Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

CCN1 compositions and methods

A kind of analog, homologous technology, applied in the field of anti-CCN1 antibody, CCN1 related peptide, its composition, extracellular matrix signal transduction molecule

Inactive Publication Date: 2006-09-20
MUNIN
View PDF5 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Targeted disruption of the CCN1 gene in mice results in embryonic death due to lack of blood vessels, whereas CCN2-null mice die perinatally due to respiratory failure due to skeletal malformations

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • CCN1 compositions and methods
  • CCN1 compositions and methods
  • CCN1 compositions and methods

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

Domain III (TSPI-homology domain) of CCN1 supports α 6 beta 1 dependent cell adhesion

[0064] Previous studies identified primary human dermal fibroblasts through integrin α 6 beta 1 and heparan sulfate proteoglycans adhere to CCN1, inducing the formation of α-containing 6 beta 1 Foci complexes and activation of focal adhesion kinases, paxillin and Rac. See Chen et al. (2000) J. Biol. Chem. 275, 24953-24961; Chen et al. (2001) J. Biol. Chem. 276, 10443-10452. Deletion analysis reveals that a C-terminally truncated CCN1 mutant containing only the first three domains is retained in smooth muscle by integrin α 6 beta 1 Ability to induce chemotaxis, thus positioning integrin binding sites in the first three domains. See Grzeszkiewicz et al (2002) Endocrinology 143, 1441-1450.

[0065] In order to define the relationship with integrin alpha 6 beta 1 interacting CCN1 domains, we expressed each of these three domains in insect cells via a baculovirus vector ( figure 1 A)....

Embodiment 2

T1 sequence in domain III of CCN1 contains integrin α 6 beta 1 binding site

[0071] We used an additional systematic screening strategy to pinpoint integrin α in CCN1 6 beta 1 binding site. A series of overlapping peptides covering the entire first three domains of CCN1 were prepared by expressing the peptides as fusion proteins linked to GST (Table 1). By polymerase chain reaction (PCR), using CCN1 cDNA as a template to amplify various peptides encoding ( image 3 ) coding sequence. The primers used corresponded to the appropriate coding sequences and contained BamHI and EcoRI restriction sites for cloning. For example, the following primers were used to generate the T1 peptide coding sequence:

5'-CGGGATCCGCGGGCCAGAAATGCATCGTT-3' and

5'-CCGGAATTCCGCTCTTGGAGCACTGGGACC-3' The PCR product was purified on a polyacrylamide gel, digested with BamHI and EcoRI, and ligated in pGEX-4T-2 vector (Amersham Pharmacia Biotech). All cloning steps were confirmed by sequence analys...

Embodiment 3

Soluble T1 peptide inhibits alpha 6 beta 1 dependent cell adhesion

[0074] We predict that soluble T1 peptides are capable of blocking cell adhesion to integrins known to bind alpha 6 beta 1 substrate. Such as Figure 5 As shown in A, addition of 0.2 mM T1 to the cell suspension effectively blocked fibroblast adhesion to CCN1 whereas T2, T3 and T4 had no effect. The inhibitory effect of T1 on cell adhesion CCN1 was dose-dependent, and the maximum inhibitory effect was obtained at 100 μM ( Figure 5 C). Other members of the CCN protein family, CCN2 (CTGF) and CCN3 (NOV), have also been shown to act through integrin α 6 beta 1 Supports fibroblast adhesion (see Chen et al. (2001) J. Biol. Chem. 276, 10443-10452; Lin et al. (2003) J. Biol. Chem. in press), corresponding among these CCN proteins There is a high degree of homology in the T1 sequence of . Figure 5 A shows that T1 also specifically inhibits cell adhesion to CCN2 and CCN3, suggesting a T1 sequence integrin α...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The angiogenesis inducer CCN1 (cysteine-rich 61, CYR61) is a secreted stromal cell protein of the CCN family, including α 6 beta 1 Ligands for various integrins. Previous studies have shown that CCN1 interacts with integrin α 6 beta 1 The interaction mediates cell adhesion of fibroblasts, endothelial cells, and smooth muscle cells, as well as migration of smooth muscle cells. Recently, we reported that CCN1-induced tubule formation in unactivated endothelial cells is also mediated by integrin α 6 beta 1 mediate. In this study, we demonstrate that human dermal fibroblasts specifically adhere to the T1 sequence (GQKCIVQTTSWSQCSKS) in domain III of CCN1, and that this process is inhibited by a 6 and anti-b 1 blocked by monoclonal antibodies. Alanine substitution mutagenesis of the T1 sequence further identified the sequence TTSWSQCSKS as mediator of α 6 beta 1 Key determinants of dependent adhesion. Soluble T1 peptide specifically inhibits fibroblast adhesion to CCN1 in a dose-dependent manner. Moreover, T1 also inhibits cell adhesion to other α 6 beta 1 Ligands, including CCN2 (CTGF), CCN3 (NOV) and laminin, but not ligands that inhibit other integrins. Furthermore, in a collagen gel matrix containing CCN1, T1 specifically inhibited the α 6 beta 1 Dependent tubule formation. To confirm that T1 directly binds integrin α 6 beta 1 , we performed affinity chromatography showing that integrin α was isolated from octyl glucoside extracts of fibroblasts on T1-conjugated Affi-gel 6 beta 1 . Taken together, these findings identify the T1 sequence in CCN1 as a novel integrin α 6 beta 1 Binding motifs for developing peptidomimetics form a detection alpha 6 beta 1 Basis for a functional role in angiogenesis.

Description

field of invention [0001] The present invention relates to materials and methods involving extracellular matrix signaling molecules involved in cellular responses to growth factors in the form of polypeptides. More specifically, the present invention relates to CCN1-related peptides, compositions thereof, and methods of using these polypeptides. The invention also relates to anti-CCN1 antibodies. Background technique [0002] Angiogenesis, or the formation of new blood vessels from existing blood vessels, is a complex process that requires the cooperation of multiple cellular events. See Risau (1997) Nature 386, 671-674. Vessel sprouting requires degradation of the basement membrane surrounding the parent vessel, migration of vascular endothelial cells toward angiogenic stimuli, proliferation and arrangement of endothelial cells into tubular structures, and fusion of new vessels into a circular circuit to provide blood supply to target tissues. See Ri...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/00C12Q1/00C07KC07K14/515G01N33/50
CPCG01N33/5088C07K14/515G01N2333/705
Inventor L·F·劳
Owner MUNIN
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com