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Partial peptide of lacritin

An amino acid and sequence technology, applied in peptide/protein components, animal/human proteins, peptide sources, etc.

Inactive Publication Date: 2011-06-01
SENJU PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] However, there has been no report so far that lacrimal protein or its fragments (partial peptides) are involved in cell-extracellular matrix adhesion
In addition, there is no report that lacrimal protein fragments have a promoting effect on the secretion of tear proteins from lacrimal acinar cells

Method used

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  • Partial peptide of lacritin
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  • Partial peptide of lacritin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] The synthesis of embodiment 1 polypeptide

[0096] Polypeptides were synthesized by solid phase synthesis. Specifically, a fluorenylmethoxycarbonyl (Fmoc) group is introduced into an amino acid and the amino acid is supported with a resin. Then, using dichloromethane as solvent, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea (HBTU) and N-methylpyrrolidone (NMP) as crosslinking The coupling agent undergoes an amide bond forming reaction. The protecting group was removed using DMF / 20% piperidine. The resulting product was purified by high performance liquid chromatography (column: ODS, solvent: water / acetonitrile / 0.05% TFA). Thus, polypeptides 1-3 consisting of the following amino acid sequences were obtained.

[0097] Polypeptide 1: VQGTAKVTSSRQELNPLKSIVEKSILLTEQALAKA (SEQ ID NO: 3)

[0098] White powder MALDI-TOF-MS calculated value: 3750.13; actual value: 3752.27; purity (HPLC A / A%) 96.207%

[0099] Peptide 2:

[0100] QGTAKVTS SRQELNPLKSIVEKSILLTEQALAKAGKGMHG...

Embodiment 2

[0105] The synthesis of embodiment 2 polypeptide

[0106] Polypeptides were synthesized by solid phase synthesis. Specifically, a fluorenylmethoxycarbonyl (Fmoc) group is introduced into an amino acid and the amino acid is supported with a resin. Also, using dichloromethane as solvent and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea (HBTU) and N-methylpyrrolidone (NMP) as crosslinking The coupling agent undergoes an amide bond forming reaction. The protecting group was removed using DMF / 20% piperidine. The resulting product was purified by high performance liquid chromatography (column: ODS, solvent: water / acetonitrile / 0.05% TFA). Thus, polypeptides 6 and 7 consisting of the following amino acid sequences were obtained.

[0107] Peptide 6:

[0108] QGTAKVTSSRQELNPLKSIVEKSILLTEQALAKA (SEQ ID NO: 1)

[0109] White powder MALDI-TOF-MS calculated value: 3653.27; actual value: 3652.28; purity (HPLC A / A%) 99.26%

[0110] Peptide 7:

[0111] EISGPAEPASPETTTTAQETSAAAVQGTAK...

experiment Embodiment 1

[0119] Experimental Example 1: Facilitation of Partial Peptides of Lacrimal Protein on Adhesion of Human Corneal Epithelial Cells to Extracellular Matrix

[0120] An extracellular matrix solution (10 μg / mL, collagen type IV: Becton, Dickinson and Company) was added to a 96-well plate (Iwaki Glass Company, Limited). The solution was incubated at 37°C for 1 hour to coat the plate with extracellular matrix. After removing the excess extracellular matrix solution, 0.1% BSA solution (Sigma-Aldrich Co.) was added to block the area not coated with the extracellular matrix. Next, the BSA solution was removed and the plate was washed twice with PBS, and the polypeptides 1-5 (concentration: 100 μg / mL) synthesized in Example 1 and Comparative Example 1 were added at 50 μL per well. Moreover, the established human corneal epithelial cells (HCE-T: can be prepared by the method described in Invest Ophthalmol Vis Sci. 4 cells / 100 μL DMEM / F12 medium / well were added to the plate. Plates wer...

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Abstract

Disclosed is a polypeptide comprising a partial sequence of lacritin, which can promote the adhesion between a cell and an extracellular matrix. Also disclosed is a polypeptide comprising a partial sequence of lacritin, which has a tear secretion-promoting activity. Specifically disclosed is a polypeptide which comprises the amino acid sequence depicted in SEQ ID NO:1 (which is a specific partial sequence of lacritin) and has an amino acid length of 70 resides or shorter. The polypeptide can promote the adhesion between a cell and an extracellular matrix. The polypeptide can also promote the tear secretion in a lacrimal glad acinar cell.

Description

technical field [0001] The present invention relates to polypeptides having a specific partial sequence of lacritin, a protein in tear fluid. The polypeptides of the present invention can promote cell adhesion, especially cell adhesion between cells and extracellular matrix. Moreover, the polypeptide of the present invention can promote tear secretion. Background technique [0002] It is known that adhesion between cells and extracellular matrix is ​​involved in various functions such as cell survival and motility. This is an essential process that controls the normal development of an individual, the maintenance of tissues, or recovery from injury or infection. Abnormalities in signaling pathways based on this cell adhesion sometimes lead to developmental abnormalities, circulatory disorders, transformation or metastasis of cells. [0003] In addition, it has been reported that when cell-extracellular matrix adhesion is inhibited, cells fall into cell death called "anoik...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/00A61L27/00C07K14/47C12N15/09
CPCA61K38/00A61K9/0048C07K14/475A61P27/02A61P43/00
Inventor 中岛毅东光佳
Owner SENJU PHARMA CO LTD
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