KR101572412B1 - Regulation of excitatory synaptic transmission by heparan sulfates and uses thereof - Google Patents

Abstract

The present invention relates to a regulation of excitatory synaptic transmission by heparan sulfate sugars and a use thereof. By investigating a role of leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs) as a co-receptor of a glypican/leucine-rich repeat transmembrane protein 4 (GPC/LRRTM4) synapse binding complex during a development of an excitatory synapse through a heparin sulfate-dependent way, it is possible to provide a treatment strategy for maximizing synaptic neuro-transmission efficiency and improving brain functions by targeting heparin sulfate.

Description

Regulation of excitatory synaptic transmission by heparan sulfate < RTI ID = 0.0 > and uses thereof < / RTI >

The present invention relates to the regulation of excitatory neurotransmission by the heparan sulfate sugar and its use.

Synaptic adhesion molecules regulate the overall pattern of synapse development, and specific synaptic adhesion proteins, called 'synaptic modulators', modulate the mammalian synaptic structure and function. As the technology in the field of mass spectrometry continues to develop in recent years, synaptic regulators, their ligands and a number of synaptic attachment pathways are being discovered.

In vertebrates, LAR-RPTPs (leukocyte common antigen-related receptor protein tyrosine phosphatases), including three components (LAR, PTPδ and PTPσ), have recently emerged as synapse modulators. They mediate presynaptic differentiation by various postsynaptic ligands. For example, all three members of the LAR-RPTP family bind netrin-G ligand 3 (NGL-3), PTPδ binds IL1RAPL1 (interleukin-1 -receptor accessory protein 1) Lt; / RTI > In addition, the PTPδ and PTPσ proteins except for LAR bind to Slitrks (Slit- and Trk-like proteins). However, the various combinations of these methods are not yet understood at the molecular level. The LAR-RPTP ligands mentioned above are present only in vertebrates and refute that their synaptic attachment pathways do not account for the entire synaptic function of evolutionarily conserved LAR-RPTPs. In fact, many studies have shown that LAR-RPTP orthologs of invertebrates (dLAR of D. melanogaster and PTP-3 of C. elegans ) are important for neural development and function in this organism. As a candidate for the evolutionarily conserved LAR-RPTP ligand, the GPC (glypican) family of HSPGs (heparan sulfate proteoglycans) is included.

GPC is connected to the cell membrane by an anchor GPI (glycosylphosphatidylinositol). GPCs contain six members (GPC-1 to -6) in mammals, two members (Dally and Dally-like proteins) in Drosophila and one member (GPN-1) in Pretty Little Nematode. In Drosophila, Dally-like protein (Dlp) and another HSPG syndecan (Sdc) work side-by-side to regulate the induction of the midline axillary process, but they function in synapse development in combination with dLAR. In vertebrates, GPCs are primarily expressed during neural development and act as induction signals for axonal prospecting and neuronal migration. Consistently, GPCs also work with Slit, an axon protrusion inducing molecule. In addition, they regulate several signaling pathways including Wnt, Hedgehog, fibroblast growth factor and bone morphogenic protein pathways. Although the mechanisms by which GPCs work in mammalian synapses are not yet known, they have recently been shown to be synaptic receptors for Leucine Rich Repeat Transmembrane Domain Protein 4 (LRRTM4). Since GPCs are attached to the GPI, pre-synaptic membrane proteins that have not yet been identified will be involved in the action of GPC / LRRTM4 through co-receptors. Since the functional interactions of Dlp and dLAR are well established, we have assumed that LAR-RPTPs in mammals are functional auxiliary receptors for the GPC-4 / LRRTM4 synaptic adhesion complex during excitatory synapse development.

As part of this effort, we have confirmed that GPC-4 is a potential ligand for PTP σ and demonstrated that GPC-4 interacts with PTP σ and LAR only in the cis -sequence with an affinity for nano-mol concentration, Formation analysis confirmed that the binding properties of PTPσ to HS were functionally required for the synaptic action of LRRTM4 and for excitatory synaptic transmission in cultured neurons and that PTPσ was a true synapse for the GPC-4 / LRRTM4 synaptic adhesion complex Receptor, thus completing the present invention.

U.S. Published Patent Application No. 2014-0045762 (Feb.

 Charlotte H. Coles et al. Science, vol. 332, pp. 484-488 (March 31, 2011)

It is an object of the present invention to identify the role of LAR-RPTPs in the heparan sulfate-dependent manner as a co-receptor of the GPC / LRRTM4 synaptic adhesion complex during excitatory synapse development and to target this to maximize synaptic transmission efficiency while promoting complex formation And to provide a kit and a method for screening low molecular substances that can be used for brain function enhancement and the like.

In order to accomplish the above object, the present invention provides a method for producing a complex of GPC-4 (glypican-4) and LAR-RPTPs (leukocyte common antigen-related receptor protein tyrosine phosphatases) through cis-cellular adhesion Which comprises a cell component and a protein component which can be used as an active ingredient,

The cell component is selected from among GPC-4 expressing cells or LAR-RPTPs expressing cells,

Wherein said protein component is a wild type or recombinant GPC-4 protein comprising heparan sulfate, or a fragment thereof, or

A wild-type or recombinant LAR-RPTPs protein comprising a heparan sulfate binding site, or fragments thereof, for the prevention or treatment of diseases associated with membrane protein dysfunction.

In addition, the present invention relates to a cell component capable of forming a complex of GPC-4 (glypican-4) and LAR-RPTPs (leukocyte common antigen-related receptor protein tyrosine phosphatases) through cis- And contacting the protein component with any candidate material under conditions to form the complex; And

If the candidate substance is assessed as promoting or inhibiting the formation of the GPC-4 / LAR-RPTPs complex and is assessed to promote GPC-4 / LAR-RPTPs complex formation, Determining a therapeutic drug,

The cell component is selected from among GPC-4 expressing cells or LAR-RPTPs expressing cells,

Wherein said protein component is a wild type or recombinant GPC-4 protein comprising heparan sulfate, or a fragment thereof, or

A wild-type or recombinant LAR-RPTPs protein comprising a heparan sulfate binding site, or fragments thereof, for screening candidate substances for the prophylaxis or treatment of diseases associated with membrane protein dysfunction.

The present invention identifies the synaptic function of LAR-RPTPs by way of binding dependent on heparan sulfate as a co-receptor of the GPC / LRRTM4 synaptic adhesion complex during excitatory synapse development, Maximizing the function of the brain, and improving the brain function.

1 shows the results of affinity purification of GPC-4 as a PTP? -Ligand in the brain of a rat, wherein A is the recombinant Ig-control used in affinity chromatography and Coomassie blue stained gel photograph of the Ig-PTP? Fusion protein And B is the result of performing pull-down analysis on IgG or Ig-PTP for dissociated rat synaptosomes, the blue box exhibiting a specific band specific for the Ig-PTPσ-binding fraction. CD extracts ion m / z 972.03 and 517.28 from liquid chromatograph (LC) separated total ion chromatogram, PTP? (34.05 min) and GPC-4 (24.88 min; D) of Ig-PTP? (F), as obtained from LC-MS / MS at 972.03 and 517.28 divided to make an MS / MS spectrum with the? - and? (E / MS), and G is HK293T cells expressing HA-PTP ?, LRRTM4-EGFP or LRRTM2-mVenus are treated with a control IgC or Ig- GPC-4 and analyzed by immunofluorescence imaging for Ig-fusion protein (red) and HA / mVenus (green). Scale bar = 15 탆 (applies to images). H shows saturation binding of Ig-GPC-4 to PTP σ expressed in HEK293T cells. HEK293T cells expressing only PTP σ or pDisplay vector with different concentrations of GPC-4 and HA were cultured and bound GPC- 4 was measured using a HRP-labeled secondary antibody. The internal graph shows the Kd calculated in three independent experiments and the Scatchard plot resulting from the linear regression of the data. Data are presented as means ± SEM.
FIG. 2 shows the results of the absence of any interaction between neurexin-1β splice variants and LRRTM4.
Figure 3 shows that GPC-4 does not interact with various cell surface proteins.
Figure 4 shows the results of GPC-4-binding domain analysis of LAR-RPTPs, where A is a diagram of the PTPσ vector used in the cell-surface binding assay, B is the PTP? HEK293T cells expressing PTP? -ΔIg were cultured with IgC or IgC-GPC-4 and analyzed by immunofluorescence imaging for Ig-fusion protein (red) and HA-PTPσ protein (green) exposed on the surface. CE was cultured with HEK293T cells expressing splice variants of LAR-RPTPs Ig1-3 with IgC or Ig-GPC-4 and immunoprecipitated with LAR-RPTPs (red ) Were analyzed by immunofluorescence. The scale is marked with bars within BE = 10 μm (applies to all images). F is a result of saturation binding of Ig-GPC-4 with a subset of PTPσ splice variants (PTP σ Ig and PTP σ Ig ^MeA ) expressed in HEK293T cells. Data were expressed as mean ± SEM.
Figure 5 shows the results of cis -interaction of PTPσ and GPC-4 in a heparan sulfate-dependent manner and AB represents representative image (A) and data quantification (B) from cell-attachment assays and only EGFP HEK293T cells expressing HEK293T cells expressing both EGFP and PTP? Were mixed with HEK293T cells expressing only DsRed (control) or HEK293T cells expressing DsRed and GPC-4 or TrkC at the same time, Were quantified. Scale bar = 100 ㎛ (applies to all images). C is a schematic diagram of the PTP σ structure used in DE, D is the PTPσ vector and HEK293T cells are transfected with vehicle (+ veh) or 1 U / mL heparinase III (+ hep III) , And staining with HA antibody (green) under non-osmotic conditions to detect and transmit surface-exposed PTPs (red), followed by staining with 3G10 antibody. E shows that transfected HEK293T cells are treated with vehicle or hep III and incubated with IgC or IgC-GPC-4 followed by Ig-fusion protein (red) and surface exposed HA-PTPσ (green) This is the result of analysis by double-immunofluorescence microscopy. F shows the results of the same analysis in E except that the cells were incubated with 10 mM EGTA (+ EGTA). The scale bar in DF is 10 μm (applies to all images). GH was the result of a pull-down analysis (5% of total) performed with IgC and Ig-GPC-4 (G) or Ig-PTP? (H) using HEK293T cells expressing the indicated vector, Numbers represent molecular mass markers (kDa). The immunoblot analysis revealed that GPCs appeared at two positions, the ~65-kDa band represents the full-length HA-GPCs (not truncated), and the ~37-kDa band represents the N-terminal truncated fragment (truncated).
Figure 6 shows that PTPσ is a functional co-receptor for GPC-4 in mediating LRRTM4-induced pre-synaptic differentiation. AB represents a representative image (A) and quantification result (B) of heterologous synaptogenic activities of LRRTM4 and LRRTM2 ). Neurons were infected with control lentivirus (sh-Control) or LAR-KD (sh-LAR) or shRNA-expressing PTPσ-KD (sh-PTPσ) or with PTPσ-KD and LAR- ), LAR / PTP σ shRNA plus PTP σ WT (+ PTP σ WT rescue), or LAR / PTP σ shRNA plus PTPσ AAAA (+ PTPσ AAAA rescue). The infected neurons are then incubated with HEK293T cells expressing EGFP alone (control), LRRTM4-EGFP (LRRTM4), or LRRTM2-mVenus (LRRTM2) for 2 days and then incubated for EGFP (blue) and cynapsin Lt; / RTI &gt; The synapse-forming activity was quantitated by EGFP fluorescence with a synapsein staining ratio. Panel D statistics were measured by ANOVA Tukey's test ( ² *, p <0.01; ³ *, p <0.001). Scale bar = 25 μm (applies to the entire image). The numbers listed on the bar chart refer to the number of HEK293T cells.
FIG. 7 is electrophysiological data showing that PTPσ / LAR double knockdown does not affect neurotransmitter release or alter the intrinsic properties of neurons.
Figure 8 shows that PIVσ is required for heparan sulfate-dependent excitatory synaptic transmission in cultured neurons. As a result, LIV / sh-PTP?, LAR / PTP? ShRNA plus PTP? WT (+ PTP? WT (A) of miniature excitatory post-synaptic currents (mEPSCs) recorded in DIV 14-16 in cultured hippocampal neurons coinfected with lentiviruses expressing LAR / PTP sigma shRNA plus PTP sigma AAAA (+ PTP sigma AAAA rescue) And a summary graph of frequency (B) and amplification (C) of mEPSCs from infected neurons. Data shown in B and C are expressed as mean ± SEM (*, using p <0.05 student's test). Numbers written in the bar graph refer to the number of neurons.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention relates to a cell component capable of forming a complex of GPC-4 (glypican-4) and LAR-RPTPs (leukocyte common antigen-related receptor protein tyrosine phosphatases) through cis- Wherein the cellular component is selected from among GPC-4 expressing cells or LAR-RPTPs expressing cells, wherein the protein component is selected from the group consisting of wild type or recombinant GPC-4 protein comprising heparan sulfate, Or a recombinant LAR-RPTPs protein comprising a heparan sulfate binding site, or a fragment thereof, to a screening kit for a disease preventing or treating drug associated with membrane protein dysfunction.

The present invention also relates to the use of a cell component capable of forming a complex of GPC-4 (glypican-4) with LAR-RPTPs (leukocyte common antigen-related receptor protein tyrosine phosphatases) through cis- And contacting the protein component with any candidate material under conditions to form the complex; And determining whether the candidate substance promotes or inhibits the formation of the GPC-4 / LAR-RPTPs complex and is believed to promote GPC-4 / LAR-RPTPs complex formation. Or a therapeutic agent, wherein the cellular component is selected from among GPC-4 expressing cells or LAR-RPTPs expressing cells, wherein the protein component is a wild type or recombinant GPC-4 protein comprising heparan sulfate, , Or a wild-type or recombinant LAR-RPTPs protein comprising a heparan sulfate binding site, or a fragment thereof, for screening candidate substances for the prophylaxis or treatment of diseases associated with membrane protein dysfunction.

Heparan sulfate is a sugar present in various blood vessels and is a target to be considered in the development of anticancer drugs such as inhibiting the growth of cancer cells by affecting cancer blood vessels in general. The present invention suggests that heparan sulfate directly regulates the neurotransmission efficiency of synapses, the basic unit of brain function. Heparansulfate binds to specific adhesive proteins present in the membrane of nerve cells to regulate neurotransmission of excitatory synapses. If heparan sulfate fails to bind, the mechanism of regulation of excitability and inhibition of neuronal cells is destroyed. It can be the main mechanism of onset. Therefore, a low molecular substance that promotes the binding of PTP σ and heparan sulfate can maximize synaptic nerve efficiency and induce brain hyperactivity, and thus can be used as a therapeutic agent for brain diseases associated with dysfunction of membrane protein.

A brain disease associated with dysfunction of the membrane protein refers to a brain disease that can be caused by dysfunction of the GPC-4 protein, for example, autism.

In addition to the above-mentioned brain diseases, the low-molecular substance may be used for the treatment of diseases associated with dysfunction of membrane protein, GPC-4 protein or LAR-RPTPs protein such as Diabetes Mellitus, Wilms tumor 1, Simpson-golabi-behmel Syndrome type 1, neoplasms, gigantism, carcinoma, cartilaginous exostosis, malignant neoplasms, anxiety (Nervousness) Genetic Diseases (X-linked), Beckwith-wiedemann Syndrome, Chromosomal Translocation, Limb Deformities, Congenital, Intraepithelial Neurodegenerative Disorders, Uterine Cervical Neoplasm, Bone Neoplasms, Hydrocephalus, and the like can be used as a therapeutic agent for neoplasia, neuropathy, neoplasia, cardiac arrhythmia, neurodegenerative disorders, cervical dysplasia, bone neoplasms or hydrocephalus.

The present inventors have found that, From a neuron The importance of synaptic protein action between LAR-RPTPs and GPCs has been explored, and this effect is likely to be influenced by the availability of heparan sulfate in neuronal membranes, but the effect of post synaptic adhesion molecule (LRRTM4) Binding activity of PTP sigma in order to act as a receptor and as a key factor for excitatory synaptic neuronal transmission.

According to one embodiment of the present invention, in order to search for PTP? Ligands of LAR-RPTPs, binding between PTP? And cell surface-protein of GPC-4 protein was confirmed to bind strongly, As the amount of GPC-4 protein was increased, binding affinity was measured and it showed high affinity depending on the concentration.

As a result of performing cell surface-protein binding analysis by including or deleting the Ig domain of PTP σ in order to search for the binding site of PTP σ and GPC-4, it was found that only GTP-4 was expressed only in the cells expressing PTP σ- , And specifically confirmed that such binding occurs at the heparan sulfate binding site of the Ig domain.

In addition, the binding of GPC-4 / PTPσ only occurred when the cell-protein sequence, that is, the cis-sequence, was not observed in the trans-cell adhesion experiment, which means cell-cell binding, - interaction.

Next, in order to clarify the role of GPC-4 and LAR-RPTPs in LRRTM4-mediated pre-synaptic differentiation of neurons, synaptic-formation analysis was performed using shRNA for PTPσ or LAR, (WT) was expressed in LAR / PTPσ-deficient neurons, the synaptic activity of LRRTM4 was regressed, and GPC-4 The heparan sulfate-dependent interaction of PTPσ is essential for induction of pre-synaptic differentiation by LRRTM4, indicating that PTPσ acts as a presynaptic receptor for LRRTM4.

In addition, in order to investigate whether the heparan sulfate binding characteristics of LAR-RPTPs are involved in pre-synaptic functions, such as neurotransmitter release or neurotransmission, paired-pulse ratio (PPR) was measured using shRNA for PTPσ or LAR As a result, no significant change in the PPR between the control and the LAR / PTPσ-deficient neurons was observed. Thus, LAR and PTPσ do not directly regulate neurotransmitter release in excitatory synapses. In addition, when miniature excitatory post synaptic currents (mEPSCs) were recorded by using shRNA for PTPσ or LAR in hippocampal neurons, the frequency and amplitude of mEPSCs were significantly decreased when PTPσ or LAR was suppressed, , And re-expression of the PTPσ wild-type (WT) in LAR / PTPσ-deficient neurons completely restores the decrease in frequency and amplitude of mEPSCs. In addition, it is believed that PTP? And LAR regulate the density of functional excitatory synapses in a heparan sulfate-dependent manner, because inhibiting PTP? Or LAR does not reduce the potential for neurotransmitter release.

The screening kit for preventing or treating disease associated with membrane protein dysfunction according to the present invention comprises a cell component and a protein component capable of forming a GPC-4 / LAR-RPTPs complex through cis-cell adhesion as an active ingredient .

The term &quot; cis-cell adhesion &quot; refers to adhesion between cell surface and protein. When a cell component and a protein component contained in the screening kit of the present invention bind to each other to form a complex, Determining whether the formation of the complex is promoted or inhibited and judging any candidate substance that promotes complex formation to be a disease prevention or treatment agent associated with membrane protein dysfunction.

The complex may be formed in a heparan sulfate dependent manner. Thus, the cell component and the protein component comprise a heparan sulfate or heparan sulfate binding site.

The cell component may comprise heparan sulfate or a GPC-4 expressing cell or a LAR-RPTP expressing cell comprising a heparan sulfate binding site. Wild-type or recombinant LAR-RPTPs, preferably wild-type or recombinant IgC-GPC-4 comprising heparan sulfate, or GPC-4 expressing cells transformed to express these fragments and a heparan sulfate binding site, Or LAR-RPTPs expressing cells transformed to express fragments thereof.

In the case where the cell component is a cell containing heparan sulfate, a protein containing a heparan sulfate binding site is selected, and when the cell component is a cell containing a heparan sulfate binding site, the protein component includes a protein containing heparan sulfate Can be selected.

Thus, the protein component may be selected from wild-type or recombinant GPC-4 protein comprising heparan sulfate, or a fragment thereof, or a wild-type or recombinant LAR-RPTPs protein comprising a heparan sulfate binding site, or fragments thereof . The fragment of the wild-type or recombinant LAR-RPTPs protein may be the PTP? Protein or the Ig domain of PTP?, But may be used without restriction if it is a fragment of a wild-type or recombinant LAR-RPTPs protein comprising a heparan sulfate binding site.

Also, the GPC-4 expressing cells or LAR-RPTPs expressing cells are transformed cells, and the host cells used for the transformation are HEK293T (Human Embryonic Kidney 293T) cells, L-cells, COS- Cells, and the like, but are not particularly limited thereto.

The substance that promotes the formation of the GPC-4 / LAR-RPTPs complex maximizes synaptic nerve efficiency, thereby inducing brain hyperactivity. Therefore, it can be used as a therapeutic agent for brain diseases associated with dysfunction of membrane protein. In addition to the brain diseases, the substance which promotes the formation of the GPC-4 / LAR-RPTPs complex can be used as a therapeutic agent for the above-mentioned diseases related to GPC-4 or LAR-RPTPs protein abnormality.

Therefore, the substance may be a low molecular substance which promotes expression of a coding gene of GPC-4 or LAR-RPTPs and / or expression or activity of a protein.

The low-molecular substance may be a chemical substance, a nucleotide, an antisense-oligonucleotide, a shRNA (short hairpin RNA), an siRNA (small interference RNA), a miRNA (microRNA) or a natural product extract.

The method for measuring whether or not the low-molecular substance promotes or inhibits the formation of the GPC-4 / LAR-RPTPs complex can be carried out by employing various methods known in the art. For example, RT-PCR, Northern blot, hybridization using a cDNA microarray, in situ (in situ hybridization, immunohistochemistry, radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, Western blotting, ELISA, capture-ELISA, sandwich assay and the like.

The present invention also provides a pharmaceutical composition for the prophylaxis or treatment of diseases associated with membrane protein dysfunction comprising a substance which increases the concentration of GPC-4 or LAR-RPTPs as an active ingredient.

The present invention also relates to a pharmaceutical composition for treating a disease associated with membrane protein dysfunction, which comprises a substance which promotes the expression of a coding gene of GPC-4 or LAR-RPTPs or which promotes the expression or activity of GPC-4 or LAR- Or a pharmaceutically acceptable salt thereof.

The pharmaceutical composition of the present invention may contain, as an active ingredient, a chemical substance, a nucleotide, an antisense-oligonucleotide, an shRNA, an siRNA, an miRNA or a natural product extract.

The pharmaceutical composition may comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier to be contained in the pharmaceutical composition is one usually used in the present invention, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, no. The pharmaceutical composition may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition may be administered orally or parenterally (for example, intravenous, intraperitoneal, intramuscular, subcutaneous, or topical).

The appropriate dosage of the pharmaceutical composition may be variously prescribed depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient have. On the other hand, the dosage of the pharmaceutical composition may preferably be 0.001-100 mg / kg (body weight) per day.

The pharmaceutical composition may be prepared in a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

EXAMPLES Example 1 Identification of the role of PTPσ as a synaptic receptor in the GPC-4 / LRRTM4 adherent complex

(Construction of expression vector)

1. Glypicans

The following glypicane structures are described in de Wit et al. Neuron. Vol. 79, pp.696-711:

pDisplay-GPC-1; pDisplay-GPC-3; pDisplay-GPC-4; pDisplay-GPC-5; pDisplay-GPC-6;

pDisplay-GPC-4 AAA: GPC-4 mutation in which three heparan sulfate GAG attachment sites were mutated to alanine;

pDisplay-GPC-4 351-AISA: protease cleavage conserved sequence R ³⁵¹ ISR ³⁵⁴ GPC-4 mutated to A ³⁵¹ ISA ³⁵⁴ ; And

p3CPro-GPC-4: Encoding residues 1-31 of rat Caspr2 and 23-528 residues of glypicane-4 of mouse fused to human IgG Fc fragments.

2. LAR-RPTPs

pDisplay-PTP σ encodes the full-length sequence of human PTPσ (Genbank accession No. BC104812.1) except for the signal peptide (amino acid [aa] 30-1501), and Bgl II 　 And a Sal I restriction enzyme site.

pDisplay-PTPσ [Delta] Ig encodes a fragment of PTP [sigma] (aa 305-1501; 3 Ig domains deleted) and Bgl II 　 And Sal I Is cloned into the pDisplay vector by restriction enzyme sites.

pDisplay-PTPσ AAAA is identical to pDisplay-PTPσ except that the four lysine residues in the Ig1 domain of PTPσ are mutated to alanine to block the GAG-binding site.

pcDNA3.1-HA-PTP σ encodes the full-length sequence of human PTP σ except for the signal peptide (aa 21-1912) (Genbank accession No. L38929.1), and the neuroligin-2 signal peptide (aa 1-14) and the HA epitope Lt ; RTI ID = 0.0 &gt; NotI & lt ; / RTI & gt ; and Xba I restriction sites.

pDisplay-LAR encodes the full-length sequence of a human LAR (Genbank accession No. AB177857.1) except for the signal peptide (aa 30-1907), and Bgl II 　 And the Sal I restriction enzyme site.

To construct a lentiviral expression vector coding for PTPσ, the human whole-length PTPσ wild type (WT) and PTPσ AAAA to which HA was attached were PCR amplified, digested with Nhe I and BsrG I , and ligated into L-313 vector, Was cloned into a lentiviral shRNA vector containing the human H1 promoter and two human U6 promoters (Ko et &lt; RTI ID = 0.0 &gt; al . J. Cell Biol . 194: 323-334, 2011). PTis-PTP σ Ig1-3 and pDis-PTPδ (SEQ ID NO: 1) inserts were generated by PCR using the following primers: MeA (ETFESTPIR for PTP σ; ESIGGTPIR for PTP δ; GGSPIR for LAR) and / or MeB (ELRE for PTP σ and PTP δ; DQRE for LAR) Splice variants of the LAR-RPTP family were produced by cloning into the Ig1-3 vector to produce splice variants, Ig1-3, Ig1-3 ^MeA , Ig1-3 ^MeB and Ig1-3 ^MeAB , respectively, for PTP ^? , PTP? ^Or LAR .

On the other hand, pDisplay-TrkC encodes the full-length region of mouse TrkC (Genbank accession No. AY336094.1) except for the signal peptide (aa 32-825) and was cloned into the pDisplay vector by Xma I and Sac II restriction enzyme sites. The following structures are described in the references: pCMV5-LRRTM2-mVenus, pEGFP-N1 LRRTM4, and pCMV5-IgC (Ko et al. Neuron . 64: 791-798, 2009); pDisplay-Slitrkl, L-309 PTP?, and L-309 LAR (Yim et al., Proc . Natl . Acad . Sci . USA, 110: 4057-4062, 2013); pGW1 myc-Syndecan-2 (received from Dr. Yiping Hsueh); And pDisplay-Syndecan-3 (received from Dr. David Guido).

Antibodies were purchased and used commercially available antibodies: mouse monoclonal anti-HA (clone HA-7; Covance), goat polyclonal anti-EGF (Rockland), rabbit polyclonal anti-synapsin I (Millipore) Monoclonal anti-heparan sulfate delta (clone 3G10; USBiological) and mouse monoclonal anti-tubulin (clone DM1A; Hybridoma Bank).

(Production and purification of recombinant protein)

For expression of recombinant proteins, HEK293T cells were cultured in a 10-cm dish to 50-70% confluence and then transfected with 10 [mu] g of cDNA corresponding to FuGene (Roche) and various Ig proteins. A medium containing soluble Ig-fusion protein was obtained 3-4 days after transfection and centrifuged at 1000 x g. The supernatant was then adjusted with 20 mM HEPES-NaOH (pH 7.4), 1 mM EDTA and a protease inhibitor cocktail (ThermoScientific) and incubated with protein A-Sepharose (GE Healthcare) overnight to bind the human IgG Fc domain. The beads were washed with PBS (phosphate buffered saline) to remove unbound protein, and the bead-bound protein was eluted with 0.1 M glycine (pH 2.5).

(Affinity chromatography and in-gel digestion)

Two frozen mouse brains were homogenized by 6-8 strokes with 20 mL of homogenization buffer [320 mM sucrose, 10 mM HEPES (pH 7.4), 1 mM PMSF and protein inhibitor cocktail] and lysed buffer [50 mM Tris , 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 0.5% Na-deoxycholate, 17 mM PMSF, protein inhibitor cocktail and 1% NP-40] and centrifuged at 100,000 × g for 1 hour at 4 ° C Insoluble matter was precipitated. Protein A-Sepharose beads (GE Healthcare) or IgC alone conjugated with 25 mg of Ig-PTPσ were equilibrated with lysis buffer and incubated overnight at 4 ° C with 2 mg of brain extract followed by centrifugation (800 × g, 5 Min) and washed five times with lysis buffer. The bead-bound protein was eluted with SDS sample buffer. The eluted proteins were digested with sequencing-grade modified trypsin (Promega) and mass spectrometry was performed. The only band observed in the Ig-PTPσ lane was directly cut from the gel and the stain was removed with 5 mg / mL dithiothreitol (DTT) dissolved in 50 mM ammonium bicarbonate (ABC) and dried in a speed vacuum concentrator. The dried gel pieces were dehydrated with 50mM ABC containing 100 占 퐂 / mL trypsin and incubated at 37 占 폚 for 16 hours. The supernatant peptide mixture was extracted with 50% ABC in 5% formic acid (FA) for 30 min, dried in a speed vacuum concentrator and used for mass spectrometry.

(Mass analysis)

The mass spectrometry was performed using an Agilent HPLC-Chip / TOF MS system equipped with an Agilent 1260 nano-LC system, a HPLC Chip-cube MS interface and a 6530 QTOF single quadrupole-TOF mass spectrometer on the trypsin treated and dried samples. Dried peptide samples were resuspended in 2% ACN / 0.1% FA and concentrated on large volume HPLC chips (Agilent Technologies). The HPLC chip was integrated into an enrichment column (9 mm; 75 urn ID; 160 nL) and then integrated into a reverse-phase column (15 cm; 75 urn ID; filled with Zorbax 300SB-C18 5 urn resin). Peptide separation was performed using a 70-minute gradient of 3-45% buffer A (0.1% FA) with 3-45% buffer B (90% ACN / 0.1% FA) at a flow rate of 300 nL / min. MS and MS / MS data were acquired in positive ion mode and data was stored in centroid mode. The chip spray voltage was set at 2100V and chip conditions were used. The gas drying temperature was set at 325 캜 at a flow rate of 3.5 L / min. A medium separation window (4m / z) was used for precursor separation. For fragmentation, the collision energy with a slope of 3.7V / 100Da and an offset of 2.5V was used. MS data were acquired over a mass range of 300-3000 m / z, while MS / MS data were obtained over a mass range of 50-2500 m / z. The reference mass compensation was activated using a 922 reference mass. The precursor was set on the exclusion list for 30 seconds after acquiring two MS / MS spectra. The MS / MS spectra were extracted using the Mass Hunter Qualitative Analysis B.05.00 software (Agilent Technologies) under the default parameters and the spectra were analyzed using Mascot v2.3 to search against the Uniprot / Swiss-Prot database (10/10/2013) (Matrix Science, London, UK). Individual ion scores of > 25 were employed when identifying or exhibiting broad homology ( p < 0.05). Database searches were performed with peptide mass tolerance of 10 ppm, MS / MS tolerance of 0.5 Da and stringent trypsin specificity allowing two deleted cleavage sites. Carbamidomethylation of Cys was set as a fixed strain, and oxidation (M) was considered as a variable strain.

(Saturated ligand binding assay)

Weakly in DMEM containing transfected (as added by the serial dilutions) of the switching HEK293T cells 50mM HEPES-NaOH (pH7.4), 2mM CaCl 2, 2mM MgCl 2, Ig fusion proteins of 0.1% BSA, and a concentration Lt; RTI ID = 0.0 &gt; 4 C &lt; / RTI &gt; for 16 hours. The cells were washed three times with cold DMEM to remove excess Ig protein and fixed on ice with 4% paraformaldehyde for 10 min. The cells were washed again with cold PBS and incubated in a blocking solution containing PBS and 3% BSA at room temperature for 15 minutes. HRP-conjugated rabbit anti-human IgG antibody was added (diluted 1: 40,000 in blocking solution) and incubated again for one hour. The cells were washed three times with blocking solution, washed again with PBS, and then subjected to a colorimetric 3,3 ', 5,5'-tetramethylbenzidine peroxidase enzyme immunoassay (Bio-Rad) according to the manufacturer's instructions.

In summary, the provided HRP substrate solution was added to each well and the samples were incubated at room temperature for 15 minutes with vigorous stirring until blue appeared. Equal amounts of 1N sulfuric acid were added to terminate each reaction to make it yellow. The absorbance at 450 nm was measured in a 24-well plate using Benchmark Plus (BioRad) and the ligand concentration for the difference in absorbance measured between the plasmid and mock-infected cells was displayed. The dissociation constant (Kd) values were calculated from the data using Scatchard analysis.

(Cell-surface binding assay)

Boucard et al., J. Biol . Chem . 287: 9399-9413, 2012 to produce IgC-fusion protein and PTP? Of GPC-4 in HEK293T cells. Soluble IgC protein was purified using protein A-sepharose beads (GE Healthcare), eluted and immediately neutralized with 1 M Tris-HCl (pH 8.0). Transformed HEK293T cells expressing full-length PTPσ or pDisplay-PTPσ, pDisplay-PTPσ, or pDisplay-LAR selective splice variants with HA attached were incubated with 0.2 μM IgC or IgC-GPC-4 as described. For heparinase treatment, heparinase III (Sigma) or vehicle [20 mM Tris-HCl (pH 7.5), 0.1 mg / mL BSA, 4 mM CaCl ₂ ] was added to the transformed HEK cells at 37 ° C for 2 hours Lt; / RTI &gt; Images were acquired in a confocal microscope (LSM 510; Zeiss).

(Cell-adhesion assay)

HEK293T cells were transformed with the expression vectors described above. After 48 hours of transformation, cells were isolated, mixed and incubated at room temperature with gentle agitation. To analyze the extent of cell fusion, they were aliquot separated and they were spotted onto a 4-well culture slide and confocal microscopy imaging was performed. Quantification is performed as described in Lee et al ., Proc . Natl . Acad . Sci . US A. 110: 336-341, 2013.

(Full-down analysis)

pDisplay-GPC-3, pDisplay-GPC-4, pDisplay-GPC-4, pDisplay-GPC-4, pDisplay-GPC-3, pDisplay-GPC- 10 μg of Ig-PTPσ, Ig-GPC-4 or IgC (control) was mixed with HEK293T cells transformed with pDisplay-GPC-6, pDisplay-GPC-4 AAA, or pDisplay- Lt; / RTI &gt; for 2 hours. Antibodies used for immunoblotting were anti-HA (1: 1000) and anti-EGF (1 μg / mL).

(Production of recombinant lentivirus)

Recombinant lentiviruses have been described previously (Um et al. Cell Rep . 3-plasmid-L-309 vector (L-309 PTP? -KD, L-309 LAR-KD or L-309 alone), psPAX and FuGene-6 (Roche) were produced in HEK293T cells transfected with triplicate using pMD2G. The psPAX and pMD2G plasmids encode essential elements for packaging viral particles. After 48 hours of transfection, the HEK293T cell supernatant was recovered and purified as previously described (Yim et &lt; RTI ID = 0.0 &gt; al ., Proc. Natl . Acad . Sci . USA . 110: 4057-4062, 2013). Cultured rat hippocampal neurons were infected in vitro for several days (DIV) and recovered in DIV12-13. Quantitative RT-PCR (qRT-PCR) was used to measure knockdown efficiency (Ko meat al . J. Cell Biol . 194: 323-334, 2011).

(Heterogeneous synapse-formation analysis)

HEK293T cells were transformed with pCMV5-LRRTM2-mVenus, pEGFP-N1-LRRTM4, or EGFP (negative control) using FuGene (Roche). Forty-eight hours later, transformed HEK293T cells were treated with trypsin, seeded into hippocampal neurons cultured in DIV9, co-cultured for 48 hours, and immunostained with DIV11 using antibodies against EGFP and synapsin I Ko et al . Neuron . 64: 791-798, 2009). Images were obtained from a confocal microscope (LSM510; Zeiss). For quantification, the outline of transformed HEK293T cells was selected as the region of interest. Using the MetaMorph Software (Molecular Devices), the fluorescence intensities of the normalized synapse points for each HEK293T cell area for both the red and green channels were quantitated.

(Primary neuron culture and immunocytochemical analysis)

Cultured hippocampal neurons were prepared from previously described embryonic 18-day-old rat brain (E18) (Ko et al ., J. Neurosci . B-27 (Invitrogen), 0.5% fetal bovine serum, 0.5 mM Glutamax (Invitrogen) and sodium pyruvate (Invitrogen) were incubated in a cover slip coated with poly-L- Lt; RTI ID = 0.0 &gt; Neurobasal &lt; / RTI &gt; For immunocytochemical analysis, the cultured neurons were fixed with 4% paraformaldehyde / 4% sucrose, permeabilized with 0.2% Triton X-100 dissolved in PBS, immunostained with primary antibody, FITC-conjugated secondary antibody (Jackson ImmunoResearch).

(Electrophysiological experiment)

Cell culture electrophysiology experiments were performed as described in the references (Um et al . Cell Rep . 6: 1096-1109, 2014). DIV14-16 rat hippocampus. For the whole-cell voltage clamp recording, a patch pipette (4-7 MΩ) was filled with the following internal solution: 90 mM Cs-gluconate, 10 mM CsCl, 10 mM HEPES, 10 mM EGTA, 5 mM NaCl, 4 mM Mg- Na-GTP, (pH 7.2). Recording was performed in Hepes buffer saline (HBS) having the following composition: 100mM NaCl, 4mM KCl, 1mM NaH 2 PO 4, 20mM HEPES, glycine 10μM, 10mM glucose, 2mM CaCl _2, and 1mM MgCl _2, (pH 7.4) . mEPSCs were recorded at a holding potential of -70 mV with 1 μM tetrodotoxin (TTX; AbcaM), 100 μM picrotoxin, and 50 μM APV. For double-pulse recording, a bipolar electrode plate (Inter Medical) was placed at 100 占 퐉 from the sole of a patched neuron and stimulated at intervals of 100 ms between stimulations. The amplification of the second EPSC was measured relative to the amplification of the first EPSC. To measure cell membrane statistics in each experiment, three 1-minute traces were collected between each trace with a -2mV voltage step. For all the whole-cell recordings, cell membrane statistics were measured after each trace. The following full-cell recording criteria were used: Ra was <MΩ, and when the Ra or Rm was changed by more than 20% throughout the course of the experiment, the cells were discarded. All records were digitized at 10 kHz and filtered at 2 kHz. The records were measured using EPC10 double USB (HEKA) and analyzed offline using the Mini Analysis Program (Synaptosoft). During all data collation and analysis, the experimenter did not see it for identification of the structure.

On the other hand, SDS-PAGE and immunoblotting were performed using standard procedures and all data were expressed as mean ± SEM. All experiments were repeated at least 3 independent cultures and data were statistically evaluated using the ANOVA Tukey's test.

Tables 1 to 3 list the specific list of GPC-4 peptides as PTP? Specific binding proteins in affinity chromatography in rat brain.

Example 1 Identification of LAR-RPTPs Ligand

To identify additional ligands for LAR-RPTPs in vivo, the PTP sigma ligand was searched because the LAR-RPTPs were most expressed in the rat brain. To this end, an expression vector encoding the PTPj extracellular domain fused to the Fc domain of human immunoglobulin (PTP? -IgC) and a human Fc domain alone coding (IgC; Figure 1A) were generated as a control protein. These Ig fusion proteins were fixed and subjected to affinity chromatography using rat brain synaptosome (FIG. 1B). Proteins from each silver-dyed gel slice showing a unique band were purified on fixed PTPs and mass spectrometry was performed (FIG. 1B). Among the identified peptides (see Tables 1 to 3), three were derived from glypicane-4 (GPC-4) (Fig. 1C-F).

To confirm whether GPC-4 directly binds to PTPσ at the cell surface, binding analysis between HEK293T cells expressing PTPσ with the recombinant Ig-fusion protein (Ig-GPC-4) of GPC-4 and HA (Fig. 1G). EGFP-fused LRRTM4 (FIG. 1G: de Wit et al., Neuron . 79: 696-711, 2013) and mVenus-fused LRRTM2 were expressed in HEK293T cells as positive and negative controls, respectively.

As a result, it was found that PTP? And LRRTM4 bind strongly to GPC-4, but LRRTM2 did not (Fig. 1G). Furthermore, it was confirmed that there was no action between LRRTM4 and neurexin-1 (Fig. 2). In addition, GPC-4 did not bind to all the cell surface-proteins used in the experiment, suggesting that GPC-4 forms a specific interaction with PTP (Fig. 3A).

In order to measure the binding affinity between GPC-4 and PTPσ, HEK293T cells expressing HA-PTPσ and control HEK293T cells were cultured with increasing amounts of Ig-GPC-4, The bound protein was measured. After identifying the nonspecific binding, Scatchard analysis was performed assuming a single independent binding site for GPC-4 at each PTP σ to obtain a Kd of 41.3 ± 3.6 nM (FIG. 1H). This result suggests that GPC-4 binds to PTPσ with a high affinity, although the dimeric GPC-4 ligand used may produce increased affinity for the action, although careful interpretation is required.

Example 2: Discovery of the PTPσ domain interacting with GPC-4

(PTP? -Lag), Ig domain alone (PTP? -Ig), or Ig-domain deleted protein (PTP? -ΔIg) to measure which PTPσ domain works with GPC-4 Cell-surface binding assay was performed using the construct (Fig. 4A).

As a result, IgC-GPC-4 binds to HEK293T cells expressing PTP? -Electric and PTP? -Ig, not PTP? -ΔIg (FIG.

In recent studies, It has been demonstrated that selective splicing events in the Ig domain of LAR-RPTPs determine their binding affinity for post-synaptic ligands. Thus, it was tested whether the GPC-4 / PTPσ interaction was modulated by a similar selective splicing insert (FIG. 4C).

GPC-4 strongly binds to four splice variants of PTP? And three splice variants of LAR (Fig. 4C), but weaker binding to four splice variants of PTP? (Fig. 4D and E).

To evaluate the affinity of these GPC-4 and PTPσ mutants for interaction, PTPσ variants were expressed on the surface of HEK293T cells and the binding affinities of other PTP isoforms were measured (FIGS. 4F and 3B). All PTPσ variants showed comparable nanomolar affinities to full-length PTPs (FIG. 4F and FIG. 3B).

These results suggest that selective splicing of LAR-RPTPs does not regulate their binding affinity to GPC-4 itself.

Example 3: Detection of GPC-4 / PTP? Binding site

GPC-4 / PTPσ interaction was observed in cell-surface binding assays (Fig. 1-4), which increased the likelihood that such binding could mediate trans-cellular adhesion, as observed in synapses. Therefore, we decided to perform cell-adhesion experiments (Figs. 5A and B). For this, L-cells (red fluorescent cells) expressing DsRed alone (control group) or DsRed and PTPσ simultaneously, and EGFP alone (control) or EGFP and GPC-4 (GPC-4) or TrkC L-cells expressing simultaneously were prepared. These cells were mixed, cultured for up to 60 minutes, and cell aggregation was measured (Fig. 5B).

As a result of quantification, cells expressing GPC-4 did not form any agglomerates with cells expressing PTP (Fig. 5B). In contrast, cells expressing TrkC formed strong aggregation with cells expressing PTPσ, consistent with previously reported trans-interactions (Figure 5A and B: Takahashi et &lt; RTI ID = 0.0 &gt; al ., Neuron . 69: 287-303, 2011).

These results suggest that the GPC-4 / PTPσ interaction only occurs in the cis-arrangement.

Example 4 Identification of GPC-4 / PTP?

It has already been shown that HS chains of HSPGs such as PTP? Agrin and collagen XVIII bind to and coexist in cells such as HSPGs in sensory neurons. Thus, we tested whether the HS chains of GPC-4 mediate their binding to PTP ?. To this end, a PTPσ construct (PTPσ-AAAA) in which four lysins (K67, K68, K70 and K71) of the primary Ig domain were all replaced with alanine to prevent HS-binding (PTPσ-AAAA) was generated (FIG. 5C). Then, the HS chain was removed from the PTP sigma by expressing either PTPσ wild type (WT) or PTPσ-AAAA in HEK293T cells and treating the cells for 2 hours with heparinase III (hep III; 1 U / mL) Cells were stained with monoclonal 3G10 antibody reacting only with the III-treated HS chain.

As a result, cells expressing PTPσ WT, but not PTPσ-AAAA, responded to the 3G10 antibody (Fig. 5D).

In addition, to test whether the HS chain attached to PTP? Is necessary to interact with GPC-4, PTP? WT-expressing HEK293T cells were treated with hep III and Ig-GPC-4 was used in the cell- (Fig. 5E).

Hep III treatment dramatically reduced Ig-GPC-4 binding to PTP (Figure 5E). This implies that the HS chain of PTPσ is required for this action. Consistent with this, PTPσ-AAAA failed to bind to GPC-4 (FIG. 5E). At the same time, these results clearly demonstrate that HS-binding ability of PTP? Is required for interaction with GPC-4 (Fig. 5E).

Finally, neurexins / neuroligins or including those mediated by neurexins / LRRTM2 many cell-adhesion interactions are extracellular Ca ^{2 +} - Because the ion requires, GPC-4 / PTPσ interaction is required for Ca ^{2 +} .

However, it was found that Ca ^{2 +} -chelator EGTA did not affect their binding (Fig. 5F). This suggests that the PTPσ interaction with GPC-4 is Ca ^{2 +} -independent, similar to the PTPσ reaction with other ligands.

<Example 5> GPC-4 / PTPσ interaction experiment using truncated GPC

To further demonstrate the GPC-4 / PTPσ interaction (Fig. 1-5), lysates of HEK293T cells expressing HA-PTPσ, HA-LAR, HA-PTP? Or NL1- 4 or IgC (negative control) was used to perform a pull-down analysis.

Ig-GPC4 captured all three LAR-RPTP isoforms, but not PTPσ-AAAA or NL1 (FIG. 5G).

In addition, Ig-PTP? Or IgC was used to perform a pull-down analysis on HEK293T cells expressing GPCs with HA attached thereto.

GPC-1 and GPC-5 vectors were not efficiently processed in HEK293T cells and no band at ~ 37 kDa was observed in HA-GPC-1 or HA-GPC-5-transformed cell lysates. In contrast, other GPCs showed both the ~65 kDa band representing the uncut form and the ~37 kDa band representing the truncated form (Figure 5H). As a result of the full-down analysis, the fixed Ig-PTPσ binds to the cleaved GPCs effectively but not to the uncleaved GPC (Fig. 5H). This binding property differs from that of GPC-4 / LRRTM4 interactions that bind to LRRTM4 both in uncut and in truncated GPCs. Surprisingly, other HSPG family SDCs (SDC-2 and SDC-3) failed to bind to PTPσ (not shown) and LAR-RPTPs appear to use evolutionarily different strategies in mediating synaptic attachment LAR-RPTPs in mammals prefer GPCs, while dLARs combine with both GPC and SDC). As a result of mutating all of the GPC-4 mutants (GPC-4 AAA, GPC-4 [S494 / S495 / S500] HS-attachment site) in a manner consistent with the above-described results (Fig. 5E) (Fig. 5H).

Example 6 Identification of the role of PTPσ in LRRT4-mediated pre-synaptic differentiation of cultured neurons

Direct reaction of GPCs with LAR-RPTPs, direct reaction of LRRTM4 with GPCs (de Wit et al. Neuron . 79: 696-711, 2013; Siddiqui et al ., Neuron . 79: 680-695, 2013) and on the biochemical properties of GPCs as GPI-anchor proteins, we hypothesized that LAR-RPTPS could be a functional pre-synaptic receptor for LRRTM4. To directly address this hypothesis, lentiviral shRNAs for PTPσ (sh-PTPσ) or LAR (sh-LAR), which have already been characterized, were used (Yim et al . Proc . Natl . Acad. Sci . USA . 110: 4057-4062, 2013). Cultured hippocampal neurons were infected with lentivirus expressing control lentivirus (control) or sh-LAR or sh-PTPσ, lentivirus (sh-LAR / sh-PTPσ) expressing LAR-KD and PTP? It was infected together. A variety of heterologous synapse-forming assays were performed using HEK293T cells expressing infected neurons and only LRRTM2, LRRTM4, or EGFP (control).

As shown in Figure 6, only PTPσ-KD significantly decreased the synaptic activity of LRRTM4, which is not LRRTM2 activity. Re-expression of PTPσ WT (+ PTPσ WT) completely reverses the deficiency in the synaptic-forming activity of LRRTM4 observed in LAR / PTPσ-deficient neurons, but re-expression of PTPσ-AAAA (+ PTPσ-AAAA) (Fig. 6). Suggesting that the HS-dependent interaction of GPC and PTP? Is essential for inducing the differentiation of pre-synapses derived by LRRTM4 (FIG. 5E and FIG. 6).

These results indicate that PTPσ acts as a synaptic receptor for LRRTM4 as a whole.

Example 7: Role of PTPσ HS-binding sequence in excitatory synaptic transmission in cultured neurons

Most LAR-RPTP ligands, when expressed in xenogeneic cells, directly interact with the individual LAR-RPTP isoforms in the axons of co-cultured neurons, proving to aggregate synaptic vesicles and neurotransmitter release devices , Suggesting that LAR-RPTP acts as a hub for pre-synaptic organization. In addition to the failure of PTPσ-AAAA to restore the synaptic activity of LRRTM4, this also indicates that the HS-binding properties of LAR-RPTP are also related to certain pre-synaptic functions (e.g., neurotransmitter release or neurotransmission) It raised questions. Surprisingly, none of the previous studies have examined whether LAR-RPTPs themselves are involved in the pre-synaptic function of mammalian neurons.

To address these questions, first neurotransmitter to identify a change in the discharge potential was generally confirmed paired-pulse ratio (PPR) the measurement method used (Branco and Staras, Nat Rev Neurosci 10:. .. 373- 383, 2009). For this purpose, control lentivirus (control) was infected with cultured hippocampal neurons, or infected with lentiviruses expressing sh-LAR / sh-PTPσ, and PPR was measured. Two stimulations were performed for 100 msec And the amplitude of the second excitatory post-synaptic current (EPSC ₂ ) was calculated by dividing by the amplitude of the first EPSC (EPSC ₁ ).

There was no significant change in PPR between control and LAR / PTPσ-deficient neurons. This suggests that LAR and PTPσ do not directly regulate the neurotransmitter release potential in excitatory synapses (FIGS. 7A and B).

Next, miniature excitatory post-synaptic currents (mEPSCs) were recorded in cultured hippocampal neurons (FIG. 8). To this end, a control lenti virus expressing EGFP alone (control), lentivirus expressing sh-LAR / sh-PTPσ together with sh-LAR / sh-PTP sigma with either PTPσ WT or PTPσ-AAAA The nerve was infected with lentivirus and mEPSCs were recorded.

Surprisingly, sh-LAR / sh-PTP sig- nificantly reduced the frequency and amplitude of mEPSCs. This reinforces our previous assumption that PTP? Is required for excitatory synaptic development (Yim et &lt; RTI ID = 0.0 &gt; al ., Proc . Natl . Acad . Sci . USA . 110: 4057-4062, 2013). The decrease in mEPSC frequency and amplitude among the LAR / PTPσ-deficient neurons was completely reversed by re-expression of PTPσ WT, rather than PTPσ-AAAA (Fig. 8).

It is believed that the experimental conditions do not affect the internal properties of the infected neurons because they could not observe changes in membrane capacitance (Cm) or input resistance (Rm) (Figs. 7C and D). Since sh-LAR / sh-PTPσ does not impair the potential for neurotransmitter release, this result suggests that LAR and PTPσ modulate the density of functional excitatory synapses in an HS-binding dependent manner.

Claims (12)

Cellular and protein components capable of forming a complex of GPC-4 (glypican-4) and LAR-RPTPs (leukocyte common antigen-related receptor protein tyrosine phosphatases) through cis-cell adhesion &Lt; / RTI &gt;
The cell component is selected from among GPC-4 expressing cells or LAR-RPTPs expressing cells,
Wherein said protein component is a wild type or recombinant GPC-4 protein comprising heparan sulfate, or a fragment thereof, or
A wild-type or recombinant LAR-RPTPs protein comprising a heparan sulfate binding site, or a fragment thereof, for the prevention or treatment of a disease associated with membrane protein dysfunction.
The method according to claim 1,
Wherein the GPC-4 expressing cell is a wild-type or recombinant IgC-GPC-4 comprising heparan sulfate, or a cell transformed to express a fragment thereof.
The method according to claim 1,
Wherein the LAR-RPTPs expressing cells are cells transformed to express wild-type or recombinant LAR-RPTPs, or fragments thereof, comprising a heparan sulfate binding site.
The method according to claim 1,
Wherein the fragment of the wild type or recombinant LAR-RPTPs protein is an Ig domain of PTP? Protein or PTP ?.
The method according to claim 2 or 3,
The cell used for transformation is selected from HEK293T (Human Embryonic Kidney 293T) cells, L-cells, COS-7 cells or HeLa cells.
The method according to claim 1,
Diseases associated with membrane protein dysfunction include, but are not limited to, autism, diabetes mellitus, Wilms tumor 1, Simpson-golabi-behmel syndrome type 1, dysplasia Neoplasms, Gigantism, Carcinoma, Cartilaginous Exostosis, Malignant Neoplasms, Anxiety, Genetic Diseases (X-linked), Beck Chromosomal Translocation, Limb Deformities, Congenital, Intraepithelial Neoplasia, Cardiac Arrhythmia, Neurodegenerative Disorders, Chronic Chromosomal Syndrome, Chromosomal Translocation, , Cervical dysplasia (Uterine Cervical Neoplasm), bone dysplasia (Bone Neoplasms), or hydrocephalus.
Cell components and protein components capable of forming a complex of GPC-4 (glypican-4) and LAR-RPTPs (leukocyte common antigen-related receptor protein tyrosine phosphatases) through cis- Contacting the substrate with any candidate material under conditions to form the complex; And
If the candidate substance is assessed as promoting or inhibiting the formation of the GPC-4 / LAR-RPTPs complex and is assessed to promote GPC-4 / LAR-RPTPs complex formation, Determining a therapeutic drug,
The cell component is selected from among GPC-4 expressing cells or LAR-RPTPs expressing cells,
Wherein said protein component is a wild type or recombinant GPC-4 protein comprising heparan sulfate, or a fragment thereof, or
A wild-type or recombinant LAR-RPTPs protein comprising a heparan sulfate binding site, or fragments thereof, for screening candidate substances for the prophylaxis or treatment of diseases associated with membrane protein dysfunction.
8. The method of claim 7,
Wherein the GPC-4 expressing cells are cells transformed to express wild-type or recombinant IgC-GPC-4, or fragments thereof, comprising heparan sulfate.
8. The method of claim 7,
Wherein the LAR-RPTPs expressing cells are cells transformed to express wild-type or recombinant LAR-RPTPs, or fragments thereof, comprising a heparan sulfate binding site.
8. The method of claim 7,
Wherein the fragment of the wild-type or recombinant LAR-RPTPs protein is an Ig domain of PTP? Protein or PTP ?.
10. The method according to claim 8 or 9,
Wherein the cells used for transformation are selected from HEK293T (Human Embryonic Kidney 293T) cells, L-cells, COS-7 cells or HeLa cells.
8. The method of claim 7,
Diseases associated with membrane protein dysfunction include, but are not limited to, autism, diabetes mellitus, Wilms tumor 1, Simpson-golabi-behmel syndrome type 1, dysplasia Neoplasms, Gigantism, Carcinoma, Cartilaginous Exostosis, Malignant Neoplasms, Anxiety, Genetic Diseases (X-linked), Beck Chromosomal Translocation, Limb Deformities, Congenital, Intraepithelial Neoplasia, Cardiac Arrhythmia, Neurodegenerative Disorders, Chronic Chromosomal Syndrome, Chromosomal Translocation, , Cervical dysplasia (Uterine Cervical Neoplasm), bone dysplasia (Bone Neoplasms), or hydrocephalus.

Images