Methods Of Producing A Secreted Protein
The invention is directed to methods of producing a polypeptide or a variant thereof, wherein the polypeptide or variant thereof is dependent on LIMP-2 for trafficking, localization, stabilization and/or sorting of the polypeptide in the cell. In general, the methods comprise culturing a lysosomal integral membrane protein II (LIMP-2) deficient cell which expresses the polypeptide or the variant thereof under conditions in which the polypeptide or the variant thereof is produced.
This application is a divisional of U.S. application Ser. No. 12/599,393, filed May 9, 2008, which is the U.S. National Stage of International Application No. PCT/US2008/005942, filed on May 9, 2008, published in English, which claims the benefit of U.S. Provisional Application No. 60/928,907, filed on May 11, 2007 and U.S. Provisional Application No. 60/967,415, filed on Sep. 4, 2007. The entire teachings of the above applications are incorporated herein by reference.
BACKGROUND OF THE INVENTIONLysosomal enzymes are synthesized as soluble or membrane-integrated glycoproteins in the rough endoplasmic reticulum (ER). In mammalian cells mannose 6-phosphate receptors (MPRs) mediate the transport of the majority of lysosomal enzymes to lysosomes. Mannose 6-phosphate (Man-6-P) terminal residues are recognized in the trans-Golgi network (TGN) by two MPRs which mediate the sorting of lysosomal enzymes from the secretory pathway and deliver them to a prelysosomal compartment from where the receptors return to the TGN and the ligands are forwarded to dense lysosomes (reviewed in (Kornfeld, 1992; Kornfeld and Mellman, 1989; Ludwig et al., 1995)). The physiological importance of the MPR-dependent transport of lysosomal enzymes is illustrated by I-cell disease (ICD). In this disorder, the deficiency of the phosphotransferase responsible for catalyzing the addition of Man-6-P results in the synthesis of lysosomal enzymes that lack Man-6-P residues leading to a failure to bind to MPRs and a strongly increased secretion of most of the lysosomal enzymes (Neufeld, 1991). Although fibroblasts of these patients have a marked deficiency of lysosomal enzymes, liver, spleen, kidney and brain tissues have nearly normal levels of lysosomal hydrolases (Kornfeld, 1986; Kornfeld and Sly, 1985). It was therefore proposed that in addition to MPR-dependent mechanisms, MPR-independent mechanisms are likely to exist for the transport of newly synthesized lysosomal enzymes to lysosomes (Ahn et al., 2002; Ginsel and Fransen, 1991; Glickman and Kornfeld, 1993; Rijnboutt et al., 1991; Tanaka et al., 2000). Also in MPR-deficient mice an ICD-like phenotype with increase of lysosomal enzymes in serum and normal activities in some tissues has been described (Dittmer et al., 1999).
In fibroblasts of ICD patients the lysosomal hydrolase β-glucocerebrosidase (βGC) has been shown to be intracellularly retained suggesting that signals other than Man-6-P are responsible for targeting this enzyme (Aerts et al., 1988; van Dongen et al., 1985). Mutations within the gene coding for human βGC are the cause of the most common lysosomal storage disorder, Gaucher Disease, in which the defective enzyme leads to an accumulation of glucosylceramide (GlcCer) (Beutler, 1991, 2006). Although the clinical course of this disease has been well described and an efficient treatment option, enzyme replacement therapy, is available little is known about how GlcCer accumulation in lysosomes leads to cellular pathology. Also the mechanism by which βGC is targeted from its site of synthesis in the ER to lysosomes is not well understood.
Thus, a greater understanding of the mechanism by which β-GC is targeted from its site of synthesis in the endoplasmic reticulum to lysosomes could lead to improved methods of treating lysosomal storage disorders such as Gaucher Disease.
SUMMARY OF THE INVENTIONβ-glucocerebrosidase, the enzyme defective in Gaucher disease, is targeted to the lysosome independently of a mannose 6-phosphate receptor. The invention is based, in part, on the identification of a protein that interacts with β-glucocerebrosidase, which has elucidated the targeting pathway of β-glucocerebrosidase. Affinity chromatography experiments revealed that the lysosomal integral membrane protein LIMP-2 is a specific binding partner of β-glucocerebrosidase and that this interaction involves a coiled coil domain within the lumenal domain. β-glucocerebrosidase activity and protein levels were severely decreased in LIMP-2 knockout mouse tissues. Analysis of fibroblasts and macrophages isolated from these mice indicated that a majority of β-glucocerebrosidase was secreted or partially retained in the ER. Missorting of β-glucocerebrosidase was also evident in vivo since protein and activity levels were significantly higher in sera from LIMP-2-deficient mice compared to wild type. Reconstitution of LIMP-2 in LIMP-2-deficient fibroblasts led to a rescue of β-glucocerebrosidase levels and distribution. LIMP-2 expression also led to lysosomal transport of a β-glucocerebrosidase endoplasmic reticulum retention mutant. These data support a role for LIMP-2 as the mannose 6-phosphate-independent trafficking receptor for β-glucocerebrosidase.
Accordingly, the invention is directed to methods of producing a polypeptide or a variant thereof, wherein the polypeptide or variant thereof is dependent on LIMP-2 for trafficking, localization, stabilization and/or sorting of the polypeptide in the cell. In general, the methods comprise culturing a lysosomal integral membrane protein II (LIMP-2) deficient cell which expresses the polypeptide or the variant thereof under conditions in which the polypeptide or the variant thereof is produced.
The invention is also directed to methods of producing a polypeptide or variant thereof for secretion, wherein the polypeptide or variant thereof is dependent on LIMP-2 for trafficking, localization, stabilization and/or sorting of the polypeptide in the cell. The method comprises culturing a LIMP-2 deficient cell or animal (e.g., a LIMP-2 knockout animal) which expresses the polypeptide under conditions in which the polypeptide is secreted from the cell into the extracellular environment, or the cells of animals into the sera.
In one embodiment, the invention is directed to a method of producing β-glucocerebrosidase or a variant thereof, comprising culturing a lysosomal integral membrane protein II (LIMP-2) deficient cell which expresses β-glucocerebrosidase or the variant thereof under conditions in which β-glucocerebrosidase or the variant thereof is produced, thereby producing β-glucocerebrosidase or the variant thereof. In a particular embodiment, the β-glucocerebrosidase or a variant thereof is secreted from the cell.
In a particular embodiment, the invention is directed to a method of producing human β-glucocerebrosidase or a variant thereof, comprising culturing a lysosomal integral membrane protein II (LIMP-2) deficient Chinese Hamster Ovary (CHO) cell which expresses β-glucocerebrosidase or the variant thereof under conditions in which β-glucocerebrosidase or the variant thereof is secreted from the CHO cell, thereby producing human β-glucocerebrosidase or the variant thereof.
Also described herein are the hamster LIMP-2 nucleotide and amino acid sequences. Thus, the invention is directed to an isolated hamster LIMP-2 nucleic acid molecule. In one embodiment, the hamster LIMP-2 nucleic acid molecule comprises SEQ ID NO: 1. In another embodiment, the nucleic acid molecule comprises a nucleotide sequence that encodes SEQ ID NO: 2.
The invention is also directed to a hamster LIMP-2 polypeptide. In one embodiment, the polypeptide has an amino acid sequence comprising SEQ ID NO: 2.
Also encompassed by the invention are expression constructs comprising the hamster LIMP-2 sequences described herein, and host cells comprising the expression constructs. Methods of producing hamster LIMP-2 using the host cells described herein, and the hamster LIMP-2 produced by the methods, are also provided.
An antibody or antigen binding fragment thereof that specifically binds to all or a portion of a hamster LIMP-2 protein is also provided. In a particular embodiment, the antibody or antigen binding fragment thereof specifically binds to all or a portion of a hamster LIMP-2 protein having the amino acid sequence of SEQ ID NO: 2.
Also encompassed by the invention is an siRNA molecule which knocks down expression of a nucleic acid that encodes a hamster LIMP-2 protein having the amino acid sequence of SEQ ID NO: 2, wherein the siRNA comprises a double stranded sequence. In the method, one strand of the siRNA molecule has sufficient sequence complementarity to a hamster LIMP-2 RNA sequence to knock down expression of the nucleic acid that encodes the hamster LIMP-2 protein.
An expression construct comprising the siRNA molecules and a host cell comprising the expression constructs are also encompassed by the invention.
The invention is also directed to a method of altering trafficking of a lysosomal polypeptide that is dependent on a LIMP-2 polypeptide for trafficking to a lysosome comprising culturing a LIMP-2 deficient cell which expresses the lysosomal polypeptide under conditions in which the trafficking of the lysosomal polypeptide to the lysosome is altered.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
As described herein, using pull-down experiments with purified β-GC the lysosomal integral membrane protein type-II (LIMP-2) has been identified as a specific binding partner for β-GC. In vitro and in vivo evidence showed that LIMP-2 acted as a receptor to bind β-GC, and that the β-GC-LIMP-2 complex was transported to the lysosomal compartment in an MPR-independent pathway.
Specifically, fractionation of tissue homogenates by affinity chromatography and subsequent mass spectrometry analysis identified LIMP-2 as a specific binding partner of β-GC. Biochemical characterization of the LIMP-2/β-GC interaction revealed the region in LIMP-2 involved in this association. Analysis and comparison of β-GC enzyme activity in tissues from wild type (WT) and LIMP-2 knockout mice indicated that that activity of the endogenous β-GC was decreased in the knockout mice and that this change directly correlated with a decrease in β-GC protein levels detected by immunoblot. Immunocytochemical staining of mouse embryonic fibroblasts (MEFs) isolated from LIMP-2 knockout mice corroborated the data. It was found that although significantly less β-GC was detected in knockout versus WT tissue homogenates, β-GC protein levels were much higher in the sera from LIMP-2 knockout mice compared to WT, and β-GC activity levels in these samples were increased over WT indicating that β-GC was missorted from cells in the absence of LIMP-2. Introduction of WT LIMP-2 into LIMP-2 knockout MEFs resulted in the rescue of β-GC protein levels and the normal localization of β-GC in these cells. Since endogenous β-GC trafficks independently of the mannose-6-phosphate receptor the data showed a role for LIMP-2 as a mannose-6-phosphate independent trafficking receptor for β-GC.
The findings described herein, particularly those relating to the missorting of β-GC in the absence of LIMP-2, provide for methods of producing a polypeptide or a variant thereof, wherein the polypeptide or variant thereof is dependent on LIMP-2 for trafficking, localization, stabilization and/or sorting of the polypeptide in the cell. In general, the methods comprise culturing a lysosomal integral membrane protein II (LIMP-2) deficient cell which expresses the polypeptide or the variant thereof under conditions in which the polypeptide or the variant thereof is produced.
As shown herein, expression of the polypeptide in a LIMP-2 deficient cell altered the localization (missorts) the polypeptide compared to the localization of the same polypeptide that was expressed in a normal or wild type cell. For example, the polypeptide can be retained in the endoplasmic reticulum of the LIMP-2 deficient cell and/or secreted from the LIMP-2 deficient cell.
Therefore, in a particular embodiment, the invention provides methods of producing a polypeptide or variant thereof for secretion (a secreted polypeptide or a variant thereof), wherein the polypeptide or variant thereof is dependent on LIMP-2 for trafficking, localization, stabilization and/or sorting of the polypeptide in the cell. The method comprises culturing a LIMP-2 deficient cell or animal (e.g., a LIMP-2 knockout animal) which expresses the polypeptide under conditions in which the polypeptide is secreted from the cell, or from the cells of the animal into the sera. Thus, in this embodiment, the method can be performed in vitro wherein the secreted protein can be obtained from the supernatant of a cell, or in vivo wherein the secreted protein can be obtained from the sera of a LIMP-2 deficient animal.
The method can be used for the production of any polypeptide (protein), referred to as a LIMP-2 ligand or LIMP-2 binding partner, that is dependent on LIMP-2 for trafficking, localization, stabilization, sorting or a combination thereof in a cell. The polypeptide can bind to all or a portion (e.g., a domain such as the lumenal domain) of LIMP-2. In another embodiment, the protein is β-glucocerebrosidase (βGC, β-GC, GC, acid β-glucocerebrosidase, acid β-glucosidase, glucosylceramidase, β-D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) or a variant thereof. β-GC is used to treat Gaucher Disease. Thus, in one embodiment, the invention is directed to a method of producing β-GC or a variant thereof, comprising culturing a LIMP-2 deficient cell or animal (e.g., a LIMP-2 knockout animal) which expresses β-GC or the variant thereof under conditions in which β-GC or the variant thereof is produced, thereby producing β-GC or the variant thereof. In a particular embodiment, the method comprises culturing a LIMP-2 deficient cell or animal which expresses β-GC under conditions in which the β-GC is secreted from the cell. In another embodiment, the method further comprises purifying the β-GC secreted from the cell (e.g. from the supernatant of the cell) or animal (e.g., from the sera of the animal).
A variant of β-GC includes a protein having an amino acid sequence that is at least 90% identical to an amino acid sequence of β-GC (e.g., SEQ ID NO: 11). In one embodiment, the variant of β-GC is imiglucerase (the active ingredient in Cerezyme®, Genzyme Corporation, Cambridge, Mass.). Imiglucerase is an oligosaccharide-modified human β-glucocerebrosidase made using recombinant cells and is used to treat patients with Gaucher disease, a rare and devastating genetic disorder caused by a deficiency or malfunction of the β-glucocerebrosidase (see, e.g., Furbish et al., Biochim. Biophys. Acta 673:425-434 (1981); U.S. Pat. No. 5,549,892 which are incorporated herein by reference).
The methods described herein can further comprise isolating and/or further manipulating or modifying the polypeptide produced by the LIMP-2 deficient cell. For example, the method can further comprise purifying (e.g., substantially purifying), concentrating, and/or remodeling the polypeptide using techniques well known to those of skill in the art. Examples of such techniques include filtration, centrifugation, chromatography (e.g., gel electrophoresis, size exclusion, ion exchange, affinity, high pressure liquid chromatography, gas chromatography), mass spectrometry, oligosaccharide remodeling (Furbish et al., Biochim. Biophys. Acta 673:425-434 (1981); U.S. Pat. No. 5,549,892) and/or lyophilization.
In a particular embodiment, the invention is directed to a method of producing β-GC or a variant thereof, comprising culturing a LIMP-2 deficient cell which expresses β-GC or the variant thereof under conditions in which β-GC or the variant thereof is produced, thereby producing β-GC or the variant thereof. The method further comprises purifying and remodeling the carbohydrate chains of the β-GC or variant thereof produced. Methods for purifying and remodeling β-GC or a variant thereof are known in the art (e.g., see U.S. Pat. No. 5,549,892 which is incorporated herein by reference).
LIMP-2 is a heavily N-glycosylated 478 residue type-III transmembrane protein (Fujita, H., et al., Biochem Biophys Res Commun 178, 444-452 (1991)) comprised of an approximately 400 amino acid lumenal domain, two transmembrane domains and a cytoplasmic domain of 20 amino acids. Based on homology, LIMP-2 has been defined as a member of the CD36 family of scavenger receptor proteins (Febbraio, M., et al., J Clin Invest 108, 785-791 (2001); Krieger, M., J Clin Invest 108, 793-797 (2001)) which also includes CLA-1 (CD36-LIMP-2 Analogous-1/Scavenger Receptor BI) (Calvo, D., and Vega, M. A., J Biol Chem 268, 18929-18935 (1993)) and the Drosophila melanogaster proteins Croquemort, (Franc, N. C., et al., Immunity 4, 431-443 (1996)) and epithelial membrane protein, emp (Hart, K., and Wilcox, M., J Mol Biol 234, 249-253 (1993)). It has been recently shown that over-expression of LIMP-2 caused an enlargement of early endosomes and late endosomes/lysosomes and an impairment of endocytotic membrane traffic out of the enlarged compartments (Kuronita, T., et al., J Cell Sci 115, 4117-4131 (2002); Kuronita, T., et al., Traffic 6, 895-906 (2005)). A deficiency of LIMP-2 in mice caused ureteric pelvic junction obstruction, deafness and peripheral neuropathy (Gamp, A., et al., Hum Mol Genet 12, 631-646 (2003)) associated with an impaired vesicular trafficking and distribution of apically expressed proteins (Knipper, M., et al., J Physiol 576, 73-86 (2006)).
A number of LIMP-2 genes have been described in the art including human (Fujita, H., et al., Biochem. Biophys. Res. Comm., 184(2):604-611 (1992)) mouse (Tabuchi, N., et al., J. Biochem., 122(4):756-763 (1997)) and rat (Vega, M. A., et al., J. Biol. Chem., 266(25):16818-16824 (1991)) LIMP-2 genes. As described herein, the isolation and characterization of the hamster LIMP-2 nucleotide and amino acid sequences have now been provided. Thus, the invention is also directed to a nucleic acid molecule comprising the nucleotide sequence of hamster LIMP-2 (SEQ ID NO: 1). In one embodiment, the nucleic acid molecule encodes an amino acid sequence comprising SEQ ID NO: 2. Accordingly, the invention is also directed to an isolated polypeptide having an amino acid sequence of hamster LIMP-2 (SEQ ID NO: 2).
Expression constructs comprising the nucleotide hamster sequence as well as host cells comprising such expression constructs are also provided herein. In addition, the expression constructs and/or host cells of the invention can be used to produce hamster LIMP-2. Thus, the invention includes methods of producing hamster LIMP-2 comprising culturing a host cell comprising an isolated hamster LIMP-2 nucleic acid described herein under conditions in which the hamster LIMP-2 polypeptide is produced. The method can further comprise isolating the hamster LIMP-2 polypeptide from the cell. The present invention also relates to an isolated hamster LIMP-2 polypeptide produced by the method.
The availability of the hamster LIMP-2 nucleotide and amino acid sequences provides for methods of identifying an agent that alters (e g., inhibits, enhances) interaction of a hamster LIMP-2 polypeptide with a LIMP-2 binding partner (e.g., β-GC). In one embodiment, the agent inhibits (e.g., partially, completely) the interaction of a hamster LIMP-2 polypeptide with a binding partner. In another embodiment, the agent enhances the interaction of a hamster LIMP-2 polypeptide with a binding partner. Such method can comprise, for example, contacting a hamster LIMP-2 polypeptide having an amino acid sequence comprising SEQ ID NO: 2 with β-GC under conditions in which the hamster LIMP-2 interacts with the β-GC, with an agent to be assessed. The extent to which the hamster LIMP-2 interacts with the β-GC in the presence of the agent to be assessed is determined, wherein if the extent to which hamster LIMP-2 interacts with β-GC is altered in the presence of the agent compared to the extent to which hamster LIMP-2 interacts with β-GC in the absence of the agent, then the agent alters interaction of a hamster LIMP-2 polypeptide with β-GC. Alternatively, the method can comprise contacting a host cell which comprises isolated nucleic acid that encodes a hamster LIMP-2 polypeptide having an amino acid sequence comprising SEQ ID NO: 2 wherein the LIMP-2 polypeptide, when expressed, interacts with β-GC in the cell, with an agent to be assessed. The secretion of β-GC from the host cell can then be assessed, wherein an altered secretion of β-GC from the host cell compared to secretion of β-GC from a control cell indicates that the agent alters interaction of a hamster LIMP-2 polypeptide with β-GC.
Example of agents (modulators) for use in the methods include nucleic acids (e.g., antisense RNA, siRNA, shRNA) peptides, peptidomimetics, small molecules such as small organic molecules or other drugs which bind to a hamster LIMP-2 polypeptide and/or inhibit or enhance (partially, completely) hamster LIMP-2 expression or activity.
Determining the ability of a hamster LIMP-2 polypeptide to bind to or interact with a binding partner can be accomplished using methods described herein and known to those of skill in the art. Moreover, in the methods of the invention, the hamster LIMP-2 polypeptide or its binding partner can be immobilized to facilitate separation of complexed from uncomplexed forms of one or both of the polypeptides, as well as to accommodate automation of the assay. Binding of an agent to a hamster LIMP-2, or interaction of a hamster LIMP-2 polypeptide with a binding partner in the presence and absence of an agent to be assessed, can be accomplished using, for example, columns, resins, microtitre plates, test tubes, and micro-centrifuge tubes.
In addition, the availability of the hamster LIMP-2 protein provides for an antibody or antigen binding fragment thereof that specifically binds to all or a portion of a hamster LIMP-2 protein having the amino acid sequence of SEQ ID NO: 1. That is, the antibody can bind to all of the hamster LIMP-2 protein of from about 8 amino acids to about 450 amino acids of the hamster LIMP-2 protein. In particular embodiments, the antibody can bind to about 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, or 425 amino acids of the LIMP-2 protein.
As used herein, the term “specific” when referring to an antibody-antigen interaction, is used to indicate that the antibody can selectively bind to hamster LIMP-2. In one embodiment, the antibody inhibits the activity of the hamster LIMP-2. In another embodiment, the antibody inhibits binding of LIMP-2 to β-glucocerebrosidase or a variant thereof. In yet another embodiment, the antibody specifically binds to all or a portion of a lumenal domain of β-glucocerebrosidase or a variant thereof.
An antibody that is specific for hamster LIMP-2 is a molecule that selectively binds to hamster LIMP-2 but does not substantially bind to other molecules in a sample, e.g., in a biological sample that contains hamster LIMP-2. The term “antibody,” as used herein, refers to an immunoglobulin or a part thereof, and encompasses any polypeptide comprising an antigen-binding site regardless of the source, method of production, and other characteristics. The term includes but is not limited to polyclonal, monoclonal, monospecific, polyspecific, humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, conjugated and CDR-grafted antibodies. The term “antigen-binding site” refers to the part of an antibody molecule that comprises the area specifically binding to or complementary to, a part or all of an antigen. An antigen-binding site may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). An antigen-binding site may be provided by one or more antibody variable domains (e.g., an Fd antibody fragment consisting of a VH domain, an Fv antibody fragment consisting of a VH domain and a VL domain, or an scFv antibody fragment consisting of a VH domain and a VL domain joined by a linker). The term “anti-hamster LIMP-2 antibody,” or “antibody against hamster LIMP-2,” refers to any antibody that specifically binds to at least one epitope of LIMP-2.
The various antibodies and portions thereof can be produced using known techniques (Kohler and Milstein, Nature 256:495-497 (1975); Current Protocols in Immunology, Coligan et al., (eds.) John Wiley & Sons, Inc., New York, N.Y. (1994); Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694 B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al., European Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Queen et al., European Patent No. 0 451 216 B1; and Padlan, E. A. et al., EP 0 519 596 A1; Newman, R. et al., BioTechnology, 10: 1455-1460 (1992); Ladner et al., U.S. Pat. No. 4,946,778; Bird, R. E. et al., Science, 242: 423-426 (1988)).
As used herein a “LIMP-2 deficient cell” or “LIMP-2 deficient animal” includes a cell or animal in which the expression and/or function (activity) of LIMP-2 is completely or partially downregulated (blocked, inhibited, disrupted). Functions of LIMP-2 include the ability of LIMP-2 to associate with one or more of its ligands, such as β-GC. Whether LIMP-2 expression and/or function in a cell is deficient can be determined using a variety of techniques described herein and known in the art such as enzyme activity assays, gel electrophoresis, immunochemistry, quantitative polymerase chain reaction (PCR) (e.g., detect mRNA levels) and mass spectrometry. The results can also be compared to the results obtained from a suitable control, e.g., a wild type cell of the same, or from a different species, as the LIMP-2 deficient cell.
Examples of methods for obtaining or producing a LIMP-2 deficient cell or animal are described herein and are known in the art. For example, a LIMP-2 deficient cell can be obtained from a LIMP-2 deficient animal (Gamp, A., et al., Hum Mol Genet 12, 631-646 (2003)). In addition, a LIMP-2 deficient cell or animal can be produced by introducing one or more targeted mutations specific for LIMP-2 into a cell, or into the cells of an animal to produce a LIMP-2 knockout animal (Gamp, A., et al., Hum Mol Genet 12, 631-646 (2003)). In one embodiment, the mutated LIMP-2 polypeptide comprises a motif that alters localization of LIMP-2. An examples of such a motif is an endoplasmic reticulum retention motif.
Alternatively, a dominant negative mutant can be introduced into a cell or animal to render the cell or animal LIMP-2 deficient. For example, a LIMP-2 mutant protein that competitively binds to a ligand of LIMP-2 (e.g., β-GC) can be introduced into a cell. The LIMP-2 mutant protein can have enhanced binding properties such that the LIMP-2 ligand(s) preferentially binds to the LIMP-2 mutant protein rather than to the wild type LIMP-2 present in the cell. In another embodiment, a LIMP-2 fragment (a LIMP-2 peptide) comprising, or consisting essentially of, a region of LIMP-2 that competitively binds to a LIMP-2 ligand, can be introduced into the cell.
Moreover, treatment of cells with one or more small molecule inhibitors or antibodies that disrupt the association of LIMP-2 and a LIMP-2 ligand can also be used to produce a LIMP-2 deficient cell.
Antisense nucleic acid molecules, that is, molecules which are complementary to a sense nucleic acid encoding a LIMP-2 polypeptide (e.g., complementary to the coding strand of a double-stranded cDNA LIMP-2 molecule or complementary to an mRNA LIMP-2 sequence) can also be used to render a cell LIMP-2 deficient. The antisense nucleic acid can be complementary to an entire LIMP-2 coding strand, or to only a portion thereof, e.g., all or part of the protein coding region (or open reading frame). An antisense nucleic acid molecule can be antisense to a noncoding region of the coding strand of a nucleotide sequence encoding LIMP-2. The noncoding regions (5′ and 3′ untranslated regions) are the 5′ and 3′ sequences which flank the coding region and are not translated into amino acids. The antisense nucleic acid molecule can be complementary to the entire coding region of LIMP-2 mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of LIMP-2 mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using procedures known in the art (e.g., using chemical synthesis and enzymatic ligation reactions).
In a particular embodiment, RNA interference (RNAi) can be used to produce a LIMP-2 deficient cell or animal (e.g., using short interfering RNA (siRNA) or short hairpin RNA (shRNA)). As known in the art RNAi is a mechanism of post-transcriptional gene silencing directed by double stranded RNA (dsRNA) (Meister G, Tuschl T., Nature. 431, 343-9, (2004)). Exogenous dsRNA molecules that have sufficient sequence complementarity to a particular mRNA sequence, are introduced into a cell to destroy a particular mRNA, thereby diminishing or abolishing expression of the mRNA sequence. The exogenous dsRNA molecules introduced into the cells are processed by the RNase III enzyme Dicer into duplexes of 21-25 nucleotides (nt) containing 5′ monophosphates and 2-nt 3′ overhangs referred to as small interfering RNAs (siRNAs) (Bernstein, E., et al., Nature. 409, 363-6 (2001); Elbashir, S. M., et al., Genes Dev. 15, 188-200 (2001)). The siRNAs are incorporated into a multi-protein RNA-induced silencing complex (RISC) that degrades RNAs with sequences complementary to the siRNA (Tomari, Y., Zamore, P. D., Genes Dev. 19, 517-29 (2005)).
Thus, one or more siRNA or shRNA that degrades a LIMP-2 RNA sequence can be introduced into a cell or animal to render the cell or animal LIMP-2 deficient. Algorithms for designing siRNA directed to a particular sequence and methods for producing such siRNA sequences are well known to those of skill in the art (e.g., Reynolds et al., Nature Biotechnology, 22(3):326-330 (2004); Takasaki, S., et al., Computational Biology and Chemistry, 30, 169-178 (2006)). In particular embodiments, the one or more siRNA or shRNA molecules are targeted to one or more domains of LIMP-2 (e.g., a transmembrane domain, a cytoplasmic domain, a lumenal domain). In a particular embodiment, the siRNA or shRNA is targeted to the lumenal domain of LIMP-2.
Thus, the invention is also directed to an siRNA molecule which knocks down expression of a nucleic acid that encodes a hamster LIMP-2 protein having the amino acid sequence of SEQ ID NO: 2, wherein the siRNA comprises a double stranded sequence and one strand of the siRNA molecule has sufficient sequence complementarity to a hamster LIMP-2 RNA sequence to knock down expression of the nucleic acid that encodes the hamster LIMP-2 protein. In one embodiment, the one strand of the siRNA molecule has sufficient sequence complementarity to a LIMP-2 RNA sequence which encodes all or a portion of a lumenal domain of the hamster LIMP2 protein. In another embodiment, the LIMP-2 RNA sequence encodes all or a portion of amino acids 27-432 of SEQ ID NO: 2. In yet another embodiment, the one strand of the siRNA molecule comprises SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or a combination thereof.
In particular embodiments, the siRNA molecules of the present invention can result in at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% knockdown of LIMP-2 protein expression.
The invention also provides an expression construct which comprises the siRNA or shRNA molecules described herein. In addition, host cells comprising such expression constructs are provided.
Appropriate siRNA or shRNA for use in the methods of the present invention can be obtained using, for example, the methods described herein or obtained from commercial sources (e.g., Ambion, Inc; Invitrogen). In one embodiment, the siRNA is double stranded and can comprise a sequence that is from about 17 nucleotides to about 35 nucleotides. In particular embodiments, the siRNA is double stranded and one or both strands (e.g., sense, antisense) can comprise a sequence of about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides. The siRNA is generally comprised of RNA, and in some embodiments, can include DNA base pairs, either at the end of or within one or more of the strands of the siRNA.
Conditions under which LIMP-2 deficient cells are maintained so that a polypeptide or variant thereof that is dependent on LIMP-2 for trafficking, localization, stabilization and/or sorting is produced, are apparent to those of skill in the art (e.g., see Basic Techniques for Mammalian Cell Tissue Culture, Mary C. Phelan, 2003, Juan S. Bonifacino, et al. (eds.), Current Protocols in Cell Biology, John Wiley & Sons, Inc). In the particular embodiment in which β-GC is produced, examples of conditions under which LIMP-2 deficient cells are maintained so that β-GC is produced are provided in Furbish et al., Biochim. Biophys. Acta 673:425-434 (1981) and U.S. Pat. No. 5,549,892.
The cells for use in the methods of the invention can include plant or animal cells. As used herein, the term “animal” includes mammals, as well as other animals, vertebrate and invertebrate (e.g., birds, fish, reptiles, insects (e.g., Drosophila species), mollusks (e.g., Aplysia). In a particular embodiment, the animal is a mammal. The terms “mammal” and “mammalian”, as used herein, refer to any vertebrate animal, including monotremes, marsupials and placental, that suckle their young and either give birth to living young (eutharian or placental mammals) or are egg-laying (metatharian or nonplacental mammals). Examples of mammalian species include primates (e.g., humans, monkeys, chimpanzees), rodents (e.g., rats, mice, guinea pigs), ruminents (e.g., cows, pigs, horses) felines and canines In particular embodiment, the cell is a fibroblast or a macrophage.
Specific examples of suitable animal cells include, but are not limited to, the Chinese Hamster Ovary (CHO) cell line, including those designated CHO-K1, DG44, DUKX (also called DXB11), and CHO-S (commercially available from Invitrogen) and the hamster cell line BHK-21; the murine cell lines designated NIH3T3, NSO, C127, the simian cell lines COS, Vero; and the human cell lines HeLa, HEK293 (also called 293), PER.C6 (commercially available from Crucell) U-937 and Hep G2.
As described herein, expression of the polypeptide in a LIMP-2 deficient cell alters the localization of (missorts) the polypeptide compared to the localization of the same polypeptide that is expressed in a normal or wild type cell. Thus, the present invention is also directed to a method of altering trafficking of a lysosomal polypeptide that is dependent on a LIMP-2 polypeptide for trafficking to a lysosome. The method comprises culturing a LIMP-2 deficient cell which expresses the lysosomal polypeptide under conditions in which the trafficking of the lysosomal polypeptide to the lysosome is altered. In one embodiment, the altered trafficking results in increased secretion of the lysosomal polypeptide from the LIMP-2 deficient cell. In another embodiment, secretion of the lysosomal polypeptide is increased at least about 1.5-fold to about 20-fold, about 3-fold to about 18-fold, about 5-fold to about 15-fold and about 8-fold to about 10-fold, compared to secretion of the lysosomal polypeptide in a control cell. In a particular embodiment, secretion of the lysosomal polypeptide is increased at least about 11-fold compared to a control cell. Any suitable control cell can be used in the method. For example, the control can be a wild type cell of the same, or from a different species, as the LIMP-2 deficient cell.
The following Examples provide illustrative embodiments of the invention. One of ordinary skill in the art will recognize the numerous modifications and variations that may be performed without altering the spirit or scope of the present invention. Such modifications and variations are encompassed within the scope of the invention. The Examples do not in any way limit the invention.
EXPERIMENTATION Example 1 LIMP-2 is a Receptor for Lysosomal Mannose 6-Phosphate Independent Targeting of β-GCIn the study described herein, the lysosomal integral membrane protein type-II (LIMP-2) has been identified as a specific binding partner for βGC. LIMP-2 is a heavily N-glycosylated 478 residue type-III transmembrane protein (Fujita et al., 1991) comprised of a ˜400 amino acid lumenal domain, two transmembrane domains and a cytoplasmic domain of 20 amino acids. Based on homology, LIMP-2 has been defined as a member of the CD36 family of scavenger receptor proteins (Febbraio et al., 2001; Krieger, 2001). It was recently shown that over-expression of LIMP-2 caused an enlargement of early endosomes and late endosomes/lysosomes and an impairment of endocytotic membrane traffic out of the enlarged compartments (Kuronita et al., 2002; Kuronita et al., 2005). A deficiency of LIMP-2 in mice caused ureteric pelvic junction obstruction, deafness and peripheral neuropathy (Gamp et al., 2003) associated with an impaired vesicular trafficking and distribution of apically expressed proteins (Knipper et al., 2006).
Presented herein is in vitro and in vivo evidence that LIMP-2 acts as a receptor to bind βGC and that the βGC-LIMP-2 complex is transported to the lysosomal compartment in an MPR-independent pathway.
Experimental Procedures
Materials
Restriction enzymes and other reagents for molecular biology were purchased from New England BioLabs (Beverly, Mass.) and Fermentas (Burlington, Canada). SDS-PAGE gels and protein standards were obtained from Invitrogen (Carlsbad, Calif., USA). BCA protein assay kits and western blotting reagents were purchased from Pierce (Rockford, Ill., USA).
Cell Lines and Mice
Mouse embryonic fibroblasts from LIMP-2-deficient and wild type mice were generated from E12.5 embryos and primary cell lines between passage 3 and 6 were used for the experiments. For rescue experiments cells were transiently transfected using Fugene 6 (Roche; Mannheim, Germany). Primary macrophages were isolated from mice 4 days after peritoneal injection of 4% thioglycolate (Huynh et al., 2007). Wild type and LIMP-2-deficient mice (C57B6x129SV) (Gamp et al., 2003) were maintained in a conventional animal facility. All experiments were performed with approval of the National Animal Care and Use Committee of Germany.
Antibodies and Antibody Generation
The following primary antibodies were used: rat anti-mouse LAMP-2 (ABL93), rat anti-mouse LAMP-1 (1D4B), mouse anti-tubulin (E7) (Developmental Studies Hybridoma Bank; Iowa City, Iowa, USA), mouse anti-protein disulphide isomerase (1D3, a gift of Stephen Fuller at EMBL, Germany), anti-mouse actin (SIGMA, Steinheim, Germany), anti-mouse cathepsin-D (Pohlmann, R., et al., J Biol Chem, 270:27311-27318 (1995)), anti-KDEL (Stressgene, Victoria, BC, Canada), anti human glucocerebrosidase antibody 8E4 (kind gift of J. M Aerts, University of Amsterdam). Antibody to the HPC4-epitope fusion tag was obtained from Roche Applied Science (Indianapolis, Ind., USA). Antibodies to the HA-epitope (3F10) were from Roche (Mannheim, Germany), antibodies against the myc-epitope tag were obtained from Abcam (Cambridge, UK).
Polyclonal antibodies to human LIMP-2 were raised in rabbits at Pine Acres Rabbit Farm antibody service facility (Norton, Mass.) using purified recombinant LIMP-2 (R27-T432) as antigen. The resulting antiserum was affinity purified over CNBr-activated agarose resin (Sigma Chemical Co.) covalently coupled with LIMP-2 (R27-T432). Anti-mouse LIMP-2 antibodies were raised in specific pathogen free rabbits at Eurogentec (Seraing, Belgium) against 2 peptides (C-RFQINTYVRKLDD (AS-382-394 of mLIMP-2) (SEQ ID NO: 20) and C-MDEGTADERAPLIRT (AS-464-478 of mLIMP-2) (SEQ ID NO: 21)). The resulting antiserum was affinity purified over a mixture of ACH- and CNBr-sepharose covalently coupled with the aforementioned peptides. Secondary conjugated antibodies for immunoblotting and immunofluorescence studies were obtained from Sigma Chemical Co. (St. Louis, Mo.) and Molecular Probes (Carlsbad, Calif.), respectively. Polyclonal antibodies to murine β-glucocerebrosidase were raised in rabbits by plasmid injection followed by a boost with purified recombinant antigen (Aldevron Inc., Fargo, N. Dak.). The resulting antisera were affinity purified on recombinant antigen covalently coupled to NHS-activated sepharose.
Expression Plasmid Generation
cDNA clones encoding full length human LIMP-2 was obtained from the ATCC (Manassas, Va.). The sequences encoding LIMP-2 (R27-T432) or CD36 (G30-K437) were amplified by PCR using primers which generated 5′ EcoRI and 3′ HindIII sites at their ends. The 5′ primers also included the sequences encoding the honeybee mellitin signal peptide and the 3′ primers included sequences encoding the HPC4 epitope fusion tag in the case of LIMP-2, or a tandem 6×his-HPC4 fusion tag in the case of CD36. The resulting products were subcloned into the pFastBac-1 expression vector, and introduced into the Bac-to-Bac baculovirus expression vector (BEV) system following the manufacturer's protocols (Invitrogen, Carlsbad, Calif.). The sequence encoding recombinant human β-glucocerebrosidase was amplified by PCR from the cDNA sequences encoding the human placental isoform using primers to generate a 5′ NheI site and a 3′ ClaI site. This product was subcloned into the pFastBac-1 expression vector (Invitrogen, Carlsbad, Calif.). The β-glucocerebrosidase N370S, G202R, and L444P mutants were derived from the wildtype construct using the Quickchange II mutagenesis kit according to the manufacturer's protocol (Stratagene, La Jolla, Calif.). Plasmid harboring the cDNA for full length murine β-glucocerebrosidase was purchased from Invitrogen and the coding sequences amplified by PCR using primers to generate a 5′ EcoRI site and a C-terminal tandem 8×his+hpc4 tag flanked by a Not I site at the 3′ end. This product was subcloned into the Invitrogen pENTR1A entry vector and an expression plasmid generated using the Gateway system to transfer the coding sequences into the pDEST 8 destination vector for use in BEV system.
cDNA clones encoding the full length murine LIMP-2 (mLIMP-2) and βGC (mGC) were obtained from RZPD (Heidelberg, Germany). The sequences encoding mLIMP-2 and mGC were amplified by PCR and subcloned into the eukaryotic expression vector pFrog3 derived from pcDNA3 (Invitrogen) (Gunther, W., et al., Proc. Natl. Acad. Sci, USA, 95:8075-8080 (1998)). The myc-epitope was inserted after the last amino acid of mLIMP-2. All recombinant sequences were determined to be free of PCR errors by nucleotide sequence analysis (Sequegen Inc. or MWG-Biotech AG). Murine LIMP-2 (Δcc and L160P, soluble LIMP-2) mutants and GC (P415R and L444P) mutants were derived from the respective wild type constructs using fusion PCR based site directed mutagenesis. The coding sequences for the C-terminally HPC4-tagged human LIMP-2 mutants, L160P and L177), were ordered from DNA 2.0 (Menlo Park, Calif.), then transferred into the pFastBac1 insect cell expression vector using the Invitrogen Gateway system. To generate LIMP-2 with a strong ER-retention signal the C-terminal 14 amino acids of the human α2C-adrenergic receptor (-KHILFRRRRRGFRQ) (SEQ ID NO: 22) (Zerangue, N., et al., Proc. natl. Acad. Sci, USA, 97:3591-3595 (2000)) were fused to the C-terminus of LIMP-2 using PCR techniques.
Plasmids, Expression and Purification of Recombinant Proteins
Expression plasmids for LIMP-2 and βGC were generated as described in the Experimental Procedures. For protein expression in the BEV system Tn-5 cells (Expression Systems, CA) were infected with recombinant virus at an MOI=1. Conditioned medium was harvested 48 hr post-infection by centrifugation at 500 g and 0.22 μm filtered. Proteins expressed as fusions to the 12 amino acid HPC4 epitope tag were purified from the medium as described by (Rezaie et al., 1992). Recombinant human βGC was purified according to the method we previously described (Sawkar et al., 2006).
Affinity Chromatography and Binding Assays
Purified recombinant βGC was covalently coupled to CNBr-activated Sepharose 4B (Sigma Chemical Co., St. Louis, Mo.) as described by (Reczek et al., 1997) or coupled using the AminoLink immobilization kit (Pierce). Mouse tissue extracts were prepared from frozen tissues (Pel-Freeze Biologicals, Rogers, AR) and small-scale affinity chromatography experiments performed essentially as described (Reczek et al., 1997). For large-scale affinity chromatography, bound proteins were eluted from the coupled resin in 50 mM sodium acetate pH 4.7, 1M sodium chloride. Antibody-based pull down experiments were done using a monoclonal antibody to the HPC4 epitope covalently coupled to NHS-activated Sepharose 4 Fast Flow agarose support resin (AP-Biotech). Resin was incubated with solutions of HPC4-epitope tagged proteins, or non-epitope tagged proteins as controls, or with a mixture of epitope tagged protein and proteins to be tested for binding by co-capture in buffers containing 1 mM calcium chloride to allow for the calcium dependent HPC4 epitope-antibody interaction. 5-10 μg of each protein was used per reaction. Following extensive washing in the presence of 1 mM calcium chloride, HPC4-tagged and co-captured proteins were eluted in buffer containing 5 mM EDTA in place of calcium chloride and sample analyzed by SDS-PAGE followed by Coomassie staining.
Enzyme Activity Assays, PNGase-F, EndoH Treatment, and GL1-Substrate Analysis
The 4MU-based activity or colorimetric assay for βGC and alpha galactosidase and the quantitation of tissue glucosylceramide levels were performed using methods described previously (Marshall et al., 2002). PNGase F and EndoH treatment of LIMP-2 or βGC were performed using kits from New England Biolabs (Beverly, Mass.) and Roche (Mannheim, Germany) respectively.
LIMP2-siRNA in Hela Cells and Pulse-Chase-Analysis
HeLa cells were transfected with 60 pmol each of the LIMP2-RNAi-Duplexes (SCARB2 Stealth Select 3 RNAi; GAAAGCCAAACUAGGAGACACGAAA (SEQ ID NO: 12); CCAAAGAGAGAUGCAACCUAUUUGU (SEQ ID NO: 13); GAUGGAGAGGCUGACAUCAUGAUCA (SEQ ID NO: 14)) using Lipofectamine 2000 (Invitrogen, Paisley, UK). After 2 days, cells were pulsed for 2 hours with 250 μCi 35S-Methionine/Cysteine ([35S]Met-label, Hartmann Analytic GmbH, Braunschweig, Germany) and chased for different time periods in DMEM containing 2% FCS. Medium was collected and cells were lysed and immunoprecipitated using mAb 8E4 anti-h βGC or anti-LIMP-2 Ab and Protein-A-Agarose (Pierce, Rockford, USA). βGC was released from Protein-A-agarose by boiling for 5 min with 100 mM citrate, 1% SDS, 0.1% B-mercaptoethanol and incubated for 16 h with 15 mU of endoglycosidase H (Roche, Mannheim, Germany). Proteins were separated on a 10% SDS-Gel and exposed for 3 days to a BAS-MP Imaging Plate (Fuji, Tokyo, Japan). The Imaging Plate was analyzed with the Fuji FLA-5000 (Fuji, Tokyo, Japan) using AIDA Version 3.10 and Multigauge.
Analysis of β-GC Membrane Association
Cos7 cells were transfected with expression vectors for β-GC, LIMP-2myc or eGFP. 48 h after transfection cells were harvested in 0.5 ml PBS including protease inhibitors, sonicated and centrifuged at 1200 g. The post nuclear supernatants were either adjusted to 1% Triton X100 (cell extract) or further centrifuged at 45,000 g for 45 min. The resulting supernatants represent the soluble fraction and were used for SDS-PAGE analysis. The pellet (membrane fraction) was resuspended in RIPA buffer (150 mM NaCl, 50 mM Tris-Cl pH 7.4, 5 mM EDTA, 0.1% SDS, 1% NP40, 0.5% deoxycholate, proteinase inhibitors), sonicated, centrifuged for 10 min at 100,000 g and the resulting supernatant was used for SDS-PAGE analysis.
SDS-PAGE and Immunoblotting
Laemmli-SDS-PAGE gels were stained with Coomassie brilliant blue R-250, or stained using the Silver Stain kit from Owl Separation Systems (Portsmouth, N.H.). For immunoblots, proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Corp, Bedford, Mass.) using a semi-dry electroblotter (Biorad, Hercules, Calif.). All blots were developed using an enhanced chemiluminescent detection system (Pierce). Immunoblots were blocked in 10% milk in PBS or TBS containing 0.1% Tween-20, then probed with 0.2 μg/mL affinity purified anti-LIMP-2, anti-βGC, anti-LAMP-2, anti-cathepsin-D, or anti-tubulin antibodies in 1% milk or 1% BSA, followed by 0.1 μg/mL of an appropriate peroxidase conjugated secondary IgG in 1% milk or 1% BSA.
Immunostaining and Immunohistochemistry on Tissue Sections
For immunostaining cells were grown on glass coverslips, fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min, and permeabilized with PBS/0.2% saponin. The primary and secondary antibodies were diluted in PBS/3% BSA/0.2% saponin and incubated on cells for 1 h. Alexa Fluor 350-, 488-, or 594-conjugated secondary antibodies were from Molecular Probes (Eugene, Oreg.). Cells were embedded in Mowiol containing Dabco (Sigma Aldrich, Steinheim, Germany) as an antifading agent.
Spleen and liver of WT and LIMP-2 KO mice (n=2, each genotype) perfused with 4% paraformaldehyde (PFA) in 0.1M phosphate buffer (PB) were incubated in 30% sucrose in 0.1M phosphate buffer (PB) ≧24 h before preparing free-floating cryo-sections (50 μm) at a Leica microtome. For immunohistochemistry, sections were rinsed in 0.1M PB and blocked in 0.1M PB with 0.2% BSA, 4% normal goat serum, 0.5% Triton-X-100 for 2 hrs at RT. After incubation with first antibody, sections were rinsed with 0.1M PB/0.5% Triton-X-100 and incubated with secondary fluorescent antibodies (Alexa fluor 488 and 594, Molecular Probes, 1:1000). Primary antibodies used: LAMP-1 (1:500, Pharmingen); LIMP-2 (1:500), β-GC (1:250). DAPI (1:1000) was used to stain nuclei. Samples were examined and photographed with a Zeiss Axiovert 200M fluorescence microscope equipped with an Apotome to generate optical sections and an Axiocam MRm Rev. 2D camera, using Axiovision Software Rel 4.2 (Zeiss; Göttingen, Germany).
Northern Blotting
Total RNA of kidney, liver and fibroblasts were prepared using RNAeasy columns from Qiagen (Hilden, Germany). Total RNA samples were separated in a formaldehyde agarose gel and blotted on Nylon filters. Filters were hybridized overnight with a mouse βGC or mouse glyceraldehyde dehydrogenase (GAPDH) cDNA probe, washed, exposed to an imaging plate and developed with the Fuji FLA-5000-Phosphoimager (Fujifilm, Japan) using AIDA software (version 3.10).
Mass Spectroscopy
LIMP-2 affinity purified from murine liver extract was resolved by SDS-PAGE, stained with Coomassie brilliant blue R-250, excised from the gel, in-gel digested with trypsin, and the resulting peptides analyzed by nano-LC/MS/MS to obtain peptide sequence data. These data were used to query the National Center for Biotechnology Information (Bethesda, Md.) non-redundant database using the Mascot program (Matrix Science Inc, Boston Mass.).
Proteasomal Inhibition Experiments
Hela cells or MEF cells were treated for 8 h with 15 μM MG-132 (Merck, Darmstadt, Germany) and 25 μM ALLN (Merck, Darmstadt, Germany) in normal cell culture or in 35S-Met/Cys medium for metabolic labeling. For metabolic labeling, MG-312 and ALLN were present throughout the labelling and the chase period. Proteasomal inhibition was confirmed by the accumulation of high-molecular weight Ubiquitin-aggregates (detected by an mouse-anti-polyUbiquitin, clone FK1, Biotrend, Cologne, Germany).
Analysis of Coiled-Coil Probability
Coiled-coil probabilities within LimpII wild-type and respective mutants were determined with the program COILS (www.ch.embnet.org).
Results
Isolation and Identification of LIMP-2 as a β-Glucocerebrosidase Interacting Protein
The intracellular trafficking and enzymatic activity of βGC, unlike that of most lysosomal hydrolases, was unaffected in mouse embryonic fibroblasts doubly deficient for both MPRs (
The Lumenal Domain of LIMP-2 is Required for Association with Glucocerebrosidase
βGC resides in lysosomes as a protein associated with the lumenal membrane (Rijnboutt et al., 1991). Topologically, the lumenal portion of the LIMP-2 polypeptide was postulated to be in close proximity to this membrane (Okazaki et al., 1992). Therefore, whether this region in LIMP-2 might be responsible for the association with βGC (
The efforts described herein to isolate and identify LIMP-2 from tissue extracts revealed that full length endogenous LIMP-2 could be released effectively from βGC affinity resin by elution with buffers at lysosomal pH. βGC passes through intracellular compartments of varying pH en route from its point of synthesis in the ER to its final destination in lysosomes. To determine compartments in which the association or dissociation of LIMP-2 and βGC may occur, the ability of βGC affinity resin to bind L2LD over a range of pH was tested via small-scale in vitro binding reactions (
Effects of LIMP-2 Deficiency on β-Glucocerebrosidase Activity, Levels, and Localization
The in vitro binding data provided herein support a situation in which βGC and LIMP-2 interact directly within intracellular trafficking compartments of cells. The effects of LIMP-2 deficiency in the LIMP-2 knockout mouse were explored to determine whether there might be βGC-specific consequences. This was started by evaluating βGC activity levels in total tissue homogenates from wild type and LIMP-2 knockout mice (
The differences in βGC enzyme activity detected between LIMP-2 wild type and knockout tissues were almost certainly a reflection of the drop in total βGC protein levels, a conclusion that was further substantiated by immunohistochemical staining (
Reduction of lysosomal βGC levels has been found to correlate with an increase in the βGC substrate glucosylceramide (GlcCer) in Gaucher disease (Brady et al., 1965; Nilsson et al., 1985; Nilsson and Svennerholm, 1982). Analysis of GlcCer levels in tissues isolated from wild type and LIMP-2 knockout mice revealed an almost two-fold increase of GlcCer in the liver and lung from LIMP-2-deficient animals (Table 1) compared to normal.
LIMP-2-Deficient Lysosomes are Depleted of β-Glucocerebrosidase
To more precisely define the molecular events leading to the reduction in βGC activity levels in LIMP-2-deficient tissues we examined the status of βGC in mouse embryonic fibroblast (MEF) cells derived from wild type and LIMP-2-deficient embryos. Both the activity (
Given the dramatic effects of LIMP-2 deficiency on the status of βGC in cells whether LIMP-2 was directly required for the correct lysosomal localization of βGC was investigated. For this myc-tagged murine LIMP-2 was transfected into LIMP-2-deficient MEF cells and assessed whether LIMP-2 expression could rescue the mis-localization of βGC. Transfected cells were identified by staining with antibodies against the lumenal domain of LIMP-2 (not shown) or against the myc-epitope (
The requirement of LIMP-2 for the lysosomal localization of βGC was also supported by an additional experiment in COS cells in which LIMP-2 expression enabled the efficient delivery of βGC to the lysosomal compartment, but the fusion of LIMP-2 to a strong ER retention motif (Zerangue et al., 2000) prevented the transport of βGC out of the ER (
A Coiled-Coil Motif within the Lumenal Domain of LIMP-2 is Needed for-Glucocerebrosidase Binding
The isolated halves of the LIMP-2 lumenal domain (residues 27-155 and 155-432) do not bind βGC (data not shown). This suggested to us that a region around amino acid residue 155 might be required for the βGC-LIMP-2 association. Exploring this hypothesis, a conserved coiled-coil domain LIMP-2 from amino acid residue 150-167 (
LIMP-2 Expression Leads to Lysosomal Localization of a Clinical Glucocerebrosidase Mutant Normally Retained in the Endoplasmic Reticulum
To analyze if any of the more common clinical mutations within βGC affected binding to LIMP-2, βGC N370S, G202R and L444P were expressed, and interaction of LIMP-2 and these mutant proteins were analyzed in an in vitro binding assay. None of these mutations in βGC abrogated the binding to LIMP-2 (
β-Glucocerebrosidase is Mis-Targeted In Vitro and In Vivo
Since MPR-deficient mice and I-cell disease patients show an increased level of several lysosomal enzymes in their serum, whether an analogous observation might be made for βGC in the serum of LIMP-2-deficient mice was examined. Sera from wild type and LIMP-2-deficient mice were collected and total normalized protein samples analyzed for βGC levels by immunoblot. Whereas no βGC was found in the sera of wild type mice a significant level of βGC was observed in the serum from LIMP-2-deficient mice (
Pulse-chase experiments in HeLa cells depleted of LIMP-2 using siRNA allowed examination of the fate of βGC under conditions of LIMP-2 deficiency over time. (
Taken together, these experiments show that LIMP-2 was required for the intracellular retention of βGC and a failure to bind LIMP-2 led to mis-targeting and a marked increase in the secretion of βGC with residual intracellular βGC localized to the ER or degraded.
Discussion
In the present study a novel role for LIMP-2 as a sorting receptor required for the delivery of glucocerebrosidase to lysosomes has been identified. The Man-6-P receptor pathway has been very well characterized as a major route for the sorting of lysosomal enzymes, however the mechanism for the intracellular targeting of βGC to lysosomes has been unclear until now.
A role for LIMP-2 in the maintenance and biogenesis of lysosomes has been suggested based on overexpression studies in cultured cells (Kuronita et al., 2005) and on the phenotypic analysis of LIMP-2 knockout mice (Gamp et al., 2003; Knipper et al., 2006). Based on the data presented herein, a number of features and qualities of LIMP-2 can be seen in the context of its role as a M6P-independent trafficking receptor for βGC. Lysosomal membrane proteins such as LIMP-2, in contrast to soluble M6PR-binding lysosomal proteins, are directed from the Golgi to lysosomes in a manner unique from that of soluble proteins destined for this same compartment. Rather than relying on the M6PR for sorting and delivery they are incorporated into clathrin coated vesicles by virtue of the interaction of distinct targeting motifs in their cytoplasmic tails with multimeric adaptor proteins; in the case of LIMP-2 the tandem leu-ile residues in its cytoplasmic domain have been suggested to mediate such an association with AP-3 (Honing et al., 1998; Le Borgne et al., 1998; Ogata and Fukuda, 1994; Sandoval et al., 1994; Tabuchi et al., 2002; Vega et al., 1991b) and AP-1 (Fujita et al., 1999)) and affect its targeting. The present findings that βGC associates with LIMP-2, that these proteins co-localize in intracellular vesicular compartments, and that the activity, levels and localization of βGC exhibit a dramatic correlation with the presence or absence of LIMP-2 described herein, reveal that βGC independence of M6P-based sorting mechanisms is almost certainly a consequence of its routing through the lysosomal membrane protein delivery pathway. However, when the status of βGC and LIMP-2 in AP-3 deficient cells from the mocha mouse was explored, missorting or altered βGC localization was not observed, indicating that there may be other targeting motifs in LIMP-2 that do not involve interactions with AP-3 (data not shown). Additionally, the deletion of the cytoplasmic-tail of LIMP-2 did not result in an altered localization of this LIMP-2 (data not shown) indicating that additional sorting motifs exist within the transmembrane or lumenal domain of this membrane protein. The evidence that the association of βGC and LIMP-2 was sensitive to changes in the range of pH values found within the intracellular compartments through which these proteins pass is also consistent with a model for LIMP-2 based trafficking (
Such an early association is supported by our co-immunoprecipitation experiments, in particular after expression of a LIMP-2 ER retention mutant. Interestingly, also observed was that a clinical mutant of βGC, L444P, which is usually retained in the ER, can be shifted to the lysosomal compartment after co-expression of LIMP-2. These data not only suggest that an increase in LIMP-2 levels may be of some therapeutic interest for selected βGC mutations but also support the other data demonstrating that interaction can occur in the endoplasmic reticulum. When the fate of newly synthesized βGC was monitored in the absence of LIMP-2 it was observed that βGC release from the ER was retarded before βGC was missorted and secreted suggesting that LIMP-2 binding to βGC is needed for efficient ER exit towards the lysosomal compartment. βGC secretion in the absence of LIMP-2 is also substantiated by our experiments with proteasomal inhibitors which did not affect the rapid intracellular loss of βGC (
The in vitro binding studies described herein indicate that βGC interacts with the lumenal but not the cytoplasmic domain of LIMP-2. This region of LIMP-2 is in a context where it would encounter an intra-vesicular protein like βGC. βGC associates with membranes in a carbohydrate-independent manner (Aerts et al., 1988; Imai, 1988; Leonova and Grabowski, 2000) and this persists from the ER through lysosomes (Rijnboutt et al., 1991). Interestingly, it was found that overexpression of LIMP-2 in Cos7 cells significantly enhanced the membrane association of co-expressed βGC compared to the expression of βGC on its own (
It is still unclear whether LIMP-2 might, similarly to the M6PR, also recycle back from endosomes to the Golgi apparatus, and we can not completely rule out the possibility that other lysosomal proteins might also bind to LIMP-2 at this point. The results from our affinity chromatography experiments with mouse liver extract and the lack of LIMP-2 association with β-galactosidase and β-glucosidase (data not shown) however, highlight a rather specific nature for the LIMP-2/βGC interaction. LIMP-2 is homologous to CD36 and defines it as a member of the CD36-scavenger receptor-like protein family (Vega et al., 1991). Despite the fact that LIMP-2 and CD36 share ˜34% sequence identity over their lumenal domains we found that βGC did not bind to this region of CD36 under conditions in which it bound the corresponding region in LIMP-2. Thrombospondin-1 (TSP-1), however, has been demonstrated to bind both CD36 and LIMP-2 (Asch et al., 1987; Crombie and Silverstein, 1998).
The lumenal domain of LIMP-2 is homologous to CD36 and defines it as a member of the CD36-scavenger receptor-like protein family (Vega et al., 1991a). Members of this family have been shown to function in receptor roles in various cellular contexts. For example, CD36 acts at the cell surface to bind different ligands such as long chain fatty acids, modified low-density lipoproteins, thrombospondin-1 (TSP-1), and Plasmodium falciparum infected erythrocytes (Ge and Elghetany, 2005). Other members of this family such as CLA-1 and the Drosophila protein Croquemort function as a cell surface receptor for high density lipoproteins (Murao et al., 1997) and in embryogenesis as a monocyte receptor for apoptotic cells (Franc et al., 1996), respectively. Despite the fact that LIMP-2 and CD36 share ˜34% sequence identity over their lumenal domains it was found that βGC did not bind to this region of CD36 under conditions in which it bound the corresponding region in LIMP-2. TSP-1, however, has been demonstrated to bind both CD36 and LIMP-2 (Asch et al., 1987; Crombie and Silverstein, 1998). The differing ability of these two family members to bind βGC indicates that as a ligand βGC has an apparently higher level of specificity for LIMP-2 than does TSP-1. Since LIMP-2 is predominantly localized on intracellular membrane bound compartments (Vega et al., 1991a) and exhibits only low levels of cell surface expression (Maeda et al., 1999; Okazaki et al., 1992; Suarez-Quian, 1987, 1988) it is possible that the interaction between LIMP-2 and TSP-1 does not predominate in the physiological setting, particularly given that TSP-1 is found in the matricellular environment. It may be that there are two subtypes to the CD36 family: proteins like LIMP-2 which function intracellularly, and others like CD36, CLA-1 and Croquemort that preferentially act at the plasma membrane.
The binding studies indicate that βGC interacts with a coiled-coil domain in the lumenal domain of LIMP-2. The disruption of the coiled-coil domain abolished interaction with βGC yet allowed normal expression and lysosomal transport of these LIMP-2 mutants. Coiled-coil domains have been shown to be involved in protein-protein interaction (Lupas et al., 1991) and the experiments revealed a critical role of this domain in LIMP-2 for βGC binding. A more detailed structural analysis however, will be necessary to completely resolve the nature of the binding of both proteins.
A large variety of mutations that occur in the gene encoding βGC result in the accumulation of its substrate glucosylceramide (GlcCer) and consequently lead to Gaucher disease in humans (for review see: (Sidransky, 2004)). Manifestation of this disease is typically due to a deficiency in enzyme activity, inappropriate βGC localization, decreased βGC stability, or a combination of these factors (Beutler and Kuhl, 1986; Liou et al., 2006; Schmitz et al., 2005; Zimmer et al., 1999). Given the significant decreases in βGC protein levels and activity that was detected in tissues of LIMP-2 knockout animals and that this was a consequence of the enzyme being mislocalized, LIMP-2 knockout mice were examined more closely for phenotypes that might correlate with Gaucher disease. It was found that the levels of GlcCer substrate were elevated in LIMP-2-deficient liver and lung but apparently unaffected in other tissues, namely, kidney, spleen and brain (data not shown). Such tissue-specific differences could result from differences in substrate availability or utilization in each tissue; alternatively they could be due to varying amounts of residual βGC activity still detectable in LIMP-2 deficient animal tissues which may be sufficient to prevent GlcCer accumulation to levels at which more significant Gaucher-like pathologies might be seen. Despite the absence of robust Gaucher-like phenotypes in LIMP-2 knockout mice it is nonetheless interesting to speculate that some of the diversity in Gaucher phenotypes observed in humans carrying identical βGC mutations might result from secondary mutations in the LIMP-2 pathway.
It was observed that in contrast to the more common βGC disease related mutant L444P which, like the N370S and G202R mutants, still bound to the LIMP-2 lumenal domain, the βGC mutant P415R could not be transported to lysosomes after co-expression with LIMP-2 suggesting that this mutation directly or indirectly affects the binding site for LIMP-2. Structural analyses of the LIMP-2/βGC complex will be required to evaluate the importance of this βGC region for LIMP-2 binding.
In conclusion, a novel function for LIMP-2 as a Man-6-P-independent trafficking receptor for βGC has been identified. The discovery of this function helps link together many of the details from previous studies of both LIMP-2 and βGC biology and, importantly, it helps clarify the role proposed for LIMP-2 in the biogenesis and maintenance of lysosomes. The nature of how the interaction between these two proteins is mediated, whether there are additional cargoes for LIMP-2, and how this finding plays into the realm of LIMP-2 and βGC related pathologies will be the exciting subjects for future studies.
Isolation of Hamster LIMP-2 (haLIMP-2) Open Reading Frame
Total RNA was prepared from the a CHO parental cell line and high fidelity reverse transcriptase (Stratagene) was used to synthesize cDNA from the RNA preparation. The resulting cDNA preparation was used as a template for PCR. Several forward and reverse PCR primers were designed based on the alignment of human, mouse, rat, and mucaca LIMP-2 cDNA sequences (
Generation of Reagents Using the haLIMP-2 Sequence
Plasmids for Mouse Injection
The haLIMP-2 open reading frame and the haLIMP-2 lumenal domain were each cloned into mammalian expression vectors. The full length open reading frame fragment was excised from the Topo-Blunt DNA vector and ligated into pcDNA3.1(−) (Invitrogen). A DNA fragment encoding the lumenal domain was PCR amplified to include a stop codon at the 3′ end. The PCR product was gel purified and ligated into pENTR/GHSS (derived from Invitrogen pENTR™, with start codon/human growth hormone signal sequence, “GHSS” added). The GHSS/haLIMP-2 lumenal domain fragment was inserted into pcDNA-DEST40 (Invitrogen) by Gateway™ recombination.
siRNA Duplexes for RNAi Knockdown Experiments
Five siRNA duplexes were designed using an algorithm based on published reports (Reynolds et al., Nature Biotechnology, 22(3):326-330 (2004); Takasaki, S., et al., Computational Biology and Chemistry, 30, 169-178 (2006)). Specifically: The haLIMP-2 open reading frame sequence was analyzed using a computer program written to evaluate 19 base pair sections for potential use in siRNA duplexes. The rules utilized by the program were adapted from an established, frequently cited publication (Reynolds et al., Nature Biotechnology, 22(3):326-330 (2004)) and are summarized in Table 2. Note: the program did not predict hairpins, thus the third rule on the list was not incorporated.
In addition, an algorithm utilizing a scoring table derived from statistical analysis was used in a similar manner to evaluate blocks of the haLIMP-2 open reading frame for utility as siRNA duplexes. This scoring method was reported by Takasaki, S., et al., Computational Biology and Chemistry, 30, 169-178 (2006)). Use of the two scoring methods provided five candidate siRNA duplex sequences. Two sequences, si976 and si1182, were the top scoring sequences from the first algorithm (Reynolds, A., et al., Nature Biotechnology, 22(3), 326-330 (2004)). Sequence si376 received the top scoring from the second method (Takasaki, S., et al., Computational Biology and Chemistry, 30, 169-178 (2006)), based on both the scoring table and GC content. The final two sequences, si202 and si1135, were selected based on combinatorial analysis of the two sets of results, described below. Sequence si1135 was also near the top of the Reynolds et al. scoring (tied with 6 other sequences for 3rd best score).
Five siRNA duplexes designed through use of these algorithms were ordered from Invitrogen (custom siRNA service); these and their sequences are in Table 3 below.
The position indicated refers to the basepair index in the haLIMP-2 ORF corresponding to the first 5′ base of the sense strand of the siRNA duplex. The [dT] designates the two deoxyribose thymines added to the 3′ ends of both strands in each duplex.
TaqMan Primer/Probe Sets
Using Applied Biosystems Primer Express software, two optimal primer/probe sets for TaqMan quantitative PCR analysis were designed using the haLIMP-2 open reading frame sequence. The primers and probes were ordered from Integrated DNA Technologies.
Experimental Conditions
Optimal siRNA transfection conditions were initially determined for a stable recombinant human β-GC expressing CHO cell line using Lipofectamine™ 2000 (Invitrogen) and the BLOCK-iT™ fluorescent oligo (Invitrogen). Also, both TaqMan primer/probe sets successfully detected haLIMP-2 in cDNA samples from the cell line and one primer/probe set was chosen for use in all subsequent work.
All optimization experiments were done in 6-well plates. For each transfection, the CHO cell line was seeded at 3.0×105 cells/well in MEMα+GlutaMAX™-I medium with ribonucleosides and deoxyribonucleosides (Invitrogen) containing 10% fetal bovine serum (Invitrogen) and incubated overnight at 37° C., 5% CO2. Cells were transfected the next day, when they were approximately 30% confluent. The siRNA and the Lipofectamine™ 2000 were each diluted in OptiMem® medium (Invitrogen) then mixed and added to the cells. Within the scope of three experiments, siRNA transfection conditions were optimized for haLIMP-2 knockdown:
1) siRNA duplex concentrations of 50, 100, and 200 pmol per 2 ml medium (per well) were compared, keeping Lipofectamine™ 2000 concentration constant (7.5 ul per 2 ml medium) and assessing knockdown efficiency through 48 hours post-transfection;
2) Lipofectamine™ 2000 concentrations of 2.5, 5.0, and 7.5 ul per 2 ml medium were compared, keeping siRNA concentration constant (100 pmol) and assessing knockdown efficiency through 48 hours post-transfection
3) siRNA concentrations of 100 and 200 pmol per 2 ml medium were compared, keeping Lipofectamine™ 2000 concentration constant (7.5 ul per 2 ml medium) and assessing knockdown through 96 hours post-transfection (without a 24 hour time point).
In a particular embodiment, the conditions were 200 pmol siRNA and 7.5 ul Lipofectamine™ 2000 per 2 ml medium (per well of 6-well plate).
haLIMP-2 RNA Knockdown Results
To determine the degree of haLIMP-2 knockdown in the rhGC cell line, cells from an individual well of 6-well plate or from an individual T25 flask were harvested at 24, 48, 72, and/or 96 hours post-transfection (each time point from an individual well of 6-well plate). For T25 transfections, all conditions (cell number, medium volume, siRNA concentration, and lipid concentration) were scaled according to the increase in surface area from the 6-well plate experiments, and as noted previously, cells were approximately 30% confluent at the time of transfection. Two control conditions were also consistently done: 1) cells that were transfected with a non-specific siRNA (designed from huLIMP-2 3′UTR sequence that is not contained in the mouse, rat, and presumably, hamster sequence) and 2) untransfected cells. In one experiment, a lipid only control was also included (Lipofectamine™ 2000 diluted in OptiMem®). Total RNA was isolated from harvested cells using the illustra RNA Spin Mini kit (GE Healthcare), and cDNA was prepared from the RNA using the Ready-To-Go™ You-Prime First Strand Beads kit (Amersham). The cDNA samples were analyzed by TaqMan in two separate plates, one using the haLIMP-2 primers and probe to determine the LIMP-2 RNA level in each sample, and the other using elongation factor 1 (EF1) primers and probe to determine the RNA level of an internal standard. To illustrate the degree of knockdown obtained with siRNA treatment, the haLIMP-2 value was normalized to the EF1 value in each sample and the normalized values were graphed as a percentage of the untransfected control sample values.
All five siRNAs resulted in haLIMP-2 RNA knockdown, ranging from approximately 60-80% knockdown. The most effective siRNAs were si976 and si1135, which reproducibly resulted in approximately >80% knockdown of haLIMP-2 RNA in an rhGC cell line. Results (FIGS. 20 and 21A-21B) demonstrate stable knockdown of LIMP-2 RNA levels through 96 hours post-transfection. Each transfection was done in duplicate and results are shown as the averages of the duplicate samples. “siMock” samples were transfected with the non-specific siRNA noted above, “UT” samples were untransfected cells, and “Lipid” samples were treated with lipid only as noted above.
Results
GC Production Levels from LIMP-2 siRNA Treated rhGC Cells
To assess GC production levels of the haLIMP-2 siRNA transfected rhGC cells and control cells, cells in T25 flasks were incubated in serum-free conditioning medium for 24 hours. Briefly, at a designated time point post-transfection (detailed below), growth medium was removed, cells were rinsed twice with phosphate buffered saline (PBS), and 1.5 ml serum-free conditioning medium was added to the cells. Cells were incubated for 24 hours at 37° C., 7.5% CO2. GC activity levels in the conditioned media harvests were determined by a 4MU-βGlc activity assay (Marshall et al., 2002). Each resulting GC value was normalized to total cell number at the time of media harvest to calculate the specific production rate (SPR) for GC from each sample.
For
For
For
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The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.
While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
Claims
1. A nucleic acid molecule comprising SEQ ID NO: 1.
2. A nucleic acid molecule that encodes an amino acid sequence comprising SEQ ID NO: 31.
3. An isolated polypeptide having an amino acid sequence comprising SEQ ID NO: 31.
4. An expression construct comprising SEQ ID NO: 1.
5. A host cell comprising the expression construct of claim 4.
Type: Application
Filed: Jan 9, 2015
Publication Date: Sep 3, 2015
Inventors: David J. Reczek (Sudbury, MA), Paul Saftig (Gettorf), Christine T. DeMaria (Franklin, MA)
Application Number: 14/593,904
