficient to elicit an immune response.
• the mice receive an initial immunization on day 0 and are rested for about 3 to about 30 weeks.
• Immunized mice are given one or more booster immunizations of by the intravenous (IV) route.
• Lymphocytes from antibody positive mice are obtained by removing spleens from immunized mice by standard procedures known in the art.
• Hybridoma cells are produced by mixing the splenic lymphocytes with an appropriate fusion partner under conditions which will allow the formation of stable hybridomas.
• the antibody producing cells and fusion partner cells are fused in polyethylene glycol at concentrations from about 30% to about 50%.
• Fused hybridoma cells are selected by growth in hypoxanthine, thymidine and aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) by procedures known in the art. Supernatant fluids are collected from growth positive wells and are screened for antibody production by an immunoassay such as solid phase immunoradioassay. Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson, Soft Agar Techniques, in Tissue Culture Methods and Applications, Kruse and Paterson, Eds., Academic Press, 1973.
• DMEM Dulbecco's Modified Eagles Medium
• Humanized monoclonal antibodies refers to monoclonal antibodies derived from a non-human monoclonal antibody, such as a mouse monoclonal antibody.
• humanized monoclonal antibodies can be derived from chimeric antibodies that retains, or substantially retains, the antigen-binding properties of the parental, non-human, monoclonal antibodies but which exhibits diminished immunogenicity as compared to the parental monoclonal antibody when administered to humans.
• chimeric monoclonal antibodies can comprise human and murine antibody fragments, generally human constant and mouse variable regions.
• Humanized monoclonal antibodies can be prepared using a variety of methods known in the art, including but not limited to (1) grafting complementarity determining regions from a non-human monoclonal antibody onto a human framework and constant region ("humanizing"), and (2) transplanting the non-human monoclonal antibody variable domains, but "cloaking" them with a human-like surface by replacement of surface residues ("veneering").
• humanizing grafting complementarity determining regions from a non-human monoclonal antibody onto a human framework and constant region
• transplanting the non-human monoclonal antibody variable domains but "cloaking" them with a human-like surface by replacement of surface residues ("veneering”).
• These methods are disclosed, for example, in, e.g., Jones et al., Nature 321 :522-525 (1986); Morrison et al., Proc. Natl. Acad. Sci., U.S.A., 81 :6851- 6855
• Monoclonal antibodies can be fragmented using conventional techniques, and the fragments screened for utility in the same manner as for whole antibodies.
• F(ab') 2 fragments can be generated by treating antibody with pepsin.
• the resulting F(ab') 2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments.
• Fab fragments can be obtained by treating an IgG antibody with papain;
• F(ab') fragments can be obtained with pepsin digestion of IgG antibody.
• a F(ab') fragment also can be produced by binding Fab' described below via a thioether bond or a disulfide bond.
• a Fab' fragment is an antibody fragment obtained by cutting a disulfide bond of the hinge region of the F(ab')2.
• a Fab' fragment can be obtained by treating a F(ab')2 fragment with a reducing agent, such as dithiothreitol.
• Antibody fragment peptides can also be generated by expression of nucleic acids encoding such peptides in recombinant cells (see, e.g., Evans et al., J. Immunol. Meth. 184: 123-38 (1995)).
• a chimeric gene encoding a portion of a F(ab')2 fragment can include DNA sequences encoding the CHl domain and hinge region of the H chain, followed by a translational stop codon to yield such a truncated antibody fragment molecule.
• monoclonal antibody fragments include (i) a Fab fragment, a monovalent fragment consisting essentially of the VL, VH, CL and CH I domains; (ii) F(ab)2 and F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the VH and CHl domains; (iv) a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists essentially of a VH domain; and (vi) one or more isolated CDRs or a functional paratope.
• a Fab fragment a monovalent fragment consisting essentially of the VL, VH, CL and CH I domains
• F(ab)2 and F(ab')2 fragments bivalent fragment
• the immunogens are typically formulated with a pharmaceutically acceptable carrier for parenteral administration.
• acceptable adjuvants include, but are not limited to, Freund's complete, Freund's incomplete, alum-precipitate, water in oil emulsion containing Corynebacterium parvum and tRNA.
• the formulation of such compositions, including the concentration of the polypeptide and the selection of the vehicle and other components, is within the skill of the art.
• the present invention provides substantially purified monoclonal antibodies that specifically binds to the polypeptide of SEQ ID NO:2 and not to the polypeptide of SEQ ID NO:4.
• Such monoclonal antibodies of the invention are useful, for example, in distinguishing 91 kD GPBP from 77 kD GPBP in assays including, but not limited to, ELIS A-based assays for GPBP detection in urine or plasma.
• Such monoclonal antibodies can be generated using methods disclosed above and the use of peptide immunogens present in the polypeptide of SEQ ID NO:2 but not present in SEQ ID NO:4. Such immunogens may be of any suitable length to generate an antibody response.
• the monoclonal antibodies are generate against an immunogen comprising or consisting of DGWKGRLPSPLVLLPRSARC (SEQ ID NO:7).
• DGWKGRLPSPLVLLPRSARC SEQ ID NO:7
• the monoclonal antibody binds to an epitope within the amino acid sequence DGWKGRLPSPLVLLPRSARC (SEQ ID NO:7).
• An exemplary such antibody, Ab24 is disclosed below.
• the present invention provides isolated hybridoma cells expressing the monoclonal antibodies of the eighth or ninth aspects of the invention.
• the invention also provides methods for making the antibodies of the invention, as disclosed above and below.
• the present invention provides methods for detecting circulating Goodpasture antigen-binding protein (GPBP), comprising
• GPBP-binding protein GPBP-binding protein
• a "plasma sample” means blood plasma, the liquid component of blood, and is prepared, for example, by centrifugation of whole blood to remove blood cells.
• a plasma sample also includes a blood serum sample, in which blood clotting factors have been removed.
• the present invention provides methods for detecting urinary Goodpasture antigen-binding protein (GPBP), comprising
• Urine samples are easily obtained, and analyte determination in urine is well known in the art.
• a "GPBP-binding molecule” is a peptide or nucleic acid molecule that binds selectively to GPBP, as opposed to one or more other biological molecules, structures, cells, tissues, etc. Exemplary embodiments of such GPBP-binding molecules include but are not limited to antibodies, aptamers or substrates.
• a "GPBP substrate” is a target of GPBP biological activity that binds to GPBP, or a fragment thereof that retains GPBP-binding activity.
• Such GPBP substrates include, but are not limited to, 1-20 (SEQ ID NO: 16), GPBP-interacting proteins (GIPs) (SEQ ID NOS: 17- 21), myelin basic protein (MBP) and derivatives thereof (SEQ ID NOS:22-25), prion protein (PrP) (SEQ ID NO:26), type IV collagen ⁇ 3 chain NCl domain ( ⁇ 3(IV)NCl) (SEQ ID NO:27), and Alzheimer's disease beta peptide (A ⁇ M2 ) (SEQ ID NO:28).
• GIPs GPBP-interacting proteins
• MBP myelin basic protein
• PrP prion protein
• SEQ ID NO:27 type IV collagen ⁇ 3 chain NCl domain
• a ⁇ M2 Alzheimer's disease beta peptide
• GPBP refers to all GPBP isoforms reactive with GPBP-selective antibodies, including but not limited to 77 kD GPBP and 91 kD GPBP, as well lower and higher molecular weight GPBP isoforms of 160-, 60-, 58-, 56- 53- 50- 46- 35 and 34-kD, and aggregates thereof.
• the "plasma sample” or “urine sample” may be obtained from any suitable subject, preferably from a mammal, including but not limited to a human, dog, cat, horse, or livestock (cow, sheep, etc.).
• the plasma sample or urine sample is obtained from a human subject, such as a human subject suspected of having an autoimmune condition including but not limited to Goodpasture Syndrome and/or immune-complex mediated glomerulonephritis.
• the inventors have isolated native circulating 77 kD GPBP from human plasma and have observed increased levels in Goodpasture patients and in animal models for immune complex-mediated glomerulonephritis, demonstrating that GPBP secretion occurs in vivo and revealing the clinical utility of serological and urinary determination of GPBP.
• the antibody can be any selective GPBP antibody, whether polyclonal, monoclonal, or humanized monoclonal as described above, although monoclonal antibodies are preferred.
• antibodies according to the eighth or ninth aspects of the invention are used.
• the methods of the tenth and eleventh aspect of the invention may comprise analyzing a specific GPBP isoform, such as 77 kD GPBP or 91 kD GPBP; in these embodiments, antibodies selective for 77 kD GPBP or selective for 91 kD GPBP can be used, including but not limited to those selective antibodies disclosed herein.
• the antibodies for use in the methods of the tenth and eleventh aspects of the invention are those that bind to native GPBP isoforms, such as those disclosed herein.
• GPBP-binding molecules such as antibodies, aptamers or substrates
• Conditions suitable to promote binding of GPBP-binding molecules, such as antibodies, aptamers or substrates, to GPBP in the plasma or urine samples can be determined by those of skill in the art based on the teachings herein and the examples provided below.
• antibody-antigen binding often depends on hydrophobic interactions (the so called hydrophobic bonds); thus, high salt concentrations, such as in the molar range can be used to reduce nonspecific binding and increase specific antigen- antibody binding.
• further steps may be included to promote selectivity and specificity, including but not limited to one or more wash steps to remove unbound or weakly bound serum proteins; inhibitors of non-specific binding to reduce binding of high concentration serum proteins, control samples known to contain GPBP isoforms and/or negative controls known not to bind to GPBP isoforms, and/or inclusion of serum or urine samples known to not possess GPBP (ex: deleted for GPBP).
• tenth and eleventh aspects of the present invention may be used to test for the presence of GPBP in the plasma or urine sample by standard techniques including, but not limited to ELISA, immunoflourescence, and chromatography (for example, lateral flow assays where the antibody is immobilized on a surface and plasma or urinary proteins are labeled and allowed to flow over the surface under conditions suitable to permit binding of the antibody to GPBP in the plasma or urine).
• functional beads Becton Dickinson technology coupled to flow cytometry are used; this technique is an emerging method to measure the levels of proteins in biological fluid or cell/tissue extracts.
• beads made of a fluorescence matrix are coated with one or more specific GPBP antibodies, mixed with the plasma sample and further incubated with a detecting antibody labeled with a phycoerythrins. Finally, beads are analyzed by a flow cytometry program which selects the beads according matrix fluorescence emission and measurement of the level of the analyte through phycoerythrin emission. There are up to thirty different types of beads that can be simultaneously detected and discriminated by the cytometer. This method couples high sensitivity and performance with versatility since a specific bead type coated with GPBP antibody can be mixed with a distinct bead type coated with binding peptides for other analyte (i.e. autoantibodies) and simultaneously measured.
• analyte i.e. autoantibodies
• the techniques may determine only the presence or absence of the GPBP isoform(s).
• the techniques may be quantitative, and provide information about the relative amount of the protein or peptide of interest in the sample. For quantitative purposes, ELISAs are preferred.
• Detection of immunocomplex formation can be accomplished by standard detection techniques. For example, detection of immunocomplexes can be accomplished by using labeled antibodies or secondary antibodies. Such methods, including the choice of label are known to those ordinarily skilled in the art. (Harlow and Lane, Supra).
• the antibodies can be coupled to a detectable substance.
• the term "coupled” is used to mean that the detectable substance is physically linked to the antibody.
• Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase.
• Suitable prosthetic- group complexes include streptavidin/biotin and avidin/biotin.
• suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin.
• An example of a luminescent material includes luminol.
• suitable radioactive material include 125 I, 131 1, 35 S or 3 H.
• the methods of this aspect of the invention can be used, for example, to detect GPBP-mediated disorder in a subject, including but not limited to an antibody-mediated disorder (including but not limited to a glomerulonephritis selected from the group consisting of IgA nephropathy, systemic lupus erythematosus and Goodpasture disease), inflammation, an ER-stress mediated disorder, and drug-resistant cancer.
• the methods would comprise comparison of GPBP levels detected in a test serum or urine sample with a control, such as a control from a serum or urine sample known to have "normal" levels of GPBP or previously determined normal values for GPBP in sera or urine from the subject from whom the serum is obtained.
• the control provides a standard curve using recombinant GPBP or a reference value.
• an increase preferably a statistically significant increase using standard statistical analysis techniques
• GPBP in the serum or urine sample relative to the control indicates the presence of one or more of the disorders noted above, or an increased risk of developing one or more of the disorders, all of which are correlated with increased GPBP expression.
• GPBP expression induces IgA nephropathy, immune complex-related glomerulonephritis; that increased GPBP expression is intimately involved in Goodpasture Syndrome pathogenesis; and that increased GPBP expression mediates resistance of cancer cells to chemotherapeutic agents that induce protein misfolding and ER stress-mediated cell death.
• the methods of the present invention thus provide methods for diagnosing these disorders by serological or urine testing for the presence of GPBP.
• the methods identify individuals either having or at risk of being stricken with one or more of an antibody- mediated disorder (including but not limited to a glomerulonephritis selected from the group consisting of IgA nephropathy, systemic lupus erythematosus and Goodpasture disease), inflammation, an ER-stress mediated disorder, and drug-resistant cancer.
• an antibody- mediated disorder including but not limited to a glomerulonephritis selected from the group consisting of IgA nephropathy, systemic lupus erythematosus and Goodpasture disease
• inflammation an ER-stress mediated disorder
• drug-resistant cancer drug-resistant cancer.
• the methods can be used to test cancer patients either prior to or after initiation of a chemotherapy regimen; those patients that test positive for increased serum levels of GPBP are at increased risk of having a drug-resistant tumor or of their tumor is developing drug-resistance, and an attending physician can assess appropriate treatment options in light thereof.
• Such patients may undergo periodic testing for serum or urine levels of GPBP to monitor potential risk of developing a drug-resistant tumor.
• patients thought to be at risk for developing, or suspected of already having developed a glomerulonephritis selected from the group consisting of IgA nephropathy, systemic lupus erythematosus and Goodpasture disease can be tested for serum or urine levels of GPBP.
• a glomerulonephritis selected from the group consisting of IgA nephropathy, systemic lupus erythematosus and Goodpasture disease
• GPBP is a circulating molecule and GBM (glomerular basement membrane) a principal component of the glomerular filtration barrier; therefore, GPBP accumulation in the glomerulus could result from local production but also from the sequestration of circulating GPBP produced elsewhere, and could also be reflected in increased GPBP in the urine.
• the local overproduction could account for primary antibody-mediated glomerulonephritis whereas increased circulating levels may induce secondary forms of this pathology and perhaps are responsible for disease recurrence upon renal transplantation. Consequently, in another embodiment, quantification of the levels of circulating or urinary GPBP is useful in discriminating primary from secondary antibody-mediated glomerulonephritis and for the clinical monitoring of renal transplantation.
• combining GPBP determination with analysis of other analytes the methods permit one to perform differential diagnosis or prognosis in the above disorders.
• IgA nephropathy patients produce anti-basement membrane autoantibodies. These circulating autoantibodies recognize the NCl domain of type IV collagen. Determination of the titer of these antibodies could help to monitor disease progression or also to distinguish different IgA nephropathy patients or to perform prognosis in these patients.
• any determination used to diagnosis of primary diseases listed in Donadio and Grande (2002) N Engl J Med 347, 738-748 associated with glomerular deposition of IgA can be used in conjunction with the methods of the invention for plasma or urinary detection of GPBP for differential diagnosis in secondary IgA nephropathy patients.
• a normal value of GPBP as a reference for an standard curve is between ⁇ 1 ng/ml-10 ng/ml in plasma and approximately 0.2 ng/ml to 1.5 ng/ml in urine, while Goodpasture patients exceed the normal by at least 2-fold; in other embodiments, by at least 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or more the normal values.
• the present invention provides methods for isolating native GPBP isoforms, comprising:
• these methods can be used, for example, to substantially purify native 77 kD GPBP from plasma, as disclosed in more detail in the examples that follow.
• the present invention provides methods for isolating native GPBP isoforms, comprising: (a) subjecting a urine sample to salt precipitation;
• double ion-exchange chromatography means carrying out two successive and distinct ion-exchange chromatography steps prior to step (c).
• IEC techniques are well known in the art, and include those disclosed in the examples that follow.
• these methods can be used, for example, to substantially purify native 91 kD GPBP from urine, as disclosed in more detail in the examples that follow.
• the present invention provides methods for isolating native GPBP isoforms, comprising:
• these methods can be used, for example, to substantially purify native 77 kD GPBP and native 91 kD GPBP from plasma and urine, as disclosed in more detail in the examples that follow.
• the GPBP-binding molecule comprise GPBP antibodies.
• the antibodies comprise the novel monoclonal antibodies of the present invention.
• the eluting step comprises use of a denaturing eluting buffer.
• GPBP Goodpasture-antigen binding protein
• GPBP is a nonconventional Ser/Thr kinase for the type IV collagen of basement membrane. More recently, we have shown that GPBP is an extracellular protein that when overexpressed induces type IV collagen disorganization and deposit of immune complexes in glomerular basement membrane (Ref. 4). Here we show that cells expressed at least two GPBP isoforms resulting from canonical (77-kDa) and noncanonical (91-kDa) mRNA translation initiation. The 77- kDa polypeptide interacted with type IV collagen and localized as a soluble form in the extracellular compartment.
• the FFAT motif and the 26-residue Ser-rich region were required for the exportation of the 77-kDa polypeptide.
• removal of the 26-residue Ser-rich region yielded the previously recognized GPBP isoform (GPBP ⁇ 26/CERT) that was cytosolic and in contrast to GPBP, sensitive to sphingomyelinase cell treatment.
• type IV collagen which includes: 1) phosphorylation and regulation of protein molecular/supramolecular organization (GPBP); and 2) inter- organelle ceramide trafficking and regulation of protein cargo transport to the plasma membrane (GPBP ⁇ 26/CERT).
• GPBP protein molecular/supramolecular organization
• GPBP ⁇ 26/CERT inter- organelle ceramide trafficking and regulation of protein cargo transport to the plasma membrane
• GBM glomerular basement membrane
• COL4A3BP also encodes for GPBP ⁇ 26, a more-abundant less-active alternatively spliced GPBP variant lacking a 26-residue Ser-rich region, which is apparently not regulated under these pathological conditions (3).
• GPBP contains multiple structural elements including N terminal pleckstrin homology (PH) domain, Ser-Xaa-Yaa region, bipartite nuclear localization signal, coiled-coil domain, two phenylalalines in an acidic track (FFAT) motif and C terminal steroidogenic acute regulatory related lipid transfer (START) domain. Additional structural features include motifs for self-interaction and phosphorylation (1, 3, 5, 6).
• the PH domains comprise a variety of poorly conserved structures present only in eukaryotes which have been proposed to mediate protein targeting to cellular membranes through interaction with phosphoinositides (7).
• a variety of proteins including several protein kinases contain PH domains (8).
• the FFAT motifs target proteins to the ER through interaction with the transmembrane cytosolic domain of the vesicle associated membrane protein-associated proteins (VAPs) (9), which have been proposed to play a role in maintaining homeostasis for protein folding in the endoplasmic reticulum (ER) and in regulating protein cargo transport to the plasma membrane (10, 11).
• VAPs vesicle associated membrane protein-associated proteins
• the START domains bind lipids including ceramide, phospholipids and sterols, and are modules present in a variety of proteins with distinct physiological and pathological functions (12, 13).
• mice and human blood samples were obtained according to institutional guidelines for human studies and animal experimentation. We used sera from New Zealand white (NZW) mice that were previously characterized (4) and which represent healthy young (4-month) and old undergoing IgA immune complex-mediated (7 -month). Human plasmapheresis and sera from control or Goodpasture patients were obtained following standard procedures.
• NZW New Zealand white mice
• Antibodies and recombinant proteins Using truncated recombinant GPBP isoforms and synthetic peptides, we have mapped the epitope of GPBP/GPBP ⁇ 26- specific mouse monoclonal antibody 14 (mAb 14) (1) to the FFAT motif (Fig. 9). Mouse mAb e26 was raised against the 26-residues characteristic of GPBP (GPBPpepl) and therefore, was not reactive with GPBP ⁇ 26/CERT (Fig. IA). Human monoclonal F(ab) 2 fragments were isolated from a recombinant F(ab) 2 expression library using a synthetic peptide representing the alternatively translated region (ATR) of GPBP (Fig.
• ATR alternatively translated region
• Polyclonal antibodies specific for GPBP and GPBP ⁇ 26/CERT were produced either in rabbits immunized with GST-FL AG-GPBP (1) following standard procedures ( ⁇ GPBPr) or in chickens immunized with a specific synthetic peptide and purchased from Abeam ( ⁇ GPBPab). Specific antibodies in ⁇ GPBPr were affinity-purified using recombinant FLAG-GPBP (see below) bound to Sepharose-CNBr (Sigma). For glyceraldehyde-3 -phosphate dehydrogenase detection, we used a mouse monoclonal antibody provided by Erwin Knecht.
• Polyclonal antibodies specific for calregulin, p65 or cathepsin D were from Santa Cruz Biotechnology Inc and those specific for pyruvate dehydrogenase (PDH) were from Molecular Probes.
• Monoclonal antibodies specific for PrP (clone 3F4) or for golgin-97 were from Clontech and Molecular Probes, respectively.
• HRP HRP
• ⁇ FLAG chicken antibodies
• ⁇ FLAG-FITC goat antibodies
• Alexa Fluor® 488- streptavidin was from Molecular Probes and secondary antibodies were from Promega (anti-mouse and anti-rabbit HRP conjugates), Jackson Immunoresearch (anti-human F(ab) 2 -HRP) and Sigma (anti-chicken HRP and other FITC and TRITC conjugates).
• Recombinant FLAG-GPBP and FLAG-GPBP ⁇ 26 were expressed in Pichia pastoris and affinity-purified as previously described (1, 3).
• Plasmid constructs The production of pc-n4', a pcDNA3 (Invitrogen)-derived construct expressing a cDNA which contained the 5' untranslated region (UTR) and coding sequence of GPBP mRNA has been reported (1). Plasmids derived from pc-n4' included pc-GPBP-Met, a deletion mutant devoid of 5'UTR, and pc-n4'-Mmut, a construct where the canonical AUG (Met) translation initiation was substituted with GGA (GIy).
• pc-FL AG-GPBP which expresses the FLAG sequence fused to the coding region of GPBP
• the pc-FLAG- GPBP ⁇ 26 expresses the FLAG sequence fused to the coding region of GPBP ⁇ 26 and has been produced similarly to pc-FL AG-GPBP.
• pc-n4'and pc-n4'-Mmut mutants by introducing stop codons at various positions in the open reading frame (ORF) upstream of iMet position.
• ORF open reading frame
• the pSilencerTM 2.1-U6 hygro was employed for transient expression of small interfering mRNAs (siRNAs) specific for GPBP or for
• GPBP/GPBP ⁇ 26 The corresponding derived constructs and cDNA target sequences were: pSi-GPBP/GPBP ⁇ 26-2, ACAGAGTATGGCTGCAGAG (SEQ ID NO: 11); pSi- GPBP/GPBP ⁇ 26-3, GTACTTTGATGCCTGTGCT (SEQ ID NO: 12); pSi-GPBP-1, GCCCTATAGTCGCTCTTCC (SEQ ID NO: 13). Selection of the target sequence and plasmid construction were based on manufacturer's recommendations. The efficiency of siRNA expressing-plasmids was assessed in a cell recombinant expression system (not shown).
• control plasmid in these studies was designed for targeting the mRNA of green fluorescence protein, a protein not expressed in human cells. All mutants were produced by standard PCR-based mutagenesis and the fidelity of all the cDNAs cloned was confirmed by nucleotide sequencing.
• HEK-293 or HeLa cells were grown with Dulbecco's modified Eagle's medium or Minimal Essential Medium Eagle respectively, supplemented with 2 mM L-glutamine, 10% (v/v) fetal bovine serum and penicillin
• Transfections were performed for 16-24 h using ProFection Mammalian Transfection System-Calcium Phosphate (Promega) or Lipofectamine 2000 (Invitrogen), following manufacturer's recommendations.
• ProFection Mammalian Transfection System-Calcium Phosphate Promega
• Lipofectamine 2000 Invitrogen
• cells were seeded on poly-L-lysine-coated cover slips in 24-well plates.
• HEK 293 cells were transfected with pc-n4'-Mmut and selected with G418 (Invitrogen) for 15 days. Resistant cells were further cloned by limiting dilution and the expression of 91-kDa GPBP in a number of individual clones was determined by Western blot analysis of cell extracts (see below). Clones expressing elevated (c8, cl4) or reduced (cl9) levels of 91-kDa were used in functional studies.
• Cell extracts and cell fractioning To obtain cell extracts, growing cultures were rinsed with ice-cold phosphate buffered-saline (PBS) and homogenized on ice bed with 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 1 mM phenylmethylsulphonyl fluoride (PMSF) and 10 ⁇ g/ml leupeptin. Mixtures were cleared by centrifugation at 500 x g for 10 min, protein concentration determined and stored at - 7O 0 C.
• PBS phosphate buffered-saline
• microsomal fraction which contains fragments of cellular membranes i.e. endoplasmic reticulum, plasma membrane and secretory vesicles (pellet) and the cytosolic fraction (supernatant). All steps were performed at 0-4 0 C and protein concentrations determined using Protein Assay reagent (Bio-Rad).
• the supernatant of 500 x g was loaded on a resource-Q FPLC column, and the bound material eluted in 0 to 1 M NaCl gradient in 10 mM Tris- HCl pH 8.0.
• the 0.55-0.6 M NaCl fractions containing the bulk of cellular GPBP were precipitated with ethanol and used as partially purified GPBP for Western blot analysis.
• cells were brought to RT by rinsing with PBS and incubated for 10 min with culture medium containing 1% formaldehyde.
• the crosslink reaction was quenched with 125 mM GIy-HCl in PBS (pH 7.4) for 10 min at RT.
• Cells were brought to 4 0 C by rinsing with ice-chilled PBS and procedure continued at 4 0 C.
• the beads were collected by centrifugation and washed twice with 1 ml of extraction buffer and once with Tris-buffered saline (TBS, 5OmM Tris-HCl pH 7.5, 150 mM NaCl). Proteins were eluted twice with 25 or 125 ⁇ l (six- well or 150-mm plate) of a 100 ⁇ g/ml solution of FLAG peptide in TBS at RT. Eluted samples were boiled with electrophoresis sample buffer (2X) for 15 min to reverse cross-linking and further analyzed by SDS-PAGE and either Coomassie blue staining or Western blot.
• TBS Tris-buffered saline
• HeLa cells transfected with pc-FLAG-GPBP or pc-FLAG- GPBP ⁇ 26 were treated or not with Bacillus cereus sphingomyelinase (Sigma) as previously described (5) and cells were either fixed with methanol/acetone and analysed by direct immunofluorescence (see below) or lysed in 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, 50 mM NaF, 1 mM sodium orthovanadate, 10 ⁇ g/ml leupeptin, 1 mM PMSF, cleared by centrifugation (500 x g for 10 min) and used for FLAG-immunoprecipitation (see above).
• the immunopurified materials from untreated cells were divided and one-half was treated with 5 U/ ⁇ l of ⁇ PPase (New England Biolabs) at 30 °C for 30 min following manufacturer's recommendations. All the samples were further analysed by Western blot using anti-FLAG antibodies.
• cells were grown in 150-mm plates, transfected with 20 ⁇ g of plasmid constructs encoding FLAG-tagged proteins and cultured for two additional days in fresh media. Twenty milliliters of media were used for FLAG- immunoprecipitation essentially as above indicated.
• Flow cytometry Cells were gently detached and dispersed in culture media. Non-specific antibody binding sites on cell surface were blocked with mouse ascites fluid containing non-relevant mAb (blocking solution). Cells were subsequently incubated in blocking solution in the presence or absence of biotinylated ⁇ GPBP with or without blocking peptide (GPBPpepl) or a non-relevant synthetic peptide. Cells were incubated with Alexa Fluor® 488-streptavidin in blocking solution and further subjected to analysis in a Cytomics FC500 flow cytometer (Beckman Coulter) to measure fluorescence emission. Cell integrity was assessed measuring forward and side scattering, using untreated fresh cells as reference. All incubations were at RT for 1 h.
• Direct and indirect immunofluorescence with fixed cell Cells were transfected and fixed with methanol-acetone (1/1) chilled at -20 0 C for 10 min. Subsequently, cells were incubated with blocking solution (rabbit serum diluted 1 :2 in PBS) for 30 min at RT, incubated with the primary antibodies (20 ⁇ g/ml in blocking solution) for 2 h at 37 0 C in a humidified chamber, followed by incubation with the secondary antibody (1 :200 in blocking solution) for 1 h at RT.
• blocking solution rabbit serum diluted 1 :2 in PBS
• COL4A3BP encodes for polypeptides of 77-, 91- and 120-kDa—
• mAb 14 previously reported to recognize GPBP and GPBP ⁇ 26 (1)
• mAb e26 a novel monoclonal antibody raised against the 26-residue Ser-rich region exclusive for GPBP (Fig IA).
• GPBP deletion mutants and synthetic peptides we have mapped mAb 14 epitope to the FFAT motif and thus, this antibody did not react with a GPBP mutant lacking the FFAT motif (GPBP ⁇ FF AT) (Fig. 9).
• pc-n4' a construct bearing the 5'UTR and coding sequence of COL4A3BP (1, 17), was used in transient gene expression assays in cultured cells.
• the expression of pc-n4' yielded three polypeptides of ⁇ 77-, 91- and 120-kDa which were detected by mAb e26.
• mAb e26 the expression of pc-n4' yielded three polypeptides of ⁇ 77-, 91- and 120-kDa which were detected by mAb e26.
• only the -77- and 91-kDa polypeptides were significantly reactive with mAb 14.
• siRNAs small interfering RNAs
• Fig ID small interfering RNAs
• GPBP isoforms of 91- and 120-kDa the later likely bearing a modified FFAT motif that prevented consistent mAb 14 binding.
• the reduction in the cellular levels of 77-kDa polypeptide when using GPBP-specific siRNAs requires further investigation since this polypeptide displayed no significant reactivity with mAb e26 (Fig. IB).
• Major cellular GPBP isoforms result from noncanonical mRNA translation initiation — To further define the origin of cellular GPBP isoforms, we produced (pc- n4')-derived constructs expressing mRNA mutants consisting of 5'UTR-deletion or iMet to GIy substitution (Fig.
• Fig. 2B protein expression assays
• pc-GPBP-Met construct representing 5'UTR-deleted mRNA
• pc-n4'-Mmut constructs representing the iMet to GIy substitution
• pc-n4'Mmut expressed only the 91- and 120-kDa polypeptides
• pc-GPBP-Met also expressed 77- kDa GPBP polypeptide but pc-n4'Mmut yielded only the 91-kDa polypeptide and no significant expression of 120-kDa polypeptide was observed (Fig. 2B, in vitro).
• GPBP mRNA contained a noncanonical translation initiation site(s) in the 5'UTR that accounted for polypeptides of 91- and 120-kDa whereas the 77-kDa polypeptide was the product of canonical translation initiation.
• our data also suggested that the 91-kDa was the primary product of noncanonical translation initiation and the 120-kDa polypeptide represented a posttranslational derived product that could not be expressed in a cell-free system devoid of cellular membranes.
• GPBP isoforms are insoluble membrane-bound polypeptides — GPBP isoform of 91-kDa was predicted to be non-classical secreted proteins when analyzed with SecretomeP 2.0 Server (18, http://www.cbs.dtu.dk/services/SecretomeP/) and to localize in mitochondria (60.9 %), nucleus (26.1 %), cytoskeleton (8.7 %) and vesicles of secretory system (4.3 %) when analyzed with PSORT II Prediction (http://psort.ims.u-tokyo.ac.jp/form2.html).
• these theoretical considerations suggested that GPBP isoforms resulting from noncanonical translation initiation were noncytosolic polypeptides that entered into cellular organelles including the secretory pathway.
• ⁇ GPBP bound to living cells in a specific manner since binding of the antibodies was efficiently abolished by a synthetic peptide representing GPBP (GPBPpepl) but not by an unrelated polypeptide (Contpep).
• the 77-kDa GPBP is a soluble extracellular protein which interacts with type W collagen — Previous reports suggested that 77-kDa GPBP interacts with type IV collagen (1, 3, 4). This was further assessed by ex vivo cross-linking and FLAG- immunoprecipitation of cells expressing or not expressing BM40-FLAG- ⁇ 3 (IV)NCl, a recombinant exportable form of the human ⁇ 3(IV)NC 1 (16), followed by SDS-PAGE analysis of immunoprecipitates (Fig. 4A).
• FLAG-specific antibodies efficiently precipitated FL AG- ⁇ 3 (IV)NCl and a 77-kDa polypeptide representing either GPBP or GPBP ⁇ 26/CERT (2) (Western) along with Grp78 and Grp94 (Coomassie), two ER resident chaperones implicated in protein folding and ER homeostasis maintenance (19, 20).
• GPBP indeed interacted with FLAG- ⁇ 3(IV) in the ER
• cells expressing or not expressing BM40-FLAG- ⁇ 3 (IV)NCl were transfected with pc- n4' and similarly analyzed (Fig. 4B).
• FLAG antibodies efficiently precipitated 77-kDa GPBP from cells expressing FL AG- ⁇ 3 (IV)NCl but not from control cells, suggesting that 77-kDa GPBP isoform enters into the secretory pathway and interacts with FLAG- ⁇ 3(IV)NCl.
• FLAG-GPBP co-localized extensively with calregulin, an ER resident protein, suggesting that, as described for GPBP ⁇ 26/CERT (21, 22), FLAG- GPBP bound to the ER through FFAT-VAP interaction. Consequently, we expressed and similarly analyzed FLAG-GPBP ⁇ F F A T, a FLAG-GPBP variant devoid of FFAT motif.
• GPBP ⁇ 26/CERT also binds to the ER in a FFAT-dependent manner (21 , 22); however, we found GPBP ⁇ 26/CERT in the cytosol and 77-kDa GPBP in the extracellular compartment, supporting that the Ser-rich 26-residue region exclusive to GPBP is also critical for GPBP secretion.
• 91-kDa GPBP regulates the extracellular levels of 77-kDa GPBP This was accomplished by recombinant expression of FLAG-GPBP in individual cell lines expressing recombinant 91-kDa GPBP to a different levels (Fig.
• 77-kDa GPBP is not sensitive to cell treatment with sphingomyelinase — Recombinant expression studies also showed that 77-kDa GPBP was a cytosolic polypeptide associated with ER that underwent translocation to the Golgi apparatus when FFAT motif was mutated (Fig. 5A). Consequently, we asked whether 77-kDa GPBP underwent dephosphorylation and translocation to the Golgi apparatus in response to sphingomyelinase cell treatment as previously reported for GPBP ⁇ 26/CERT (5).
• Circulating levels of 77-kDa GPBP are upregulated in Goodpasture patients and in animal models of immune complex-mediated glomerulonephritis — Evidence suggested that 77-kDa GPBP was secreted as a soluble protein in vivo was first investigated by immunoaffinity chromatography to isolate circulating human 77-kDa GPBP (Fig. 8A). We used plasmapheresis obtained by standard therapeutic procedures from Goodpasture patients, which were predicted to express higher levels of GPBP (3).
• the ELISA displayed a linear range between 0.5 ng and 10 ng/ml when measuring recombinant GPBP (Fig. 8B) and detected comparatively more circulating 77-kDa GPBP in Goodpasture patients than in control individuals (Fig. 8C).
• Fig. 8B young (4- month) and aged (7-month) NZW mice
• Fig. 8D a mouse strain that develops GPBP- dependent IgA immune complex-mediated glomerulonephritis and lupus-prone autoantibody production commencing at 7 months of age (4).
• AK096854 bears an alternative canonical translation initiation site (iMet) that extends the ORF of the 91-kDa polypeptide upstream by 45 residues.
• iMet canonical translation initiation site
• noncanonical GPBP isoforms are molecular species associated with cellular membranes (Fig. 3); 2) noncanonical GPBP isoforms are the predominant GPBP species in the cell (Fig. 1) and GPBP-specific antibodies bound to the external surface of intact living cells (Fig. 3); 3) 120-kDa polypeptide is not expressed from the mRNA when translation occurs in a cell-free system devoid of cellular membranes (Fig. 2); and 4) 91-kDa GPBP isoform regulates the levels of the 77-kDa GPBP at the extracellular compartment (Fig. 6).
• 91-kDa polypeptide is the primary product of noncanonical translation initiation. This isoform enters into the secretory pathway where undergoes covalent modification to yield the 120-kDa polypeptide and remains bound to membranes reaching the external surface of the plasma membrane.
• the mechanism by which 91- kDa GPBP regulates the extracellular levels of 77-kDa GPBP remains unknown.
• GPBP polypeptides accumulate in the cytosol (Fig. 10), revealing that GPBP transportation into the ER is a saturable process.
• mAb e26 displayed more reactivity for the cytosolic 77-kDa polypeptide than for this isoform when residing in the extracellular compartment (Fig. 10 and 11).
• mAb 14 reacted comparatively more with recombinant than with native 91-kDa GPBP and did not react significantly with native or recombinant 120-kDa product (Fig. 1).
• cytosolic 77-kDa polypeptide representing GPBP ⁇ 26/CERT were significantly reduced in cells expressing GPBP-specific siRNA (Fig. ID). This suggests that either siRNA is also targeting the pre-mRNA or that the mRNA of GPBP is to some extent a precursor of GPBP ⁇ 26 mRNA.
• siRNA is also targeting the pre-mRNA or that the mRNA of GPBP is to some extent a precursor of GPBP ⁇ 26 mRNA.
• cells expressing recombinant GPBP also expressed limited amounts of recombinant GPBP ⁇ 26/CERT (unpublished observations). This reveals that mature GPBP mRNA is subjected to a nonclassical processing, similarly to that reported for XBPl in response to ER stress signals (25).
• GPBP species bearing covalently modified 26- residue Ser-rich region which co-migrate with GPBP ⁇ 26/CERT could also account for this observation.
• the 77-kDa GPBP is a nonconventional Ser/Thr kinase that binds and phosphorylates the ⁇ 3 (IV)NCl domain at sites (1) that are also phosphorylated in vivo (26); 2)
• the 77-kDa GPBP is mainly found in the extracellular compartment both soluble (Fig. 5 and Fig. 8) or associated with GBM collagen (4), and is not expressed at significant levels in the cytosol of cultured cells
• Figs. 1 and 3 Cellular GPBP isoforms localize at the external surface of the plasma membrane (Fig. 3); 4) The 91-kDa GPBP isoform is associated with cellular membranes (Fig. 3) and regulates the extracellular levels of the 77-kDa GPBP isoform (Fig.
• the ⁇ 3(IV)NCl domain undergoes unique structural diversification and at least two distinct conformational isoforms (conformers) assemble in basement membranes (27); 6) An increased expression of the 77-kDa GPBP perturbs the quaternary structure of type IV collagen, suggesting that the elevated GPBP levels interferes with the conformational diversification program (tertiary structure) of the ⁇ 3 (IV)NCl domain (4); 7) Increased serum levels of GPBP correlates with type IV- collagen based glomerulonephritis (Fig. 8); 8) The FFAT motif is a structural requirement for 77-kDa GPBP secretion (Fig.
• phosphate transfer and ceramide trafficking may be molecular strategies through which COL4A3BP regulates protein secretion (i.e. type IV collagen). Consistent with this, it has been shown that VAP is also critical for regulating protein cargo transport to the plasma membrane (11).
• GPBP is a circulating molecule and GBM a principal component of the glomerular filtration barrier; therefore, pathogenic GPBP accumulation in the glomerulus could result from local production but also from the sequestration of circulating GPBP produced elsewhere.
• the local overproduction could account for primary antibody-mediated glomerulonephritis whereas increased circulating levels may induce secondary forms of this pathology and perhaps are responsible for disease recurrence upon renal transplantation. Consequently, quantification of the levels of circulating GPBP might be useful in discriminating primary from secondary antibody-mediated glomerulonephritis and for the clinical monitoring of renal transplantation.
• the 77-kDa polypeptide can be resolved as a doublet representing phosphorylated (higher) and dephosphorylated (lower) versions of GPBP ⁇ 26/CERT (5)
• ⁇ 3 (IV)NCl the NCl domain of the ⁇ 3 chain of type IV collagen
• ATR alternative translated region
• CERT and CERT L short and large isoforms of the ceramide transfer protein
• COL4A3BP the gene encoding for GPBP (CERT L ) and GPBP ⁇ 26 (CERT) which was named collagen IV ⁇ 3 -binding protein
• EDTA ethylenediaminetetraacetic acid
• ER endoplasmic reticulum
• FFAT two phenylalalines in an acidic track
• GBM glomerular basement membrane
• GPBP and GPBP ⁇ 26 large and short alternatively spliced variants of the Goodpasture antigen- binding protein
• HRP horseradish peroxidase
• mAb monoclonal antibody
• NCl noncollagenous-1 domain
• ORF open reading frame
• NZW new Zealand white
• PBS phosphate buffered-saline
• NZW new Zealand white
• GPBP was purified from 50 ml of frozen control plasma using a combination of salting-out precipitation, ion exchange chromatography and gel filtration.
• Proteins precipitated by freezing were first removed by plasma centrifugation at 8200 x g for 10 min at 4 0 C. Since the specific properties for purification of plasma GPBP are not known, proteins were sequentially precipitated from the original sample with growing (NH 4 ) 2 SO 4 saturations (20%, 40%, 60% and 80%). Sequential precipitations were performed by centrifugation at 8200 x g for 10 min at 4 0 C and precipitates were dissolved in 5 ml of 50 mM Tris-HCl, pH 7.5. Protein mixtures were desalted by dialysis against 50 mM Tris-HCl, pH 7.5 using membrane bags with 3.5- kDa cut-off. The final supernatant of (NH-O 2 SC) 4 precipitations was similarly dialyzed and further used for purification as the final fraction yielded by the precipitation process.
• the fractions rendered by the salting-out were subsequently analyzed by ion exchange chromatography (IEC) using a HiTrap Q-sepharose anion exchange column.
• the column was first equilibrated with buffer A (50 mM Tris/HCl, pH 7.5, 2OmM NaCl), further loaded with each individual sample and washed with 10 volumes of buffer A.
• Bound proteins were eluted with a gradient from buffer A to buffer B (50 mM Tris/HCl, pH 7.5, 1 M NaCl) and collected in 0.6 ml fractions.
• IEC fractions containing GPBP material were detected by Western blot with GPBP-specific biotinylated N27 monoclonal antibody.
• GPBP-containing IEC fractions were pooled, concentrated to 0.5 ml, and subsequently subjected to gel filtration chromatography with SuperdexTM 200 10/300 CL.
• the column was first equilibrated with TBS (50 mM Tris/HCl pH 7.5, 150 mM NaCl), the sample was injected into the column and proteins separated by size.
• the gel filtration fractions were analysed by Western blot for detection of GPBP material with biotinylated N27 monoclonal antibody.
• the fractions containing GPBP were pooled, precipitated with 80% acetone and resuspended in 50 mM Tris-HCl pH 7.5, 8M urea.
• polypeptides not represented in significant amounts in the Western blot in Figure 12 that were identified in Western blot analysis performed during the purification process. These included polypeptides of approximately: 368-kDa [20% (NH 4 ) 2 SO 4 ], 40-, HO-, 120- and 311-kDa [40% (NILO 2 SO 4 ], and 91-, 146-, 171- and 300-kDa polypeptides [60% (NH 4 ⁇ SO 4 ] (data not shown). Finally, the size of each chromatographic peak in gel filtration analysis, which represented individual GPBP quaternary structures was also estimated.
• the major circulating GPBP isoform includes the previously recognized 77-kDa and derived polypeptides of lower M r .
• GPBP is a normal component of the urine which can be both measured by simple immunological-based procedures (i.e. ELISA) and isolated by chemical and immunochemical procedures.
• ELISA simple immunological-based procedures
• chemical and immunochemical procedures The evidence indicates that 91-kDa polypeptide and derived polypeptides are the major urinary GPBP products.
• GPBP was extracted from urine of a control donor using Sepharose 4B loaded with GPBP-specific rabbit polyclonal antibodies.
• the column-bound material was eluted and analyzed by Western blot using GPBP-specific chicken polyclonal antibodies (Fig. 13).
• a number of polypeptides displaying a broad range of MW were detected with GPBP-specific antibodies in the immunoaffinity purified sample.
• Antibody specificity was confirmed by staining a control lane loaded with the same material with secondary antibody (Cont). Bars and numbers or arrows and numbers indicate the position and size (kDa) of MW standards (left) or GPBP polypeptides (right), respectively
• C is represented raw data obtained analyzing donor samples with: secondary antibody (Cont), nonspecific chicken IgY and secondary antibody (IgY), or with ⁇ GPBPch and secondary antibody ( ⁇ GPBPch).
• D the table shows corresponding transformed data using the curve obtained in B.
• CM resin was eluted with IM NaCl, 50 mM Tris pH 7.5 (CM, IM NaCl), and DEAE resin was subsequently eluted with 0.35M NaCl, 50 mM Tris pH 7.5 (DEAE, 0.35M NaCl) and IM NaCl, 50 mM Tris pH 7.5 (DEAE, IM NaCl).
• Equivalent amounts of each sample including the supernatant of the DEAE extraction (Spt CM/DEAE) were analyzed by Western blot with GPBP-specific chicken polyclonal antibodies and HRP-labelled anti-chicken IgY ( ⁇ GPBPch).
• Nonspecific reactive polypeptides were identified by staining an in-parallel analysis using only HRP-labelled anti-chicken IgY (Cont). Bars and numbers or arrows and numbers indicate the position and size (kDa) of MW standards (left) or polypeptides specifically reacting with anti- GPBP antibodies and that were detected only in SptCM/DEAE (right), respectively. (Fig. 15). Western blot analysis using GPBP-specific chicken polyclonal antibodies revealed that most of GPBP material was precipitated by salt and did not bound to either CM or DEAE. A major GPBP polypeptide of 91-kDa was detected along with significant amounts of GPBP polypeptide of 77-kDa and only traces of GPBP-related polypeptides of 60- and 50-kDa. Conclusions
• GPBP polypeptides can be isolated from urine either by affinity chromatography or by salting-out precipitation followed by ion-exchange chromatography.
• GPBP levels in urine can be assessed either by indirect ELISA or sandwich ELISA using specific anti-GPBP antibodies.
• the antibodies N4, N7, Nl 1 , N14 and N27 also recognized an additional GPBP-related polypeptide of ⁇ 88-kDa, which may represent a phosphorylated version of the 77-kDa canonical polypeptide (Raya et al 1999 J. Biol Chem. 274, 12642-12649).
• the N26 antibody recognizes a 91- kDa polypeptide which co-migrated with the recently characterized 91-kDa GPBP isoform (Revert et al. 2008 J. Biol. Chem. 283:30246-55) targeted by mAb e26 (Fig. 17).
• Table 2 The relative efficiencies of the new monoclonal antibodies for detection of GPBP isoforms (native or recombinant) have been estimated and summarized in Table 2.
• Deletion mutants ⁇ 1- ⁇ 4 were obtained by standard procedures using two consecutive PCRs and specific primers to introduce the corresponding deletions (Fig. 18B). Interestingly, all the antibodies failed to react with ⁇ 2 and ⁇ 3 internal deletion FLAG-GPBP mutants but reacted with ⁇ l and ⁇ 4 mutants (Fig. 18C). Data indicate that the sequence represented by residues 305-344 of GPBP (GGPDYEEGPNSLINEEEFFDAVEAALDRQDKIEEQ SQSEK, SEQ ID NO: 10) conforms a highly immunogenic epitope cluster . Consistently, previously characterized mAb 14 was found to react with this region at the FATT motif (Revert et al. 2008 J. Biol. Chem. 283:30246-55).
• N1-N28 monoclonal antibodies by indirect immunofluorescence analysis of HeLa cells expressing recombinant GPBP.
• HeLa cells were transfected with pcDNA3 -FLAG-GPBP, cultured for 24 additional hours, and fixed with methanol/acetone (50%-50%). After fixation, cells were blocked with 3% BSA in PBS (blocking solution) and incubated with the indicated antibodies (cultured media) diluted 1 :2 in blocking solution. Subsequently, cells were washed with PBS and incubated with FITC-labeled anti-mouse IgG, washed again, mounted and observed with an inverted fluorescence microscope. Images were acquired with a 40 x objective using identical exposition times and gains.
• N1-N28 monoclonal antibodies Characterization of N1-N28 monoclonal antibodies by indirect immunofluorescence analysis of HeLa cells.
• HeLa cells were seeded and cultured on crystal slides, processed as above, and analyzed with an inverted fluorescence microscope using a 40 x objective and an image amplification of 1.63.
• All antibodies showed endoplasmic reticulum distribution similar to that yielded by antibody N27 shown at the left of the composite.
• Some cells showed also a peri-nuclear and focal reinforcements typical of the Golgi apparatus (white arrow).
• the pattern unveiled by N26 mixes the previously described reticular distribution with nuclear and peri-nuclear punctuate clusters, and linear decoration of plasma membrane.
• N1-N28 monoclonal antibodies Characterization of N1-N28 monoclonal antibodies by immunohistochemical analysis of paraffin-embedded human kidney tissue. Individual monoclonal antibodies were used for standard immunohistochemical analysis of paraffin-embedded human kidney samples. All the reactive antibodies stained mainly convoluted and collecting tubules with significant staining also within glomeruli at mesangial cells, podocytes, mesangial matrix and capillary walls. In the later case with a linear pattern at the endothelium surface and with a granular-like distribution within the capillary wall. In capillary walls, immunostaining was less frequent, being N5, N6, N7, N8, NlO and N26 the best antibodies for these purposes. The antibodies rendering better GPBP detection using immunohistochemical techniques were N5, N6, N7, N8, N9, NlO, N12, N26 and N27 (see Table 2).
• Bound GPBP was detected with chicken polyclonal anti-GPBP and HRP-labelled anti-chicken IgY. Development was performed with a fluorescent reagent (Amplex).
• b " Capture assays for human circulating GPBP (plasma * ). Anti-mouse-coated ELISA plates were loaded and blocked as above indicated and further incubated with a
• Table 2 Summary of the relative efficiency of the 28 monoclonal antibodies as detection antibodies in Western blot, immunofluorescence (IF) and immunohistochemistry (IHC), and as capture antibodies in sandwich ELISA

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